Expression of Ovalbumin and its Natural Variants
20230212634 · 2023-07-06
Assignee
Inventors
- Joyce Josephine Elisabeth Arnouts (Achtmaal, NL)
- María Vázquez Vitali (Gent, BE)
- Lucie Parenicová (The Hague, NL)
- Niels Lauret (Prinsenbeek, NL)
- Maloe Kleine Haar (Nijmegen, NL)
- Jos Reijngoud (Alphen aan den Rijn, NL)
- Stefan Robert Marsden (The Hague, NL)
- Wilhelmus Theodorus Antonius Maria De Laat (Breda, NL)
Cpc classification
C12N15/00
CHEMISTRY; METALLURGY
C12Y302/01003
CHEMISTRY; METALLURGY
C12P21/02
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to the animal-free production of animal-derived proteins for human consumption by expression of such proteins, e.g. ovalbumin, in fungal cells. The invention relates to fungal cells modified for the production of animal-derived proteins, such as ovalbumin and to methods wherein these cells are used for the production of animal-derived proteins.
Claims
1. A fungal host cell comprising an expression cassette comprising a nucleotide sequence coding for an animal-derived food-protein of interest, which nucleotide sequence is operably linked to at least one regulatory sequence that is capable of effecting expression of the encoded protein of interest by the fungal host cell.
2. The fungal host cell according to claim 1, wherein the regulatory sequence is the promoter of a highly expressed fungal protein.
3. The fungal host cell according to claim 1, wherein the nucleotide sequence coding for the animal-derived food-protein of interest is codon optimized with reference to the native codon usage of the highly expressed fungal protein from which the promoter is derived.
4. The fungal host cell according to claim 1, wherein the expression cassette is integrated in a locus of a gene coding for a highly expressed fungal protein.
5. The fungal host cell according to claim 1, wherein the expression cassette comprises a nucleotide sequence encoding a signal sequence from a highly expressed secreted fungal protein, and optionally a pro-sequence, operably linked in frame to the nucleotide sequence coding for the animal-derived food-protein of interest.
6. The fungal host cell according to claim 5, wherein the expression cassette encodes a fusion protein comprising, in a N- to C-terminal direction: an A. niger glucoamylase pre-pro sequence; optionally, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of the mature A. niger glucoamylase amino acid sequence; optionally, a cleavable linker polypeptide; fused to the N-terminus of the protein of interest.
7. The fungal host cell according to claim 1, wherein the host cell is a yeast or a filamentous fungus, preferably, a filamentous fungal host cell belong to a species selected from Alternaria alternata, Apophysomyces variabilis, Aspergillus spp., Aspergillus fumigatus, Aspergillus flavus, Aspergillus oryzae, Aspergillus niger, Aspergillus awamori, Aspergillus nidulans, Aspergillus terreus, Cladosphialophora spp., Fonsecaea pedrosoi, Fusarium spp., Fusarium oxysporum, Fusarium solani, Lichtheimia spp., Lichtheimia corymbifera, Lichtheimia ramosa, Myceliophthora spp., Myceliophthora thermophila, Rhizopus spp., Rhizopus microsporus, Rhizomucor spp., Rhizomucor pusillus, Rhizomucor miehei, Trichoderma spp., Trichoderma reesei, Trichophyton spp., Trichophyton interdigitale, and Trichophyton rubrum.
8. The fungal host cell according to claim 7, wherein the host cell is a strain of a filamentous fungus that has the ability to grow in a yeast-like morphology.
9. The fungal host cell according to claim 1, wherein animal-derived food-protein of interest is a hemeprotein, a milk protein or an egg protein.
10. The fungal host cell according to claim 9, wherein food-protein of interest is ovalbumin comprising an amino acid sequence with at least 50% identity to the amino acid sequence of an ovalbumin from a bird selected from the group consisting of chicken, pelican, quail, pigeon, ostrich, plover, turkey, duck, goose, gull, guineafowl, junglefowl, peafowl, partridge, pheasant, emu, rhea and kiwi.
11. A process for producing an animal-derived food-protein of interest, the process comprising the steps of: a) culturing the fungal host cell as defined in claim 1 in a medium in a fermenter under condition conducive to the expression of the protein of interest; and, b) optionally, recovery of the protein of interest.
12. The process according to claim 11, wherein in step a) the fungal host cell is cultured at a pH that is equal to or higher than pH 5.0, 5.5, 6.0, 6.5, 7.0 or 7.5, or that is equal to or lower than pH 9.0, 8.5 or 8.0.
13. The process according to claim 11, wherein in step a) the input of mechanical power into the medium in the fermenter is no more than 2.5, 2.0, 1.8, 1.6, 1.4, 1.0, 0.5, 0.2 or 0.1 kW/m3.
14. The process according to claim 11, wherein in step b) the recovery of the protein of interest comprises at least a first anion exchange step wherein the culture medium obtained in step a) is applied to an anion exchange column at an ionic strength that is less than the equivalent of 0.08 M NaCl at a pH in the range of pH 6 to 9 and wherein the protein of interest is collected in the flow-through, wherein, optionally the flow-through obtained in the first anion exchange step is subjected to a second anion exchange step under identical conditions as the first step and wherein the protein of interest is collected in the flow-through from the second anion exchange step.
15. A process for purifying an animal-derived food-protein of interest from a spent culture medium of a fungal host cell wherein the protein has been expressed, the process comprising the steps of: a) a first anion exchange step wherein the spent culture medium is applied to an anion exchange column at an ionic strength that is less than the equivalent of 0.08 M NaCl at a pH in the range of pH 6 to 9 and wherein the protein of interest is collected in the flow-through, and, b) optionally, a second anion exchange step wherein the flow-through obtained in step a) is subjected to anion exchange under identical conditions as the first step, and wherein the protein of interest is collected in the flow-through.
16. (canceled)
17. The fungal host cell according to claim 2, wherein the highly expressed fungal protein is selected from acid α-amylase, α-amylase, TAKA-amylase, glucoamylase, xylanase, cellobiohydrolase, pyruvate kinase, glyceraldehyde-phosphate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, sucrase, acetamidase, superoxide dismutase.
18. The fungal host cell according to claim 17, the promoter is an A. niger glucoamylase promoter.
19. The fungal host cell according to claim 3, wherein the highly expressed fungal protein is an A. niger glucoamylase.
20. The fungal host cell according to claim 8, wherein the strain is Aspergillus niger strain CICC2462, or a strain that is a single colony isolate and/or a derivative of strain CICC2462.
21. The process according to claim 11, wherein the food-protein of interest is ovalbumin.
Description
DESCRIPTION OF THE FIGURES
[0141]
[0142]
[0143]
[0144] A) Lanes contain the A. niger wild type, the A. niger OVA transformants carrying different expression constructs and the OVA standard as follows : 1. BZASNI.22a (shake flask); 2. GLA502 carrier OVA hen (CO glaA) with DDDK site (shake flask); 3. OVA hen (CO glaA, DWP); 4. OVA hen (CO whole genome, DWP); 5. OVA Pelican (CO whole genome, DWP); 6. OVA Quail (CO whole genome, DWP); 7. BZASNI.48 (shake flask); 8. OVA hen (CO glaA, DWP); 9. OVA Pelican (CO glaA, DWP); 10. OVA standard (0.05 g/l); 11. OVA standard (0.1 g/l); and 12. OVA standard (0.3 g/l). Samples in lanes 2 to 6 were produced in a BZASNI.22a background and sample in lanes 8 and 9 in a BZASNI.48 background. OVA stands for ovalbumin, CO for codon optimized.
[0145] B) Lanes contain the A. niger wild type, the A. niger OVA transformants carrying different expression constructs and the OVA standard as follows: 1. BZASNI.22a (shake flask); 2. GLA502 carrier OVA hen (CO glaA, shake flask) with DDDK site; 3. OVA hen (CO glaA, DWP); 4. BZASNI.48 (shake flask); 5. OVA hen (CO glaA, shake flask); 6. GLA54 OVA hen (CO glaA, shake flask) transformant 1; 7. GLA54 OVA hen (CO glaA, shake flask) transformant 2; 8. GLA100 OVA hen (CO glaA, shake flask) transformant 1; 9. GLA100 OVA hen (CO glaA, shake flask) transformant 2; 10. GLA502 OVA hen (CO glaA, shake flask) transformant 1; 11. GLA502 OVA hen (CO glaA, shake flask) transformant 2; 12. OVA standard (0.05 g/l); 13. OVA standard (0.1 g/l); and 14. OVA standard (0.3 g/l). Samples in lanes 2 and 3 were produced in a BZASNI.22a background and sample in lanes 6 to 11 in a BZASNI.48 background.
[0146]
[0147]
[0148]
EXAMPLES
General Molecular Biology Techniques
[0149] Unless indicated otherwise, the methods used are standard biochemical techniques. Examples of suitable general methodology textbooks include Sambrook et al., Molecular Cloning, a Laboratory Manual (1989) and Ausubel et al., Current Protocols in Molecular Biology (1995), John Wiley & Sons, Inc.
Strains
[0150] Aspergillus niger strain BZASNI.22a is a faster growing single colony isolate of CICC2462. BZASNI.22a was characterized by rDNA ITS sequencing and by resequencing of the genome (BaseClear, the Netherlands). Strain A. niger BZASNI.33 is a high-copy transformant of A. niger BZASNI.22a with the GLA carrier-OVA expression cassette (cloned in BZESCO.23).
[0151] BZASNI.48 is a single colony isolate of BZASNI.22a. BZASNI.60 is a high-copy transformant of A. niger BZASNI.48 with the GLA pre-pro-peptide OVA expression cassette.
[0152] For cloning purposes NEB 10-beta Competent Escherichia coli ( E. coli) cells and NEB 5-alpha Competent E. coli cells from New England Biolabs were used, which were are obtained from Bioke.
[0153] Alternative strains include, A. niger CBS513.88 (wild type), the NRRL3 strain (wild type) and the A. niger CICC2462.
Plasmids and Oligonucleotide Primers
[0154] Plasmids used in the Examples are listed in Table 1. Primers used in the Examples are listed in Table 2.
TABLE-US-00004 Listing of plasmids used in examples Name Characteristics Origin and/or SEQ ID NO pUC57-kan neomycin/kanamycin resistance marker, E. coli plasmid delivery plasmid for all synthetic genes, from Genscript (SEQ ID NO. 13) pCR™Blunt ll-TOPO® neomycin/kanamycin resistance marker, E. coli plasmid plasmid for ligation purified PCR products, from Zero Blunt™ TOPO™ PCR Cloning Kit (SEQ ID NO. 21) pGGA chloramphenicol resistance marker, E. coli plasmid Golden Gate vector, from NEB Golden Gate Assembly Kit (SEQ ID NO. 22) pBZ0024 vector with glaAp (from A. niger) SEQ ID 23 pBZ0025 vector with glaAt (from A. niger) SEQ ID 24 pBZ0028 vector with cassette glaAp -OVA (Ostrich) - glaAt SEQ ID 25 pBZ0029 Vector with cassette glaAp -OVA (Plover) - glaAt SEQ ID 26 pBZ0030 vector with cassette glaAp -OVA (Pelican) - glaAt SEQ ID 27 pBZ0031 vector with cassette glaAp -OVA (Pigeon) - glaAt SEQ ID 28 pBZ0032 vector with cassette glaAp -OVA (Quail) - glaAt SEQ ID 29 pUC57 Ampicillin resistance marker, E. coli plasmid delivery plasmid for synthetic genes, from Genscript (SEQ ID NO. 31) FXESCO.12 Hen ovalbumin (codon optimized with codon usage of A. niger glaA gene) in pUC57 SEQ ID 32 pUC57-Brick Ampicillin resistance marker, E. coli plasmid delivery plasmid for synthetic genes, from Genscript (SEQ ID NO. 42) FXESCO.14 Hen OVA construct (codon usage glaA) in pUC57-Brick SEQ ID 43 FXESCO.15 GLA carrier fusion + Hen OVA construct (codon usage glaA) in pUC57-Brick SEQ ID 44 pCNS43 hygromycin selection marker between Aspergillus nidulans tryptophan C promoter and terminator (trpC) ordered at BCCM Belgian Coordinated Collections of Microorganisms (SEQ ID NO. 45) pBZ0026 Aspergillus nidulans gpdA promoter, amdS ORF and trpC terminator SEQ ID NO. 62 pBZ0041 vector with cassette glaAp -OVA (Ostrich, CPO glaA) -glaAt SEQ ID NO. 70 pBZ0042 glaAp - OVA (Plover, CPO glaA) - glaAt SEQ ID NO. 71 pBZ0043 glaAp - OVA (Pelican, CPO glaA) - glaAt SEQ ID NO. 72 pBZ0044 glaAp - OVA (Pigeon, CPO glaA) - glaAt SEQ ID NO. 73 pBZ0045 glaAp - OVA (Quail, CPO glaA) - glaAt SEQ ID NO. 74 pUC19 ampicillin resistance marker, E. coli plasmid Cloning vector from New England Bioloabs; SEQ ID NO. 79 pBZ0061 pUC19 - 3″glaA - glaAp - OVA (hen, FXESCO.14) - glaAt SEQ ID NO. 82 pBZ0062 pUC19 - 3″glaA - glaAp - OVA (Ostrich, CPO glaA) - glaAt SEQ ID NO. 83 pBZ0063 pUC19 - 3″glaA - glaAp - OVA (Plover, CPO glaA) - glaAt SEQ ID NO. 84 pBZ0064 pUC19 - 3″glaA - glaAp - OVA (Pelican, CPO glaA) - glaAt SEQ ID NO. 85 pBZ0065 pUC19 - 3″glaA - glaAp - OVA (Pigeon, CPO glaA) - glaAt SEQ ID NO. 86 pBZ0066 pUC19 - 3″glaA - glaAp - OVA (Quail, CPO glaA) - glaAt SEQ ID NO. 87 pBZ0046 glaAp - GLA54 - OVA (Ostrich, CPO glaA) - glaAt SEQ ID NO. 102 pBZ0047 glaAp - GLA54 - OVA (Plover, CPO glaA) - glaAt SEQ ID NO. 103 pBZ0048 glaAp - GLA54 - OVA (Pelican, CPO glaA) - glaAt SEQ ID NO. 104 pBZ0049 glaAp - GLA54 - OVA (Pigeon, CPO glaA) - glaAt SEQ ID NO. 105 pBZ0050 glaAp - GLA54 - OVA (Quail, CPO glaA) - glaAt SEQ ID NO. 106 pBZ0051 glaAp - GLA100 - OVA (Ostrich, CPO glaA) - glaAt SEQ ID NO. 107 pBZ0052 glaAp - GLA100 - OVA (Plover, CPO glaA) - glaAt SEQ ID NO. 108 pBZ0053 glaAp - GLA100 - OVA (Pelican, CPO glaA) - glaAt SEQ ID NO. 109 pBZ0054 glaAp - GLA100 - OVA (Pigeon, CPO glaA) - glaAt SEQ ID NO. 110 pBZ0055 glaAp - GLA100 - OVA (Quail, CPO glaA) - glaAt SEQ ID NO. 111 pBZ0056 glaAp - GLA502 - OVA (Ostrich, CPO glaA) - glaAt SEQ ID NO. 112 pBZ0057 glaAp - GLA502 - OVA (Plover, CPO glaA) - glaAt SEQ ID NO. 113 pBZ0058 glaAp - GLA502 - OVA (Pelican, CPO glaA) - glaAt SEQ ID NO. 114 pBZ0059 glaAp - GLA502 - OVA (Pigeon, CPO glaA) - glaAt SEQ ID NO. 115 pBZ0060 glaAp - GLA502 - OVA (Quail, CPO glaA) - glaAt SEQ ID NO. 116 pBZ0067 pUC19 - 3″glaA - glaAp -GLA54 - OVA (hen, FXESCO.14) - glaAt SEQ ID NO. 125 pBZ0068 pUC19 - 3″glaA - glaAp -GLA100 - OVA (hen, FXESCO.14) - glaAt SEQ ID NO. 126 pBZ0069 pUC19 - 3″glaA - glaAp -GLA502 - OVA (hen, FXESCO.14) - glaAt SEQ ID NO. 127
TABLE-US-00005 Listing of oligonucleotide primers used in the examples with sequences Primer code SEQ ID Comment 406 SEQ ID NO. 17 glaA promoter amplification, glaAp - OVA (all different) -glaAt amplification 408 SEQ ID NO. 18 glaA promoter amplification 409 SEQ ID NO. 19 glaA terminator amplification 410 SEQ ID NO. 20 glaA terminator amplification, glaAp - OVA (all different) -glaAt amplification 276 SEQ ID NO. 34 glaA promoter amplification 286 SEQ ID NO. 35 glaA promoter amplification + truncated glaA 277 SEQ ID NO. 36 glaA promoter amplification 280 SEQ ID NO. 37 glaAt amplification 281 SEQ ID NO. 38 glaAt amplification 278 SEQ ID NO. 39 Hen OVA amplification 279 SEQ ID NO. 40 Hen OVA amplification 287 SEQ ID NO. 41 Hen OVA amplification 404 SEQ ID NO. 46 hygromycin marker amplification 405 SEQ ID NO. 47 hygromycin marker amplification 553 SEQ ID NO. 48 diagnostic primer OVA ostrich 554 SEQ ID NO. 49 diagnostic primer OVA ostrich 555 SEQ ID NO. 50 diagnostic primer OVA plover 556 SEQ ID NO. 51 diagnostic primer OVA plover 557 SEQ ID NO. 52 diagnostic primer OVA pelican 558 SEQ ID NO. 53 diagnostic primer OVA pelican 559 SEQ ID NO. 54 diagnostic primer OVA pigeon 560 SEQ ID NO. 55 diagnostic primer OVA pigeon 561 SEQ ID NO. 56 diagnostic primer OVA quail 562 SEQ ID NO. 57 diagnostic primer OVA quail 336 SEQ ID NO. 58 diagnostic primer hygromycin 337 SEQ ID NO. 59 diagnostic primer hygromycin 400 SEQ ID NO. 60 diagnostic primer amdS 401 SEQ ID NO. 61 diagnostic primer amdS 411 SEQ ID NO. 63 amdS marker amplification 415 SEQ ID NO. 64 amdS marker amplification 728 SEQ ID NO. 75 3″ glaA amplification 729 SEQ ID NO. 76 3″ glaA amplification 733 SEQ ID NO. 77 glaA promoter amplification, glaAp - OVA (all different) -glaAt amplification 734 SEQ ID NO. 78 glaA terminator amplification, glaAp - OVA (all different) -glaAt amplification 431 SEQ ID NO. 80 Ampicillin + ori from pUC19 432 SEQ ID NO. 81 Ampicillin + ori from pUC19 715 SEQ ID NO. 92 glaA promoter amplification, glaAp - OVA Ostrich - glaAt amplification 422 SEQ ID NO.93 glaA terminator amplification, glaAp - OVA Ostrich - glaAt amplification 716 SEQ ID NO. 94 glaA promoter amplification, glaAp - OVA Plover - glaAt amplification 424 SEQ ID NO. 95 glaA terminator amplification, glaAp - OVA Plover - glaAt amplification 717 SEQ ID NO. 96 glaA promoter amplification, glaAp - OVA Pelican - glaAt amplification 426 SEQ ID NO. 97 glaA terminator amplification, glaAp - OVA Pelican - glaAt amplification 718 SEQ ID NO. 98 glaA promoter amplification, glaAp - OVA Pigeon - glaAt amplification 428 SEQ ID NO. 99 glaA terminator amplification, glaAp - OVA Pigeon - glaAt amplification 719 SEQ ID NO. 100 glaA promoter amplification, glaAp - OVA Quail - glaAt amplification 430 SEQ ID NO. 101 glaA terminator amplification, glaAp - OVA Quail - glaAt amplification 823 SEQ ID NO. 117 glaA promoter amplification, glaA - GLA54 amplification 824 SEQ ID NO. 118 GLA54 amplification, glaA -GLA54 amplification 825 SEQ ID NO. 119 GLA100 amplification, glaA -GLA100 amplification 826 SEQ ID NO. 120 GLA502 amplification, glaA -GLA502 amplification 827 SEQ ID NO. 121 GLA 54 - OVA hen amplification, OVA (hen, CPO glaA) - glaA terminator -backbone - 3″ glaA amplification 828 SEQ ID NO. 122 GLA 100 - OVA hen amplification, OVA (hen, CPO glaA) - glaA terminator -backbone - 3″ glaA amplification 829 SEQ ID NO. 123 GLA 502 - OVA hen amplification, OVA (hen, CPO glaA) - glaA terminator -backbone - 3″ glaA amplification 830 SEQ ID NO. 124 3″ glaA part amplification -OVA (hen, CPO glaA) - glaA terminator - backbone - 3″ glaA amplification 787 SEQ ID NO. 128 3″ glaA amplification for transformation, 3″ glaA -(GLA54/100/502) - glaA promoter - OVA (selected ovalbumins) - glaA terminator 789 SEQ ID NO. 129 glaA terminator for transformation, 3″ glaA -(GLA54/100/502) - glaA promoter - OVA (selected ovalbumins) - glaA terminator
Kits
[0155] For the purification of PCR fragments and extraction of DNA fragments from agarose gel the Wizard® SV Gel and PCR clean-up system (Promega) was used. Purified PCR products were transformed in the pCR™Blunt II-TOPO® vector from the Zero Blunt™ TOPO™ PCR Cloning Kit (Thermo Fisher Scientific). Amplified plasmids were isolated with the Qiaprep spin miniprep kit (Qiagen). Golden Gate Reactions were carried out with the NEB Golden Gate Assembly Kit (New England Biolabs). Gibson Cloning Reactions were carried out with the NEB Gibson Assembly Cloning Kit (New England Biolabs).
Enzymes
[0156] Enzymes for DNA manipulations (e.g. digestions, Golden Gate Reactions) were obtainable from New England Biolabs and were used according to the manufacturer’s protocols.
Media
[0157] Media used in the construction of ovalbumin expression cassettes: liquid LB (10 g/l Tryptone, 5 g/l Yeast extract, 5 g/l NaCl) and solid LB (addition of 15 g/l agar) with the appropriate antibiotic (Neomycin 50 .Math.g/ml or chloramphenicol 25 .Math.g/ml).
[0158] Media used for transformation and selection of the A. niger transformants: Bottom agar (187.36 g/L D-Saccharose; 0.5 g/kg KCI; 4 g/kg KH2PO4; 1.1 g/kg Na2HPO4; 1.5 g/kg Citric acid; 2 g/kg MgSO4.7 aq; 0.01 g/kg FeSO4.7 aq; 0.1 g/kg CaCl2.2 aq; 0.0125 g/kg ZnSO4.7 aq; 0.012 g/kg MnCl2.4 aq; 0.0016 g/kg CuSO4.5 aq; 0.0009 g/kg KI, 9 g/L agarose and initial pH of 6) with the appropriate selection (antibiotic hygromycin 400 .Math.g/ml or 10 mM acetamide in a combination with 15 mM caesium chloride), Top agar (0.5 g/kg KCI; 4 g/kg KH2PO4; 1.1 g/kg Na2HPO4; 1.5 g/kg Citric acid; 2 g/kg MgSO4.7 aq; 0.01 g/kg FeSO4.7 aq; 0.1 g/kg CaCl2.2 aq; 0.0125 g/kg ZnSO4.7 aq; 0.012 g/kg MnCl2.4 aq; 0.0016 g/kg CuSO4.5 aq; 0.0009 g/kg KI, 11 g/L D-glucose, 9 g/L agar and initial pH of 6) with the appropriate selection (antibiotic hygromycin 500 .Math.g/ml or 10 mM acetamide in a combination with 15 mM caesium chloride) and minimal medium (0.5 g/kg KCI; 4 g/kg KH2PO4; 1.1 g/kg Na2HPO4; 1.5 g/kg Citric acid; 2 g/kg MgSO4.7 aq; 0.01 g/kg FeSO4.7 aq; 0.1 g/kg CaCl2.2 aq; 0.0125 g/kg ZnSO4.7 aq; 0.012 g/kg MnCl2.4 aq; 0.0016 g/kg CuSO4.5 aq; 0.0009 g/kg KI, 11 g/L D-glucose, 9 g/L agar and initial pH of 6) with the appropriate selection (antibiotic hygromycin 500 .Math.g/ml or 10 mM acetamide in a combination with 15 mM caesium chloride). If hygromycine was used for the selection of transformants the transformation plates and the minimal plates were containing 0.54 g/L NH.sub.4Cl as the nitrogen source. Sporulation agar (0.54 g/L NH.sub.4Cl, 0.5 g/kg KCI; 4 g/kg KH2PO4; 1.1 g/kg Na2HPO4; 1.5 g/kg Citric acid; 2 g/kg MgSO4.7 aq; 0.01 g/kg FeSO4.7 aq; 0.1 g/kg CaCl2.2 aq; 0.0125 g/kg ZnSO4.7 aq; 0.012 g/kg MnCl2.4 aq; 0.0016 g/kg CuSO4.5 aq; 0.0009 g/kg KI , 11 g/L D-glucose, 14 g/L agar and 44.73 g/L KCL and initial pH of 6) was used to purify the A. niger transformants.
[0159] Media used for screening of ovalbumin transformants in deep-well plates: PDA (20 g/L dextrose, 15 g/L agar, and 4 g/L potato starch). Production medium — PM — (0.5 g/kg KCI; 4 g/kg KH2PO4; 1.1 g/kg Na2HPO4; 1.5 g/kg Citric acid; 2 g/kg MgSO4.7 aq; 0.01 g/kg FeSO4.7 aq; 0.1 g/kg CaCl2.2 aq; 0.0125 g/kg ZnSO4.7 aq; 0.012 g/kg MnCl2.4 aq; 0.0016 g/kg CuSO4.5 aq; 0.0009 g/kg KI, 88 g/L D-glucose, 70 g/L citric acid, 12.5 g/L L-glutamine, 11 g/L tri-ammonium citrate and initial pH of 5.25).
[0160] Media used for screening of ovalbumin transformants in shake flasks: PDA. PM. GYE medium (20 g/L yeast extract, 22 g/L D-glucose and initial pH of 5). KM3.0 (5 g/L urea, 50 g/L D-glucose, 2 g/L KH2PO4, 0.55 g/L Na2HPO4, 1 g/L MgSO4.7 aq, 0.000125 ZnSO4.7 aq, 0.0001 g/L FeSO4.7 aq, 0.006 g/L MnCl2.4 aq, 0.05 g/L CaCl2.2 aq, 0.3 g/l KCI, 1.5 g/L Citric acid and initial pH of 5).
[0161] Fermentation medium used in 5 L scale fermenters: the defined fermentation medium for production of ovalbumin was used as described in US2014/0342396 A1 while using 5.5 g/L (NH.sub.4).sub.2SO.sub.4, feeding glucose (90%) together with maltodextrines (10%) and titrating to control pH using 12.5% ammonium solution. 0.23 g/L antifoam BT03 (van Meeuwen, The Netherlands).
Fermentation of A. Niger
[0162] The medium for the fermentation in deep-well plates (DWP) is described above. The inoculum and fermentation were carried out in the following way. 200 .Math.l of a glycerol strain stock was put on a PDA plate, 3 ml PM was added and the plate was first shaken to spread out the mycelium, and then incubated for 3 days at 32° C. The whole PDA+PM medium plate was harvested by adding 5 ml of PM medium on the top of the grown fungal mycelium, which was scratched with a T-spatula to loosen the mycelium. The whole mycelium was collected in a sterile tray to make more homogenic inoculum. 0.5 ml of the homogenized mycelium was transferred to an DWP. The DWP was incubated for 6 days at 34° C. at 350 rpm in an incubator (Infors minitron, 2.5 cm stroke). The supernatant was used for analysis using SDS-PAGE.
[0163] The medium for the fermentation in shake flasks (KM 3.0) is described above. The inoculum and fermentation were carried out in the following way. 200 .Math.l of a glycerol strain stock was put on a PDA plate, 3 ml PM was added and the plate was first shaken to spread out the mycelium, and then incubated for 3 days at 32° C. The whole PDA+PM medium plate was harvested, which was scratched with a T-spatula to loosen the mycelium and the mycelium was inoculated in 300 ml baffled shake flasks with steristoppers with 35 ml GYE medium, and then incubated for 2 days at 32° C., 220 rpm in an incubator (Infors multritron, 2.5 cm stroke). 1 ml of the GYE culture is inoculated in 100 ml non-baffled shake flasks with steristoppers with 22 ml KM3.0 medium, and then incubated for 6 days at 32° C., 120 rpm in an incubator (Infors multritron, 2.5 cm stroke). The supernatant was used for analysis using SDS-PAGE.
[0164] The medium in the 5 L fermenter was prepared as described above. Glucose was fed to control glucose > 10 g/L. Stirring was done to mix the fermenter and provide oxygen enough to produce optimal protein amounts. pH was set at 6.5 pH and the fermenter was kept at 32° C. pH was controlled between 5 and 7 pH and temperature between 31 to 33° C.
Purification of A. Niger Transformants
[0165] The primary transformants were re-streaked on minimal medium with hygromycin or acetamide and the growing (positive) transformants were analysed by colony PCR for the presence of the ovalbumin gene and hygromycin or acetamidase resistance encoding gene. The positive transformants after the colony PCR were transferred to sporulation agar for sporulation. Single sporulating colonies were selected and streaked on minimal medium with hygromycin or acetamide. Genomic DNA was isolated of single colonies for confirmation of the presence of the ovalbumin gene and hygromycin resistance or acetamidase encoding gene.
SDS-PAGE and Western Blot Analysis of A.Niger Transformants
[0166] SDS-PAGE was carried out after the small-scale fermentation. Sample preparation was 7.8 .Math.l supernatant, 3 .Math.l NuPAGE® LDS Sample Buffer (4x) and 1.2 .Math.l NuPAGE® Reducing Agent (10x), which was denaturated for 10 min at 95° C. 10 .Math.l sample was loaded in a Bolt™ 8% Bis-Tris Plus SDS-PAGE and 5 .Math.l BenchMark™ Unstained Protein Ladder was added. 1 liter 1x NuPAGE® running buffer was fresh prepared with 20x NuPAGE® SDS running buffer MOPS, 200 ml 1x NuPAGE® running buffer with 0.5 ml NuPAGE® antioxidant was added in the inner chamber of the system, the rest of the 1x NuPAGE® running buffer was added in the outer chamber. The SDS-PAGE ran according to manufacturing protocol. Staining of the SDS-PAGE was carried out with SimplyBlue Safestain according to manufacturing protocol. All chemicals used in the SDS-PAGE protocol are from Invitrogen/Thermo Fisher Scientific.
[0167] The supernatant of 5 (d5) and 6 (d6) days-old cultures grown in a 96-deep well plate (the small-scale fermentation) was analysed on the SDS-PAGE (left panel in
Selection of Ovalbumin Sequences From NCBI Database and Their in Silico Analysis
[0168] The hen ovalbumin protein sequence (GenBank accession number AAB59956.1) was used to Blast (Altschul., S. F., et al (1997) “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs” Nucleic Acids Res. 25:3389-3402) the NCBI database in order to obtain alternative ovalbumin’s from different bird species. Multiple sequences were found, from which, a selection of five alternative hen ovalbumin was chosen according to the following criteria: [0169] i) a history of human use of eggs of selected birds (information based on search in Wikipedia); [0170] ii) the absence of hen egg allergenic epitopes (Mine Y. and Rupa P., (2003) Clin Exp Immunol 103, 446-453); allergen databases: IEDB - http:/www.iedb.org/home_v3.php and SEDB -http://sedb.bicpu.edu.in/home.php); and [0171] iii) phylogenetic distance of birds (Jarvis E.D. et al., (2014), Whole-genome analyses resolve early branches in the tree of life of modern birds, Science 346 (6215), 1320-1331). A selection of the ovalbumin sequences that fulfil these criteria, but are not limited to this selection, are from ostrich ( Struthio camelus australis)-, pelican ( Pelecanus crispus); pigeon ( Columba livia); quail ( Coturnix coturnix); and plover ( Charadrius vociferus) (see
Construction of Ovalbumin Expression Cassettes with Whole Genome Based Codon Usage
[0172] To overexpress selected ovalbumin in A. niger we constructed the expression cassettes in the following way: [0173] i) the protein sequences (SEQ ID NOs. 1 to 6) were used as the input for an in-vitro synthesis of the corresponding DNA coding sequence. The codon optimization for expression in A. niger was based on the complete codon usage of A. niger CBS513.88 (See
[0176] The regulatory sequences of the glucoamylase gene (GenBank: An03g06550) were used for driving a high expression of the gene of interest and ensuring an efficient transcript termination. The glaA promoter and the glaA terminator were PCR amplified from the host A. niger BZASNI.22a strain with primers 406 and 408 (SEQ ID NOs. 17 and 18) and primers 409 and 410 (SEQ ID NOs. 19 and 20), respectively, and purified with the Wizard® SV Gel and PCR clean-up system (Promega). These purified PCR products were ligated in the pCR™Blunt II-TOPO® vector (SEQ ID NO. 21) from the Zero Blunt™ TOPO™ PCR Cloning Kit (Thermo Fisher Scientific) and transformed to NEB 10-beta Competent E. coli cells. The plasmid from the positive E. coli transformants was checked by a restriction enzyme analysis and by DNA sequencing for the correct sequence and orientation of the inserts. See Table 1 for all constructed plasmids.
[0177] The expression cassette consisting of the 1000 bp glaA promoter, the pre-pro-peptide or the carrier protein, the ovalbumin coding sequence and the 600 bp glaA terminator were assembled using the NEB Golden Gate Assembly Kit (New England Biolabs, with plasmid pGGA, SEQ ID NO. 22) and transformed to NEB 10-beta Competent E. coli cells. The plasmid from the positive E. coli transformants was checked by a restriction enzyme analysis and by DNA sequencing for the correct sequence and orientation of the inserts. See Table 1 for all constructed plasmids. The construction of GLA protein fusions was carried out as mentioned above.
Construction of Ovalbumin Expression Cassettes With Gene Specific Codon Usage
[0178] In order to test another approach for a codon usage optimization for the expression of heterologous proteins in A. niger we selected the highly expressed A. niger gene glaA, and based the optimisation of the codon usage on its codon preference. We constructed the expression cassettes in the following way:
[0179] The cDNA of hen ovalbumin (Genbank accession number MF321659.1) was used as the input for the optimization of the codon usage for expression of ovalbumin in A. niger. The A. niger glucoamylase (glaA) cDNA (NCBI reference number XM_001 390493.2) codon usage was analysed using the on-line available analysis tool, the Sequence Manipulation Suite (http://www.bioinformatics.org/sms2/codon usage.html) (see Table 3A.). Subsequently, the hen ovalbumin cDNA was codon-optimized using the codon-usage table of the glaA cDNA of A.niger CBS513.88 (Table 3A.) as the input using an on-line available DNA optimizing tool OPTIMISER (http://genomes.urv.es/OPTIMISER/; Puigbo P. et al., 2007; Nucl Acids Res 35; W126-W131). The resulting hen ovalbumin cDNA was further manually corrected to make the closest match to the glaA cDNA codon usage table (see Table 3.B, SEQ ID NO.32), taking into the account an equal distribution of changes throughout the whole cDNA sequence. The gene synthesis was done by GenScript (US). The methionine of the ovalbumin protein sequence was replaced by the glucoamylase (GLA) pre-pro sequence of A. niger (SEQ ID NO.14) to direct the secretion of the protein extracellularly (Note that ovalbumin comprises an internal signal sequence (residues 21-47), which is not cleaved off, but remains as part of the mature protein). The corresponding DNA coding sequence is mentioned in the sequence listing under the SEQ ID NO.30. Genscript synthesized the hen ovalbumin coding sequence in the destination plasmid pUC57 (SEQ ID NO.31), which was named BZESCO.21 (SEQ ID NO.32).
[0180] Alternatively, the ovalbumin coding sequence was fused to the truncated glucoamylase gene sequence (SEQ ID NO. 33), which is fulfilling the role of a carrier through the A. niger secretory pathway. To release the secreted ovalbumin from the GLA carrier, the enterokinase cleavage site (DDDDK) was added between the carrier protein and the start of the native ovalbumin. The truncated glucoamylase gene, with the added enterokinase cleavage site, was PCR amplified with primers 276 and 286 (SEQ ID NOs. 34 and 35) from genomic DNA of A. niger BZASNI.22a.
[0181] Construction of both ovalbumin constructs (with and without the GLA carrier fusion) were made using fusion PCR. For the construct without the GLA carrier fusion, the promoter of glaA was PCR amplified with primers 276 and 277 (SEQ ID NOs. 34 and 36) and the terminator with primers 280 and 281 (SEQ ID NOs. 37 and 38), both from genomic DNA of A. niger BZASNI.22a. The hen ovalbumin ORF was PCR amplified with the primers 278 and 279 (SEQ ID NOs. 39 and 40) using the plasmid BZESCO.21 (SEQ ID NO. 32) as a template. For the GLA carrier - ovalbumin fusion construct, the promoter of glaA and the truncated glaA gene corresponding to the GLA carrier protein were PCR amplified with the primers 276 and 286 (SEQ ID NOs. 34 and 35) as well as the terminator (primers 280 and 281, corresponding to the SEQ ID NO. 37 and 38, respectively), both from the genomic DNA of A.niger BZASNI.22a. The hen ovalbumin ORF was PCR amplified with the primers 287 and 279 (SEQ ID NOs. 41 and 40) from BZESCO.21 (SEQ ID NO. 32). The expression cassette parts described above were assembled using an overlap extension PCR with primers aligning at the 5′ and 3′ ends of the cassette, respectively. The obtained products were cloned into pUC57-Brick vector (SEQ ID NO. 42) by restriction-ligation. The complete construct without the GLA carrier fusion was named BZESCO.22 (SEQ ID NO. 43) and the GLA carrier fusion construct was named BZESCO.23 (SEQ ID NO. 44).The hen ovalbumin expression cassettes (with and without the GLA carrier fusion) were PCR amplified with primers 276 and 281 (SEQ ID NOs. 34 and 38) and transformed with the DNA fragment containing the hygromycin selection marker PCR amplified from pCNS43 (SEQ ID NO. 45) with primers 404 and 405 (SEQ ID NOs. 46 and 47) to the A. niger strain BZASNI.22a.
[0182] To overexpress the other selected ovalbumin encoding genes based on the codon usage optimization of A. niger highly expressed gene glaA, we constructed the expression cassettes in the following way: i) the protein sequences of ostrich, pelican, pigeon, quail and plover ovalbumins (SEQ ID NOs. 2 to 6), were used as the input for gene synthesis by GenScript (US) using the codon-usage table of the glaA cDNA of A. niger CBS513.88 (Table 3A.) and their in house gene synthesis, codon optimization algorithm. The methionine at the position +1 of the ovalbumin protein sequences was replaced by the glucoamylase (GLA) pre-pro-peptide sequence of A. niger (SEQ ID NO.14) to direct the secretion of the protein extracellularly (Note that ovalbumin comprises an internal signal sequence (residues 21-47), which is not cleaved off, but remains as part of the mature protein). The corresponding DNA coding sequences are mentioned in the sequence listing under the SEQ ID NOs. 65 to 69. The sequences were delivered by GenScript in pUC57-kan (SEQ ID NO.13) and were further modified at the 5′ and the 3′ end by adding the Bsal enzyme restriction sites to facilitate the Golden Gate Reaction. The glaA promoter and the glaA terminator were PCR amplified from the host A. niger BZASNI.22a strain with primers 406 and 408 (SEQ ID NOs. 17 and 18) and primers 409 and 410 (SEQ ID NOs.19 and 20), respectively, and purified with the Wizard® SV Gel and PCR clean-up system (Promega). These purified PCR products were ligated in the pCR™Blunt II-TOPO® vector (SEQ ID NO. 21) from the Zero Blunt™ TOPO™ PCR Cloning Kit (Thermo Fisher Scientific) and transformed to NEB 10-beta Competent E. coli cells. The plasmid from the positive E. coli transformants was checked by a restriction enzyme analysis and by DNA sequencing for the correct sequence and orientation of the inserts. See Table 1 for all constructed plasmids.
[0183] The expression cassette consisting of the 1000 bp glaA promoter, the glucoamylase pre-pro-peptide with the ovalbumin coding sequence and the 600 bp glaA terminator were assembled using the NEB Golden Gate Assembly Kit (New England Biolabs, with plasmid pGGA, SEQ ID NO.22) and transformed to NEB 10-beta Competent E. coli cells. The plasmid from the positive E. coli transformants was checked by a restriction enzyme analysis and by DNA sequencing for the correct sequence and orientation of the inserts. See Table 1 for all constructed plasmids (SEQ ID NO.70 to 74).
[0184] For the A. niger genome targeted single cross-over integration construct, a 1500 bp region downstream of the glaA locus was used (marked as 3″ glaA). This 3″ glaA part was PCR amplified from the host A. niger BZASNI.22a with primers 728 and 729 (SEQ ID NO. 75 and 76). The different expression cassettes (glaA promoter — codon optimized ovalbumin gene — glaA terminator) were PCR amplified from Golden Gate constructs (SEQ ID NO. 70 to 74) and BZESCO.22 (SEQ ID NO.43) with primers 733 and 734 (SEQ ID NO. 77 and 78). The plasmid backbone part was PCR amplified from plasmid pUC19 (SEQ ID NO. 79) with primers 431 and 432 (SEQ ID NO. 80 and 81). All PCR products were purified with the Wizard® SV Gel and PCR clean-up system (Promega). The integration construct consisting of the 1500 bp 3″ glaA part, the different expression cassettes and the plasmid backbone were assembled using the NEB Gibson Assembly Cloning Kit (New England Biolabs) and transformed to NEB 5-alpha Competent E. coli cells. The plasmids from the positive E. coli transformants were checked by a restriction enzyme analysis and by DNA sequencing for the correct sequence and orientation of the inserts. See Table 1 for all constructed plasmids (SEQ ID NO.82 to 87).
[0185] Table 3. Codon usage table (frequency of the codon usage for a certain amino acid, e.g. for instance: UUU - F - 0.18, means 18% of all F are coded by UUU) of the glucoamylase (glaA) cDNA, from Aspergillus niger (A) and the glaA codon-based optimized hen ovalbumin cDNA (B).
TABLE-US-00006 A. codon usage table of gla A from A.niger CBS513.88 UUU F 0.18 UCU S 0.18 UAU Y 0.22 UGU C 0.3 UUC F 0.82 UCC S 0.22 UAC Y 0.78 UGC C 0.7 UUA L 0 UCA S 0.05 UAA * 0 UGA * 0 UUG L 0.13 UCG S 0.16 UAG * 1 UGG W 1 CUU L 0.06 CCU P 0.18 CAU H 0 CGU R 0.2 CUC L 0.35 CCC P 0.45 CAC H 1 CGC R 0.35 CUA L 0.04 CCA P 0 CAA Q 0.18 CGA R 0.2 CUG L 0.42 CCG P 0.36 CAG Q 0.82 CGG R 0.15 AUU I 0.5 ACU T 0.27 AAU N 0.24 AGU S 0.14 AUC I 0.46 ACC T 0.53 AAC N 0.76 AGC S 0.26 AUA I 0.04 ACA T 0.07 AAA K 0 AGA R 0.05 AUG M 1 ACG T 0.14 AAG K 1 AGG R 0.05 GUU V 0.14 GCU A 0.38 GAU D 0.48 GGU G 0.3 GUC V 0.36 GCC A 0.29 GAC D 0.52 GGC G 0.47 GUA V 0.05 GCA A 0.15 GAA E 0.35 GGA G 0.15 GUG V 0.45 GCG A 0.17 GAG E 0.65 GGG G 0.09
TABLE-US-00007 B. codon usage table hen OVA optimized for expression in A.niger based on the gla A A.niger sequence UUU F 0.2 UCU S 0.18 UAU Y 0.2 UGU C 0.33 UUC F 0.8 UCC S 0.21 UAC Y 0.8 UGC C 0.67 UUA L 0 UCA S 0.05 UAA * 0 UGA * 0 UUG L(s) 0.13 UCG S 0.16 UAG * 1 UGG W 1 CUU L 0.06 CCU P 0.21 CAU H 0 CGU R 0.2 CUC L 0.34 CCC P 0.43 CAC H 1 CGC R 0.33 CUA L 0.06 CCA P 0 CAA Q 0.2 CGA R 0.2 CUG L(s) 0.41 CCG P 0.36 CAG Q 0.8 CGG R 0.13 AUU I 0.48 ACU T 0.27 AAU N 0.24 AGU S 0.13 AUC I 0.48 ACC T 0.53 AAC N 0.76 AGC S 0.26 AUA I 0.04 ACA T 0.07 AAA K 0 AGA R 0.07 AUG M(s) 1 ACG T 0.13 AAG K 1 AGG R 0.07 GUU V 0.16 GCU A 0.4 GAU D 0.5 GGU G 0.26 GUC V 0.35 GCC A 0.29 GAC D 0.5 GGC G 0.47 GUA V 0.03 GCA A 0.14 GAA E 0.36 GGA G 0.16 GUG V 0.45 GCG A 0.17 GAG E 0.64 GGG G 0.11
Construction of Alternative Gla Carrier Fusion Ovalbumin Expression Cassettes With Gene Specific Codon Usage
[0186] To overexpress selected ovalbumin in A. niger we constructed an alternative ovalbumin expression cassette with different size of truncated glucoamylase from A. niger. The selection of the truncated carrier sequence was based on the tertiary structure of glucoamylase, the pl and the Mw of the truncated protein was taken into account to facilitate the downstream processing of expressed ovalbumin. The optimized ovalbumin coding sequence was fused to 3 different truncated glucoamylase gene sequences, coding for its first 54, 100 and 502 amino acids of GLA, respectively. All these glucoamylase sequences included its own secretory pre-pro-peptide sequence at the N-terminal end.
[0187] The glucoamylase sequence, which consist of the first 54 amino acids (GLA54, SEQ ID NOs 88 and 89) translates to the pre-pro-peptide sequence, the first alpha helix of the glucoamylase protein and ends with the addition of KEX2 (Lys-Arg) cleavage site. The glucoamylase sequence, which consist of the first 100 amino acids (GLA100, SEQ ID NOs 90 and 91) translates to the pre-pro-peptide sequence and the first 3 alpha helixes of the glucoamylase protein and ends with the addition of KEX2 (Lys-Arg) cleavage site. The glucoamylase sequence, which consist of the first 502 amino acids (SEQ ID NOs. 130 and 16) translates to the pre-pro-peptide sequence and the glucoamylase protein without the starch binding domain. Between the 502 amino acids of glucoamylase and the start of the mature ovalbumin sequence, a synthetic peptide of 8 amino acids, which includes the KEX2 (Lys-Arg) cleavage site, was inserted. The corresponding DNA coding sequences of the 3 different truncated glucoamylase variants were synthesized by GenScript (US) in the destination plasmid pUC57-kan (SEQ ID NO.13). At the 5′ and the 3′ end of each sequence Bsal enzyme restriction sites were added to facilitate the Golden Gate Reaction.
[0188] A similar truncated glucoamylase protein sequence, that was used as a homologous carrier through the secretory pathway of A. niger, was reported to increase significantly production of a heterologous protein (Jeenes, D.J. et al., 1993 supra). The regulatory sequences of the glucoamylase gene (GenBank: An03g06550) were used for driving a high expression of the gene of interest and ensuring an efficient transcript termination.
[0189] For constructing of the expression cassettes, the glaA promoter and the glaA terminator were PCR amplified from the host A. niger BZASNI.22a strain with primers 406 and 408 (SEQ ID NOs. 17 and 18) and primers 409 and 410 (SEQ ID NOs.19 and 20), respectively, and purified with the Wizard® SV Gel and PCR clean-up system (Promega). These purified PCR products were ligated in the pCR™Blunt ll-TOPO® vector (SEQ ID NO. 21) from the Zero Blunt™ TOPO™ PCR Cloning Kit (Thermo Fisher Scientific) and transformed to NEB 10-beta Competent E. coli cells. The plasmid from the positive E. coli transformants was checked by a restriction enzyme analysis and by DNA sequencing for the correct sequence and orientation of the inserts. See Table 1 for all constructed plasmids.
[0190] The ovalbumin coding sequences were PCR amplified with primers 715 and 422 for OVA ostrich (SEQ ID NO.92 and 93) from the GenScript (US) synthesized DNA coding sequence with SEQ ID NO.65, primers 716 and 424 for OVA Plover (SEQ ID NO94 and 95) from the GenScript (US) synthesized DNA coding sequence with SEQ ID NO.69, primers 717 and 426 for OVA Pelican (SEQ ID NO.96 and 97) from the GenScript (US) synthesized DNA coding sequence with SEQ ID NO.66, primers 718 and 728 for OVA Pigeon (SEQ ID NO.98 and 99) from the GenScript (US) synthesized DNA coding sequence with SEQ ID NO.67 and primers 719 and 430 for OVA Quail (SEQ ID NO.100 and 101) from the GenScript (US) synthesized DNA coding sequence with SEQ ID NO.68. All PCR amplified ovalbumin genes were purified with the Wizard® SV Gel and PCR clean-up system (Promega). The expression cassette consisting of the 1000 bp glaA promoter, the 3 different glucoamylase variants, codon optimized ovalbumin coding sequence and the 600 bp glaA terminator were assembled using the NEB Golden Gate Assembly Kit (New England Biolabs, with plasmid pGGA, SEQ ID NO.22) and transformed to NEB 10-beta Competent E. coli cells. The plasmid from the positive E. coli transformants was checked by a restriction enzyme analysis and by DNA sequencing for the correct sequence and orientation of the inserts. See Table 1 for all constructed plasmids (SEQ ID NO.102 to 116).
[0191] For the construction of the GLA carrier fusion constructs with the hen ovalbumin (SEQ ID NO.30), already constructed plasmids were used as PCR templates to PCR amplify: i) the glaA promoter with the 3 different glucoamylase variants and ii) to PCR amplify the codon optimized hen ovalbumin, glaA terminator, the plasmid backbone and the single cross-over integration part - 3″ glaA. In Table 4 is described which part of the GLA carrier expression cassette is PCR amplified from which template construct with a primer combination.
TABLE-US-00008 Primer combinations for the GLA carrier fusion hen ovalbumin constructs PCR target PCR template Primer combination glaA promoter-GLA54 pBZ0050 (SEQ ID NO.106) 823 and 824 (SEQ ID NO. 117 and 118) glaA promoter-GLA100 pBZ0055 (SEQ ID NO.111) 823 and 825 (SEQ ID NO. 117 and 119) glaA promoter-GLA502 pBZ0060 (SEQ ID NO. 116) 823 and 826 (SEQ ID NO. 117 and 120) OVA-glaA terminator pBZ0061 (SEQ ID NO.82) 827 and 830 (SEQ ID NO. 121 and backbone-3″ glaA (for GLA54 fusion) 124) OVA-glaA terminator pBZ0061 (SEQ ID NO.82) 828 and 830 (SEQ ID NO. 122 and backbone-3″ glaA (for GLA100 fusion) 124) OVA-glaA terminator pBZ0061 (SEQ ID NO.82) 829 and 830 (SEQ ID NO. 123 and backbone-3″ glaA (for GLA100 fusion) 124)
[0192] All PCR products were purified with the Wizard® SV Gel and PCR clean-up system (Promega), assembled using the NEB Gibson Assembly Cloning Kit (New England Biolabs) and transformed to NEB 5-alpha Competent E. coli cells. The plasmids from the positive E. coli transformants were checked by a restriction enzyme analysis and by DNA sequencing for the correct sequence and orientation of the inserts. See Table 1 for all constructed plasmids (SEQ ID NO. 125 to 127).
Transformation of Overexpression Cassettes and Selection of A. Niger Transformants
[0193] The transformation of A. niger was based on the protocol of Balance et al. (Ballance D.J. et al., (1983), Transformation of Aspergillus nidulans by the orotidine-5′-phosphate decarboxylase gene of Neurospora crassa. Biochem. Biophys. Res. Comm. 112,284-289). For the transformation, the entire expression cassette, containing the ovalbumin-encoding gene and the glaA regulatory sequences, and the various GLA based carrier sequences, was PCR amplified using the primers aligning at the 5′ glaA promoter (forward primer 406, SEQ ID NO. 17) and the 3′gla A terminator (reverse primer 410, SEQ ID NO. 20) DNA sequence. This DNA fragment was co-transformed with the hygromycin selection marker, which was PCR amplified from pCNS43 (see Table 1) with primers 404 and 405 (SEQ ID NOs. 46 and 47) and the transformants were selected on plates containing minimal medium supplemented with the antibiotic hygromycin. The primary transformants were re-streaked on minimal medium with hygromycin and the growing (positive) transformants were analysed by colony PCR for the presence of the ovalbumin gene and hygromycin (See Table 5 for the primer combinations, SEQ ID NOs. 48 to 61). The positive colonies were purified via a single-spore purification procedure and used to prepare an inoculum for a small-scale fermentation.
[0194] The transformation can also be carried out with a DNA fragment encoding the A.nidulans acetamidase gene as the selection marker. The corresponding DNA fragment can be PCR amplified from pBZ0026 (SEQ ID NO. 62) with primers 411 and 415 (SEQ ID NOs. 63 and 64). The transformants were selected on plates containing minimal medium supplemented with acetamide as the sole nitrogen source. The single cross-over integration constructs with codon optimization of ovalbumin based on the glaA gene and the different GLA fusion constructs with hen ovalbumin were transformed with the acetamide selection marker and were PCR amplified for transformation with primers 787 and 789 (SEQ ID NO.128 & 129). The primary transformants were analysed by colony PCR for the presence of the ovalbumin-encoding gene and the further analysis proceeded as described above for the hygromycin-ovalbumin co-transformants.
TABLE-US-00009 Primer combinations diagnostic PCRs Target Forward primer Reverse primer OVA ostrich 553 554 OVA plover 555 556 OVA pelican 557 558 OVA pigeon 559 560 OVA quail 561 562 hygromycin 336 337 amdS 400 401
Small Scale Fermentation in Deep-Well Plates
[0195] The fermentation was carried out up to the glucose consumption (in total about 6 days) and the supernatant of the cultures was harvested in different days of the fermentation. It was analysed on SDS-PAGE (see
[0196] Based on the SDS-PAGE analysis of the various ovalbumin overexpressing transformants, the highest expression was obtained for the glaA codon optimized hen ovalbumin compared to the whole A. niger genome based codon optimisation of hen or other ovalbumin. Furthermore, the overexpressed hen ovalbumin appeared as two different Mw protein bands (
Small Scale Fermentation in Shake Flasks
[0197] The fermentation was carried out up to the glucose consumption (in total about 6 days) and the supernatant of the cultures was harvested in different days of the fermentation. The rounds per minute (rpm) of the incubator (Infors Multritron, 2.5 cm stroke) was critical for the production of ovalbumin in shake flasks. With 120 rpm production was very good, however with 220 rpm no production of ovalbumin was seen. It was analysed on SDS-PAGE (see
Fermentation in 5 L Fermenter
[0198] One of the best hen ovalbumin producing transformants, A.niger BZASNI.33, was grown in a 5 L fermenter.
Stirring Albumen and Albumen Broth
[0199] The properties of hen egg white (with 54% ovalbumin) was assessed in a stirred fermenter. Above 600 rpm in a Cplus fermenter of Sartorius at standard configuration no solid egg white was observed, but from 900 rpm and higher, solid egg white appeared. At 600 rpm the power input is 1.4 kW/m3. Egg solids appearing after 22 hrs stirring at 32° C. in Cplus fermenter Sartorius.
TABLE-US-00010 Rpm 300 600 900 1200 1500 Egg solids appearing - - + ++ +++
Purification of the Ovalbumin
[0200] Anion exchange chromatography was used as an example for purification of ovalbumin. The buffer for binding and eluting the protein(s), such as potassium phosphate or Tris-HCI buffer, had a molarity ranging from 20 to 50 mM. The pH of the buffer varied from 6 to 9. The salt concentration (such as NaCl), at which the ovalbumin eluted, ranges between 0.0 and 0.3 M.
[0201] Alternatively, the size exclusion chromatography was used to separate proteins with a substantially different size. The molarity of the buffer (such as potassium phosphate or Tris-HCl buffer) ranges between 10 and 50 mM. The pH of the buffer ranges between 6 and 9. The molarity of the salt (such as NaCl) ranges between 0.1 and 0.3 M. The flow rate of the isocratic elution ranges from 10 to 200 cm/h.
[0202] As an example, a purification of overexpressed hen ovalbumin from A. niger (BZASNI.60) microfiltrate using Anion Exchange Chromatography is shown in
Testing Product
A) Foaming Capacity
[0203] A protein content ranging from 10 to 150 mg/mL is used. The pH of the testing solutions typically ranges from 2 to 11. The agitation time ranges from 1 to 20 min using a suitable method (e.g. homogenization, blending), after which the foam is transferred to a suitable measuring cylinder. The total foam volume after 30 s is observed using a measuring cylinder and defines the foam capacity. The total foam volume is observed over a time ranging from 1 to 24 h and the rate at which the foam volume decreases defines the foam stability.
B) Solubility
[0204] A protein content ranging from 1 to 10 g is added to an amount ranging from 10 to 1000 mL of buffer solution. The pH of the buffer solution ranges from 2 to 12, more preferably from 3 to 7. The protein solution is stirred for a time ranging from 30 to 120 minutes. Hereafter, the solution is centrifuged for a time of 10 to 60 minutes at a speed of 2000 to 20000 xg. Hereafter, the protein content of the supernatant is measured.
C) Emulsification
[0205] A protein content ranging from 1 to 100 mg/mL, is suspended in a solution with a pH ranging from 2 to 10. Hereafter the solution is mixed with a suitable oil. The wt% of the protein solution in the final mixture ranges from 50 wt% to 100 wt%. The wt% of the oil in the final mixture ranges from 0 wt% to 50 wt%. The mixture is agitated for a time ranging from 1 to 10 min. The total volume of the emulsified phase, the water phase and the oil phase are observed using a measuring cylinder. The ratio between the phases defines the emulsion ability. The total volume of the emulsified phase, the water phase and the oil phase are recorded for a time ranging from 1 to 100 h. The rate at which the volumes change defines the emulsion stability.
[0206] Alternatively, the emulsifying properties of the protein of interest is based on the turbidity of the emulsion. After the emulsion is prepared, as described, a sample is taken and diluted with a suitable solution (e.g. sodium dodecyl sulfate) in a range of 10-500 times dilution. The turbidity of the dilution is measured at a suitable absorbance (e.g. for sodium dodecyl sulfate: 500 nm). The emulsifying activity index is then calculated using the absorbance, light path of the spectrophotometer, volume of the oil phase and concentration of protein before the emulsion is formed. The emulsion stability can be determined similarly by taking emulsion samples at time points in a range of 0-24 h, after which the turbidity is measured as described above. The emulsifying activity index over time indicates the emulsion stability. Next, the emulsion is heated for a time range of 10 to 60 min at a temperature range of 60 to 100° C. After the emulsion has cooled, a sample is taken, and its turbidity measured as described previously. After calculating the emulsifying activity index of these samples, the values can be used to calculate the emulsion stability.
D) Gelling
[0207] A protein content ranging from 30 to 200 mg/mL is used. The salt (e.g. sodium chloride) molarity ranges between 0 and 200 mM. The pH of the solution ranges between 3 and 9. The solution is heated for a time range of 45 to 120 minutes at a temperature range of 60 to 100° C. Hereafter, the gel is cooled to room temperature and ready to undergo rheological measurements.
Testing Product - Allergenicity
[0208] Sera of sensitized or allergic people containing IgE antibodies are used to perform in vitro allergenicity studies and to examine the allergenicity of a protein.
[0209] ELISA (Enzyme Linked Immunosorbent Assay) and immunoblotting are used to investigate the capability of a protein to bind allergen specific IgE antibodies. IgE antibodies from people allergic to Gal d 2 (the main hen ovalbumin allergen) are also used in immunoblotting assays to characterize Gal d 2 epitopes. The capability of a protein to bind IgE antibodies does not always mean that the binding will cause the release of inflammatory mediators and, therefore, an allergic reaction.
[0210] The capability of a protein to induce the release of inflammatory mediators is usually explored through the Basophilic Activation Test (BAT). In BAT basophil cells from allergic individuals are exposed to the allergen and the production of histamine upon allergen exposure is observed.
REFERENCES
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