EP-4735472-A1 - RECOMBINANT FUNGAL CELLS AND METHODS THEREOF FOR INDUSTRIAL SCALE PRODUCTION OF LECTINS

Abstract

The present disclosure is generally related to methods and compositions for producing heterologous lectin proteins of interest in recombinant filamentous fungal cells. Certain embodiments are therefore, directed to compositions and methods for the production of lectin proteins, recombinant filamentous fungal strains comprising enhanced lectin protein productivity phenotypes, polynucleotides ( e.g ., expression constructs) encoding one or more lectin proteins, industrial scale fermentation, lectin protein recovery processes and the like.

Inventors

• ESTELL, DAVID A.
• HENG, MENG HONG
• HUANG, LIXUAN
• Bhate, Manasi
• ZHAO, QIANG
• LEI, Chunli

Assignees

• Danisco US Inc.

Dates

Publication Date

20260506

Application Date

20240701

Claims (1)

1. NB42133-WO-PCT[2] CLAIMS 1. A recombinant filamentous fungal cell expressing a polynucleotide encoding a heterologous lectin. 2. The recombinant cell of claim 1, wherein the lectin is secreted into the broth when fermented under suitable conditions for the production of the lectin. 3. The recombinant cell of claim 1, wherein the lectin is derived from a plant cell, a cyanobacterial cell, an algae cell, a bacterial cell, a fungal cell, an insect cell, or an animal cell. 4. The recombinant cell claim 1, wherein polynucleotide encoding the lectin is an introduced expression cassette comprising an upstream promoter sequence operably linked to a downstream nucleic acid encoding a signal sequence operably linked to a downstream nucleic acid encoding the lectin. 5. The recombinant cell of claim 1, wherein the polynucleotide encoding the lectin is integrated into the genome of cell. 6. The recombinant cell of claim 1, wherein the cell is an Ascomycete filamentous fungal cell. 7. The recombinant cell of claim 1, wherein the lectin is a monomer. 8. The recombinant cell of claim 1, wherein the lectin is a dimer. 9. The recombinant cell claim 1, wherein the lectin is a fusion protein. 10. The recombinant cell of claim 1, comprising a genetic modification rendering the cell deficient in the production of one or more endogenous enzymes. 11. An expression cassette comprising an upstream promoter sequence functional in an Ascomycete cell operably linked to a downstream nucleic acid encoding a secretion sequence functional in an Ascomycete cell operably linked to a downstream nucleic acid encoding a heterologous lectin. 12. The cassette of claim 11, wherein the encoded lectin is a monomer or a dimer. 13. The cassette of claim 11, wherein the encoded lectin is a fusion protein. 14. A method for producing a heterologous lectin in a filamentous fungal cell comprising: (a) obtaining a filamentous fungal cell and introducing into the cell an expression cassette comprising an upstream promoter operably linked to a downstream nucleic acid encoding a secretion sequence operably linked to a downstream nucleic acid encoding the lectin, and (b) fermenting the modified cell under suitable conditions for the production of the lectin, wherein the lectin is secreted into the fermentation broth. NB42133-WO-PCT[2] 15. The method of claim 14, wherein the lectin is derived from a plant cell, a cyanobacterial cell, an algae cell, a bacterial cell, a fungal cell, an insect cell, or an animal cell. 16. The method of claim 14, wherein the cassette is integrated into the genome of cell. 17. The method of claim 14, wherein the cell is an Ascomycete filamentous fungal cell. 18. The method of claim 14, wherein the lectin is a monomer. 19. The method of claim 14, wherein the lectin is a dimer. 20. The method of claim 14, wherein the lectin is a fusion protein. 21. The method of claim 19, wherein the lectin dimer comprises an amino acid linker sequence between the first and second lectin sequences, wherein the linker sequence comprises about four (4) to about six (6) amino acid residues. 22. The method of claim 20, wherein the lectin fusion protein comprises an N-terminal protein fusion and/or comprises a C-terminal protein fusion. 23. The method of claim 14, wherein the cell comprises a genetic modification rendering the cell deficient in the production of one or more endogenous enzymes. 24. The method of claim 14, comprising harvesting the end of fermentation broth comprising the secreted lectin. 25. The method of claim 24, wherein the harvested broth is subjected to a clarification process 26. The method of claim 25, wherein the clarified broth is subjected to a concentration process. 27. The method of claim 26, wherein the lectin is recovered from the clarified and concentrated broth. 28. A lectin protein preparation obtained by the method of claim 14.

Description

NB42133-WO-PCT[2] RECOMBIANT FUNGAL CELLS AND METHODS THEREOF FOR INDUSTRIAL SCALE PRODUCTION OF LECTINS CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims benefit International Application No. PCT/CN2023/104906, filed June 30, 2023, which is hereby incorporated by reference in its entirety. FIELD [0002] The present disclosure is generally related to the fields of microbial cells, molecular biology, fermentation, protein production, protein recovery, protein purification, protein preparations, and the like. Certain aspects of the disclosure are related to the industrial scale production and recovery of lectin proteins, recombinant filamentous fungal cells producing heterologous lectins, compositions and methods for recovering and/or purifying one or more lectins, enhanced purity lectin preparations thereof and the like. REFERENCE TO A SEQUENCE LISTING [0003] The contents of the electronic submission of the text file Sequence Listing, named “NB42133-WO- PCT_SequenceListing.xml” was created on June 21, 2023, and is 185 KB in size, which is hereby incorporated by reference in its entirety. BACKGROUND [0004] Lectins are generally defined as carbohydrate binding proteins that can recognize and bind simple or complex carbohydrates in a reversible and highly specific manner, while displaying no catalytic activity (Lagarda-Diaz et al., 2017). Lectin proteins were originally named hemagglutinins, due to their ability to agglutinate red blood cells (and other cells). More recently, lectins such as the red algae (Griffithsia sp.) griffithsin (GRFT) protein, the red algae (Kappaphycus alvarezii) KAA-2 protein, the concanavalin A (ConA) protein from jack-beans, the jacalin protein from jackfruit (A. heterophyllus), the cyanovirin-N (CV-N) protein from cyanobacteria (N. ellipsosporum) and the like, have been evaluated for their anti-viral activities (Whitley et al., 2013). [0005] For example, PCT Publications WO2005/118627 and WO2007/064844, describe methods for isolating the native griffithsin (GRFT) lectin from red algae (Griffithsia sp.), cloning the wild-type (grft) gene thereof, generating recombinant polynucleotides thereof, fermenting and producing the same in E. coli host cells, followed by isolating the recombinant His-tagged GRFT protein from the E. coli host, and characterizing its anti-viral activity. PCT Publication No. WO2010/01424 generally describes methods of inhibiting a hepatitis C viral infection of a host comprising administering to the host an effective amount of a glycosylation resistant GRFT (variant) protein (or a polypeptide conjugate thereof) in combination with NB42133-WO-PCT[2] another anti-viral protein. For example, as described in this publication, the inventors noted that the anti- viral protein combination of scytovirin (SVN) and griffithsin (GRFT) have (nanomolar) activity against the Hepatitis C virus (HCV). [0006] U.S. Patent Publication No. US20110263485 describes methods of inhibiting a human immunodeficiency virus (HIV) infection of a host comprising administering to the host an effective amount of a gp120 Griffithsin and a peptide selected from a gp41-binding protein, a CCR5-binding protein, a gp120-binding protein, or another griffithsin, which combinations are potent inhibitors to HIV infection. PCT Publication No. WO2016/130628 discloses variant griffithsin proteins having mutations that change the isoelectric point of the GRFT protein, which are reported to alter its solubility in various pH ranges allowing for improved product release. PCT Publication No. WO2019/108656 generally describes microbicidal compositions comprising an endosperm extract and an anti-HIV lectin, an anti-HIV antibody, or antigen binding antibody fragment thereof. [0007] The recombinant production of GRFT in tobacco plants (Nicotiana benthamiana) has been described by O’Keefe et al. (2009), wherein the GRFT accumulates to a level of about 1 gram of recombinant GRFT per kilogram of Nicotiana benthamiana leaf material, when expressed via an infectious tobacco mosaic virus (TMV) based vector. For example, as contemplated in the O’Keefe et al. publication, despite the promise that biologics such as griffithsin have as HIV prophylactics, their practical application as topical microbicides is hampered by high production costs, wherein it is unlikely that any manufacturing system reliant on growth in sterile conditions can be competitive with the price of a male condom, which is necessary if the product is to be available for use by those at risk for sexual transmission of HIV. [0008] Hirayama et al. (2016) have described the elucidated primary structure of KAA-2 lectin using peptide mapping and complementary DNA (cDNA) cloning and prepared its active recombinants using an E. coli expression system. Gengenbach et al. (2019) have described the transient expression of the mistletoe lectin named “viscumin” (Viscum album) in intact Nicotiana benthamiana plants, and purification of the recombin