Chimeric proteins and engineered host cells for producing recombinant proteins

Abstract

Aspects of the present disclosure include chimeric proteins and engineered host cells that comprise the chimeric proteins. Another aspect of the present disclosure include methods of reducing porphyrins or derivatives thereof from recombinant proteins expressed or secreted from the engineered host cell. Another aspect of the present disclosure includes methods of making recombinant proteins from the engineered host cells.

Description

Attorney Ref.: 41522-65386 / 418Client Ref.: 770 / 01 WO CHIMERIC PROTEINS AND ENGINEERED HOST CELLS FOR PRODUCING RECOMBINANT PROTEINS1. CROSS REFERENCE TO RELATED APPLICATIONS|0001] This application claims benefit of U. S. Provisional Patent Application No.:63 / 741,337, filed on January 2, 2025, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.2. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in XML format via USPTO Patent Center and is hereby incorporated by reference in its entirety. Said XML copy, created on December 30, 2025, is named 41522-65386_WO_SEQLISTING and is 16.9 kilobytes in size.3. BACKGROUND

[0003] Pichia pastoris is known to produce protoporphyrin, the precursor of porphyrin compounds, and it has known enzymes in the heme metabolite pathway. It has also been shown that blue biliverdin derivatives are expressed in Pichia. However, heme is typically linked with red color and biliverdin with blue color.

[0004] There is a need for alternative methods of identifying heme or biliverdin in the fermentation process in yeast. There is also a need for methods of removing or reducing colorants produced during fermentation of tire recombinant protein.4. SUMMARY

[0005] Prior to the present disclosure, it was previously hypothesized that the green color of recombinant proteins was one of two classes of compounds: flavin and derivatives thereof, or a reaction product between iron and sulfite as they are commonly observed in eggs. An example of the “green” color around an egg yolk is shown as FIG. 1.

[0006] The present inventors surprisingly identified that an engineered Pichia pastoris comprising a recombinant protein ovalbumin produces a heme derivative that is bound or tightly associated to the recombinant ovalbumin, resulting in a green color production in the liquid media after the fermentation process. The heme derivative resulted in a green color of the fermentation liquid containing the recombinant ovalbumin. The present inventors found that the green color was a result of the colorants absorbing both blue and yellow light.41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO Therefore, tire present inventors sought to engineer Pichia pastoris to divert the green or blue molecule into a different color spectrum to reduce the overall production of the colorants from the heme derivatives. Pichia pastoris is also known as Komagataella pastoris or as Komagataella phaffi, and any of these names may be used throughout.|0007] Tlie present inventors engineered recombinant host cell strains with enzymes involved in heme metabolism that were shown to divert the blue molecule into a different color spectrum or reduce the overall production of the colorants. Solutions to reduce the green color impact at strain level are also described. The engineered Pichia pastoris developed by the inventors reduces the color impact for recombinant protein products expressed or secreted from Pichia pastoris. Tire color impact is addressed at the strain level, rather than merely downstream purification processes. Tire engineered Pichi pastoris was also able to localize the recombinant enzyme biliverdin inside the yeast strain.

[0008] The present inventors identified a green color generated from Pichi pastoris fermentation and classified the green colorant as a porphyrin or derivative thereof of Pichia pastoris, as part of a heme metabolite pathway. The present inventors found that Pichia pastoris produced porphyrin derivatives during heme metabolism that are green in color but that absorbs both the blue and yellow spectrum. They cannot be removed by common size¬ based purification strategy (such as ultrafiltration) as they are tightly bound to larger molecules such as proteins,

[0009] 'Iliis present disclosure addresses the problem observed during some large scale fermentations using ovalbumin (OVA) production strains designed herein, wherein functional Chicken OVA powder was “stained” with an inseparable green color following fermentation and downstream processing. This present disclosure describes the successful identification and removal of green color using advanced identification methods, where biliverdin reductase (BVR) is introduced into the OVA production strain (e.g., engineered strain) such that the BVR electrochemically converts biliverdin to bilirubin, thereby shifting the absorption of the green color to a yellow, red and / or orange color spectrum.

[0010] An aspect of the present disclosure includes a chimeric protein comprising: a biliverdin reductase; one or more linkers; and a peroxisomal targeting signal.

[0011] In some embodiments, the biliverdin reductase comprises an amino acid sequence having at least 80%, at least 85%, or at least 90% sequence identity to SEQ ID NO: 6. In2 41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO some embodiments, the biliverdin reductase comprises an amino acid sequence of SEQ ID NO: 6.

[0012] In some embodiments, the linker comprises a flex linker. In some embodiments, the linker further comprises an alpha helix linker. In some embodiments, the linker comprises an amino acid sequence having at least 80%, at least 85%, or at least 90% sequence identity to any one of SEQ ID NOs: 7 and 13-15. In some embodiments, the linker comprises an amino acid sequence of any one of SEQ ID NOs: 7 and 13-15. In some embodiments, the peroxisomal targeting signal comprises an amino acid sequence having at least 80%, at least 85%, or at least 90% sequence identity to SEQ ID NO: 8. In some embodiments, the peroxisomal targeting signal comprises an amino acid sequence of SEQ ID NO: 8.

[0013] In some embodiments, the chimeric protein comprises an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 5, In some embodiments, the chimeric protein comprises an amino acid sequence of SEQ ID NO: 5.

[0014] Another aspect of the present disclosure includes a polynucleotide encoding the chimeric protein of the present disclosure. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the polynucleotide comprises a nucleotide sequence of SEQ ID NO: 1.

[0015] Another aspect of the present disclosure includes a vector comprising the polynucleotide of the present disclosure.

[0016] Another aspect of the present disclosure includes an engineered host cell comprising one or more vectors comprising: a nucleotide sequence encoding the chimeric protein of the present disclosure, wherein the engineered host cell reduces the production of porphyrins or derivatives thereof from the host cell thereof compared to a host cell that does not comprise the chimeric protein. In some embodiments, the engineered host cell further comprises a vector comprising a nucleotide sequence encoding a recombinant protein, wherein the host cell that does not comprise the chimeric protein comprises porphyrins or derivatives thereof bound to the recombinant protein upon expression of the recombinant protein,

[0017] In some embodiments, the engineered host cell reduces the amount of porphyrins or derivatives thereof bound to the recombinant protein compared to the host cell that does not. comprise the chimeric protein.3 41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO

[0018] Another aspect of the present disclosure includes an engineered host cell comprising one or more vectors comprising: a nucleotide sequence encoding a recombinant protein; and a nucleotide sequence encoding a biliverdin reductase, wherein the engineered host cell reduces the production of porphyrins or derivatives thereof from the host cell thereof compared to a host cell that does not comprise the biliverdin reductase.

[0019] In some embodiments, the host cell that does not comprise the biliverdin reductase comprises porphyrins or derivatives thereof bound to the protein upon expression of the recombinant protein. In some embodiments, the engineered host cell reduces the amount of porphyrins or derivatives thereof bound to the recombinant protein compared to the host cell that does not compose the biliverdin reductase.

[0020] In some embodiments, the one or more vectors further comprise a nucleotide sequence encoding a flex linker having at least 90% sequence identity to SEQ ID NO: 10. In some embodiments, the one or more vectors further comprises a nucleotide sequence encoding an alpha helix linker having at least 90% sequence identity to SEQ ID ON: 11. In some embodiments, the one or more vectors further comprises a nucleotide sequence encoding a third linker having at least 90% sequence identity to SEQ ID NO: 12.

[0021] In some embodiments, the one or more vectors further comprise a nucleotide sequence encoding a peroxisomal targeting sequence.

[0022] In some embodiments, at least one of the one or more vectors comprises: (a) the nucleotide sequence encoding the recombinant protein; and (b) the nucleotide sequence encoding the biliverdin reductase; (c) the nucleotide sequence encoding the flex linker; (d) the nucleotide sequence encoding the alpha helix linker; (e) the nucleotide sequence encoding the third linker; and (f) the nucleotide sequence encoding the peroxisomal targeting signal.

[0023] In some embodiments, the biliverdin reductase is a codon optimized biliverdin reductase, comprising a nucleotide sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 2. In some embodiments, the biliverdin reductase is a codon optimized biliverdin reductase, comprising a nucleotide sequence of SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding the alpha helix linker and flex linker comprises a nucleotide sequence having at least 80% or at least 90% sequence identity to SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding the one or more linkers comprise a nucleotide sequence of any one of SEQ ID NOs: 3 and 10-4 41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO 12. In some embodiments, the nucleotide sequence encoding the peroxisomal targeting sequence comprises a nucleotide sequence having at least 80% or at least 90% sequence identity to SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the peroxisomal targeting sequence comprises a nucleotide sequence of SEQ ID NO: 4.|0024] In some embodiments, the nucleotide sequence encoding the recombinant protein is on the same vector as the biliverdin reductase or on a different vector.

[0025] In some embodiments, one or more vectors comprises: a first vector comprising:(b) the nucleotide sequence encoding the biliverdin reductase; (c) the nucleotide sequence encoding the flex linker; (d) the nucleotide sequence encoding the alpha helix linker; (e) the nucleotide sequence encoding the third linker; and (f) the nucleotide sequence encoding the peroxisomal targeting sequence sequence; and a second vector comprising the nucleotide sequence encoding the recombinant protein,

[0026] In some embodiments, the porphyrins or derivatives thereof comprise biliverdin or analogues thereof, or wherein the porphyrins or derivatives thereof comprise a precursor of porphyrins comprising protoporphyrin.

[0027] In some embodiments, the porphyrins or derivatives thereof comprise phytochromobilin (PphiB) or analogues thereof. In some embodiments, the porphyrins or derivatives thereof comprise phycocyanobilin (PCB) or analogues thereof

[0028] In some embodiments, at least one of the one or more vectors comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, at least one of the one or more vectors comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, at least one of the one or more vectors comprises a nucleotide sequence of SEQ ID NO: 1.

[0029] In some embodiments, the recombinant protein is a recombinant ovalbumin or recombinant ovomucoid. In some embodiments, the recombinant protein is recombinant ovalbumin having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 9.

[0030] In some embodiments, the recombinant protein is a recombinant ovalbumin comprising the amino acid sequence of SEQ ID NO: 9.5 41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO

[0031] In some embodiments, the host cell is a yeast cell. In some embodiments, the yeast cell is Pichia pastoris.

[0032] Another aspect of the present disclosure includes a recombinant protein expressed or secreted by an engineered host cell of the present disclosure. In some embodiments, the recombinant protein is ovalbumin or ovomucoid.

[0033] Another aspect of the present disclosure includes a method for producing a recombinant protein in a host cell, the method comprising: (a) culturing the engineered host cell of any one of claim s 16-45 under conditions sufficient to secrete or express the recombinant protein, wherein the recombinant protein is secreted or expressed by the host cell into a liquid media; (b) harvesting the liquid media containing the secreted or expressed recombinant protein; (c) purifying the secreted or expressed recombinant protein by separating the recombinant protein from the liquid media.

[0034] In some embodiments, the liquid media does not comprise secreted biliverdin reductase. In some embodiments, the method comprises reduction or removal of porphyrins or derivati ves thereof.

[0035] Another aspect of the present disclosure includes a method of diverting or reducing the expression of porphyrins or derivatives thereof in an engineered host cell that expresses a recombinant protein, the method comprising: introducing a vector of the present disclosure encoding the chimeric protein into the engineered host cell, wherein tire engineered host cell reduces the production of porphyrins or derivatives thereof from the host cell thereof compared to a host cell that does not comprise the chimeric protein.

[0036] Another aspect of the present disclosure includes a method of reducing an amount of green colorants in a liquid media comprising recombinant ovalbumin secreted or expressed by an engineered host, cell, the method comprising: introducing a vector of the present, disclosure encoding the chimeric protein into the engineered host cell, wherein the engineered host cell reduces the production of porphyrins or derivati ves thereof from the engineered host cell thereof, thereby reducing the amount of green colorant.

[0037] In some embodiments, the method further comprises harvesting the liquid media containing the secreted or expressed recombinant ovalbumin. In some embodiments, the method further comprises purifying the secreted or expressed recombinant ovalbumin by6 41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO separating the recombinant ovalbumin from the liquid media. In some embodiments, the method further comprises spray drying the recombinant ovalbumin to formulate the recombinant ovalbumin into a powder.

[0038] In some embodiments, the powder does not comprise porphyrins or derivatives thereof bound to the recombinant ovalbumin. In some embodiments, the powder comprises a reduction of porphyrins or derivatives thereof bound to the recombinant ovalbumin compared to a recombinant ovalbumin produced from a host cell that does not compose the vector of the present disclosure.

[0039] In some embodiments, the liquid media comprising the recombinant ovalbumin comprises a yellow hue compared to a green hue of a liquid media comprising a recombinant ovalbumin produced from a host ceil that does not comprise the vector of the present disclosure,5. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0040] These and other features, aspects, and advantages of the present inven tion will become better understood with regard to the follow ing description, and accompanying drawings, where:

[0041] FIG. 1 provides a photo showing the green color of an egg.

[0042] FIG. 2 shows identification and isolation of the green compound. SDS PAGE gel analysis of green P2 sample (ovalbumin; “OVA”) suggested that the green molecule is bound to the P2 (OVA) molecule in some percentage of the green species population.

[0043] FIGs. 3A-3B chromatographic approaches to identify the isolation of green from P2 production. FIG. 1A show's SEC 200 of P2. FIG. 3B show's SEC 200 of P2 shows 650nm peak (blue region) overlaps with A280 (protein) peak. This suggests that the green molecule has a high affinity for P2.

[0044] FIG. 4 shows anion exchange chromatography with resolution of green peak from main P2 peak showing that these 2 species are separable using charge-based separation techniques.

[0045] FIG. 5 shows conversion of the unknown green molecule from green to brown using bilirubin oxidase.7 41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO

[0046] FIG. 6 shows gene expression data from different strains showed increasing expression of HEM 1 at the end of fermentation, w hen the color increases the most. This could be relevant to color production in K. phaffii since biological pathw ays are often regulated (by transcription, translation, post-translational modifications) at branch points between pathways. In this case, the heme biosynthesis pathway is limited by the first step, catalyzed by HEM 1. Metabolic network image was taken from SGD (https: / / www.yeastgenome.org / ) based on the known enzymes in the well studied yeast S. cerevisiae.

[0047] FIG. 7 shows gene expression data from a certain P2 strains in two different fermentation conditions show ed a dramatic increase in expression of HEM14 after induction and stayed high during later fermentation, when the color increases the most. This gene expression pattern could be consistent w ith increased production of protoporphyrin IX, the precursor to the final step in heme biosynthesis before iron is added to create ferroheme.

[0048] FIG. 8 shows blue molecule purification and the appearance. Purification of the unknown colorant that appeared to be greenish blue.

[0049] FIG. 9 provides a photo of the MF permeate for both strains (control strain 1512 and engineered strain (CS2729) fermented at 2L scale following the protocol described in Example 1.

[0050] FIG. 10 provides quantification of the color for the supernatant after broth centrifugation and after the second microfiltration. Absorbance at 450 nm was recorded using water as blank.

[0051] FIG. 11 provides quantification of the color for the supernatant after broth centrifugation and after the second microfiltration. Absorbance at 660 nm was recorded using water as blank.

[0052] FIG. 12 provides a polynucleotide map of the chimeric construct. This construct is a codon-optimized chimera. Tire structural outline is: GgBVR (chicken biliverdin) - alpha helix linker - peroxisomal targeting sequence (PTS).

[0053] FIG. 13 provides analysis of ion exchange chromatogram for Pl protein. For this, protein w as found to initiate elution at 95 mM sodium phosphate and the chromophore eluted8 41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO at 230 mM sodium phosphate. For P2, protein elution initiates at 40 mM sodium phosphate and the chromophore elutes at 225 mM sodium phosphate. Tire difference in protein elution for Pl and P2 is expected based on distinct isoelectric points. Pl has a lower isoelectric point and thus binds tighter to the resin. In contrast, the chromophore elutes at essentially the same sodium phosphate concentration for P l and P2 samples. This result is consistent with an identical chromophore bound to the different proteins.6. DETAILED DESCRIPTION|0054] Aspects of the present disclosure include chimeric proteins, engineered host cells that produce recombinant proteins, and methods of reducing unwanted colorants in recombinant proteins in engineered host cells that comprise the chimeric protein as described herein.6.1. Chimeric Proteins

[0055] An aspect of the present disclosure includes a chimeric protein comprising: a biliverdin reductase and a peroxisomal targeting signal.|0056] In some embodiments, the chimeric protein further comprises one or more linkers.

[0057] In some embodiments, the one or more linkers is configured to link the biliverdin reductase and the peroxisomal targeting signal.|0058] In some embodiments, the biliverdin reductase (BVR) is isolated from a Gallus galhis chicken. In some embodiments, the B VR is isolated from a duck or goose. In some embodiments, the BVR nucleotide sequence is codon optimized,

[0059] In some embodiments, the biliverdin reductase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 6. In some embodiments, the biliverdin reductase comprises an amino acid sequence of SEQ ID NO: 6.

[0060] In some embodiments, the linker comprises a flex linker. In some embodiments, the linker further comprises an alpha helix linker. A “flex linker”, as used herein, refers to a linker without secondary’ structure. For example, in some embodiments, the flex linker can comprise amino acid residues glycine and serine. The flex linker can link different functional domains.9 41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO

[0061] In some embodiments, the linker comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 7. In some embodiments, the linker comprises an amino acid sequence of SEQ ID NO: 7.

[0062] In some embodiments, the peroxisomal targeting signal comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 8. In some embodiments, the peroxisomal targeting signal comprises an amino acid sequence of SEQ ID NO: 8.

[0063] In some embodiments, the chimeric protein comprises a BVR, a linker, and a peroxisomal targeting signal, wherein the chimeric protein comprises an amino acid sequence having at least 80?% at least 85?%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 5. In some embodiments, the chimeric protein comprises an amino acid sequence of SEQ ID NO: 5.6.2. Polynucleotides encoding the chimeric protein

[0064] Another aspect of the present disclosure includes a polynucleotide encoding a chimeric protein described herein.

[0065] In some embodiments, the polynucleotide comprises: a nucleotide sequence encoding a biliverdin reductase, and a nucleotide sequence encoding a peroxisomal targeting signal. In some embodiments, the polynucleotide further comprises a nucleotide sequence encoding one or more linkers. In some embodiments, the one or more linkers are between the B VR and peroxisomal targeting signal. In some embodiments, the polynucleotide comprises: (a) the nucleotide sequence encoding tire biliverdin reductase; (b) the nucleotide sequence encoding a flex linker; (c) the nucleotide sequence encoding a alpha helix linker; (d) the nucleotide sequence encoding the third linker; and (e) the nucleotide sequence encoding the peroxisomal targeting sequence

[0066] In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80?% at least 85%, at least 90%, at least 95%, at least 96?% at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the polynucleotide comprises a nucleotide sequence of SEQ ID NO: 1.10 41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO

[0067] In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2. In some embodiments, the polynucleotide comprises a nucleotide sequence of SEQ ID NO: 2.

[0068] Another aspect of the present disclosure comprises a vector encoding the polynucleotide described herein.

[0069] Another aspect of the present disclosure comprises a construct comprising one or more vectors, wherein at least one vector comprises a polynucleotide encoding the chimeric protein described herein. In some embodiments, a second vector comprises a polynucleotide encoding a recombinant protein. In some embodiments, the recombinant protein is ovalbumin or ovomucoid. In some embodiments, the recombinant protein is ovalbumin. In some embodiments, the recombinant protein is ovomucoid. In some embodiments, the ovalbumin comprises a nucleotide sequence encoding a ovalbumin, wherein the ovalbumin comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 9.

[0070] In some embodiments, the nucleotide sequence encoding a recombinant ovalbumin and protein sequence of the recombinant oval bumin can be found in PCT application publication No,: W02021034980, which is hereby incorporated by reference in its entirety.

[0071] In some embodiments, the one or more vectors comprises one or more plasmids. In some embodiments, the one or more vectors are non-viral vectors. In some embodiments, the one or more vectors are viral vectors.

[0072] In some embodiments, the one or more vectors comprise a polynucleotide as shown in FIG. 12. This construct is a codon-optimized chimera. The structural outline is: BVR - alpha helix linker - peroxisomal targeting signal (PTS).6.3. Engineered Host cells

[0073] Another aspect of the present disclosure includes an engineered host cell comprising the chimeric protein described herein.

[0074] Another aspect of the present disclosure includes an engineered host cell comprising one or more vectors comprising: a polynucleotide comprising a nucleotide acid sequence encoding the chimeric protein described herein.11 41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO

[0075] In some embodiments, the host cell comprises a first vector comprising a polynucleotide comprising a nucleotide acid sequence encoding the chimeric protein. In some embodiments, the host cell further comprises a second vector comprising a nucleotide sequence encoding a recombinant protein. In some embodiments, the first vector comprises a polynucleotide comprising a nucleotide acid sequence encoding the chimeric protein and a nucleotide sequence encoding the recombinant protein. For example, in some embodiments, the chimeric protein and the recombinant protein are on the same vector. In some embodiments, the chimeric protein and the recombinant protein are on different vectors.

[0076] Another aspect of the present disclosure includes an engineered host cell comprising one or more vectors comprising: a nucleotide sequence encoding a recombinant protein; and a nucleotide sequence encoding a biliverdin reductase.

[0077] In some embodiments, the one or more vectors further comprise a nucleotide sequence encoding a flex linker. In some embodiments, the one or more vectors further comprises a nucleotide sequence encoding an alpha helix linker. In some embodiments, the one or more vectors further comprises a nucleotide sequence encoding a third linker.

[0078] In some embodiments, the one or more vectors further comprise a nucleotide sequence encoding a peroxisomal targeting sequence,

[0079] In some embodiments, the at least one of the one or more vectors comprises: (a) the nucleotide sequence encoding the recombinant protein; and (b) the nucleotide sequence encoding the biliverdin reductase; (c) the nucleotide sequence encoding the flex linker; (d) the nucleotide sequence encoding the alpha helix linker; (e) the nucleotide sequence encoding the third linker; and (f) the nucleotide sequence encoding the peroxisomal targeting sequence.

[0080] In some embodiments, a first vector comprises (a) the nucleotide sequence encoding the biliverdin reductase; (b) the nucleotide sequence encoding the flex linker; (c) the nucleotide sequence encoding the alpha helix linker; (d) the nucleotide sequence encoding the third linker; and (e) the nucleotide sequence encoding the peroxisomal targeting sequence. In some embodiments, a second vector comprises the nucleotide sequence encoding the recombinant protein.

[0081] In some embodiments, the engineered host cell comprises a first vector encoding (a) the nucleotide sequence encoding the recombinant protein; and (b) the nucleotide sequence12 41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO encoding the biliverdin reductase; (c) the nucleotide sequence encoding the flex linker; (d) the nucleotide sequence encoding the alpha helix linker; (e) the nucleotide sequence encoding the third linker; and (f) the nucleotide sequence encoding the peroxisomal targeting sequence.

[0082] In some embodiments, the nucleotide sequence encoding the recombinant protein is on the same vector as the biliverdin reductase or on a different vector.

[0083] In some embodiments, the engineered host cell comprises a first vector comprising: (b) the nucleotide sequence encoding the biliverdin reductase; (c) the nucleotide sequence encoding the flex linker; (d) the nucleotide sequence encoding the alpha helix linker; (e) the nucleotide sequence encoding the third linker; and (f) the nucleotide sequence encoding the peroxisomal targeting sequence signal; and a second vector comprising the nucleotide sequence encoding the recombinant protein.

[0084] In some embodiments, the biliverdin reductase is a codon optimized biliverdin reductase, comprising a nucleotide sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2. In some embodiments, the biliverdin reductase is a codon optimized biliverdin reductase, comprising a nucleotide sequence of SEQ ID NO: 2.

[0085] In some embodiments, the one or more linkers is a flex linker that comprises a nucleotide sequence having at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10. In some embodiments, the nucleotide sequence encoding the flex linker comprises a nucleotide sequence of SEQ ID NO: 10.

[0086] In some embodiments, the one or more linkers is an alpha helix linker that comprises a nucleotide sequence having at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11. In some embodiments, the nucleotide sequence encoding the alpha helix linker comprises a nucleotide sequence of SEQ ID NO: 11.

[0087] In some embodiments, the one or more linkers is third linker that comprises a nucleotide sequence having at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 12. In13 41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO some embodiments, the nucleotide sequence encoding the third linker comprises a nucleotide sequence of SEQ ID NO: 12.

[0088] In some embodiments, the one or more linkers comprise a nucleotide sequence having at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding the one or more linkers comprise a nucleotide sequence of SEQ ID NO: 3.

[0089] In some embodiments, the nucleotide sequence encoding the peroxisomal targeting sequence comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the peroxisomal targeting sequence comprises a nucleotide sequence of SEQ ID NO: 4.

[0090] In some embodiments, at least one of tire one or more vectors comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%,, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, at least one of the one or more vectors comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 1.

[0091] Another aspect of the present disclosure comprises an engineered host cell comprising a chimeric protein described herein. In some embodiments, the engineered host cell further comprises a recombinant protein described herein.

[0092] In some embodiments, chimeric protein comprising: a biliverdin reductase and a peroxisomal targeting signal. In some embodiments, the chimeric protein further comprises one or more linkers.

[0093] In some embodiments, the one or more linkers is configured to link the biliverdin reductase and the peroxisomal targeting signal. In some embodiments, the biliverdin reductase (BVR) is isolated from a Gallus gallus chicken. In some embodiments, the BVR is isolated from a duck or goose. In some embodiments, the BVR is codon optimized.

[0094] In some embodiments, the biliverdin reductase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at14 41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO least 98%, or at least 99% sequence identity to SEQ ID NO: 6. In some embodiments, the biliverdin reductase comprises an amino acid sequence of SEQ ID NO: 6.

[0095] In some embodiments, the linker comprises a flex linker. In some embodiments, the flex linker comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13. In some embodiments, the flex linker comprises an amino acid sequence of SEQ ID NO: 13.

[0096] In some embodiments, the linker further comprises an alpha helix linker. In some embodiments, the alpha helix linker comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14. In some embodiments, the alpha helix linker comprises an amino acid sequence of SEQ ID NO: 14.

[0097] In some embodiments, the linker further comprises a third linker. In some embodiments, the third linker is a second flex linker. In some embodiments, the third linker comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15. In some embodiments, the third linker comprises an amino acid sequence of SEQ ID NO: 15.

[0098] In some embodiments, the linker comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 7. In some embodiments, the linker comprises an amino acid sequence of SEQ ID NO: 7.

[0099] In some embodiments, the peroxisomal targeting signal comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 8. In some embodiments, the peroxisomal targeting signal comprises an amino acid sequence of SEQ ID NO: 8.

[0100] In some embodiments, the chimeric protein comprises a BVR, a linker, and a peroxisomal targeting signal, wherein the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at15 41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO least 98%, or at least 99% sequence identity to SEQ ID NO: 5. In some embodiments, the chimeric protein comprises an amino acid sequence of SEQ ID NO: 5.

[0101] In some embodiments, the engineered host cell described herein is a eukaryotic host cell. In some embodiments, the host cell is a yeast cell. In some embodiments, the yeast cell is pichia pastoris. In some embodiments, the yeast cell is Komagataella phaffi.6.3.1. Herne derivatives

[0102] In some embodiments, the engineered host cell reduces the production of heme derivatives.

[0103] In some embodiments, the heme derivative comprises a porphyrin or derivatives thereof.

[0104] In some embodiments, the engineered host cell reduces the production of porphyrins or derivatives thereof from the host cell thereof compared to a host cell that does not comprise the chimeric protein.

[0105] In some embodiments, the engineered host cell reduces the production of porphyrins or derivatives thereof from the host cell thereof compared to a host cell that does not comprise the biliverdin reductase.

[0106] In some embodiments, the host cell that does not comprise the biliverdin reductase comprises porphyrins or derivatives thereof bound to the recombinant protein upon expression of the recombinant protein. In some embodiments, the host cell comprises a reduction of porphyrins or derivatives thereof bound to the recombinant protein upon expression of the recombinant protein.

[0107] In some embodiments, the engineered host cell reduces the amount of prophyrins or derivatives thereof bound to the recombinant protein compared to the host cell that does not. comprise the biliverdin reductase.

[0108] In some embodiments, the host cell that does not comprise the chimeric protein comprises porphyrins or derivatives thereof bound to the recombinant protein upon expression of the recombinant protein. In some embodiments, the porphyrins or derivatives thereof are in a liquid media following a fermentation process to produce the recombinant protein from the engineered host cell.16 41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO

[0109] In some embodiments, the porphyrins or derivatives are bound to the recombinant protein in a host cell that does not comprise the chimeric protein described herein.

[0110] In some embodiments, the host cell reduces the amount, of prophyrins or derivatives thereof bound to the recombinant protein compared to the host cell that does not comprise the chimeric protein.

[0111] In some embodiments, the porphyrin or derivative thereof comprise biliverdin or analogues thereof. In some embodiments, the porphyrins or derivatives thereof comprise a precursor of porphyrins comprising protoporphyrin.

[0112] In some embodiments, the porphyrins or derivatives thereof comprise phytochromobilin (PphiB) or analogues thereof. In some embodiments, the porphyrins or derivatives thereof comprise phycocyanobilin (PCB) or analogues thereof.6.4. Recombinant proteins

[0113] Another aspect of the present disclosure comprises a recombinant protein expressed or secreted by an engineered host cell described herein.

[0114] In some embodiments, the recombinant protein is a recombinant ovalbumin or recombinant ovomucoid described herein. In some embodiments, the recombinant protein is a recombinant ovalbumin described herein. In some embodiments, the recombinant protein is a recombinant ovomucoid described herein.

[0115] In some embodiments, the recombinant ovalbumin that can be expressed or secreted by the engineered host cell described herein can be found in PCT application publication No.: WO2021034980, which is hereby incorporated by reference in its entirety'.6.5. Methods of expressing recombinant proteins with reduced colorants

[0116] Another aspect of the present disclosure includes methods of expressing recombinant proteins with reduced colorants by engineering the host cells described herein with the chimeric protein described herein to produce recombinant proteins that have a reduced amount of colorants produced by the heme deri vatives described herein.

[0117] For example, an aspect of the present disclosure includes a method for producing a recombinant protein in a host cell, tire method comprising: (a) culturing the engineered host17 41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO cell under conditions sufficient to secrete or express the recombinant protein, wherein the recombinant protein is secreted or expressed by the host cell into a liquid media; (b) harvesting the liquid media containing the secreted or expressed recombinant protein; (c) purifying the secreted or expressed recombinant protein by separating the recombinant protein from the liquid media.

[0118] In some embodiments, the method further comprises filtering the liquid media to purify the recombinant ovalbumin. In some embodiments, the method comprises diafiltering. In some embodiments, the method comprises ultrafiltration techniques. In some embodiments, the method comprises ultrafiltration and diafiltration techniques.

[0119] In some embodiments, the liquid media does not comprise secreted biliverdin reductase.

[0120] In some embodiments, the liquid media comprises reduction or removal of heme derivatives described herein. In some embodiments, the heme derivatives comprise porphyrins or derivatives thereof. In some embodiments, the liquid media comprises a reduction or removal of porphyrins or derivatives thereof.

[0121] Another aspect of the present disclosure includes a method of diverting or reducing the expression of porphyrins or derivatives thereof in an engineered host cell that expresses a recombinant protein, the method comprising: introducing a vector comprising a polynucleotide described herein encoding the chimeric protein into the engineered host cell.

[0122] In some embodiments, the engineered host cell reduces the production of heme derivatives from the host cell thereof compared to a host cell that does not comprise the chimeric protein.

[0123] Another aspect of the present disclosure includes a method of reducing an amount of green colorants in a liquid media comprising recombinant ovalbumin secreted or expressed by an engineered host cell, the method comprising: introducing a vector encoding a chimeric protein described herein into the engineered host cell, wherein the engineered host cell reduces the production of heme derivatives from the engineered host cell thereof, thereby reducing the amount of green colorant.18 41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO

[0124] In some embodiments, a host cell that does not comprise the chimeric protein described herein results in, following a fermentation process the host cell, a green color production in the fermentation liquid.

[0125] In some embodiments, the methods described herein further comprises harvesting the liquid media containing the secreted or expressed recombinant ovalbumin. In some embodiments, the methods described herein comprises purifying the secreted or expressed recombinant ovalbumin by separating the recombinant ovalbumin from the liquid media. In some embodiments, the methods described herein further comprises spray drying the recombinant ovalbumin to formulate the recombinant ovalbumin into a powder. In some embodiments, the powder does not comprise porphyrins or derivatives thereof bound to the recombinant ovalbumin.

[0126] In some embodiments, the powder comprises a reduction of porphyrins or derivatives thereof bound to the recombinant ovalbumin compared to a recombinant ovalbumin produced from a host cell that does not comprise the vector encoding the chimeric protein described herein.

[0127] In some embodiments, the liquid media comprising the recombinant ovalbumin comprises a yellow hue compared to a green hue of a liquid media comprising a recombinant ovalbumin produced from a host cell that does not comprise the vector of the present disclosure.

[0128] In some embodiments, the chimeric protein comprising a peroxisomal targeting sequence to ensure the recombinant biliverdin reductase (BVR) is not secreted into supernatant (liquid media) with a desired recombinant protein product,

[0129] In some embodiments, the present disclosure describe methods of shifting the color impact for recombinant protein products from Pichia pastoris from green to a yellow or reddish / orange color. The present methods take a systems approach to reduce color impact at the strain level (for example, not through downstream purification which is more typical in the industry’).

[0130] In some embodiments, the green molecule belongs to the porphyrin class. The present inventors performed UV-Visible spectrum of a recombinant protein powder product. The spectrum showed characteristic absorbance peaks at 650-660 nm that is known to result in19 41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO visual blue spectrum. The spectrum also showed intense absorbance peaks between 400-450 nm that typically result in visual yellow spectrum.

[0131] Another aspect of the present disclosure include electrochemical conversion of biliverdin to bilirubin using a chimeric, peroxisomal -targeted biliverdin reductase (BVR) in a host cell described herein. In some embodiments, recombinant biliverdin reductase (BVR) is not secreted into supernatant liquid media with the desired recombinant product.

[0132] Another aspect of the present disclosure includes a biliverdin reductase (BVR) protein used in a chimeric construct for producing engineered yeast strains that produce recombinant proteins.7. TABLE OF SEQUENCESName Sequence SEQ ID NO: Chimeric Protein ATGGCAGTAGTCAAAATAAACAAAATGTTTGGCACTGTAG I of FIG. 12 TGGTCGGCGTGGGCATCGCAGGCCTTGCAAGGATTAGGGABVR-linkers- CTTGATGAATCCCATGCCCTCCTCTCCCTCAGAGCACCTAA Peroxisomal AACTGC ITGGCTTCG ITAGTAGAAGGTCITTCGGAAATATAtargeting signal GATGAGGCAGAGCAAATTTCTCTAGAAGACGCCTTACGTT(PTS) CAAAGGAAATTCACGCTGCCTTCATTAGTACGGAAAACAG GTCCCATGAAGAAACAATAAGAATGTTCCTAGAGGCAGGANucleotide AAACATGTTCTTGTCGAATATCCAATGGCCCTAAGTGCTGAsequence AGCAGCTCACGAGCTATGGGAGACAGCCGAACAGAAAGG AAAGGTACTACACGTGGAACACATAGAGCTACTTACAGAG GAATACAAACAGITGAAGAAGGAAGTTGCCGGTAAAGACC TTGTAAAGGGAACGCTTCATTTCACAGGATCTGTATTGGAC GAGACAAAGGCCGG IT 1 TCCCGCTTTTTCTGGAATCGCACGTTTGAC TTGGCTG ATCGACC 1 I 1 I CGGAGATTTGACGATTACTTC AG CATCAAGAGAGGAACAGAAAGATAAGAATTATAGTCGTAT GACTGTACATTTCCAGACGGCCAACAAGAAGCCATTAACT TGGATCGAAGAAAGAGGCCCTGGTA TGCGTCG TGAAAAAA AGATAAATTTCTGCTTTACgAGTGGATGTCTGGAAAACTTT CCACAAGCCCCCAGGCCAGCCGTGGGATTGTTCATGCAGG ACCAGAATCTTTTTGCCAAAAAACTTCTTGGACAAGTTAGT AAGGAGGAACTTGCTgctGAGAAGTGGAGAATCCTGAGATG CCTGGATCTGGCTGGAATGATTCAACAGCAATGCGAGCAA CCCGAGAAAATA TGCTCAGGTTC A TCAGGGI CC TCAGGAT CATCCGGI AGTAGTGGTTCA TCCGGTTC ATCCGGAI CAAGT GGCTCCTCTGAAGCTGCAGCAAGGGAGGCTGCAGCCCGTG AGGCAGCCGCTAGAGAAGCCGCCGCTAGGGGTGGTGGCGG CTCTGGCGGAGGCGGTTCCGGTGGCGGAGGCTCTTTCATCCCTAGACGTGCTCATAGAAGTTCTAAACTATAA20 41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO Codon optimized ATGGCAGTAGTCAAAATAAACAAAATGTTTGGCACTGTAG 2 Biliverdin TGGTCGGCGTGGGCATCGCAGGCCTTGCAAGGATTAGGGA reductase (gallus CTTGATGAATCCCATGCCCTCCTCTCCCTCAGAGCACCTAA gallus) AACTGCTTGGCTTCGTTAGTAGAAGGTCTTTCGGAAATATA Nucleotide GATGAGGCAGAGCAAATTTCTCTAGAAGACGCCTTACGTT sequence CAAAGGAAATTCACGCTGCCTTCATTAGTACGGAAAACAG GTCCCATGAAGAAACAATAAGAATGTTCCTAGAGGCAGGA AAACATGTTCTTGTCGAATATCCAATGGCCCTAAGTGCTGA AGCAGCTCACGAGCTATGGGAGACAGCCGAACAGAAAGG AAAGGTACTACACGTGGAACACATAGAGCTACTTACAGAG GAATACAAACAGTTGAAGAAGGAAGTTGCCGGTAAAGACC TTGTAAAGGGAACGC TTC AITIC AC AGGATCI GTATTGGAC GAGACAAAGGCCGGITITCCCGCTTTTTCTGGAATCGCACG TTTGACTTGGCTGATCGACC 1111 CGGAGATTTGACGATTA CTTCAGCATCAAGAGAGGAACAGAAAGATAAGAATTATAG TCGTATGACTGTACATTTCCAGACGGCCAACAAGAAGCCA TTAACTTGGATCGAAGAAAGAGGCCCTGGTATGCGTCGTG AAAAAAAGATAAATTTCTGCTTTACgAGTGGATGTCTGGAA AACTTTCCACAAGCCCCCAGGCCAGCCGTGGGATTGTTCAT GCAGGACCAGAATCTTTTTGCCAAAAAACTTCTTGGACAA GTTAGTAAGGAGGAACTTGCTgctGAGAAGTGGAGAATCCT GAGATGCCTGGATCTGGCTGGAATGATTCAACAGCAATGC GAGCAACCCGAGAAAATATGCTCALinkers GGTTCATCAGGGTCCTCAGGATCATCCGGTAGTAGTGGTTC 3 Flex linker - ATCCGGTTCATCCGGATCAAGTGGCTCCTCTGAAGCTGCAG Alpha helix CAAGGGAGGCTGCAGCCCGTGAGGCAGCCGCTAGAGAAGC linker - third CGCCGCTAGGGGTGGTGGCGGCTCTGGCGGAGGCGGTTCC linker GGTGGCGGAGGCTCTNucleotidesequencePeroxisomal TTCATCCCTAGACGTGCTCATAGAAGTTCTAAACTATAA 4 targeting signalNucleotidesequenceChimeric Protein MAVVKINKMFGTVVVGVGIAGLARIRDLMNPMPSSPSEFILKL 5 of FIG. 12 LGFVSRRSFGNIDEAEQISLEDALRSKEIHAAFISTENRSHEETI BVR-linkers- RMFLEAGKHVLVEYPMALSAEAAHELWETAEQKGKVLHVE Peroxisomal H1ELLTEEYKQLKKEVAGKDLVKGTLHFTGSVLDETKAGFPA targeting signal FSGIARLTWLIDLFGDLTITSASREEQKDKNYSRMTVHFQTAN (PTS) KKPLTWIEERGPGMRREKKINFCFTSGCLENFPQAPRPAVGLF MQDQNI-FAKKLLGQVSKEELAAEKWRILRCLDLAGMIQQQC Amino acid EQPEKICSGSSGSSGSSGSSGSSGSSGSSGSSEAAAREAAAREA sequence AAREAAARGGGGSGGGGSGGGGSFIPRRAHRSSKL*BVR (codon MAVVKINKMFGWVVGVGIAGLARIRDIA'INPMPSSPSEHLKI. 6 optimized) LGFVSRRSFGNIDEAEQISLEDALRSKEIHAAFISTENRSHEETI Amino acid RMFLEAGKHVLVEYPJVLALSAEAAHELWErAEQKGKVLHVE sequence HIELLTEE YKQLKKEVAGKDL VKGTLHF TGS VLDE TKAGFP AFSGIARLTWLIDLFGDLTITSASREEQKDKNYSRMTVHFQTANKKPLTWIEERGPGMRREKKINFCFTSGCLENFPQAPRPAVGLF21 41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO MQDQNLFAKKLLGQVSKEELAAEKWRILRCLDLAGMIQQQC EQPEKICSLinkers GSSGSSGSSGSSGSSGSSGSSGSSEAAAREAAAREAAAREAAA 7 Flex linker - RGGGGSGGGGSGGGGSAlpha helixlinker - thirdlinkerAmino acidsequencePeroxisomal FIPRRAHRSSKL* 8 targeting signalAmino acidsequenceRecombinant GSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYL 9 ovalbumin GAKDSTRTQINKVVRFDKLPGFGDSIEAQCGTSVNVHSSLRDI LNQITKPNDWSFSLASRLYAEERYPILPEYLQCVKELYRGGLAmino acid EPINFQTAADQARELINSWVESQTNG11RNVLQPSSVDSQTAM sequence VLYNAIVFKGLWEKAFKDEDTQAMPFRVTEQESKPVQMMY QIGLFRVASMASEKMKILELPFASGTMSMLVLLPDEVSGLEQL ESIINFEKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVL MAMGITDVFSSSANLSGISSAESLKISQAVHAAHAEINEAGRE WGSAEAGVDAASVSEEFRADHPFLFCIKHIATNAVLFFGRCV SPFlex linker GGTTCATCAGGGTCCTCAGGATCATCCGGTAGTAGTGGTTC 10 Nucleotide ATCCGGTTCATCCGGATCAAGTGGCTCCTCTsequenceAlpha helix GAAGCTGCAGCAAGGGAGGCTGCAGCCCGTGAGGCAGCCG 11 linker nucleotide CTAGAGAAGCCGCCGCTAGGsequence3rdlinker GGTGGTGGCGGCTCTGGCGGAGGCGGTTCCGGTGGCGGAG 12 Nucleotide GCTCTsequenceFlex linker GSSGSSGSSGSSGSSGSSGSSGSS 13 Amino acidsequenceAlpha helix EAAAREAAAREAAAREAAAR 14 linker aminoacid sequence3rd linker amino GGGGSGGGGSGGGGS 15acid sequence8. EXAMPLES8.1. Example 1: Identification of heme derivatives in pichia pastoris expressing recombinant ovalbumin22 41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO

[0133] The purpose of this study was to isolate and characterize a green molecule that was found to be bound or tightly associated with a recombinant ovalbumin developed by the present inventors expressed in Pichia pastoris.

[0134] FIG. 2 show s identification and isolation of tire green compound. SDS PAGE gel analysis of green P2 sample (ovalbumin; “OVA”) suggested that the green molecule is bound to the P2 (OVA) molecule in some percentage of the green species population.

[0135] Strong anion exchange resin (specifically Q resins as produced by Cytiva) w ill tightly bind free heme on a charge basis. Therefore, chromatographic approaches were taken to attempt the separation and isolation of green molecule from recombinant ovalbumin production.

[0136] A UV-Visible spectrum was performed with a recombinant ovalbumin protein powder product produced from Pichia pastoris. As shown in FIG. 3, the spectrum showed characteristic absorbance peaks at 650-660 nm that is known to result in visual blue spectrum. The spectrum also showed intense absorbance peaks between 400-450 nm that typically result in visual yellow spectrum. The present inventors determined the nature of the colorants to be porphyrin compounds.

[0137] FIG. 4 shows anion exchange chromatography with resolution of green peak from main P2 peak showing that these 2 species are separable using charge-based separation techniques. Purified material from FIG. 4 was attempted in a reaction catalyzed by bilirubin oxidase. Bilirubin oxidase changed the green color to red / brown (FIG. 5). Bilirubin was shown to be a degradation product of heme that is red / yellow with a canonical absorption peak at 450 nm. Its precursor molecule in the degradation pathway. The results show that biliverdin is greenish blue and is canonically measured by its characteristic absorption peak at 650 nm.

[0138] A blue residual layer w'as observed on the column and was then further investigated. The present inventors found that the blue molecule was the free form of the impurity chromophore and that it appeared green when bound / associated to / with the recombinant ovalbumin. Use of acidic -pH buffer below' the pKa of the carboxylic heme substituent groups was hypothesized to allow this molecule to unbind from the Q resin and become soluble enough to isolate. This molecule was isolated and appeared insoluble in water in concentrate which is consistent with its identity as a biliverdin analog / derivative as showm in FIG. 8.23 41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO

[0139] Additional evidence regarding the presence of relevant enzymes in the Pichia genome performed by the present inventors revealed that Pichia has the enzyme Heme Oxidase (which can perform the role of Bilirubin Oxidase, but not of Biliverdin Reductase) further strengthening the interest in inserting this gene to see its effect on the metabolic cycling of heme derivatives that results in green color.

[0140] FIG. 6 shows gene expression data from different strains showed increasing expression of HEM 1 at the end of fermentation, when the color increases the most. This could be relevant to color production in K. phaffii since biological pathways are often regulated (by transcription, translation, post-translational modifications) at branch points between pathways. In this case, the heme biosynthesis pathway is limited by the first step, catalyzed by HEM 1.

[0141] FIG. 7 shows gene expression data from a certain P2 strains in two different fermentation conditions showed a dramatic increase in expression of HEM14 after induction and stayed high during later fermentation, when the color increases the most. This gene expression pattern could be consistent with increased production of protoporphyrin IX, the precursor to the final step in heme biosynthesis before iron is added to create ferroheme.

[0142] Based on these experiments, it was determined that the compound included biliverdin analogues.

[0143] Additionally, as shown in FIG. 13, a green color was observed in protein samples of both recombinant ovalbumin proteins Pl and P2 recombinant ovalbumin products generated from Pichia pastoris fermentation. The common sources of protein production lead to consideration that the green species is the same for the two sets of samples.

[0144] Ion exchange chromatography was performed to compare the elution pattern of the species in Pl and P2 recombinant, ovalbumin solutions, -10 mg solutions of 10-50 mg / ml protein were diluted into 20 mM sodium phosphate pH 6.7 and loaded onto a column containing 5ml CaptoQ resin equilibrated in 20 mM sodium phosphate pH 6.7. Protein and chromophore were eluted over gradient from 20 mM sodium phosphate to 500 mM sodium phosphate. In each case, the absorbance at 280 nm was employed to monitor protein elution and 650 nm to monitor chromophore elution. Shown below is the chromatogram for Pl protein. For this, protein was found to initiate elution at 95 mM sodium phosphate and the chromophore eluted at 230 mM sodium phosphate. For P2, protein elution initiates at.40 mM24 41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO sodium phosphate and the chromophore elutes at 225 mM sodium phosphate. The difference in protein elution for Pl and P2 was expected based on distinct isoelectric points. Pl had a lower isoelectric point and thus binds tighter to the resin. In contrast, the chromophore elutes at essentially the same sodium phosphate concentration for Pl and P2 samples.|0145] This result was consistent with an identical chromophore bound to the different proteins8.2. Example 2: Pichia pastoris engineered to produce the chimeric protein

[0146] This study introduces the chimeric construct of SEQ ID NO: 1 containing the biliverdin reductase into an engineered pichia pastoris host cell that expresses a recombinant ovalbumin to determine whether a reduction in colorant is observed in the fermentation media following fermentation of the host cell.

[0147] FIG. 12 shows the chimeric protein construct of SEQ ID NO: 1. This construct is a codon-optimized chimera. Tire structural outline is: GgBVR (chicken biliverdin reductase) - alpha helix linker - peroxisomal targeting signal sequence (PTS).Methods:

[0148] Control strain (CS 1512) and engineered strain (CS 2729) were fermented at 2 L scale following an existing protocol. After harvest, the broth was centrifuged at 14,000 RCF for 20 min at 10C.

[0149] The Supernatant, which contains the recombinant protein ovalbumin, was decanted and filtered through 0.2 micron PES filter. Next, the filtrate was concentrated by a 5-fold (by¬ volume) using 50 kDa PES filter (Pall, OMEGA T-Series) through the ultrafiltration process using bench-top tangential flow filtration (TFF) unit (Pall, model FILTRATE).

[0150] Diafiltration was performed using the same TFF unit with 10 diafiltration volume of deionized water (as UF / DF process). The UF retentate, which contained the recombinant protein ovalbumin, -was filtered again using 0.2 micron PES filter. A photo of the MF permeate for both strains are shown in FIG. 9.

[0151] FIG. 9 shows the control strain 1512 on the left and strain 2729 on the right with visible shift from green to yellow color.25 41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO

[0152] The color was quantified for the supernatant after broth centrifugation and after the second microfiltration as shown in FIG 9. Absorbance at 450 nm (FIG. 9) and 660 nm (FIG.11) was recorded using water as blank, based on the identification work that was described previously. Hie data are shown in FIGs. 10-11.Results

[0153] As shown in FIGs. 10-11, there was a reduction of absorbance at 660 nm (“blue”) for strain 2729, indicating the reduction of biliverdin analogue with the engineered strain containing BVR.9. EQUIVALENTS AND INCORPORATION BY REFERENCE

[0154] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention,

[0155] All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.26 41522 / 65386 / FW / 20093036.1

Claims

Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO What is Claimed is:

1. A chimeric protein comprising:a biliverdin reductase;one or more linkers; anda peroxisomal targeting signal.

2. The chimeric protein of claim 1, wherein the biliverdin reductase comprises an amino acid sequence having at least 80%, at least 85?% or at least 90? A sequence identity to SEQ ID NO: 6.

3. Tire chimeric protein of claim 1, wherein the biliverdin reductase comprises an amino acid sequence of SEQ ID NO: 6.

4. The chimeric protein of any one of claims 1-3, wherein the linker comprises a flex linker.

5. The chimeric protein of any one of claims 1-4, wherein the linker further comprises an alpha helix linker.

6. The chimeric protein of any one of claims 1-5, wherein the linker comprises an amino acid sequence having at least 80%, at least 85?% or at least 90% sequence identity to any one of SEQ ID NOs: 7 and 13-15.

7. The chimeric protein of claim 6, wherein the linker comprises an amino acid sequence of any one of SEQ ID NOs: 7 and 13-15.

8. Tlie chimeric protein of any one of claims 1-7, wherein the peroxisomal targeting signal comprises an amino acid sequence having at least 80%, at least 85%, or at least 90% sequence identity to SEQ ID NO: 8.

9. The chimeric protein of any one of claims 1-8, wdrerein the peroxisomal targeting signal comprises an amino acid sequence of SEQ ID NO: 8,27 41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO 10. The chimeric protein of any one of claims 1-9, wherein the chimeric protein comprises an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 5.

11. The chimeric protein of any one of claims 1-10, wherein the chimeric protein comprises an amino acid sequence of SEQ ID NO: 5.

12. A polynucleotide encoding the chimeric protein of any one of claims 1-11.

13. The polynucleotide of claim 10, comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 1.

14. The polynucleotide of claim 11, comprising a nucleotide sequence of SEQ ID NO: 1.

15. A vector comprising the polynucleotide of any one of claims 12.-13.

16. An engineered host cell comprising one or more vectors comprising:a nucleotide sequence encoding the chimeric protein of any one of claims 1- 11, wherein the engineered host cell reduces the production of porphyrins or derivatives thereof from the host cell thereof compared to a host cell that does not compose the chimeric protein.

17. The engineered host cell of claim 16, wherein the engineered host cell further comprises a vector comprising a nucleotide sequence encoding a recombinant protein, wherein the host cell that does not comprise the chimeric protein comprises porphyrins or derivatives thereof bound to the recombinant protein upon expression of the recombinant protein.

18. The engineered host cell of claim 16, wherein engineered host cell reduces the amount of porphyrins or derivatives thereof bound to the recombinant protein compared to the host cell that does not comprise tire chimeric protein.

19. An engineered host cell comprising one or more vectors comprising:a nucleotide sequence encoding a recombinant protein; anda nucleotide sequence encoding a biliverdin reductase, wherein the engineered host cell reduces the production of porphyrins or derivatives thereof from the host cell thereof compared to a host cell that does not comprise the biliverdin reductase.28 41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO 20. The engineered host cell of claim 19, wherein the host cell that does not comprise the biliverdin reductase comprises porphyrins or derivatives thereof bound to the protein upon expression of the recombinant protein.

21. Tire engineered host cell of any one of claim s 19-20, wherein engineered host cell reduces the amount of porphyrins or derivatives thereof bound to the recombinant protein compared to the host cell that does not comprise the biliverdin reductase.

22. The engineered host cell of any one of claims 19-21, wherein the one or more vectors further comprise a nucleotide sequence encoding a flex linker having at least 90% sequence identity to SEQ ID NO: 10.

23. The engineered host cell of any one of claims 19-22, wherein the one or more vectors further comprises a nucleotide sequence encoding an alpha helix linker having at least 90% sequence identity to SEQ ID ON: 11.

24. Tire engineered host cell of any one of claims 19-23, wherein the one or more vectors further comprises a nucleotide sequence encoding a third linker having at least 90% sequence identity to SEQ ID NO: 12.

25. The engineered host cell of any one of claims 19-24, wherein the one or more vectors further comprise a nucleotide sequence encoding a peroxisomal targeting sequence.

26. The engineered host cell of claim 25, wherein at least one of the one or more vectors comprises:(a) the nucleotide sequence encoding the recombinant protein; and(b) the nucleotide sequence encoding the biliverdin reductase;(c) the nucleotide sequence encoding the flex linker;(d) the nucleotide sequence encoding the alpha helix linker;(e) the nucleotide sequence encoding the third linker; and(f) the nucleotide sequence encoding the peroxisomal targeting signal.

27. The engineered host cell of any one of claims 19-26, wherein the biliverdin reductase is a codon optimized biliverdin reductase, comprising a nucleotide sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 2.29 41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO 28. The engineered host cell of any one of claims 19-27, wherein the biliverdin reductase is a codon optimized biliverdin reductase, comprising a nucleotide sequence of SEQ ID NO:

229. The engineered host cell of any one of claims 22-2.8, wherein the nucleotide sequence encoding the alpha helix linker and flex linker comprises a nucleotide sequence having at least 80% or at least 90% sequence identity to SEQ ID NO: 3.

30. The engineered host cell of any one of claims 2.2-29, wherein the nucleotide sequence encoding the one or more linkers comprise a nucleotide sequence of any one of SEQ ID NOs: 3 and 10-12.

31. Tire engineered host cell of any one of claims 26-30, wherein the nucleotide sequence encoding the peroxisomal targeting sequence comprises a nucleotide sequence having at least 80% or at least 90% sequence identity to SEQ ID NO: 4.

32. The engineered host, cell of claim 31, wherein the nucleotide sequence encoding the peroxisomal targeting sequence comprises a nucleotide sequence of SEQ ID NO: 4.

33. Tire engineered host cell of any one of claims 19-32, wherein the nucleotide sequence encoding the recombinant protein is on the same vector as the biliverdin reductase or on a different vector.

34. The engineered host cell of any one of claims 26-33, wherein one or more vectors comprises:a first vector comprising:(b) the nucleotide sequence encoding the biliverdin reductase;(c) the nucleotide sequence encoding the flex linker;(d) the nucleotide sequence encoding the alpha helix linker;(e) the nucleotide sequence encoding the third linker; and(f) the nucleotide sequence encoding the peroxisomal targeting sequence sequence; anda second vector comprising the nucleotide sequence encoding the recombinant protein.30 41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO35. The engineered host cell of any one of claims 19-34, wherein the porphyrins or derivatives thereof comprise biliverdin or analogues thereof, or wherein the porphyrins or derivatives thereof comprise a precursor of porphyrins comprising protoporphyrin.

36. The engineered host cell of any one of claims 19-35, wherein the porphyrins or derivatives thereof comprise phytochromobilin (PphiB) or analogues thereof.

37. The engineered host, cell of any one of claims 19-35, wherein the porphyrins or derivatives thereof comprise phycocyanobilin (PCB) or analogues thereof.

38. The engineered host cell of any one of claims 6-37, wherein at least one of the one or more vectors comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 1.

39. The engineered host cell of claim 25, wherein at least one of the one or more vectors comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 1.

40. The engineered host cell of claim 25, wherein at least one of the one or more vectors comprises a nucleotide sequence of SEQ ID NO: 1.

41. The engineered host cell of any one of claims 19-40, wherein the recombinant protein is a recombinant ovalbumin or recombinant ovomucoid.

42. The engineered host cell of any one of claims 19-41, wherein the recombinant protein is recombinant ovalbumin having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 9.

43. The engineered host cell of any one of claims 19-42, wherein the recombinant protein is a recombinant ovalbumin comprising the amino acid sequence of GSIGAASMEFCFDVFKEI. VHHANFJS1IFYCPIAIMSAI MVYLGAKDSTRTQINKVV RFDKLPGFGDSIEAQCGTSVNVIISSLRDILNQITKPNDVYSFSLASRLYAEERYPILPE YLQCVKELYRGGLEPINFQTAADQARELINSWVESQTNGIIRNVLQPSSVDSQTAMV LVNAIVFKGLWEKAFKDEDTQAMPFRVTEQESKPVQMMYQIGLFRVASMASEKMK ILELPFASGTMSMLVLLTDEVSGLEQLESIINFEKLTEWTSSNVMEERKIKVYLTRMK31 41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO MEEKYNLTSVLMAMGITOVFSSSANLSGISSAESLKISQAVHAAHAEINEAGREWG SAEAGVDAASVSEEFRADHPFLFCIKHIATNAVLFFGRCVSP (SEQ ID NO: 9; rOVA).

44. The engineered host cell of any one of claims 16-43, wherein the host cell is a yeast cell.

45. The engineered host cell of claim 44, wherein the yeast cell is Pi chi a pastoris.

46. A recombinant protein expressed or secreted by an engineered host cell of any one of claims 16-45.

47. The recombinant protein of any one of claims 19-29, wherein the recombinant protein is ovalbumin or ovomucoid.

48. A method for producing a recombinant protein in a host cell, the method comprising:(a) culturing the engineered host cell of any one of claims 16-45 under conditions sufficient to secrete or express the recombinant protein, wherein the recombinant protein is secreted or expressed by the host cell into a liquid media;(b) harvesting the liquid media containing the secreted or expressed recombinant protein;(c) purifying the secreted or expressed recombinant protein by separating the recombinant protein from the liquid media.

49. The method of claim 48, wherein the liquid media does not comprise secreted biliverdin reductase.

50. The method of claim 49, wherein the method comprises reduction or removal of porphyrins or derivatives thereof.

51. A method of diverting or reducing tlie expression of porphyrins or derivatives thereof in an engineered host cell that expresses a recombinant protein, the method comprising: introducing a vector of claim 15 encoding the chimeric protein into the engineered host cell, wherein the engineered host cell reduces the production of porphyrins or derivatives thereof from the host cell thereof compared to a host cell that does not comprise the chimeric protein.32 41522 / 65386 / FW / 20093036.1Attorney Ref.: 41522- 65386 / WO Client Ref: 770 / 01 WO 51. A method of reducing an amount of green colorants in a liquid media comprising recombinant ovalbumin secreted or expressed by an engineered host cell, the method comprising:introducing a vector of claim 15 encoding the chimeric protein into the engineered host cell, wherein the engineered host cell reduces the production of porphyrins or derivatives thereof from the engineered host cell thereof, thereby reducing the amount of green colorant.

52. The method of claim 51, wherein the method further comprises harvesting the liquid media containing the secreted or expressed recombinant ovalbumin.

53. The method of claim 52, wherein the method further comprises purifying the secreted or expressed recombinant ovalbumin by separating the recombinant ovalbumin from the liquid media.

54. The method of claim 53, wherein the method further comprises spray drying the recombinant ovalbumin to formulate the recombinant ovalbumin into a powder,55. The method of claim 54, wherein the powder does not comprise porphyrins or derivatives thereof bound to tire recombinant ovalbumin.

56. The method of claim 54, wherein the powder comprises a reduction of porphyrins or derivatives thereof bound to the recombinant ovalbumin compared to a recombinant ovalbumin produced from a host cell that does not comprise the vector of claim 15.

57. The method of any one of claims 51-53, wherein the liquid media comprising the recombinant ovalbumin comprises a yellow hue compared to a green hue of a liquid media comprising a recombinant ovalbumin produced from a host cell that does not comprise the vector of claim 15.33 41522 / 65386 / FW / 20093036.1

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