Compositions and methods for producing recombinant proteins with high secretion yields

Abstract

Methods for producing recombinant proteins in high secreted yields are provided, as well as recombinant vectors, recombinant host cells, and fermentations for use in such methods. The present invention provides a recombinant host cell comprising at least two polynucleotide sequences encoding recombinant proteins operably linked to at least two distinct secretion signals, preferably wherein at least one of the distinct secretion signals comprises a signal peptide but no leader peptide.

Description

【Technical Field】 【0001】 Cross - reference to related applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 470,153, filed Mar. 10, 2017, the disclosure of which is incorporated herein by reference. 【0002】 Field of the Invention The present disclosure relates to methods for producing recombinant proteins, and to compositions used in such methods and produced by such methods. Specifically, the present disclosure relates to methods for producing recombinant proteins with high secretion yields, as well as to recombinant vectors, recombinant host cells, and fermentations used in such methods. 【Background Art】 【0003】 Background of the Invention Many proteins (e.g., enzymes, vaccines, hormones, and biopharmaceutical proteins) required for research, industrial, or therapeutic purposes are produced industrially within recombinant host cells. Yeast, particularly Saccharomyces cerevisiae, is a preferred eukaryotic host organism for such uses. Yeast cells grow rapidly to high cell densities in inexpensive media and contain cellular machinery for protein folding and post - translational modifications (e.g., proteolytic maturation, disulfide bond formation, phosphorylation, O - linked and N - linked glycosylation). The yeast species most commonly used for the production of recombinant proteins include Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, and Kluyveromyces lactis. Of these, Pichia pastoris is suitable for applications where recombinant proteins are to be produced on a larger (e.g., industrial) scale, as it can achieve high - density cell growth. 【0004】 Because secreted proteins are easily separated from intact cells, when recombinant proteins are secreted from cells, industrial-scale production of recombinant proteins in recombinant host cells is promoted, eliminating the need for cell lysis and subsequent separation of proteins from cell debris. Pichia pastris is particularly suitable for the production of secreted recombinant proteins because it can be grown in minimal salt media that allow for the isolation of secreted proteins by filtration and low-conductivity chromatography, and because Pichia pastris naturally secretes relatively small amounts of fermentation products (i.e., small proteins), which further promote the isolation and purification of secreted recombinant proteins. 【0005】 Recombinant host cells used for the production of secreted recombinant proteins ideally produce large quantities of recombinant proteins and secrete a large fraction of the produced recombinant proteins. The former is usually achieved by employing methods well known in the art, such as codon optimization of polynucleotide sequences engineered within recombinant host cells to encode recombinant proteins, placing transcription of such polynucleotide sequences under the control of strong promoters and effective terminators, optimizing translation by introducing suitable ribose-binding sites, and increasing the copy number of polynucleotide sequences in recombinant host cells (for example, by engineering host cells containing two or more copies of a particular polynucleotide sequence). However, these strategies tend to reach natural limits in effectiveness because high copy numbers genetically destabilize recombinant host cells, and strong promoters produce higher levels of recombinant protein than the recombinant host cells can properly fold and / or secrete (Damasceno et al.

[2012] Appl Microbiol Biotechnol 93:31-39 (Non-patent Literature 1); Parekh et al.

[1995] Protein Expr Purif. 6(4):537-45 (Non-patent Literature 2); Zhu et al.

[2009] J Appl Microbiol 107:954-963 (Non-patent Literature 3); Liu et al.

[2003] Protein Expr. Purif. 30:262-274 (Non-patent Literature 4)).As a result, recombinant protein yields tend to plateau or even decrease because unfolded or misfolded recombinant proteins accumulate within recombinant host cells, and recombinant host cells activate molecular stress responses (e.g., unfolded protein response [UPR] or ER-associated proteolytic pathway [ERAD]) (Hohenblum et al.

[2004] Biotechnol Bioeng. 12:367-375 (Non-patent Literature 5); Vassileva et al.

[2001] J Biotechnol. 12:21-35 (Non-patent Literature 6); Inan et al.

[2006] Biotechnol Bioeng. 12:771-778 (Non-patent Literature 7); Zhu et al.

[2009] J Appl Microbiol. 12(3):954-963 (Non-patent Literature 3)). Indeed, upregulation of chaperone proteins or key UPR transcription regulators (Hac1p) has been shown to reduce the effects of UPR and increase recombinant protein yield (Zhang et al.

[2006] Biotechnol Prog. 12:1090-1095 (Non-patent Literature 8); Lee et al.

[2012] Process Biochem. 12:2300-2305 (Non-patent Literature 9); Valkonen et al.

[2003] Appl Environ Microbiol. 12:6979-6986 (Non-patent Literature 10)). However, such methods have yielded mixed results (Guerfal et al.

[2010] Microb Cell Fact. 12:49 (Non-Patent Literature 11)) and still do not completely resolve the saturation of the secretory pathways of recombinant host cells (Inan et al.

[2006] Biotechnol Bioeng. 12:771-778 (Non-Patent Literature 7)). Therefore, the capacity of the secretory mechanisms of recombinant host cells remains a major bottleneck for the production of recombinant proteins. [Prior art documents] [Non-patent literature] 【0006】 [Non-Patent Document 1] Damasceno et al. 【2012】 Appl Microbiol Biotechnol 93:31-39 【Non-Patent Document 2】 Parekh et al. 【1995】 Protein Expr Purif. 6(4):537-45 【Non-Patent Document 3】 Zhu et al. 【2009】 J Appl Microbiol 107:954-963 【Non-Patent Document 4】 Liu et al. 【2003】 Protein Expr. Purif. 30:262-274 【Non-Patent Document 5】 Hohenblum et al. 【2004】 Biotechnol Bioeng. 12:367-375 【Non-Patent Document 6】 [[ID=2,0]]Vassileva et al. 【2001】 J Biotechnol. 12:21-35 【Non-Patent Document 7】 Inan et al. 【2006】 Biotechnol Bioeng. 12:771-778 【Non-Patent Document 8】 Zhang et al. 【2006】 Biotechnol Prog. 12:1090-1095 【Non-Patent Document 9】 Lee et al. 【2012】 Process Biochem. 12:2300-2305 【Non-Patent Document 10】 Valkonen et al. 【2003】 Appl Environ Microbiol. 12:6979-6986 【Non-Patent Document 11】 Guerfal et al. 【2010】 Microb Cell Fact. 12:49 【Summary of the Invention】 【0007】 Therefore, what is needed are methods and compositions that enable increased expression of a desired recombinant protein while mitigating the negative effects of overexpression in recombinant host cells. [Invention 1001] A recombinant host cell containing at least two polynucleotide sequences encoding recombinant proteins that are operably linked to at least two distinct secretory signals. [Invention 1002] Recombinant host cell of the present invention 1001, wherein at least one of the aforementioned separate secretory signals contains a signal peptide but does not contain a leader peptide. [Invention 1003] Recombinant host cell of the present invention 1001, wherein at least one of the aforementioned separate secretory signals comprises a signal peptide and a leader peptide. [Invention 1004] The recombinant host cell of the present invention 1001, wherein at least one of the aforementioned distinct secretory signals is a native secretory signal, or a functional variant having at least 80% amino acid sequence identity with the native secretory signal. [Invention 1005] Recombinant host cell of the present invention 1001, wherein at least one of the aforementioned separate secretory signals is a recombinant secretory signal. [Invention 1006] Recombinant host cell of the present invention 1001, wherein at least one of the aforementioned separate secretory signals includes a signal peptide selected from Table 1, or a functional variant having at least 80% amino acid sequence identity with the signal peptide selected from Table 1. [Invention 1007] Recombinant host cells of the present invention 1001, wherein at least one of the aforementioned distinct secretory signals comprises a leader peptide that is native pro-αMF(sc), or a functional variant having at least 80% amino acid sequence identity with SEQ ID NO: 1. [Invention 1008] The recombinant host cell of the present invention 1005, wherein the recombinant secretion signal comprises a leader peptide having at least 80% amino acid sequence identity with SEQ ID NO: 1, and a signal peptide not containing pre-αMF(sc). [The present invention 1009] The recombinant host cell of the present invention 1001, wherein at least one of the separate secretion signals is a functional variant having at least 80% amino acid identity with native pre-αMF(sc) / pro-αMF(sc) or SEQ ID NO: 7, and at least one other of the separate secretion signals is a recombinant secretion signal comprising a functional variant having at least 80% amino acid sequence identity with native pro-αMF(sc) or SEQ ID NO: 1 and a signal peptide not containing pre-αMF(sc). [The present invention 1010] The recombinant host cell of the present invention 1009, wherein the functional variant of native pre-αMF(sc) / pro-αMF(sc) has at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity with SEQ ID NO: 7. [The present invention 1011] The recombinant host cell of the present invention 1009, wherein the functional variant of native pre-αMF(sc) / pro-αMF(sc) is native pre-αMF(sc) / pro-αMF(sc) containing one or two substituted amino acid changes. [The present invention 1012] The recombinant host cell of the present invention 1009, wherein the functional variant of native pre-αMF(sc) / pro-αMF(sc) is SEQ ID NO: 8. [The present invention 1013] The recombinant host cell of the present invention 1009, wherein the functional variant of native pro-αMF(sc) has at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity with SEQ ID NO: 1. [The present invention 1014] The recombinant host cell of the present invention 1009, wherein the functional variant of native pro-αMF(sc) is native pro-αMF(sc) containing one or two substituted amino acid changes. [The present invention 1015] The recombinant host cell of the present invention 1009, wherein the functional variant of the native pro-αMF(sc) is SEQ ID NO: 2. [The present invention 1016] The recombinant host cell of the present invention 1009, wherein the signal peptide is selected from Table 1 or is a functional variant having at least 80% amino acid sequence identity with the signal peptide selected from Table 1. [The present invention 1017] The recombinant host cell of the present invention 1016, wherein the functional variant of the signal peptide has at least 85%, at least 90%, at least 95%, or at least 99% amino acid identity with the signal peptide selected from Table 1. [The present invention 1018] The recombinant host cell of the present invention 1009, wherein the recombinant secretion signal is selected from SEQ ID NOs: 9-12 or is a functional variant having at least 80% amino acid sequence identity with the recombinant secretion signal selected from SEQ ID NOs: 9-12. [The present invention 1019] The recombinant host cell of the present invention 1018, wherein the functional variant has at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity with the recombinant secretion signal selected from SEQ ID NOs: 9-12. [The present invention 1020] The recombinant host cell of the present invention 1001, wherein at least one of the polynucleotide sequences is stably integrated into the genome of the recombinant host cell. [The present invention 1021] The recombinant host cell of the present invention 1001, wherein at least one of the polynucleotide sequences is maintained extrachromosomally in the recombinant host cell. [The present invention 1022] The recombinant host cell of the present invention 1001, wherein the protein is silk or a silk-like protein. [The present invention 1023] The recombinant host cell of the present invention 1022, wherein the protein comprises SEQ ID NO: 13. [Invention 1024] The recombinant host cell of the present invention 1022, wherein the protein contains multiple copies of SEQ ID NO: 13. [Invention 1025] A recombinant host cell of the present invention 1001, wherein at least one of the polynucleotide sequences is operably linked to a promoter. [Invention 1026] The recombinant host cell of the present invention 1025, wherein the promoter is the pGCW14 promoter of Pichia pastoris. [Invention 1027] Recombinant host cells of the present invention 1025, wherein the promoter is the pGAP promoter of Pichia pastris. [Invention 1028] Recombinant host cells of the present invention 1025, wherein the promoter is an inducible promoter. [Invention 1029] A recombinant host cell of the present invention 1001, which is a yeast cell. [Invention 1030] A recombinant host cell of the present invention 1029, which is a budding yeast cell. [Invention 1031] A recombinant host cell of the present invention 1029, which is a methylotrope yeast cell. [Invention 1032] A recombinant host cell of the present invention 1030, which is a Pichia species. [Invention 1033] A recombinant host cell of the present invention 1032, which is Pichia pastrius. [Invention 1034] A recombinant host cell according to the present invention 1001, which produces the recombinant protein in a secreted yield of at least 30% by weight of the total yield of the recombinant protein produced by the recombinant host cell. [Invention 1035] Fermentation comprising recombinant host cells and culture medium according to any of invention 1001 to 1034. [Invention 1036] Fermentation according to the present invention 1035, comprising at least 30% by weight of the total yield of the recombinant protein produced by the recombinant host cell as secreted recombinant protein. [Invention 1037] Fermentation according to the present invention 1035, wherein the culture medium contains at least 0.5 g / L of the recombinant protein. [Invention 1038] A method for producing recombinant proteins, (a) A step of culturing recombinant host cells according to any of the invention 1001 to 1034 in a culture medium to obtain fermentation according to any of the invention 1035 to 1037, and (b) Step of extracting the recombinant protein from the culture medium. The method, including the method described above. [Brief explanation of the drawing] 【0008】 [Figure 1] This is a flowchart of a method for producing recombinant proteins with high secretion yield. [Figure 2A] This is an exemplary map of a recombinant vector containing a single polynucleotide sequence encoding a silk-like protein operably linked to an N-terminal secretory signal containing a functional variant of the secretory signal of the α-conjugation factor (pre-αMF(sc) / *pro-αMF(sc)) of Saccharomyces cerevisiae, or a recombinant secretory signal consisting of a functional variant of the leader peptide and a signal peptide of the α-conjugation factor (*pro-αMF(sc)) of Saccharomyces cerevisiae. This is an exemplary map of a recombinant vector used for introducing a single copy of a polynucleotide sequence. [Figure 2B]This is an exemplary map of a recombinant vector containing three polynucleotide sequences encoding a silk-like protein operably linked to an N-terminal secretory signal containing a functional variant of the secretory signal of the α-conjugation factor (pre-αMF(sc) / *pro-αMF(sc)) of Saccharomyces cerevisiae, or a recombinant secretory signal consisting of a functional variant of the leader peptide and a signal peptide of the α-conjugation factor (*pro-αMF(sc)) of Saccharomyces cerevisiae. This is an exemplary map of a recombinant vector used to introduce three copies of a polynucleotide sequence. [Figure 3] This shows the intracellular (non-secretory) and extracellular (secretory) yields of recombinant silk-like protein produced by various Pichia pastris recombinant host cells, as assayed by ELISA. Legend: 4 / 5 / 6xαMF = Recombinant host strain containing a 4 / 5 / 6 polynucleotide sequence encoding silk-like protein operably linked to the signal sequence pre-αMF(sc) / *pro-αMF(sc) (SEQ ID NO: 8); 4xαMF+3xPep4 = Recombinant host strain 4xαMF further containing three polynucleotide sequences encoding silk-like protein operably linked to the signal sequence pre-PEP4(sc) / *pro-αMF(sc) (SEQ ID NO: 9); 4xαMF+3xDse4 = Recombinant host strain 4xαMF further containing three polynucleotide sequences encoding silk-like protein operably linked to the signal sequence pre-DSE4(pp) / *pro-αMF(sc) (SEQ ID NO: 10). [Figure 4] This shows the intracellular (non-secretory) and extracellular (secretory) yields of recombinant silk-like protein produced by various Pichia pastris recombinant host cells, as assayed by ELISA. Legend: 6xαMF = recombinant host strain containing a 6-polynucleotide sequence encoding silk-like protein operably linked to the signal sequence pre-αMF(sc) / *pro-αMF(sc) (SEQ ID NO: 8); 4xαMF+2xEpx1 = recombinant host strain 4xαMF further containing two polynucleotide sequences encoding silk-like protein operably linked to the signal sequence pre-EPX1(pp) / *pro-αMF(sc) (SEQ ID NO: 11). [Figure 5] This shows the intracellular (non-secretory) and extracellular (secretory) yields of recombinant silk-like protein produced by various Pichia pastris recombinant host cells, as assayed by ELISA. Legend: 4 / 5 / 6xαMF = Recombinant host strain containing a 4 / 5 / 6 polynucleotide sequence encoding silk-like protein operably linked to the signal sequence pre-αMF(sc) / *pro-αMF(sc) (SEQ ID NO: 8); 4xαMF+2xEpx1 = Recombinant host strain 4xαMF further containing two polynucleotide sequences encoding silk-like protein operably linked to the signal sequence pre-EPX1(pp) / *pro-αMF(sc) (SEQ ID NO: 11); 4xαMF+2xEpx1+1xCLSP = Recombinant host strain 4xαMF+2xEpx1 further containing one polynucleotide sequence encoding silk-like protein operably linked to the signal sequence pre-CLSP(gg) / *pro-αMF(sc) (SEQ ID NO: 12). [Figure 6] This is a diagram of a recombinant vector, according to one embodiment of the present invention, which includes an expression construct for expressing a polypeptide via a recombinant secretory signal. [Figure 7] This shows the secretion levels of α-amylase derived from Pichia pastris transformed to express recombinant α-amylase in response to various recombinant secretion signals. [Figure 8] This shows the secretion levels of fluorescent proteins derived from Pichia pastris transformed to express recombinant fluorescent proteins in response to various recombinant secretion signals. 【0009】 The figures illustrate various embodiments of the present disclosure for illustrative purposes only. Those skilled in the art will readily recognize from the following considerations that alternative embodiments of the structures and methods described herein may be used without departing from the principles described herein. [Modes for carrying out the invention] 【0010】 Detailed description of the invention definition Unless otherwise stated, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to whom this disclosure relates. 【0011】 As used herein, the terms "a," "an," and "the," and similar references, refer to both singular and plural nouns, unless otherwise indicated herein or unless the context clearly indicates otherwise. 【0012】 Amino acids can be referred to by a one-letter code or a three-letter code. The one-letter codes, amino acid names, and three-letter codes are as follows: G-glycine (Gly), P-proline (Pro), A-alanine (Ala), V-valine (Val), L-leucine (Leu), I-isoleucine (Ile), M-methionine (Met), C-cysteine ​​(Cys), F-phenylalanine (Phe), Y-tyrosine (Tyr), W-tryptophan (Trp), H-histidine (His), K-lysine (Lys), R-arginine (Arg), Q-glutamine (Gln), N-asparagine (Asn), E-glutamic acid (Glu), D-aspartic acid (Asp), S-serine (Ser), T-threonine (Thr). 【0013】 As used herein, the term “functional variant” refers to a protein that differs in composition from a native protein, with functional properties conserved within 10% of those properties. In some embodiments, the difference between the functional variant and the native protein may be in the primary amino acid sequence (e.g., one or more amino acids are removed, inserted, or substituted) or in post-translational modifications (e.g., glycosylation, phosphorylation). Amino acid insertions may include N-terminal and / or C-terminal fusions, as well as intrasequential insertions of single or multiple amino acids. Amino acid substitutions include non-conservative and conservative substitutions, and a table of conservative amino acid substitutions is well known in the art (see, for example, Creighton (1984) Proteins. WH Freeman and Company (Eds)). In some embodiments, the functional variant and the native protein have at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% amino acid or nucleotide sequence identity. 【0014】 In the context of nucleic acids or amino acid sequences as used herein, the terms “identity” or “identical” refer to nucleotide or amino acid residues in two sequences that are identical when aligned to the greatest extent possible. Depending on the application, percentage “identity” may exist over an entire region of the sequences being compared (i.e., a subsequence [e.g., across a functional domain]) or, instead, over the entire length of the sequence. A “region” can be considered a contiguous stretch of at least 9, 20, 24, 28, 32, or 36 nucleotides, or at least 6 amino acids. In sequence comparison, typically one sequence serves as the reference sequence over which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, and, if necessary, the coordinates of the subsequence and the sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the percentage sequence identity of the test sequence(s) to the reference sequence based on the specified program parameters. Optimal alignment of sequences for comparison can be achieved, for example, by the local homology algorithm of Smith & Waterman, Adv.Appl.Math.2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J.Mol.Biol.48:443 (1970), the similarity search method of Pearson & Lipman, Proc.Nat'l.Acad.Sci.USA 85:2444 (1988), by running these algorithms on a computer (Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., GAP, BESTFIT, FASTA, and TFASTA in Madison, Wisconsin), or by visual inspection (see Ausubel et al. below in general).One example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm (see, for example, Altschul et al.

[1990] J.Mol.Biol.215:403-410; Gish & States.

[1993] Nature Genet.3:266-272; Madden et al.

[1996] Meth.Enzymol.266:131-141; Altschul et al.

[1997] Nucleic Acids Res.25:3389-3402; Zhang 7 Madden.

[1997] Genome Res.7:649-656). Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information. Such software can also be used to determine the molar percentage of any identified amino acid within a polypeptide sequence or within a domain of such a sequence. Those skilled in the art will recognize that such percentages can also be determined by inspection and manual calculation. 【0015】 The terms "including," "includes," "having," "has," and "with," or variations thereof, are intended to be as comprehensive as the term "comprising." 【0016】 As used herein, the term “microorganism” refers to a single-celled organism. As used herein, this term includes all bacteria, all archaea, single-celled protists, single-celled animals, single-celled plants, single-celled fungi, single-celled algae, all protists, and all Chromista. 【0017】 As used herein, the term “natural” refers to something found in nature in its natural, unaltered state. 【0018】 As used herein, the term “operably linked” means a polynucleotide or amino acid sequence that is sequentially linked to a protein-coding polynucleotide sequence or a protein, and a polynucleotide or amino acid sequence that acts trans- or at a distance to a protein-coding polynucleotide sequence and controls the transcription, translation, folding, secretion, or other functional aspects of the protein-coding polynucleotide or protein. 【0019】 The terms “optional” or “optional” mean that a feature or structure may or may not exist, or that an event or situation may or may not occur, and that this specification includes cases where a particular feature or structure exists and cases where a feature or structure does not exist, or cases where an event or situation occurs and cases where an event or situation does not occur. 【0020】 As used herein, the term "protein" refers to both polypeptides that do not have a functional structure and polypeptides that fold into an active structure. 【0021】 As used herein, the term “recombinant protein” refers to a protein produced in a recombinant host cell or a protein synthesized from recombinant nucleic acid. 【0022】 As used herein, the term “recombinant host cell” refers to a host cell containing recombinant nucleic acid. 【0023】 As used herein, the term “recombinant nucleic acid” refers to nucleic acids extracted from the natural environment, or nucleic acids that, when found in nature, do not associate with all or part of the nucleic acids adjacent to or nearest to them, or nucleic acids that are effectively linked to nucleic acids that are not naturally linked, or nucleic acids that do not occur naturally, or nucleic acids that contain modifications not found in natural nucleic acids (e.g., insertions, deletions, or point mutations introduced artificially, e.g., by human intervention), or nucleic acids that are integrated into chromosomes at heterologous sites. This term includes cloned DNA isolates and nucleic acids, including chemically synthesized nucleotide analogs. 【0024】 As used herein, the term “recombinant secretory signal” refers to a secretory signal that includes a non-natural combination of signal peptides and leader peptides. 【0025】 As used herein, the term “recombinant vector” refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it is ligated. This term includes circular double-stranded DNA loops to which additional DNA segments can be ligated, and “plasmids” in general, referring to linear double-stranded molecules such as those resulting from polymerization by polymerase chain reaction (PCR) or processing of plasmids using restriction enzymes. Other non-limiting examples of vectors include bacteriophages, cosmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and viral vectors (i.e., complete or partial viral genomes to which additional DNA segments are ligated). Certain vectors can autonomously replicate in the recombinant host cell into which they are introduced (e.g., vectors with an origin of replication that functions within the cell). Other vectors, upon introduction, can be integrated into the genome of the recombinant host cell and thereby replicate together with the cellular genome. 【0026】 As used herein, the term “secreted recombinant protein” refers to recombinant proteins exported across the cell membrane and / or cell wall of recombinant host cells that produce recombinant proteins. 【0027】 As used herein, the term "secretion yield" refers to the amount of secreted protein produced by host cells, based on the fixed amount of carbon supplied to the fermentation process, including the host cells. 【0028】 As used herein, the term "total yield" refers to the total amount of protein produced by host cells, based on the amount of fixed carbon supplied to the fermentation process, including the host cells. 【0029】 As used herein, the term “cleaved” refers to a protein sequence that is shorter in length than the native protein. In some embodiments, the cleaved protein may be more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the length of the native protein. 【0030】 Exemplary methods and materials are described below. However, similar or equivalent methods and materials may also be used in carrying out the invention, and this will be apparent to those skilled in the art. All publications and other references referenced herein are incorporated by reference in their entirety. In case of any conflict, this specification, including definitions, shall prevail. Materials, methods, and examples are illustrative and not limiting. 【0031】 Whenever a range of values ​​is listed, that range includes all values ​​within that range, as if it were explicitly written, and also includes the boundary values ​​of the range. Therefore, the range "X~Y" includes all values ​​that fall between X and Y, and also includes X and Y. 【0032】 Compositions and methods for producing recombinant proteins with high secretion yields Recombinant host cells and fermentation, as well as methods for using such recombinant host cells and fermentation to produce recombinant proteins with high secretion yields, are provided herein. 【0033】 The advantages of the compositions and methods provided herein include providing a cost-effective means for producing large quantities of recombinant proteins. Large quantities of the product are obtained using recombinant host cells that secrete recombinant proteins via secretory pathways. Such secretion of recombinant proteins (a) avoids the toxicity of intracellular accumulation of recombinant proteins, (b) simplifies purification by eliminating the processes of cell disruption, separation from cellular components, and protein refolding, and (c) provides recombinant proteins that are properly folded with post-translational modifications that may be important for the activity / function of the recombinant proteins. 【0034】 Recombinant host cells The recombinant host cells provided herein are host cells that employ multiple distinct secretory signals to produce recombinant proteins. 【0035】 Secretion markers For a protein to be secreted, it must be transported through the intracellular secretory pathway of the cell producing it. The protein is directed towards this pathway, rather than to a specific cell destination, by an N-terminal secretory signal. At a minimum, the secretory signal includes a signal peptide. The signal peptide typically consists of 13–36 primarily hydrophobic amino acids flanking the N-terminal basic amino acid and the C-terminal polar amino acid. The signal peptide interacts with signal recognition particles (SRPs) or other transport proteins (e.g., SNDs, GETs) that mediate the co-translation or post-translational translocation of the nascent protein from the cytosol to the lumen of the ER. In the ER, the signal peptide is typically cleaved, the protein folds, and undergoes post-translational modifications. The protein is then delivered from the ER to the Golgi apparatus, then to secretory vesicles, and finally to the extracellular space. In addition to the signal peptide, a subset of nascent proteins destined for secretion also possesses a secretory signal that includes a leader peptide. The leader peptide typically consists of hydrophobic amino acids interspersed with charged or polar amino acids. While we do not wish to be bound by theory, leader peptides are thought to stall protein transport, ensure proper folding, and / or facilitate protein transport from the ER to the Golgi apparatus, where the leader peptide is normally cleaved. 【0036】 The amount of protein secreted from cells varies greatly among proteins and is partially dependent on secretory signals operably linked to developing proteins. Many secretory signals are known in the art, and some are commonly used in the production of recombinant secretory proteins. Among these, the secretory signal of Saccharomyces cerevisiae α-conjugation factor (αMF), consisting of an N-terminal 19-amino acid signal peptide (also known herein as pre-αMF(sc)), followed by a 70-amino acid leader peptide (also known herein as pro-αMF(sc); SEQ ID NO: 1). The inclusion of pro-αMF(sc) in the αMF secretion signal of Saccharomyces cerevisiae (also referred herein as pre-αMF(sc) / pro-αMF(sc) (SEQ ID NO: 7)) has been found to be important for achieving high secretion yields of protein (see, for example, Fitzgerald & Glick

[2014] Microb Cell Fact 28;13(1):125; Fahnestock et al.

[2000] J Biotechnol 74(2):105). Adding pro-αMF(sc) or its functional variants to signal peptides other than pre-αMF(sc) has been investigated as a means of achieving recombinant protein secretion, but it has shown varying degrees of effectiveness, increasing the secretion of specific recombinant proteins in certain recombinant host cells, while having no effect on or decreasing the secretion of other recombinant proteins (Fitzgerald & Glick.

[2014] Microb Cell Fact 28;13(1):125; Liu et al.

[2005] Biochem Biophys Res Commun. 326(4):817-24; Obst et al.

[2017] ACS Synth Biol. 2017 Mar 2). 【0037】 The present invention, as provided herein, is based on the remarkable discovery made by the inventors that the secretion yield of recombinant proteins can be improved by using multiple distinct secretion signals. Specifically, the inventors have found that recombinant host cells containing the same number of polynucleotide sequences encoding recombinant proteins operably linked to at least two distinct secretion signals produce recombinant proteins with higher secretion yields compared to recombinant host cells containing multiple polynucleotide sequences encoding recombinant proteins operably linked to only one secretion signal (e.g., pre-αMF(sc) / pro-αMF(sc)). While not wishing to be bound by theory, the use of at least two distinct secretion signals may allow recombinant host cells to engage in the secretory pathways of distinct cells that achieve efficient secretion of recombinant proteins, and thus prevent oversaturation of either secretory pathway. 【0038】 Accordingly, the recombinant host cells provided herein are host cells comprising at least two polynucleotide sequences encoding recombinant proteins that are operably ligated to at least two distinct secretory signals. The secretory signals are typically ligated to the N-terminus of the protein. 【0039】 In some embodiments, at least one of the distinct secretory signals includes a signal peptide but does not include a leader peptide. In some embodiments, at least one of the distinct secretory signals includes both a signal peptide and a leader peptide. In some embodiments, at least one of the distinct secretory signals is a native secretory signal or a functional variant having at least 80% amino acid sequence identity with the native secretory signal. In some embodiments, at least one of the distinct secretory signals is a recombinant secretory signal. 【0040】 In some embodiments, at least one of the distinct secretory signals comprises a signal peptide selected from Table 1, or a functional variant having at least 80% amino acid sequence identity with the signal peptide selected from Table 1. In some embodiments, the functional variant is a signal peptide selected from Table 1 containing one or two substituted amino acids. In some such embodiments, the functional variant has at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity with the signal peptide selected from Table 1. In some embodiments, the signal peptide mediates the posttranslational translocation of the nascent recombinant protein to the ER (i.e., protein synthesis precedes the translocation so that the nascent recombinant protein is present in the cytosol before translocation to the ER). In other embodiments, the signal peptide mediates the cotranslational translocation of the nascent recombinant protein to the ER (i.e., protein synthesis and translocation to the ER occur simultaneously). The advantage of using a signal peptide that mediates cotranslational translocation to the ER is that it prevents recombinant proteins that tend to fold rapidly from adopting conformations that hinder translocation to the ER, and therefore secretion. 【0041】 (Table 1) Signal Peptides TIFF0007872575000001.tif49165 【0042】 In some embodiments, at least one of the distinct secretory signals includes a leader peptide which is native pro-αMF(sc) (SEQ ID NO: 1), or a functional variant having at least 80% amino acid sequence identity with SEQ ID NO: 1. In some embodiments, the functional variant is native pro-αMF(sc) containing one or two substituted amino acids. In some embodiments, the functional variant is *pro-αMF (SEQ ID NO: 2). In some embodiments, the functional variant has at least 85%, at least 90%, at least 95%, or at least 99% amino acid identity with SEQ ID NO: 1. In some embodiments, the functional variant is αMF_no_EAEA or αMFΔ or αMFΔ_no_Kex (Obst et al.

[2017] ACS Synth Biol. 2017 Mar 2). 【0043】 In some embodiments, at least one of the separate secretory signals comprises a leader peptide which is native pro-αMF(sc) (SEQ ID NO: 1), or a functional variant having at least 80% amino acid sequence identity with SEQ ID NO: 1, and a signal peptide which does not contain pre-αMF(sc). In some embodiments, the functional variant is native pro-αMF(sc) which has at least 85%, at least 90%, at least 95%, or at least 99% amino acid identity with SEQ ID NO: 1, or is *pro-αMF (SEQ ID NO: 2), or is αMF_no_EAEA, αMFΔ, or αMFΔ_no_Kex (Obst et al.

[2017] ACS Synth Biol. 2017 Mar 2). In some embodiments, the signal peptide is selected from Table 1, or is a signal peptide selected from Table 1 which contains one or two substituted amino acids, or has at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity with a signal peptide selected from Table 1. In some embodiments, at least one of the separate secretory signals is a functional variant having at least 80% amino acid sequence identity with native pre-αMF(sc) / pro-αMF(sc) (SEQ ID NO: 7) or SEQ ID NO: 7, and at least one of the separate secretory signals is a recombinant secretory signal comprising a functional variant having at least 80% amino acid sequence identity with native pro-αMF(sc) (SEQ ID NO: 1) or SEQ ID NO: 1 and a signal peptide that is not pre-αMF(sc). In some embodiments, the functional variant of native pre-αMF(sc) / pro-αMF(sc) is either native pre-αMF(sc) / pro-αMF(sc) containing 2 to 4 substituted amino acid changes, or pre-αMF(sc) / *pro-αMF(sc) (SEQ ID NO: 8), or has at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity with SEQ ID NO: 7.In some such embodiments, a functional variant of native pro-αMF(sc) is native pro-αMF(sc) containing one or two substituted amino acid changes, *pro-αMF (SEQ ID NO: 2), having at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity with SEQ ID NO: 1, or αMF_no_EAEA, αMFΔ, or αMFΔ_no_Kex (Obst et al.

[2017] ACS Synth Biol. 2017 Mar 2). In some embodiments, a signal peptide that is not pre-αMF(sc) is selected from Table 1, or a functional variant having at least 80% amino acid sequence identity with a signal peptide selected from Table 1. In some such embodiments, the signal peptide is a signal peptide selected from Table 1 containing one or two substituted amino acids, or has at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity with a signal peptide selected from Table 1. In some embodiments, the recombinant secretory signal is selected from SEQ ID NOs: 9-12 in Table 2, or is a functional variant having at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity with the recombinant secretory signal selected from SEQ ID NOs: 9-12. 【0044】 (Table 2) Recombinant secretory signals TIFF0007872575000002.tif97165 【0045】 Suitable separate secretion signals and combinations of secretion signals can be identified by using the methods disclosed herein. Such methods are (a) A step of measuring the secretion yield of recombinant protein produced by recombinant host cell A containing z copies of a first polynucleotide sequence encoding a recombinant protein operably linked to a first secretion signal, (b) Measuring the secretion yield of recombinant protein produced by recombinant host cell B, which contains m copies of a first polynucleotide sequence and n copies of a second polynucleotide sequence encoding a recombinant protein operably linked to a second secretion signal (recombinant host cell A and recombinant host cell B are identical except for the copy number of the first and second polynucleotide sequences, m+n=z, and the first and second secretion signals are not identical), and (c) Selecting a combination of first and second secretion signals that provides a secretion yield of recombinant protein in recombinant host cell B that is at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% greater than that achieved in recombinant host cell A. Includes. 【0046】 In some embodiments, recombinant host cell B contains y copies of a third polynucleotide sequence encoding a recombinant protein operably linked to a third secretory signal, and recombinant host cell A and recombinant host cell B are identical except for the copy number of the first, second, and third polynucleotide sequences, m+n+y=z, and the first, second, and third secretory signals are not identical. In further embodiments, recombinant host cell B contains an additional polynucleotide sequence encoding a recombinant protein operably linked to an additional distinct secretory signal. In embodiments where recombinant host cell A containing the same number of polynucleotide sequences as recombinant host cell B cannot be isolated, step (c) may use recombinant host cell A containing fewer copies of the polynucleotide sequence. Although we do not wish to be bound by theory, the inability to isolate recombinant host cell A having a certain number of polynucleotide sequences may be due to genetic instability resulting from an increase in the copy number of identical polynucleotide sequences or to an inadequate health state of the host cell. 【0047】 In some embodiments, the expression construct is stably integrated into the genome (e.g., chromosomes) of the recombinant host cell, for example, via homologous recombination or targeted integration. Non-limited examples of sites suitable for genome integration include the Ty1 locus in the Saccharomyces cerevisiae genome, the rDNA and HSP82 loci in the Pichia pastris genome, and transposition factors whose copies are scattered throughout the genome of the recombinant host cell. In other embodiments, the expression construct is maintained outside the chromosome (e.g., on a plasmid) rather than being stably integrated into the genome of the recombinant host cell. The polynucleotide sequence may be located at a single location within the genome of the recombinant host cell or distributed throughout the genome of the recombinant host cell. In some embodiments, the polynucleotide sequence is located in the genome of the recombinant host cell as a head-to-tail expression cassette multimer. 【0048】 High secretion yield recombinant proteins are provided by the use of at least two distinct secretion signals, as provided herein. Thus, in various embodiments, the recombinant host cell provides at least 1% by weight, at least 5% by weight, at least 10% by weight, at least 20% by weight, or at least 30% by weight of the total yield of recombinant proteins produced by the recombinant host cell; 1% to 100% by weight, ~90% by weight, ~80% by weight, ~70% by weight, ~60% by weight, ~50% by weight, ~40% by weight, ~30% by weight, ~20% by weight, or 10% by weight; 10% to 100% by weight, ~90% by weight, ~80% by weight, ~70% by weight, ~60% by weight, ~50% by weight, ~40% by weight, ~30% by weight, or ~20% by weight; 20% to 100% by weight, ~90% by weight, ~80% by weight, ~70% by weight, ~60% by weight, ~50% by weight It produces recombinant protein in secretion yields of % by weight, ~40% by weight, or ~30% by weight; 30% by weight ~ 100% by weight, ~90% by weight, ~80% by weight, ~70% by weight, ~60% by weight, ~50% by weight, or ~40% by weight; 40% by weight ~ 100% by weight, ~90% by weight, ~80% by weight, ~70% by weight, ~60% by weight, or ~50% by weight; 50% by weight ~ 100% by weight, ~90% by weight, ~80% by weight, ~70% by weight, or ~60% by weight; 60% by weight ~ 100% by weight, ~90% by weight, ~80% by weight, or ~70% by weight; 70% by weight ~ 100% by weight, ~90% by weight, or ~80% by weight; 80% by weight ~ 100% by weight, or ~90% by weight; or 90% by weight ~ 100% by weight. The identification of the produced proteins can be confirmed by HPLC quantification, Western blot analysis, polyacrylamide gel electrophoresis, and two-dimensional mass spectrometry (2D-MS / MS) sequence identification. 【0049】 Recombinant protein The recombinant protein encoded by at least two polynucleotide sequences contained in the recombinant host cell provided herein may be any protein. 【0050】 In some embodiments, the recombinant protein is silk or a silk-like protein. Such silk or silk-like proteins may be selected from a wide range of full-length or cleaved native silk proteins, or functional variants of a wide range of full-length or cleaved native silk proteins, or may contain domains of native silk proteins or functional variants of silk proteins. Putative native silk proteins can be identified by searching sequence databases (e.g., GenBank) for appropriate terms (e.g., silkworm silk, spider silk, spidoin, fibroin, MaSp) and translating any nucleotide sequence into an amino acid sequence. 【0051】 In some embodiments, silk or silk or silk-like protein is the full-length or cleaved natural silk protein of silkworms, or a functional variant of the full-length or cleaved natural silk protein of silkworms, or comprises a domain of the natural silk protein of silkworms or a functional variant of the natural silk protein. In some such embodiments, the silkworm is Bombyx mori. 【0052】 In some embodiments, the silk or silk or silk-like protein is either the full-length or truncated natural silk protein of the spider, or a functional variant of the full-length or truncated natural silk protein of the spider, or comprises a domain of the natural silk protein of the spider or a functional variant of the natural silk protein. In some embodiments, the natural silk protein is selected from the group consisting of the large-vial spider fibroin (MaSp, also called dragline; e.g., MaSp1, MaSp2) silk protein, the small-vial spider fibroin (MiSp) silk protein, the flagellated spider fibroin (Flag) silk protein, the grape-like spider fibroin (AcSp) silk protein, the tubular spider fibroin (TuSp) silk protein, and the pear-like spider fibroin (PySp) silk protein of the golden orb-weaver spider. In some embodiments, the spiders are Agelenopsis aperta, Aliatipus gulosus, Aphonopelma seemanni, Aptostichus sp. AS217, Aptostichus sp. AS220, Araneus diadematus, Araneus gemmoides, Araneus ventricosus, Argiope amoena, Argiope argentata, Argiope bruennichi, Argiope trifasciata trifasciata), Atypoides riversi, Avicularia juruensis, Bothriocyrtum californicum, Deinopis Spinosa, Diguetia canities, Dolomedes tenebrosus, Euagrus xoseusNephila chisoseus), Euprosthenops australis, Gasteracantha mammosa, Hypochilus thorelli, Kukulcania hibernalis, Latrodectus hesperus, Megahexura fulva, Metepeira grandiosa, Nephila antipodiana, Nephila clavata, Nephila clavipes, Nephila madagascariensis, Nephila pipepes The group is selected from the following: pilipes, Nephilengys cruentata, Parawixia bistriata, Peucetia viridans, Plectreurys tristis, Poecilotheria regalis, Tetragnatha kauaiensis, or Uloborus diversus. 【0053】 Typically, silk proteins are large proteins (over 150 kDa, over 1000 amino acids) that can be divided into three domains: an N-terminal non-repeat domain (NTD), a repeat domain (REP), and a C-terminal non-repeat domain (CTD). REPs contain blocks of amino acid sequences ("repeat units") that are at least 12 amino acids long, repeat either completely ("complete repeat units") or incompletely ("semi-repeat units"), and can contain sequence motifs of 2–10 amino acids long (see Figure 1). REPs usually make up about 90% of natural spider silk proteins and, though we do not wish to be bound by theory, are assembled into alanine-rich nanocrystalline (less than 10 nm) domains (likely composed of alternating β-sheets) and glycine-rich amorphous domains (which may contain α-helices and / or β-turns), respectively, which are thought to give spider silk fibers strength and flexibility. The length and composition of REPs are known to vary between different spider silk proteins and across different spider species, resulting in a wide range of silk fibers with specific properties. 【0054】 In some embodiments, silk or silk-like protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8) native REPs or functional variants of native REPs, zero or more (e.g., 0, 1) native or functional variants of NTDs, and zero or more (e.g., 0, 1) native CTDs or functional variants of native CTDs. In some embodiments, silk or silk-like protein comprises one or more NTDs, each containing 75 to 350 amino acids. In some embodiments, silk or silk or silk-like protein comprises one or more CTDs, each containing 75 to 350 amino acids. In some embodiments, silk or silk or silk-like protein is such that each has a value greater than 60, greater than 100, greater than 150, greater than 200, greater than 250, greater than 300, greater than 350, greater than 400, greater than 450, greater than 500, greater than 600, greater than 700, greater than 800, greater than 900, greater than 1000, greater than 1250, greater than 1500, and greater than 1750. 1 or over 2000; 60-2000, ~1750, ~1500, ~1250, ~1000, ~900, ~800, ~700, ~600, ~500, ~450, ~400, ~350, ~300, ~250, ~200, ~150, or ~100; 100-2000, ~1750, ~1500, ~1250, ~1000, ~900, ~800, ~700, ~600, ~500, ~450, ~400 ~350, ~300, ~250, ~200, or ~150; 150~2000, ~1750, ~1500, ~1250, ~1000, ~900, ~800, ~700, ~600, ~500, ~450, ~400, ~350, ~300, ~250, or ~200; 200~2000, ~1750, ~1500, ~1250, ~1000, ~900, ~800, ~700, ~600, ~500, ~450 , ~400, ~350, ~300, or ~250; 250~2000, ~1750, ~1500, ~1250, ~1000, ~900, ~800, ~700, ~600, ~500, ~450, ~400, ~350, or ~300; 300~2000, ~1750, ~1500, ~1250, ~1000, ~900, ~800, ~700, ~600, ~500, ~450, ~400, or ~350;350~2000, ~1750, ~1500, ~1250, ~1000, ~900, ~800, ~700, ~600, ~500, ~450, or ~400; 400~2000, ~1750, ~1500, ~1250, ~1000, ~900, ~800, ~700, ~600, ~500, or ~450 450~2000, ~1750, ~1500, ~1250, ~1000, ~900, ~800, ~700, ~600, or ~500; 500~2000, ~1750, ~1500, ~1250, ~1000, ~900, ~800, ~700, or ~600; 600~2000, ~1750, ~150 Contains one or more REPs containing repeating units with amino acid residues of 0, ~1250, ~1000, ~900, ~800, or ~700; 700~2000, ~1750, ~1500, ~1250, ~1000, ~900, or ~800; 800~2000, ~1750, ~1500, ~1250, ~1000, or ~900; 900~2000, ~1750, ~1500, ~1250, or ~1000; 1000~2000, ~1750, ~1500, or ~1250; 1250~2000, ~1750, or ~1500; 1500~2000, or ~1750; or 1750~2000. 【0055】 In some embodiments, silk or silky or silky protein is defined as having a size greater than 5kDa, greater than 10kDa, greater than 20kDa, greater than 30kDa, greater than 40kDa, greater than 50kDa, greater than 60kDa, greater than 70kDa, greater than 80kDa, or greater than 90kDa; 5kDa to 100kDa, greater than 90kDa, greater than 80kDa, greater than 70kDa, greater than 60kDa, greater than90kDa, greater than 5kDa to 100kDa, greater than 90kDa, greater than 90kDa, greater than 5kDa to 100kDa, greater than 90kDa, greater than 90kDa, greater than 5kDa to 100kDa, greater than 90kDa, greater than 8 ~70kDa, ~60kDa, ~50kDa, ~40kDa, ~30kDa, or ~20kDa; 20kDa ~100kDa, ~90kDa, ~80kDa, ~70kDa, ~60kDa, ~50kDa, ~40kDa, or ~30kDa; 30kDa ~100kDa, ~90kDa, ~80kDa, ~70kDa, ~60kDa, ~50kDa, or ~40kDa; 40kDa ~100kDa, ~90kDa, ~80kDa, ~70kDa, ~60kDa, or ~50kDa; 50kDa ~100kDa, ~90kDa, ~80k Da, ~70kDa, or ~60kDa; 60kDa~100kDa, ~90kDa, ~80kDa, or ~70kDa; 70kDa~100kDa, ~90kDa, or ~80kDa; 80kDa~100kDa, or ~90kDa; or having a molecular weight of 90kDa~100kDa, greater than 2, greater than 4, greater than 6, greater than 8, greater than 10, greater than 12, greater than 14, greater than 16, greater than 18, greater than 20, greater than 22, greater than 24, greater than 26, greater than 28, or greater than 30; 2~30, ~ 28, ~26, ~24, ~22, ~20, ~18, ~16, ~14, ~12, ~10, ~8, ~6, or ~4; 4~30, ~28, ~26, ~24, ~22, ~20, ~18, ~16, ~14, ~12, ~10, ~8, or ~6; 6~30, ~28, ~26, ~24, ~22, ~20, ~18, ~16, ~14, ~12, ~10, or ~8; 8~30, ~28, ~26, ~24, ~22, ~20, ~18, ~16, ~14, ~12, or ~10; 10~30, ~28, ~26, ~24, ~22, ~20, ~18, ~16, ~14, or ~12;Includes complete and / or quasi-repeating units of 12-30, ~28, ~26, ~24, ~22, ~20, ~18, ~16, or ~14; 14-30, ~28, ~26, ~24, ~22, ~20, ~18, or ~16; 16-30, ~28, ~26, ~24, ~22, ~20, or ~18; 18-30, ~28, ~26, ~24, ~22, or ~20; 20-30, ~28, ~26, ~24, or ~22; 22-30, ~28, ~26, or ~24; 24-30, ~28, or ~26; 26-30, or ~28; 28-30. In some such embodiments, the order of two or more complete or quasi-repeating units in silk or silky or silky proteins is not natural. ; 【0056】 In some embodiments, silk or silk or silk-like protein is greater than 1, greater than 2, greater than 4, greater than 6, greater than 8, greater than 10, greater than 15, greater than 20, or greater than 25; 1-30, ~25, ~20, ~15, ~10, ~8, ~6, ~4, or ~2; 2-30, ~25, ~20, ~15, ~10, ~8, ~6, or ~4; 4-30 , ~25, ~20, ~15, ~10, ~8, or ~6; 6~30, ~25, ~20, ~15, ~10, or ~8; 8~30, ~25, ~20, ~15, or ~10; 10~30, ~25, ~20, or ~15; 15~30, ~25, or ~20; 20~30, or ~25; or 25~30, comprising glycine-rich complete repeats and / or quasi-repeat units. In some such embodiments, one or more of the glycine-rich complete repeats and / or quasi-repeat units are greater than 30%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 70%, or greater than 80%; 30%~100%, ~90%, ~80%, ~70%, ~60%, ~55%, ~50%, ~ 45%, or ~40%; 40%~100%, ~90%, ~80%, ~70%, ~60%, ~55%, ~50%, or ~45%; 45%~100%, ~90%, ~80%, ~70%, ~60%, ~55%, or ~50%; 50%~100%, ~90%, ~80%, ~70%, ~60%, or ~55%; 55%~100%, ~90%, ~ 80%, ~70%, or ~60%; 60%~100%, ~90%, ~80%, or ~70%; 70%~100%, ~90%, or ~80%; 80%~100%, or ~90%; or 90%~100% glycine, greater than 4, greater than 6, greater than 8, greater than 10, greater than 12, greater than 15, greater than 18, greater than 20, greater than 25, greater than 30, greater than 40, greater than 50, greater than 60, greater than 70, greater than 80, greater than 90, greater than 100, or greater than 150; 4~200, ~150, ~100, ~90, ~80, ~70, ~60, ~50, ~40, ~30, ~25, ~20, ~18, ~15, ~12, ~10, ~8, or ~6;6~200, ~150, ~100, ~90, ~80, ~70, ~60, ~50, ~40, ~30, ~25, ~20, ~18, ~15, ~12, ~10, or ~8;8~200, ~150, ~100, ~90, ~80, ~70, ~60, ~50, ~40, ~30, ~25, ~20, ~18, ~15, ~12, or ~10;10~200, ~150, ~100, ~90, ~80, ~70, ~60, ~50, ~40, ~30, ~ 25, ~20, ~18, ~15, or ~12; 12~200, ~150, ~100, ~90, ~80, ~70, ~60, ~50, ~40, ~30, ~25, ~20, ~18, or ~15; 15~200, ~150, ~100, ~90, ~80, ~70, ~60, ~50, ~40, ~30, ~25, ~20, or ~18; 18~200, ~150, ~100, ~90, ~80, ~70, ~60, ~50, ~40, ~30 , ~25, or ~20; 20~200, ~150, ~100, ~90, ~80, ~70, ~60, ~50, ~40, ~30, or ~25; 25~200, ~150, ~100, ~90, ~80, ~70, ~60, ~50, ~40, or ~30; 30~200, ~150, ~100, ~90, ~80, ~70, ~60, ~50, or ~40; 40~200, ~150, ~100, ~90, ~80, ~70, ~60, Or containing consecutive amino acids of ~50; 50~200, ~150, ~100, ~90, ~80, ~70, or ~60; 60~200, ~150, ~100, ~90, ~80, or ~70; 70~200, ~150, ~100, ~90, or ~80; 80~200, ~150, ~100, or ~90; 90~200, ~150, or ~100; 100~200, or ~150; or 150~200. 【0057】 In some embodiments, silk or silk or silk-like protein is greater than 1, greater than 2, greater than 4, greater than 6, greater than 8, greater than 10, greater than 15, greater than 20, or greater than 25; 1-30, ~25, ~20, ~15, ~10, ~8, ~6, ~4, or ~2; 2-30, ~25, ~20, ~15, ~10, ~8, ~6, or ~4; 4-30 Includes alanine-rich complete and / or quasi-repeating units of ~25, ~20, ~15, ~10, ~8, or ~6; 6~30, ~25, ~20, ~15, ~10, or ~8; 8~30, ~25, ~20, ~15, or ~10; 10~30, ~25, ~20, or ~15; 15~30, ~25, or ~20; 20~30, or ~25; or 25~30. In some such embodiments, one or more of the alanine-rich full repeats and / or quasi-repeat units are greater than 70%, greater than 75%, greater than 80%, greater than 85%, or greater than 90%; 70%-100%,-90%,-85%,-80%, or-75%; 75%-100%,-90%,-85%, or-80%; 80%-100%,-90%, or-85%; 85%-100%, or-90%; or 90%-100% of alanine-rich alanine. It is a amino acid that is greater than 4, greater than 6, greater than 8, greater than 10, greater than 12, greater than 15, or greater than 18; 4-20, ~18, ~15, ~12, ~10, ~8, or ~6; 6-20, ~18, ~15, ~12, ~10, or ~8; 8-20, ~18, ~15, ~12, or ~10; 10-18, ~15, or ~12; 12-20, ~18, or ~15; 15-20, or ~18; or contains a sequence of 18-20 amino acids. 【0058】 In some embodiments, silk or silk-like protein comprises one or more glycine-rich complete repeats and / or quasi-repeat units that are 20 to 100 amino acid long and linked to a polyalanine-rich region that is 4 to 20 amino acid long. In some embodiments, silk or silk-like protein comprises 5 to 25% polyalanine region (4 to 20 polyalanine residues). In some embodiments, silk or silk-like protein comprises 25 to 50% glycine. In some embodiments, silk or silk-like protein comprises 15 to 35% GGX, where X is any amino acid. In some embodiments, silk or silk-like protein comprises 15 to 60% GPG. In some embodiments, silk or silk-like protein comprises 10 to 40% alanine. In some embodiments, silk or silk-like protein comprises 0 to 20% proline. In some embodiments, silk or silk-like protein comprises 10 to 50% β-turns. In some embodiments, the silk or silky or silk-like protein contains 10–50% α-helix composition. In some embodiments, all of these compositional ranges apply to the same silk or silky or silk-like protein. In some embodiments, two or more of these compositional ranges apply to the same silk or silky or silk-like protein. 【0059】 In some embodiments, the structure of silk or silk or silk-like protein forms a β-sheet structure, a β-turn structure, or an α-helix structure. In some embodiments, the secondary, tertiary, and quaternary structures of silk or silk or silk-like protein have nanocrystalline β-sheet regions, amorphous β-turn regions, amorphous α-helix regions, randomly spatially distributed nanocrystalline regions embedded in an amorphous matrix, or randomly oriented nanocrystalline regions embedded in an amorphous matrix. In some embodiments, silk or silk or silk-like protein is highly crystalline. In other embodiments, silk or silk or silk-like protein is highly amorphous. In some embodiments, silk or silk or silk-like protein contains both crystalline and amorphous regions. In some embodiments, silk or silk or silk-like protein contains 10% to 40% crystalline material by volume. 【0060】 In some embodiments, silk or silk or silk-like protein comprises one or more complete repeat or semi-repeat units having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity with the repeat units of natural spider silk protein. In some embodiments, silk or silk or silk-like protein comprises one or more complete repeat or semi-repeat units having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity with the repeat units of natural spider dragline silk protein. In some embodiments, silk or silk or silk-like protein comprises one or more complete repeat or semi-repeat units having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity with the repeat units of natural MA dragline silk protein. In some embodiments, the silk or silk or silk-like protein comprises one or more complete repeat or quasi-repeat units having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity with the repeat units of the natural MaSp2 dragline silk protein. 【0061】 In some embodiments, silk or silk or silk-like protein comprises one or more quasi-repeat units, the amino acid sequence of each quasi-repeat unit being described by formula 1, and the amino acid sequence of X1 (referred to as the "motif") being described by formula 2, which may vary randomly within each quasi-repeat unit. Sequence [GPG-X1] n1 This is called the "first region" and is glycine-rich. Sequence (A) n2This is called the "second region" and is alanine-rich. In some embodiments, the value of n1 is one of 4, 5, 6, 7, or 8. In some embodiments, the value of n2 is one of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the value of n3 is one of 2 to 20. In some embodiments, silk or silk or silk-like protein comprises one or more quasi-repeat units having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity with the quasi-repeat units described in formulas 1 and 2. {GGY-[GPG-X1] n1 -GPS-(A) n2} n3 (Formula 1) X1 = SGGQQ or GAGQQ or GQGPY or AGQQ or SQ (Equation 2) 【0062】 In some embodiments, the silk or silk or silk-like protein comprises quasi-repeating units described in formulas 1 and 2, where n1 is 4 or 5 for at least half of the quasi-repeating units. In some embodiments, the silk or silk or silk-like protein comprises quasi-repeating units described in formulas 1 and 2, where n2 is 5 to 8 for at least half of the quasi-repeating units. 【0063】 As used herein, the term “short quasi-repetitive unit” refers to a repetitive unit where n1 is 4 or 5 (as shown in Formula 1). As used herein, the term “long quasi-repetitive unit” refers to a repetition where n1 is 6, 7, or 8 (as shown in Formula 1). In some embodiments, n1 is 4 to 5 for at least half of the quasi-repetitive units. In some embodiments, n2 is 5 to 8 for at least half of the quasi-repetitive units. In some embodiments, silk or silk or silk-like proteins include three “long quasi-repetitive units,” followed by three “short quasi-repetitive units.” In some embodiments, silk or silk or silk-like proteins include quasi-repetitive units within a single quasi-repetition that do not have the same X1 motif more than twice consecutively or more than twice. In some embodiments, silk or silk or silk-like proteins include quasi-repetitive units that contain the same X1 motif in the same location. In some embodiments, silk or silk or silk-like proteins include quasi-repetitive units that contain the same Formula 2 sequence in the same location. In some embodiments, silk or silky or silky proteins contain quasi-repeat units, where no more than three of the six quasi-repeat units share the same X1. 【0064】 In some embodiments, silk or silk or silk-like protein comprises Xqr quasi-repeat units, Xqr=Xsqr+Xlqr (formula 3), (wherein Xqr is a number from 2 to 20, Xsqr is the number of short quasi-iterations and a number from 1 to (Xqr-1), and Xlqr is the number of long quasi-iterations and a number from 1 to (Xqr-1)). In some embodiments, X qr The number is between 2 and 20. Table 3 shows non-restrictive examples of amino acid sequences for repeating units. 【0065】 (Table 3) Exemplary repeating units of silk or silk-like proteins TIFF0007872575000003.tif227140TIFF0007872575000004.tif227146TIFF0007872575000005.tif227143 TIFF0007872575000006.tif227157TIFF0007872575000007.tif227160TIFF0007872575000008.tif227159 TIFF0007872575000009.tif227148TIFF0007872575000010.tif227157TIFF0007872575000011.tif227157 TIFF0007872575000012.tif227156TIFF0007872575000013.tif227160TIFF0007872575000014.tif227107 【0066】 In some embodiments, silk or silk or silk-like protein comprises one or more repeating units, including SEQ ID NO: 13. This repeating unit contains six quasi-repeating units. The quasi-repeating units can be linked together 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times to form a polypeptide molecule of about 50 kDal to about 1,000 kDal. This repeating unit also contains a polyalanine region associated with a nanocrystalline region, and a glycine-rich region associated with a β-turn containing a less crystalline region. 【0067】 Non-limiting examples of additional suitable silk or silk or silk-like proteins include, for example, International Patent Publication No. WO / 2016 / 201369, published on 15 December 2016; U.S. Patent Application No. 62 / 394,683, filed on 14 September 2016; U.S. Patent Application No. 15 / 705,185, filed on 14 September 2017; and U.S. Patent Publication No. 15 / 705,185, published on 4 August 2016. These are provided in International Patent Publication No. US20160222174, International Patent Publication No. WO2016 / 149414 published on March 16, 2016, International Patent Publication No. WO2014 / 066374 published on January 5, 2014, and International Patent Publication No. WO2015 / 042164 published on March 26, 2015, each of which is incorporated herein by reference in whole. 【0068】 Typically, functional linking of recombinant proteins to secretory signals requires the removal of the start codon of the polynucleotide sequence encoding the recombinant protein. 【0069】 Other components In some embodiments, at least one of the polynucleotide sequences contained in the recombinant host cell further encodes a tag peptide or polypeptide that is operably ligated to the C-terminus of the protein. Such tag peptides or polypeptides can be useful in purifying recombinant proteins. Non-limiting examples of tag peptides or polypeptides include affinity tags (i.e., peptides or polypeptides that bind to a particular drug or matrix), solubilization tags (i.e., peptides or polypeptides that help the proper folding of a protein and prevent precipitation), chromatography tags (i.e., peptides or polypeptides that modify the chromatographic properties of a protein to obtain different resolutions for a particular separation technique), epitope tags (i.e., peptides or polypeptides that are bound by an antibody), fluorescent tags, chromogenic tags, enzyme substrate tags (i.e., peptides or polypeptides that are substrates for a particular enzymatic reaction), chemical substrate tags (i.e., peptides or polypeptides that are substrates for a specific chemical modification), or combinations thereof. Non-limiting examples of preferred affinity tags include maltose-binding proteins (MBPs), glutathione-S-transferase (GST), poly(His) tags, SBP tags, Strep tags, and calmodulin tags. Non-limiting examples of suitable solubility tags include thioredoxin (TRX), poly(NANP), MBP, and GST. Non-limiting examples of chromatography tags include polyanionic amino acids (e.g., FLAG tags) and polyglutamic acid tags. Non-limiting examples of epitope tags include V5 tags, VSV tags, Myc tags, HA tags, E tags, NE tags, Ha tags, Myc tags, and FLAG tags. Non-limiting examples of fluorescent tags include green fluorescent protein (GFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), orange-red fluorescent protein (OFP), red fluorescent protein (RFP), and their derivatives.Non-limiting examples of enzyme substrate tags include peptides or polypeptides containing lysine within a sequence suitable for biotinylation (e.g., AviTag, biotin carboxyl carrier protein [BCCP]). Non-limiting examples of chemical substrate tags include substrates suitable for reaction with FIAsH-EDT2. Fusion of peptides or polypeptides with recombinant proteins of the C-terminal tag may or may not be cleavable (e.g., by TEV proteases, thrombin, factor Xa, or enteropeptidases). 【0070】 In some embodiments, at least one of the polynucleotide sequences contained in the recombinant host cell further encodes a linker peptide that is operably linked between the recombinant protein and the secretory signal. The linker peptide may have a variety of sizes. In some such embodiments, the polynucleotide sequence encoding the linker peptide includes restriction enzyme sites that allow for the substitution or addition of other polynucleotide sequences. 【0071】 In some embodiments, polynucleotide sequences encoding recombinant proteins contained in recombinant host cells are operably ligated to a promoter so that they drive the transcription of the polynucleotide sequences. The promoter may be a constitutive promoter or an inductive promoter. Induction may occur, for example, via glucose repression, galactose repression, sucrose repression, phosphate repression, thiamine repression, or methanol repression. A preferred promoter is one that mediates the expression of proteins in recombinant host cells provided herein. Non-limiting examples of suitable promoters include the alcohol oxidase (AOX1) promoter (pAOX1) of Pichia pastris, the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter (pGAP) of Pichia pastris, the YPT1 promoter, the 3-phosphoglycerate kinase 1 (PGK1) promoter (pPKG1) of Saccharomyces cerevisiae, the SSA4 promoter, the HSP82 promoter, the GPM1 promoter, the KAR2 promoter, the triose phosphate isomerase 1 (TPI1) promoter (pTPI1) of Pichia pastris, the enolase 1 (ENO1) promoter (pENO1) of Pichia pastris, the PET9 promoter, the PEX8 (PER3) promoter, the AOX2 promoter, the AOD promoter, and the THI11 promoter. Examples of promoters include the DAS promoter, FLD1 promoter, PHO89 promoter, CUP1 promoter, GTH1 promoter, ICL1 promoter, TEF1 promoter, LAC4-PBI promoter, T7 promoter, TAC promoter, GCW14 promoter, GAL1 promoter, λPL promoter, λPR promoter, β-lactamase promoter, spa promoter, CYC1 promoter, TDH3 promoter, GPD promoter, Saccharomyces cerevisiae translation initiation factor 1 (TEF1) promoter, ENO2 promoter, PGL1 promoter, GAP promoter, SUC2 promoter, ADH1 promoter, ADH2 promoter, HXT7 promoter, PHO5 promoter, and CLB1 promoter. Additional promoters that can be used are known in the art. 【0072】 In some embodiments, polynucleotide sequences encoding recombinant proteins contained in recombinant host cells are operably ligated to terminators so as to result in the termination of transcription of the recombinant polynucleotide sequences. Preferred terminators are those that terminate transcription of recombinant host cells, as provided herein. Non-limiting examples of preferred terminators include the Pichia pastris AOX1 terminator (tAOX1), PGK1 terminator, and TPS1 terminator. Additional terminators are known in the art. 【0073】 A polynucleotide sequence encoding a recombinant protein contained in a recombinant host cell can be operably ligated to the same promoter and / or terminator, or to at least two different promoters and / or terminators. 【0074】 In some embodiments, the polynucleotide sequences encoding recombinant proteins contained in the recombinant host cells further include selection markers (e.g., antibiotic resistance genes, nutrient requirement markers). Selection markers are known in the art. In some embodiments, the selection marker is a drug resistance marker. Drug resistance markers enable cells to detoxify exogenously added drugs that would otherwise kill the cells. Exemplary examples of drug resistance markers include, but are not limited to, those for resistance to antibiotics such as ampicillin, tetracycline, kanamycin, bleomycin, streptomycin, hygromycin, neomycin, and Zeocin®. In other embodiments, the selection marker is a nutrient requirement marker. Nutrient requirement markers enable cells to synthesize essential components (usually amino acids) while growing in a medium lacking those essential components. Selectable nutrient requirement markers include, for example, hisD, which enables growth in histidine-free medium in the presence of histidinol. Other selectable markers include the bleomycin resistance gene, metallothionein gene, hygromycin B-phosphotransferase gene, AURI gene, adenosine deaminase gene, aminoglycoside phosphotransferase gene, dihydrofolate reductase gene, thymidine kinase gene, and xanthine guanine phosphoribosyltransferase gene. 【0075】 host cell Recombinant host cells can be from mammals, plants, algae, fungi, or microorganisms. Non-limiting examples of suitable fungi include methylotrope yeast, filamentous yeast, Arxula adeninivorans, Aspergillus niger, Aspergillus niger var. awamori, Aspergillus oryzae, Candida etchellsii, Candida guilliermondii, Candida humilis, Candida lipolytica, Candida pseudotropicalis, Candida utilis, and Candida versatilus. versatilis), Debaryomyces hansenii, Endothia parasitica, Eremothecium ashbyii, Fusarium moniliforme, Hansenula polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces thermotolerans, Morteirella vinaceae var. raffinoseutilizer, Mucor miehei, Mucor miehei var. cornietoemersonCooney et Emerson), Mucor pusillus Lindt, Penicillium roqueforti, Pichia methanolica, Pichia (Komagataella) pastoris, Pichia (Scheffersomyces) stipitis, Rhizopus niveus, Rhodotorula sp.Saccharomyces bayanus, Saccharomyces beticus, Saccharomyces cerevisiae, Saccharomyces chevalieri, Saccharomyces diastaticus, Saccharomyces ellipsoideus, Saccharomyces exiguus, Saccharomyces florentinus, Saccharomyces fragilis, Saccharomyces pastorianus, Saccharomyces pombe Examples include *Saccharomyces pombe*, *Saccharomyces sake*, *Saccharomyces uvarum*, *Sporidiobolus johnsonii*, *Sporidiobolus salmonicolor*, *Sporobolomyces roseus*, *Trichoderma reesi*, *Xanthophyllomyces dendrorhous*, *Yarrowia lipolytica*, *Zygosaccharomyces rouxii*, and their derivatives and hybrids. 【0076】 Non-limiting examples of suitable microorganisms include Acetobacter suboxydans, Acetobacter xylinum, Actinoplane missouriensis, Arthrospira platensis, Arthrospira maxima, Bacillus cereus, Bacillus coagulans, Bacillus licheniformis, Bacillus stearothermophilus, Bacillus subtilis, Escherichia coli, and Lactobacillus acidophilus. Lactobacillus acidophilus), Lactobacillus bulgaricus, Lactobacillus reuteri, Lactococcus lactis, Lactococcus lactis Lancefield N group, Leuconostoc citrovorum, Leuconostoc dextranicum, Leuconostoc mesenteroides NRRL B-512(F) strain, Micrococcus lysodeikticus, Spirulina, Streptococcus thermophilus, Streptococcus lactis Streptococcus lactis, a subspecies of Streptococcus lactis, diacetylactis, streptococcus, Streptomyces chatanogenicsisExamples include Streptomyces chattanoogensis, Streptomyces griseus, Streptomyces natalensis, Streptomyces olivaceus, Streptomyces olivochromogenes, Streptomyces rubiginosus, Xanthomonas campestris, and their derivatives and hybrids. 【0077】 Additional strains that can be used as recombinant host cells are known in the art. The term “recombinant host cell” should be understood to refer not only to specific target cells but also to the offspring of such cells. Such offspring may not be identical to the parent cells in practice, as certain changes may occur in subsequent generations, either due to mutation or environmental influences, but they are still included within the scope of the term “recombinant host cell” as used herein. 【0078】 fermentation The fermentation provided herein comprises recombinant host cells and culture media suitable for growing the recombinant host cells. Fermentation is obtained by culturing recombinant host cells in a culture medium that provides the recombinant host cells with the nutrients necessary for cell survival and / or growth. Such culture media typically contain an excess carbon source. Non-limiting examples of suitable carbon sources include monosaccharides, disaccharides, polysaccharides, alcohols, and combinations thereof. Non-limiting examples of suitable monosaccharides include glucose, galactose, mannose, fructose, ribose, xylose, arabinose, and combinations thereof. Non-limiting examples of suitable disaccharides include sucrose, lactose, maltose, tehalose, cellobiose, and combinations thereof. Non-limiting examples of suitable polysaccharides include raffinose, starch, glycogen, glycan, cellulose, chitin, and combinations thereof. Non-limiting examples of suitable alcohols include methanol and glycol. 【0079】 The use of at least two distinct secretion signals, as provided herein, promotes a high secretion yield of recombinant protein. Thus, in various embodiments, the fermentation provided herein promotes a secretion yield of at least 1% by weight, 5% by weight, 10% by weight, 20% by weight, or 30% by weight of the total yield of recombinant protein produced by the recombinant host cell as secreted recombinant protein; 1% to 100% by weight, 90% by weight, 80% by weight, 70% by weight, 60% by weight, 50% by weight, 40% by weight, 30% by weight, 20% by weight, or 10% by weight; 10% to 100% by weight, 90% by weight, 80% by weight, 70% by weight, 60% by weight, 50% by weight, 40% by weight, 30% by weight, or 20% by weight; 20% to 100% by weight, 90% by weight, 80% by weight, 70% by weight, Including 60% by weight, 50% by weight, 40% by weight, or 30% by weight; 30% to 100% by weight, 90% by weight, 80% by weight, 70% by weight, 60% by weight, 50% by weight, or 40% by weight; 40% to 100% by weight, 90% by weight, 80% by weight, 70% by weight, 60% by weight, or 50% by weight; 50% to 100% by weight, 90% by weight, 80% by weight, 70% by weight, or 60% by weight; 60% to 100% by weight, 90% by weight, 80% by weight, or 70% by weight; 70% to 100% by weight, 90% by weight, or 80% by weight; 80% to 100% by weight, or 90% by weight; or including 90% to 100% by weight.In some embodiments, the culture medium for fermentation contains recombinant proteins produced by recombinant host cells in concentrations of at least 0.1 g / L, at least 0.5 g / L, at least 1 g / L, at least 2 g / L, at least 5 g / L, at least 7 g / L, at least 10 g / L, at least 15 g / L, or at least 20 g / L; 0.1 g / L to 30 g / L, ~25 g / L, ~20 g / L, ~15 g / L, ~10 g / L, ~7 g / L, ~5 g / L, ~2 g / L, ~1 g / L, or ~0.5 g / L; 0.5 g / L to 30 g / L, ~25 g / L, ~20 g / L, ~15 g / L, ~10 g / L, ~7 g / L, ~5 g / L, ~2 g / L, or ~1 g / L; 1 g / L to 30 g / L, ~25g / L, ~20g / L, ~15g / L, ~10g / L, ~7g / L, ~5g / L, or ~2g / L; 2g / L ~30g / L, ~25g / L, ~20g / L, ~15g / L, ~10g / L, ~7g / L, or ~5g / L; 5g / L ~30g / L, ~25g / L, ~20g / L, ~15g / L, ~10g / L, or ~7g / L; 7g / L~30g / L, ~25g / L, ~20g / L, ~15g / L, or ~10g / L; 10g / L~30g / L, ~25g / L, ~20g / L, or ~15g / L; 15g / L~30g / L, ~25g / L, or ~20g / L; 20g / L~30g / L, or ~25g / L; or 25g / L~30g / L. 【0080】 A method for producing recombinant proteins with high secretion yield. Methods for producing recombinant proteins with high secretion yields are provided herein. Unless otherwise indicated, the methods are generally carried out according to conventional methods described in various general and more specific references in the art that are well known and discussed herein. For example, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; Ausubel et al. Current Protocols in Molecular Biology, Greene Publishing Associates, 1992, and Supplements to 2002); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring. Harbor, NY,1990;Taylor and Drickamer,Introduction to Glycobiology,Oxford Univ.Press,2003;Worthington Enzyme Manual,Worthington Biochemical Corp.,Freehold,NJ;Handbook of Biochemistry:Section A Proteins,Vol I,CRC Press,1976;Handbook of Biochemistry:Section A Proteins,Vol II,CRC Press,1976;Essentials of Glycobiology,Cold Spring Harbor Laboratory See Press, 1999. 【0081】 The methods provided herein include the step of culturing recombinant host cells provided herein in a culture medium under conditions suitable for obtaining the fermentation provided herein (step 1003 in Figure 1). Suitable culture media for use in these methods are known in the art, as are suitable culture conditions. Details of culturing yeast host cells are described, for example, in Idiris et al. (2010) Appl. Microbiol. Biotechnol. 86:403-417; Zhang et al. (2000) Biotechnol. Bioprocess. Eng. 5:275-287; Zhu (2012) Biotechnol. Adv. 30:1158-1170; and Li et al. (2010) MAbs 2:466-477. 【0082】 In some embodiments, the method further includes the step of constructing a recombinant vector comprising a polynucleotide sequence encoding a recombinant protein operably linked to a secretory signal (step 1001 in Figure 1). Methods for constructing recombinant vectors are known in the art. In some embodiments, the recombinant vector is produced by synthesis. In other embodiments, the recombinant vector is isolated or PCR-amplified from an organism, cell, tissue, or plasmid construct by standard procedures. In some embodiments, the polynucleotide sequence encoding the recombinant protein is codon-optimized for expression in a specific host cell. 【0083】 In some embodiments, the method includes a step of balancing the expression of the recombinant protein (e.g., by increasing or decreasing the number of polynucleotide sequences and / or the strength of promoters operably linked to the polynucleotide sequences) with the efficiency of the recombinant protein secretion (e.g., by selecting separate secretion signals or combinations of separate secretion signals). 【0084】 In some embodiments, the method further includes the step of transforming cells with a recombinant vector to obtain recombinant host cells provided herein (step 1002 in Figure 1). In the case of such transformation, the recombinant vector may be cyclic or linearized. Methods for transforming cells are well known in the art. Non-limiting examples of such methods include calcium phosphate transfection, dendrimer transfection, liposome transfection (e.g., cationic liposome transfection), cationic polymer transfection, electroporation, cell squeezing, sonoporation, optical transfection, plasmofusion, impalefection, hydrodynamic delivery, gene gun, magnetic infection, spheroblast generation, polyethylene glycol (PEP) treatment, and viral transduction. Those skilled in the art can select one or more preferred methods for transforming cells with expression constructs or recombinant vectors provided herein, based on the knowledge in the art that certain techniques for introducing vectors work better with certain types of cells. Transformation of recombinant host cells comprising expression constructs or recombinant vectors provided herein can be readily identified, for example, by expressing drug resistance or nutritional requirement markers encoded by the recombinant vector, which enables selection for cell growth or selection against growth, or by other means (e.g., detection of luminescent peptides contained in the expression construct or recombinant vector, molecular analysis of individual recombinant host cell colonies [e.g., restriction enzyme mapping, PCR amplification, or sequence analysis of isolated extrachromosomal vectors or chromosomal integration sites]). In some embodiments, the method comprises sequential transformation of host cells using two or more recombinant vectors. 【0085】 In some embodiments, the method further includes a step of extracting secreted recombinant proteins from fermentation provided herein (step 1004 in Figure 1). Extraction can be achieved by various methods known in the art for purifying secreted proteins. Common steps in such methods include centrifugation at a rate that causes cell pelletization and removal of the cell pellet containing recombinant host cells and cell debris, followed by precipitation of recombinant proteins using a precipitating agent (e.g., 5-60% saturated ammonium sulfate, followed by centrifugation) or affinity separation (e.g., by immunological interaction with antibodies that specifically bind to the recombinant proteins or their C-terminal tags [e.g., FLAG, hemagglutinin], or by binding to a nickel column for isolation of polypeptides tagged with 6-8 histidine residues). The suspended recombinant proteins can be dialyzed to remove dissolved salts. In addition, the dialyzed recombinant proteins can be heated to denature other proteins, and the denatured proteins can be removed by centrifugation. [Examples] 【0086】 Example 1: Generation of Pichia pastris recombinant host cells producing high-yield secreted recombinant protein. Recombinant host cells of Pichia pastris (Komagataella phaffii) that secrete silk-like protein were generated by transforming cells with a HIS+ derivative of GS115 (NRRL Y15851) using a first recombinant vector and a second recombinant vector. 【0087】 The recombinant vector (see Figure 2) contained a polynucleotide sequence encoding a silk-like protein (SEQ ID NO: 110) operably linked to one of several recombinant secretory signals consisting of pre-αMF(sc) / *pro-αMF(sc) (SEQ ID NO: 8) or *pro-αMF(sc) (SEQ ID NO: 2) and a signal peptide. The silk-like protein was further operably linked to a C-terminal FLAG tag. 【0088】 The polynucleotide sequences encoding silk-like proteins within the recombinant vector were flanked by the promoter (pGCW14) and terminator (tAOX1 pA signaling pathway). The recombinant vector further contained dominant resistance markers for the selection of bacterial and yeast transformants, as well as bacterial origins of replication. The recombinant vector also contained target-directed regions intended for the integration of polynucleotide sequences flanked 3' of the HIS4, HSP82, AOX2, TEF1, MAE1, or ICL1 loci within the Pichia pastris genome. 【0089】 Recombinant host cells were generated by sequentially transforming Pichia pastris host cells with recombinant vectors via electroporation. The transformants were plated on either a histidine-deficient minimal agar plate or an antibiotic-supplemented YPD agar plate and incubated at 30°C for 48 hours. 【0090】 The obtained clones were inoculated into 400 μL of buffered glycerol complex medium (BMGY) in a 96-well block and incubated at 30°C for 48 hours with stirring at 1,000 rpm. After 48 hours of incubation, 4 μL of each culture was inoculated into 400 μL of minimal medium in a 96-well block, and this was then incubated at 30°C for 48 hours. 【0091】 To extract recombinant proteins for ELISA analysis, guanidine thiocyanate was added to the cell culture to a final concentration of 2.5 M. After incubation for 5 minutes, the solution was centrifuged and the supernatant was sampled (data labeled as extracellular are shown in Figures 3-5). The supernatant was removed by inversion, and the remaining cell pellet was resuspended in guanidine thiocyanate to its original volume. The cells were lysed by mechanical disruption in a bead mill, and the resulting lysate was clarified by centrifugation and sampled (data labeled as intracellular are shown in Figures 3-5). 【0092】 As shown in Figure 3, increasing the number of polynucleotide sequences encoding silk-like proteins operably linked to pre-αMF(sc) / *pro-αMF(sc) from 4 to 6 resulted in higher overall production of silk-like proteins, although the secretory yield did not increase as significantly. Further shown in Figure 3, recombinant host cells containing 7 copies of polynucleotide sequences encoding silk-like proteins operably linked to two distinct secretory signals (i.e., pre-αMF(sc) / *pro-αMF(sc) in 4 polynucleotide sequences and pre-DSE4(pp) / *pro-αMF(sc) or pre-PEP4(sc) / *pro-αMF(sc) in 3 polynucleotide sequences) produced a higher secretory yield than recombinant host cells containing 6 copies of polynucleotide sequences encoding silk-like proteins operably linked to a single type of secretory signal (i.e., pre-αMF(sc) / *pro-αMF(sc)). While we were unable to obtain recombinant host cells containing seven copies of a polynucleotide sequence encoding a silk-like protein operably linked to the pre-αMF(sc) / *pro-αMF(sc) secretion signal for direct comparison, it should be noted that this may indicate that such host cells are unstable and / or unviable. 【0093】 As shown in Figure 4, recombinant host cells containing six copies of polynucleotide sequences encoding silk-like proteins operably linked to two distinct secretory signals (i.e., pre-αMF(sc) / *pro-αMF(sc) in four polynucleotide sequences and pre-EPX1(pp) / *pro-αMF(sc) in two polynucleotide sequences) produced higher secretory yields and percentage secretions of silk-like proteins than recombinant host cells containing the same number of polynucleotide sequences encoding silk-like proteins operably linked to a single type of secretory signal (i.e., pre-αMF(sc) / *pro-αMF(sc)). 【0094】 As shown in Figure 5, the addition of a third recombinant secretory signal (i.e., pre-CLSP(gg) / *pro-αMF(sc)) to the 4+2xEPX1(pp) strain of Figure 4 further improved the secretory yield of silk-like protein. Cells containing seven copies of the polynucleotide sequence encoding silk-like protein operably linked to the pre-αMF(sc) / *pro-αMF(sc) secretory signal, or cells containing four copies of the polynucleotide sequence encoding silk-like protein operably linked to the pre-αMF(sc) / *pro-αMF(sc) secretory signal and three copies of the polynucleotide sequence encoding silk-like protein operably linked to the pre-EPX1(pp) / *pro-αMF(sc) secretory signal, were not available for direct comparison, but it should be noted that this may indicate that such host cells are unstable and / or unviable. 【0095】 Example 2: Generation of Pichia pastris recombinant host cells producing high secretion yields of α-amylase or green fluorescent protein. Recombinant host cells of Pichia pastris (Chomagataera fafi) secreting either α-amylase or green fluorescent protein were generated by transforming them with HIS+ derivatives of GS115 (NRRL Y15851) using various recombinant vectors. 【0096】 The recombinant vector (see Figure 6) contained an expression construct comprising a polynucleotide sequence encoding either α-amylase (SEQ ID NO: 111) or green fluorescent protein (SEQ ID NO: 112), operably linked to various N-terminal recombinant secretory signals. The recombinant secretory signal consisted of an N-terminal signal peptide operably linked to *pro-αMF(sc) (SEQ ID NO: 2). The α-amylase or green fluorescent protein was further operably linked to a C-terminal FLAG tag. Each polynucleotide sequence was facile to a promoter (pGCW14) and a terminator (tAOX1 pA signal). The recombinant vector further contained a target-directed region intended for integration of the expression construct into the region facile to the 3' of the THI4 locus in the Pichia pastris genome, a dominant resistance marker for selection of bacterial and yeast transformants, and a bacterial origin of replication. 【0097】 (Table 4) Expressed recombinant proteins TIFF0007872575000015.tif79165 【0098】 Recombinant vectors were used to transform Pichia pastris host cells via electroporation to generate recombinant host strains. The transformants were plated onto YPD agar plates supplemented with antibiotics and incubated at 30°C for 48–96 hours. 【0099】 Clones from each of the final transformations were inoculated into 400 μL of buffered glycerol complex medium (BMGY) in a 96-well block and incubated at 30°C for 48 hours with stirring at 1,000 rpm. After 48 hours of incubation, 4 μL of each culture was inoculated into 400 μL of minimal medium in a 96-well block, and this was then incubated at 30°C for 48 hours. 【0100】 To extract recombinant proteins for ELISA analysis, guanidine thiocyanate was added to the cell culture to a final concentration of 2.5 M. After incubation for 5 minutes, the solution was centrifuged and the supernatant was sampled. 【0101】 As shown in Figure 7, the pre-EPX1(pp) / *pro-αMF(sc) and pre-PEP4(sc) / *pro-αMF(sc) recombinant secretion signals produced higher secretion yields of amylase than the pre-αMF(sc) / *pro-αMF(sc) recombinant secretion signal, while the pre-DSE4(pp) / *pro-αMF(sc) secretion signal produced almost the same amount of secretion amylase. 【0102】 As shown in Figure 8, the pre-EPX1(pp) / *pro-αMF(sc) recombinant secretion signal produced a higher secretion yield of green fluorescent protein than the pre-αMF(sc) / *pro-αMF(sc) recombinant secretion signal, while the pre-PEP4(sc) / *pro-αMF(sc) and pre-DSE4(pp) / *pro-αMF(sc) secretion signals produced lower secretion yields of fluorescent protein. 【0103】 The above description of embodiments of this disclosure is provided for illustrative purposes only and is not exhaustive or intended to limit the scope of the invention. 【0104】 While efforts have been made to ensure accuracy with respect to the numerical values ​​used in the examples (e.g., quantities, temperatures, etc.), some experimental errors and deviations are, of course, to be acceptable. The reagents used in the examples are generally commercially available or can be prepared using commercially available instruments, methods, or reagents known in the art. The examples do not provide an exhaustive description of the many different embodiments of the present invention. Those skilled in the art will readily understand that many changes and modifications can be made to the embodiments presented in the examples without departing from the spirit or scope of the appended claims. 【0105】 (Table 5) pro-αMF array TIFF0007872575000016.tif34165

Claims

[Claim 1] A recombinant yeast host cell comprising a first polynucleotide sequence and a second polynucleotide sequence encoding a structurally identical recombinant protein, which are fused to separate secretory signals, The recombinant protein encoded by the first polynucleotide sequence is operably ligated to a first secretory signal comprising the pre region of the α-conjugation factor signal sequence of S. cerevisiae and the pro region of the α-conjugation factor signal sequence of S. cerevisiae, wherein the first secretory signal comprises SEQ ID NO: 8, or a functional secretory signal variant that is at least 90% identical to SEQ ID NO: 8, and The recombinant protein encoded by the second polynucleotide sequence is operably ligated to a second secretory signal comprising the pre region of the DSE4 signal sequence of P. pastoris and the pro region of the α-conjugation factor signal sequence of S. cerevisiae, wherein the second secretory signal comprises SEQ ID NO: 10, or a functional secretory signal variant that is at least 90% identical to SEQ ID NO: 10, in the recombinant yeast host cell. [Claim 2] The recombinant yeast host cell according to claim 2, wherein the yeast host cell is P. pastris. [Claim 3] The recombinant yeast host cell according to claim 1 or 2, wherein the recombinant protein comprises silk protein. [Claim 4] The recombinant yeast host cell according to claim 1 or 2, wherein the recombinant protein comprises repeating units of silk protein. [Claim 5] The recombinant yeast host cell according to claim 1 or 2, wherein the recombinant protein comprises a sequence selected from the group consisting of SEQ ID NOs: 13 to 110. [Claim 6] The recombinant yeast host cell according to claim 5, wherein the recombinant protein contains the sequence of SEQ ID NO:

13. [Claim 7] The recombinant yeast host cell according to claim 5, wherein the recombinant protein contains the sequence of SEQ ID NO:

110. [Claim 8] A method for producing recombinant proteins, including the following steps: (a) A step of culturing recombinant yeast host cells according to any one of claims 1 to 7 in a culture medium to obtain fermentation containing the recombinant yeast host cells, and (b) Step of extracting the recombinant protein from the culture medium. [Claim 9] The method according to claim 8, wherein the recombinant protein comprises repeating units of silk protein. [Claim 10] The method according to claim 8, wherein the recombinant protein comprises silk protein. [Claim 11] The method according to claim 8, wherein the recombinant protein comprises a sequence selected from the group consisting of SEQ ID NOs: 13 to 110. [Claim 12] The method according to claim 8, wherein the recombinant protein comprises the sequence of SEQ ID NO:

13. [Claim 13] The method according to claim 8, wherein the recombinant protein comprises the sequence of SEQ ID NO:

110. [Claim 14] A composition comprising recombinant yeast host cells and culture medium according to any one of claims 1 to 7. [Claim 15] The composition according to claim 14, wherein the recombinant protein comprises repeating units of silk protein. [Claim 16] The composition according to claim 14, wherein the recombinant protein comprises silk protein. [Claim 17] The composition according to claim 14, wherein the recombinant protein comprises a sequence selected from the group consisting of SEQ ID NOs: 13 to 110. [Claim 18] The composition according to claim 14, wherein the recombinant protein comprises the sequence of SEQ ID NO:

13. [Claim 19] The composition according to claim 14, wherein the recombinant protein comprises the sequence of SEQ ID NO: 110.

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