SEC engineered strains for improved secretion of recombinant proteins

Abstract

Disclosed herein are engineered strains with improved growth and productivity characteristics for improved secretion of recombinantly expressed products secreted from host organisms, as well as methods of using the engineered strains. [Selection diagram] Figure 12B

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 673,001, filed May 17, 2018, the contents of which are incorporated herein by reference in their entirety.

[0002] FIELD OF THEINVENTION The present disclosure relates to methods of optimizing strains to enhance secretion of proteins or metabolites from cells. The disclosure also relates to compositions resulting from those methods. In particular, the disclosure relates to yeast cells that have been selected or engineered to enhance secretion of recombinant proteins expressed by the yeast cells, and to methods of culturing the yeast cells to produce useful compounds. [Background technology]

[0003] 2. Background of the Invention The methylotrophic yeast Pichia pastoris has been widely used for recombinant protein production. P. pastoris can grow to high cell densities, provide tightly controlled, methanol-inducible transgene expression, and has the ability to secrete recombinant proteins into synthetic media.

[0004] However, a significant portion of the expressed recombinant protein remains intracellularly, which can make recovery difficult and can adversely affect cell growth and life span. Furthermore, recombinantly expressed proteins can be degraded in cells before being secreted, resulting in a protein mixture that contains recombinantly expressed protein fragments and low yields of full-length recombinant protein. Thus, there is a need for a means and engineered strains that enhance secretion in P. pastoris while attenuating any loss in recombinant protein productivity or improving the productivity.

[0005] Thus, there is a need for engineered organisms, and methods of using these organisms, that produce and secrete recombinant proteins while reducing any adverse effects on cellular growth and productivity. Summary of the Invention

[0006] In some embodiments, provided herein is a Pichia pastoris microorganism having abolished SEC72 activity or a deleted sec72 gene, the microorganism expressing a recombinant protein, in some embodiments, the microorganism further comprises a recombinantly expressed SSH1 translocon complex.

[0007] In some embodiments, SEC72 comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 1. In some embodiments, the SEC72 comprises SEQ ID NO: 1. In some embodiments, the SEC72 is encoded by the sec72 gene. In some embodiments, the sec72 gene comprises a polynucleotide sequence at least 95% identical to SEQ ID NO: 2. .stomach In some embodiments, the sec72 gene comprises SEQ ID NO: 2. In some embodiments, the sec72 gene is present at locus PAS_chr2-1_0448 of the microorganism.

[0008] In some embodiments, the SSH1 translocon complex comprises a first polypeptide sequence at least 95% identical to SEQ ID NO:4, a second polypeptide sequence at least 95% identical to SEQ ID NO:6, and a third polypeptide sequence at least 95% identical to SEQ ID NO:8. In some embodiments, the SSH1 translocon complex comprises a first polypeptide comprising SEQ ID NO:4, a second polypeptide comprising SEQ ID NO:6, and a third polypeptide comprising SEQ ID NO:8.

[0009] In some embodiments, the microorganism further comprises a recombinantly expressed translocon complex, hi some embodiments, the translocon complex is expressed from a recombinant SSH1 gene, a recombinant SSS1 gene, and a recombinant SBH2 gene.

[0010] In some embodiments, the SSH1 gene comprises SEQ ID NO: 3. In some embodiments, the SSH1 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO: 3. 。

[0011] In some embodiments, the SSH1 gene comprises SEQ ID NO: 5. In some embodiments, the SSH1 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO: 5. 。

[0012] In some embodiments, the SBH2 gene comprises SEQ ID NO: 7. In some embodiments, the SBH2 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO: 7. 。

[0013] In some embodiments, expression of the recombinant SSH1 gene to increase the level of the SSH1 translocon in the microorganism above the level of the SSH1 translocon expressed by the native organism improves the growth rate and / or fermentation capacity of the microorganism.

[0014] In some embodiments, the translocon complex comprises an SSH1 protein, an SSS1 protein, and an SBH2 protein. In some embodiments, the SSH1 protein comprises a polypeptide sequence at least 95% identical to SEQ ID NO:4. In some embodiments, the SSH1 protein comprises SEQ ID NO:4. In some embodiments, the SSS1 protein comprises a polypeptide sequence at least 95% identical to SEQ ID NO:6. In some embodiments, the SSS1 protein comprises SEQ ID NO:6. In some embodiments, the SBH2 protein comprises a polypeptide sequence at least 95% identical to SEQ ID NO:8. In some embodiments, the SBH2 protein comprises SEQ ID NO:8.

[0015] In some embodiments, the activity of YPS1-1 protease and YPS1-2 protease in the microorganism is reduced or eliminated.

[0016] In some embodiments, the YPS1-1 protease comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 10. In some embodiments, the YPS1-1 protease comprises SEQ ID NO: 10. In some embodiments, the YPS1-1 protease is encoded by the YPS1-1 gene. In some embodiments, the YPS1-1 gene comprises a polynucleotide sequence at least 95% identical to SEQ ID NO: 9. .stomach In some embodiments, the YPS1-1 gene comprises SEQ ID NO: 9. In some embodiments, the YPS1-1 gene is present at locus PAS_chr4_0584 of the microorganism.

[0017] In some embodiments, the YPS1-2 protease comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 12. In some embodiments, the YPS1-2 protease comprises SEQ ID NO: 12. In some embodiments, the YPS1-2 protease is encoded by the YPS1-2 gene. In some embodiments, the YPS1-2 gene comprises a polynucleotide sequence at least 95% identical to SEQ ID NO: 11. .stomach In some embodiments, the YPS1-2 gene comprises SEQ ID NO: 11. In some embodiments, the YPS1-2 gene is present at locus PAS_chr3_1157 of the microorganism.

[0018] In some embodiments, the YPS1-1 gene or the YPS1-2 gene, or both, of the microorganism are mutated or knocked out. In some embodiments, the activity of one or more additional proteases of the microorganism is reduced or eliminated.

[0019] In some embodiments, the recombinant protein comprises at least one block polypeptide sequence from a silk protein. In some embodiments, the recombinant protein comprises a silk-like polypeptide. In some embodiments, the silk-like polypeptide comprises one or more repeat sequences. TIFF0007623680000001.tif4128 (SEQ ID NO:13) wherein X1=SGGQQ (SEQ ID NO:14) or GAGQQ (SEQ ID NO:15) or GQGPY (SEQ ID NO:16) or AGQQ (SEQ ID NO:17) or SQ, wherein n1 is 4 to 8, n2 is 6 to 20, and n3 is 2 to 20. In some embodiments, the silk-like polypeptide comprises a polypeptide sequence encoded by SEQ ID NO:21.

[0020] In some embodiments, the recombinant protein comprises a secretory signal peptide. In some embodiments, the secretory signal peptide is selected from the group consisting of a PEP4 signal sequence, a CPY+4 signal sequence, a DAP2 signal sequence, and an MFα1 signal sequence. In some embodiments, the secretory signal peptide is selected from the group consisting of SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, and SEQ ID NO:90.

[0021] Also provided herein, according to some embodiments, is a microorganism called Pichia pastoris, in which the SEC72 activity of the microorganism is eliminated or the sec72 gene comprising SEQ ID NO:1 is knocked out, and the microorganism comprises a recombinantly expressed SSH1 gene comprising SEQ ID NO:3, a recombinantly expressed SSS1 gene comprising SEQ ID NO:5, and a recombinantly expressed SBH2 gene comprising SEQ ID NO:7, and the microorganism comprises a silk-like polypeptide comprising a polypeptide encoded by SEQ ID NO:21.

[0022] Also provided herein, according to some embodiments, is a cell culture comprising a recombinant microorganism described herein.

[0023] Also provided herein, according to some embodiments, are cell cultures comprising a recombinant microorganism as described herein, which exhibit improved strain growth rate and fermentation capacity under standard cell culture conditions compared to cell cultures that do not contain a recombinantly expressed SSH1 translocon complex.

[0024] Also provided herein, according to some embodiments, is a cell culture comprising a recombinant microorganism as described herein, which cell culture, under standard cell culture conditions, has improved yield or specific productivity of the recombinant protein compared to a cell culture of the same microorganism except that the microorganism contains a functional sec72 gene and does not contain a recombinantly expressed SSH1 translocon complex, each of which has the same number of copies of a recombinant silk polypeptide gene.

[0025] Also provided herein, according to some embodiments, is a method for producing a recombinant protein, comprising culturing a recombinant microorganism as described herein in a medium under conditions suitable for expression of said recombinantly expressed protein, and isolating said recombinant protein from said microorganism or from said medium.

[0026] In some embodiments, the recombinant protein is secreted from the microorganism, and isolating the recombinant protein comprises recovering medium containing the secreted recombinant protein.

[0027] In some embodiments, the yield or specific productivity of the recombinant protein is increased in the microorganism as compared to an otherwise identical microorganism in which the sec72 gene has not been deleted.

[0028] In some embodiments, the yield or specific productivity of the recombinant protein is increased in the microorganism compared to an otherwise identical microorganism that does not contain the recombinantly expressed SSH1 translocon complex and that does not have a deletion of the sec72 gene.

[0029] Also provided herein, according to some embodiments, is a method of modifying Pichia pastoris to improve secretion of a recombinantly expressed protein, the method comprising knocking out a gene encoding a SEC72 protein. In some embodiments, the method of modifying Pichia pastoris to improve secretion of a recombinantly expressed protein further comprises transforming the Pichia pastoris with a vector comprising a gene encoding a recombinantly expressed SSH1 translocon complex.

[0030] In some embodiments, the recombinantly expressed protein comprises a silk-like polypeptide. In some embodiments, the silk-like polypeptide comprises one or more repeat sequences {GGY-[GPG-X1] n1 -GPS-(A) n2} n3 (SEQ ID NO:13) wherein X1=SGGQQ (SEQ ID NO:14) or GAGQQ (SEQ ID NO:15) or GQGPY (SEQ ID NO:16) or AGQQ (SEQ ID NO:17)or SQ, wherein n1 is 4 to 8, n2 is 6 to 20, and n3 is 2 to 20. In some embodiments, the recombinantly expressed protein comprises a polypeptide sequence encoded by SEQ ID NO:21.

[0031] [The present invention 1001] A microorganism called Pichia pastoris, in which SEC72 activity has been eliminated or the sec72 gene has been deleted, said microorganism expressing a recombinant protein. [The present invention 1002] 1001. The microorganism of claim 10, wherein said SEC72 comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:1. [The present invention 1003] 1001. The microorganism of the present invention, wherein said SEC72 comprises SEQ ID NO:1. [The present invention 1004] The microorganism of the present invention, wherein said SEC72 is encoded by the sec72 gene. [The present invention 1005] The microorganism of the present invention, wherein the sec72 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO:2. 。 [ The present invention 1007 The microorganism of the present invention, wherein the sec72 gene comprises SEQ ID NO:2. [The present invention 1008] 1001. The microorganism of the present invention, wherein said sec72 gene is present at locus PAS_chr2-1_0448 of said microorganism. [The present invention 1009] The microorganism of any of the preceding inventions, further comprising a recombinantly expressed SSH1 translocon complex. [The present invention 1010] The microorganism of the present invention 1009, wherein the SSH1 translocon complex comprises a first polypeptide sequence at least 95% identical to SEQ ID NO: 4, a second polypeptide sequence at least 95% identical to SEQ ID NO: 6, and a third polypeptide sequence at least 95% identical to SEQ ID NO: 8. [The present invention 1011] 1009. The microorganism of the present invention, wherein the SSH1 translocon complex comprises a first polypeptide comprising SEQ ID NO:4, a second polypeptide comprising SEQ ID NO:6, and a third polypeptide comprising SEQ ID NO:8. [The present invention 1012] The microorganism of any of the preceding inventions, further comprising a recombinantly expressed translocon complex. [The present invention 1013] The microorganism of the present invention, wherein the translocon complex is expressed from a recombinant SSH1 gene, a recombinant SSS1 gene, and a recombinant SBH2 gene. [The present invention 1014] The microorganism of the present invention, wherein the SSH1 gene comprises SEQ ID NO:3. [The present invention 1015] The microorganism of the present invention, wherein the SSH1 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO:3. 。 [ The present invention 1017 The microorganism of the present invention, wherein the SSH1 gene comprises SEQ ID NO:5. [The present invention 1018] The microorganism of the present invention, wherein the SSH1 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO:5. 。 [ The present invention 1020 The microorganism of the present invention, wherein the SBH2 gene comprises SEQ ID NO:7. [The present invention 1021] The microorganism of the present invention, wherein the SBH2 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO:7. 。 [ The present invention 1023 A microorganism of the present invention, wherein expression of the recombinant SSH1 gene to increase the level of the SSH1 translocon in the microorganism beyond the level of the SSH1 translocon expressed by a natural organism improves the growth rate and / or fermentation capacity of the microorganism. [The present invention 1024] The microorganism of the present invention, wherein the translocon complex comprises an SSH1 protein, an SSS1 protein, and an SBH2 protein. [The present invention 1025] The microorganism of the present invention, wherein the SSH1 protein comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:4. [The present invention 1026] The microorganism of the present invention, wherein the SSH1 protein comprises SEQ ID NO:4. [The present invention 1027] The microorganism of the present invention, wherein the SSS1 protein comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:6. [The present invention 1028] The microorganism of the present invention, wherein the SSS1 protein comprises SEQ ID NO:6. [The present invention 1029] The microorganism of the present invention, wherein the SBH2 protein comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:8. [The present invention 1030] The microorganism of the present invention, wherein the SBH2 protein comprises SEQ ID NO:8. [The present invention 1031] Any of the microorganisms of the prior invention, in which the activity of YPS1-1 protease and YPS1-2 protease is reduced or eliminated. [The present invention 1032] 1031. The microorganism of the present invention, wherein the YPS1-1 protease comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:10. [The present invention 1033] 1031. The microorganism of the present invention, wherein the YPS1-1 protease comprises SEQ ID NO:10. [The present invention 1034] The microorganism of the present invention, wherein the YPS1-1 protease is encoded by the YPS1-1 gene. [The present invention 1035] The microorganism according to claim 1034, wherein the YPS1-1 gene comprises a polynucleotide sequence which is at least 95% identical to SEQ ID NO:9.。 [ The present invention 1037 The microorganism of the present invention, wherein the YPS1-1 gene comprises SEQ ID NO:9. [The present invention 1038] The microorganism of the present invention, wherein the YPS1-1 gene is present at locus PAS_chr4_0584 of said microorganism. [The present invention 1039] Any of the prior microorganisms of the present invention, wherein the YPS1-2 protease comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:12. [The present invention 1040] The microorganism of the present invention, wherein the YPS1-2 protease comprises SEQ ID NO:12. [The present invention 1041] The microorganism of the present invention, wherein the YPS1-2 protease is encoded by the YPS1-2 gene. [The present invention 1042] The microorganism of the present invention, wherein the YPS1-2 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO: 11. 。 [ The present invention 1044 The microorganism of the present invention, wherein the YPS1-2 gene comprises SEQ ID NO:11. [The present invention 1045] 1041. The microorganism of the invention, wherein said YPS1-2 gene is present at locus PAS_chr3_1157 of said microorganism. [The present invention 1046] The microorganism of any of claims 1031 to 1045, wherein the YPS1-1 gene or the YPS1-2 gene, or both, are mutated or knocked out. [The present invention 1047] The microorganism of any of claims 1031 to 1046, wherein the activity of one or more additional proteases is weakened or eliminated. [The present invention 1048] 20. The method of claim 19, wherein the recombinant protein comprises at least one blocked polypeptide sequence derived from a silk protein. [The present invention 1049] The microorganism of any of the preceding inventions, wherein said recombinant protein comprises a silk-like polypeptide. [The present invention 1050] The silk-like polypeptide comprises one or more repeat sequences Contains TIFF0007623680000002.tif4128, During the ceremony, X1=SGGQQ or GAGQQ or GQGPY or AGQQ or SQ; n1 is 4 to 8, n2 is 6 to 20, and n3 is 2 to 20; The microorganism of the present invention 1049. [The present invention 1051] The microorganism of the present invention, wherein the silk-like polypeptide comprises a polypeptide sequence encoded by SEQ ID NO:21. [The present invention 1052] Any of the prior microbial organisms of the invention, wherein said recombinant protein comprises a secretory signal peptide. [The present invention 1053] The microorganism of the present invention, wherein the secretory signal peptide is selected from the group consisting of a PEP4 signal sequence, a CPY+4 signal sequence, a DAP2 signal sequence, and an MFα1 signal sequence. [The present invention 1054] The microorganism of the present invention, wherein the secretory signal peptide is selected from the group consisting of SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, and SEQ ID NO:90. [The present invention 1055] Pichia pastoris is a microorganism that The microorganism has a deleted SEC72 activity or a knockout of the sec72 gene comprising SEQ ID NO:1; a recombinantly expressed SSH1 gene comprising SEQ ID NO:3, a recombinantly expressed SSS1 gene comprising SEQ ID NO:5, and a recombinantly expressed SBH2 gene comprising SEQ ID NO:7; and A silk-like polypeptide comprising a polypeptide encoded by SEQ ID NO: 21, The microorganism. [The present invention 1056] A cell culture comprising the microorganism according to any one of claims 1001 to 1055 of the present invention. [The present invention 1057] A cell culture comprising any of the microorganisms of the present invention 1009 to 1055, wherein the growth rate and fermentation capacity of the strain are improved under standard cell culture conditions compared to a cell culture not containing a recombinantly expressed SSH1 translocon complex. [The present invention 1058] A cell culture comprising any one of the microorganisms according to the present invention 1009 to 1055, Under standard cell culture conditions, identical microorganisms except that they contain a functional sec72 gene and do not contain a recombinantly expressed SSH1 translocon complex, each of which has the same number of copies of a recombinant silk polypeptide gene. the yield or specific productivity of the recombinant protein is increased compared to a cell culture of The cell culture. [The present invention 1059] Cultivating the microorganism according to any one of claims 1001 to 1055 in a medium under conditions suitable for expression of the recombinantly expressed protein; isolating said recombinant protein from said microorganism or from said culture medium. A method for producing a recombinant protein comprising the steps of: [The present invention 1060] The method of claim 1059, wherein said recombinant protein is secreted from said microorganism and said step of isolating said recombinant protein comprises recovering the culture medium containing the secreted recombinant protein. [The present invention 1061] The method of claim 1059, wherein the yield or specific productivity of said recombinant protein in said microorganism is increased compared to an otherwise identical microorganism in which the sec72 gene is not deleted. [The present invention 1062] an identical microorganism except that it does not contain the recombinantly expressed SSH1 translocon complex and the sec72 gene is not deleted. The method of claim 1059, wherein the yield or specific productivity of said recombinant protein in said microorganism is increased compared to [The present invention 1063] 1. A method for modifying Pichia pastoris to improve secretion of a recombinantly expressed protein, comprising knocking out a gene encoding a SEC72 protein. [The present invention 1064] The method of claim 1063, further comprising the step of transforming said Pichia pastoris with a vector comprising a gene encoding a recombinantly expressed SSH1 translocon complex. [The present invention 1065] The method of claim 1063, wherein said recombinantly expressed protein comprises a silk-like polypeptide. [The present invention 1066] The silk-like polypeptide has one or more repeat sequences {GGY-[GPG-X1] n1 -GPS-(A) n2} n3 Including, During the ceremony, X1=SGGQQ or GAGQQ or GQGPY or AGQQ or SQ; n1 is 4 to 8, n2 is 6 to 20, and n3 is 2 to 20; The method of the present invention 1063. [The present invention 1067] The method of claim 1063, wherein said recombinantly expressed protein comprises a polypeptide sequence encoded by SEQ ID NO:21. The foregoing and other objects, features and advantages will become apparent from the following description of specific embodiments of the invention, as illustrated in the accompanying drawings, in which like reference characters refer to the same parts throughout the different views, and in which the drawings are not necessarily drawn to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the invention. [Brief description of the drawings]

[0032] [Figure 1] 1 shows the classification of genes in the secretion complexes SEC, SEC61, SEC63, and SSH1, and also shows the homology values ​​between the S. cerevisiae and P. pastoris translocon complexes. [Diagram 2] 13 is a plasmid map of a vector for SEC72 deletion containing a yeast selectable marker flanked by SEC72 homology arms. [Diagram 3] 1 is a plasmid map of a vector for overexpression encoding proteins comprising the SSH1 complex, including promoters, open reading frames (ORFs), and terminators for SBH2, SSS1, and SSH1, as well as a yeast selectable marker. [Figure 4-1] FIG. 4A is a plasmid map of a vector containing the nourseothricin marker for use with homology arms for targeted protease gene deletion. [Figure 4-2] Figure 4B shows a cassette for protease knockout with homology arms targeting a desired protease gene adjacent to a nourseothricin resistance marker. Figure 4C shows a cassette for protease knockout with homology arms targeting a desired protease gene adjacent to a nourseothricin resistance marker. [Diagram 5] 1 is a plasmid map of a vector containing an open reading frame (ORF) for expressing a silk-like polypeptide, including a secretion signal and a FLAG tag. The ORF is operably linked to a pGCW14 promoter and a tAOX1 terminator. The vector also contains a selection marker for selecting successfully transformed cells. [Figure 6]FIG. 1 shows secretion of recombinant 18B silk-like polypeptide expressed by wild-type (WT) and Δsec72 (Δ) strains expressing 2X, 4X, or 6X copies of 18B as measured by enzyme-linked immunosorbent assay (i.e., ELISA). [Figure 7] Figure 1 shows secretion of recombinant proteins containing PEP4 (sc), CPY+4 (sc), DAP2 (sc), or MFα1 (sc) signal peptides expressed by wild-type (WT) and Δsec72 (Δ) strains and measured by ELISA (sc) indicates the signal peptide from S. cerevisiae. [Figure 8] Figure 1 shows secretion of long silk polypeptides, Argiope bruennichi major ampullate gland spidroin 2 (i.e., Ab MaSp2) (106 kDa) and Latrodectus hesperus major ampullate gland spidroin 1 (i.e., Lh MaSp1 (55 kDa)), expressed by wild-type (WT) and Δsec72 (Δ) strains and measured by ELISA. [Figure 9A] FIG. 1 shows secretion of recombinant 18B silk-like polypeptide expressed by wild-type (WT), Δsec72 (Δ) strains, strains overexpressing sec72 under the control of pTHI11, and strains in which SEC72 was knocked down by DAmP (Decreased Abundance by mRNA Perturbation) and measured by ELISA. [Figure 9B] FIG. 1 shows secretion of recombinant 18B silk-like polypeptide expressed by wild-type (WT) strains, strains overexpressing non-SEC72 proteins from the SEC63 complex (i.e., sec62, sec63, and sec66), and strains overexpressing sec72, as measured by ELISA. [Figure 10A] FIG. 1 shows expression normalized to reference in Δsec72 strains as fold change from the WT strain. Shading indicates range, and the middle line represents the median across n=3 biological replicates (except for n=2 for Δsec72). [Figure 10B] Projections of centered, log-scaled, reference-normalized expression data onto principal component space (left panel = PC1 vs. PC3, right panel = PC2 vs. PC3) are shown. Replicate data are color-coded by strain genotype, indicating low intra-genotype variability compared to inter-genotype variability. [Figure 11] FIG. 1 shows the growth rates observed for the WT and Δsec72 strains and a comparison of their respective growth rates with those of strains engineered to overexpress the SSH1 translocon complex or SEC61. [Figure 12A] FIG. 1 shows the production (yield) of 18B as a function of glucose for wild-type (WT) strains expressing four (4x) or six (4+2x) copies of 18B compared to a Δsec72 strain (4x, Δsec72) and a Δsec72 strain overexpressing the SSH1 translocon complex (4x, Δsec72, 2N SSH1). [Figure 12B] FIG. 1 shows the specific productivity (product per biomass) of each strain. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Detailed Description Details of various embodiments of the invention are set forth in the following description. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0034] definition Unless otherwise defined herein, scientific and technical terms used in the context of the present invention shall have the meaning commonly understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. The terms "a" and "an" include plural referents unless otherwise indicated by context. In general, the terms used in the context of biochemistry, enzymology, molecular cell biology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization and the techniques related thereto described herein are those commonly used and well known in the art.

[0035] Unless otherwise indicated, the following terms shall be understood to have the following meanings:

[0036] The term "polynucleotide" or "nucleic acid molecule" refers to a polymeric form of nucleotides having a length of at least 10 bases. Such terms include DNA molecules (e.g., cDNA or genomic DNA or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA that contain non-natural nucleotide analogs, non-natural internucleoside linkages, or both. Nucleic acids can be in any topological conformation. For example, nucleic acids can be in single-stranded, double-stranded, triple-stranded, quadruplexed, partially double-stranded, branched, hairpinned, circular, or padlocked conformations.

[0037] Unless otherwise indicated, as an example for any sequence described herein in the general format of "SEQ ID NO:", a "nucleic acid comprising SEQ ID NO: 1" refers to a nucleic acid having, at least in part, (i) a sequence as set forth in SEQ ID NO: 1, or (ii) a sequence complementary to SEQ ID NO: 1. The alternative is dictated by the context. For example, if the nucleic acid is being used as a probe, the alternative is dictated by the requirement that the probe be complementary to the desired target.

[0038] The term "recombinant" refers to a biological molecule, such as a gene or protein, that is (1) removed from its naturally occurring environment, (2) not associated with all or a portion of a polynucleotide with which the gene is found in nature, (3) operably linked to a polynucleotide with which it is not linked in nature, or (4) not occurring in nature. The term "recombinant" may be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs biologically synthesized in heterologous systems, as well as proteins and / or mRNAs encoded by such nucleic acids.

[0039] An endogenous nucleic acid sequence is considered "recombinant" herein when a heterologous sequence is placed adjacent to an endogenous nucleic acid sequence of an organism's genome such that the expression of the endogenous nucleic acid sequence (or the protein product encoded by the sequence) is altered. In this case, the heterologous sequence is a sequence that is not naturally adjacent to the endogenous nucleic acid sequence, regardless of whether the heterologous sequence is itself endogenous (from the same host cell or its descendants) or exogenous (from a different host cell or its descendants). As an example, a promoter sequence can replace (e.g., by homologous recombination) the native promoter of a gene present in the genome of a host cell, such that the expression pattern of the gene is altered. The gene would now be considered "recombinant" because it has been separated from at least some of the sequences that naturally flank it.

[0040] A nucleic acid is also considered to be "recombinant" if it contains any modification relative to the corresponding nucleic acid in a genome that does not occur naturally. For example, an endogenous coding sequence is considered to be "recombinant" if it contains an insertion, deletion, or point mutation that has been artificially introduced, such as by human intervention. "Recombinant nucleic acid" also includes a nucleic acid integrated into a host cell chromosome at a heterologous site, and a nucleic acid construct that exists as an episome.

[0041] As used herein, the phrase "degenerate variant" of a reference nucleic acid sequence encompasses a nucleic acid sequence that can be translated according to the standard genetic code to provide an amino acid sequence identical to the amino acid sequence translated from the reference nucleic acid sequence. The term "degenerate oligonucleotide" or "degenerate primer" is used to mean an oligonucleotide that can hybridize to target nucleic acid sequences that are not necessarily identical in sequence but are homologous to each other within one or more specific segments.

[0042] The term "sequence identity (%)" or "identical" in nucleic acid sequences refers to the residues in two sequences that are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a region of at least about 9 nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, usually at least about 28 nucleotides, more usually at least about 32 nucleotides, and preferably at least about 36 or more nucleotides. There are many different algorithms known in the art that can be used to measure nucleotide sequence identity. For example, polynucleotide sequences can be compared using FASTA, Gap, or Bestfit, which are programs included in the Wisconsin Package version 10.0 (Genetics Computer Group (GCG), Madison, Wis.). FASTA provides alignment and sequence identity (%) of the regions of the most overlap between the query and search sequences. Pearson, Methods Enzymol. 183:63-98 (1990), which is incorporated herein by reference in its entirety. For example, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (string size of 6 and a factor of the scoring matrix of NOPAM) or Gap with its default parameters, as provided in GCG version 6.1, which is incorporated herein by reference.Alternatively, sequences can be compared using the computer program BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), in particular blastp or tblastn (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).

[0043] The terms "substantial homology" or "substantial similarity" when referring to a nucleic acid or a fragment thereof indicates that when optimally aligned with another nucleic acid (or its complementary strand) using appropriate insertion or deletion of nucleotides, the nucleotide sequence identity, as measured by any of the well-known sequence identity algorithms such as FASTA, BLAST or Gap as described above, is at least about 75%, 80%, 85%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases.

[0044] The nucleic acids (also called polynucleotides) of the present invention may include both sense and antisense strands of RNA, cDNA, genomic DNA, as well as synthetic forms and mixed polymers of the above. They may be chemically or biochemically modified and may contain non-natural or derivatized nucleotide bases, as will be readily understood by those skilled in the art. Such modifications include, for example, labels, methylation, substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications, such as uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), pendant moieties (e.g., polypeptides), intercalators (e.g., acridines, psoralens, etc.), chelators, alkylators, and modified bonds (e.g., alpha anomeric nucleic acids, etc.). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonds and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages replace the phosphate linkages in the backbone of the molecule. Other modifications can include, for example, analogs in which the ribose ring contains bridging moieties or other structures, such as those found in "locked" nucleic acids.

[0045] The term "mutated," as applied to a nucleic acid sequence, means that nucleotides within a nucleic acid sequence have been inserted, deleted, or changed as compared to a reference nucleic acid sequence. A single change (point mutation) may be made at a locus, or multiple nucleotides may be inserted, deleted, or changed at a single locus. Furthermore, one or more changes may be made at any number of loci within a nucleic acid sequence. Nucleic acid sequences may be mutated by any method known in the art, including, but not limited to, mutagenesis techniques such as "error-prone PCR" (a method in which PCR is performed under conditions of low fidelity of DNA polymerase replication to yield a high rate of point mutations throughout the length of the PCR product; see, e.g., Leung et al., Technique, 1:11-15 (1989) and Caldwell and Joyce, PCR Methods Applic. 2:28-33 (1992)) and "oligonucleotide-directed mutagenesis" (a method that allows for the generation of site-specific mutations in any cloned DNA segment of interest; see, e.g., Reidhaar-Olson and Sauer, Science 241:53-57 (1988)).

[0046] As used herein, the term "attenuate" generally refers to functional deletion, including mutation, partial or complete deletion, insertion, or other alteration made to a gene sequence or the sequence controlling the transcription of the gene sequence, thereby reducing or inhibiting the production of the gene product or making the gene product non-functional. In some examples, functional deletion is represented as a knockout mutation. Attenuation also includes changing the nucleic acid sequence, placing the gene under the control of a less active promoter, down-regulating, modifying the amino acid sequence by expressing an interfering RNA, ribozyme, or antisense sequence that targets the gene of interest, or modifying the amino acid sequence via any other technique known in the art. In one example, a particular enzyme is made less sensitive to feedback inhibition or inhibition caused by a composition that is neither a product nor a reactant (pathway non-specific feedback), so that the enzyme activity is not affected by the presence of a compound. In other cases, an enzyme that is altered to have less activity may be said to be attenuated.

[0047] As used herein, the term "deletion" refers to the removal of one or more nucleotides from a nucleic acid molecule, or one or more amino acids from a protein, with the regions on either side joined to each other.

[0048] As used herein, the term "knockout" is intended to refer to a gene whose expression or activity level is reduced to zero.In some instances, a gene is knocked out by deleting part or all of its coding sequence.In other instances, a gene is knocked out by introducing one or more nucleotides into its open reading frame, thereby resulting in the translation of a nonsense protein product, or an otherwise non-functional protein product.

[0049] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which generally refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated, but also includes linear double-stranded molecules, such as those resulting from amplification by polymerase chain reaction (PCR) or from treatment of a circular plasmid with a restriction enzyme. Other vectors include cosmids, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs). Another type of vector is a viral vector, in which additional DNA segments may be ligated into the viral genome (discussed in more detail below). Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors with an origin of replication that functions in the host cell). Other vectors may be integrated into the genome of a host cell when introduced into the host cell, and thus are replicated along with the host genome. Additionally, certain preferred vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors").

[0050] "Operatively linked" or "operably linked" expression control sequences refer to a linkage in which the expression control sequences are adjacent to a gene of interest and control such a gene of interest, as well as expression control sequences that act in trans or at a distance to control a gene of interest.

[0051] The term "expression control sequence" refers to a polynucleotide sequence necessary to affect the expression of a coding sequence to which it is operably linked. Expression control sequences are sequences that control the transcription, post-transcriptional events and translation of a nucleic acid sequence. Expression control sequences include appropriate sequences for transcription initiation, transcription termination, promoters and enhancers; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and, where necessary, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences generally include promoters, ribosome binding sites, and transcription termination sequences. The term "control sequence" is intended to include, at a minimum, all components the presence of which is essential for expression, and may include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0052] The term "regulatory element" refers to any element that affects the transcription or translation of a nucleic acid molecule. These include, but are not limited to, regulatory proteins (e.g., transcription factors), chaperones, signal transduction proteins, RNAi molecules, antisense RNA molecules, microRNAs, and RNA aptamers, by way of example. Regulatory elements may be endogenous to the host organism. Regulatory elements may be exogenous to the host organism. Regulatory elements may be synthetically generated regulatory elements.

[0053] As used herein, the term "promoter", "promoter element" or "promoter sequence" refers to a DNA sequence that, when linked to a nucleotide sequence of interest, is capable of controlling the transcription of the nucleotide sequence of interest into mRNA. A promoter is generally, but not necessarily, located 5' (i.e., upstream) of the nucleotide sequence of interest whose transcription it controls into mRNA, and provides a site for specific binding of RNA polymerase and other transcription factors for transcription initiation. A promoter may be endogenous to the host organism. A promoter may be exogenous to the host organism. A promoter may be a synthetically generated regulatory element.

[0054] Promoters useful for expressing the recombinant genes described herein include both constitutive and inducible / repressible promoters. When multiple recombinant genes are expressed in the genetically engineered organisms of the invention, such different genes can be controlled by different promoters or by the same promoter in separate operons, or expression of two or more genes may be controlled by a single promoter as part of an operon.

[0055] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell into which a recombinant vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Such progeny may not in fact be identical to the parent cell, since certain modifications may occur with passage due to mutation or environmental influences, but are still included within the scope of the term "host cell" as used herein. A recombinant host cell may be an isolated cell or a cell line grown in culture, or may be a cell present in a living tissue or organism.

[0056] As used herein, the term "wild type" (i.e., "WT") refers to a comparative strain that does not have the modification under discussion. Wild type does not refer to a natural, unmodified strain, but rather to a strain that does not have the selected modification. For example, when comparing two versions of a recombinant strain that has been modified to express a recombinant silk polypeptide, one of which is a sec72 KO, the KO strain may be referred to as a Δsec72 strain, while the other version is referred to as "WT."

[0057] As used herein, the term "peptide" refers to short polypeptides, e.g., generally less than about 50 amino acids in length, more generally less than about 30 amino acids in length. As used herein, the term encompasses analogs as well as mimetics that mimic structure-function and thereby biological function.

[0058] The term "polypeptide" encompasses both naturally occurring and non-naturally occurring proteins, as well as fragments, variants, derivatives, and analogs thereof. A polypeptide may be monomeric or polymeric. Furthermore, a polypeptide may contain multiple distinct domains, each having one or more distinct activities.

[0059] The term "isolated protein" or "isolated polypeptide" refers to a protein or polypeptide that is (1) not associated with naturally associated components that accompany it in its native state, (2) that exists in a purity not found in nature, where the purity can be determined with respect to the presence of other cellular material (e.g., free of other proteins from the same species), (3) that is expressed by cells from a different species, or (4) that does not occur in nature (e.g., it is a fragment of a naturally occurring polypeptide or contains amino acid analogs or derivatives not found in nature or bonds other than standard peptide bonds), in terms of its origin or source, and is "isolated" from its naturally associated components. A polypeptide or protein may be rendered substantially free of naturally associated components by isolation using protein purification techniques well known in the art. When defined in this way, "isolated" does not necessarily require that the protein, polypeptide, peptide, or oligopeptide so described has been physically removed from its natural environment.

[0060] The term "polypeptide fragment" refers to a polypeptide having a deletion compared to a full-length polypeptide, e.g., a deletion at the amino terminus and / or carboxy terminus. In preferred embodiments, a polypeptide fragment is a contiguous sequence in which the amino acid sequence of the fragment is identical to the corresponding positions in the naturally occurring sequence. Fragments are generally at least 5, 6, 7, 8, 9 or 10 amino acids in length, preferably at least 12, 14, 16 or 18 amino acids in length, more preferably at least 20 amino acids in length, more preferably at least 25, 30, 35, 40 or 45 amino acids in length, even more preferably at least 50 or 60 amino acids in length, and even more preferably at least 70 amino acids in length.

[0061] A protein has "homology" or is "homologous" to a second protein if the nucleic acid sequence encoding the protein has a similar sequence to the nucleic acid sequence encoding the second protein. Alternatively, a protein has homology to a second protein if the two proteins have "similar" amino acid sequences. (Thus, the term "homologous proteins" is defined to mean that two proteins have similar amino acid sequences). As used herein, homology between two amino acid sequence regions (especially with respect to predicted structural similarities) is interpreted to mean similarity in function.

[0062] When "homologous" is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making such adjustments are well known to those of skill in the art. See, e.g., Pearson, 1994, Methods Mol. Biol. 24:307-31 and 25:365-89, incorporated herein by reference.

[0063] The twenty conventional amino acids and their abbreviations follow convention. nded. 1991). Stereoisomers of the twenty conventional amino acids (e.g., D-amino acids), non-naturally occurring amino acids, such as α-,α-disubstituted amino acids, N-alkylamino acids, and other non-conventional amino acids may also be suitable components of the polypeptides of the invention. Examples of non-conventional amino acids include 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand end corresponds to the amino terminus and the right-hand end corresponds to the carboxy terminus, in accordance with standard usage and convention.

[0064] Each of the following six groups contains amino acids that are conservative substitutions for one another: 1) serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), alanine (A), valine (V), and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

[0065] Sequence homology of polypeptides, sometimes referred to as percent sequence identity, is typically measured using sequence analysis software. See, e.g., Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software matches similar sequences using homology measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG comes with programs such as "Gap" and "Bestfit," which can be used with default parameters to determine sequence homology or identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutant protein. See, e.g., GCG version 6.1.

[0066] A useful algorithm for comparing a particular polypeptide sequence to a database containing a large number of sequences from different organisms is the computer program BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), in particular blastp or tblastn (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).

[0067] Preferred parameters for BLASTp are: expectation: 10 (default); filter: seg (default); gap opening cost: 11 (default); gap extension cost: 1 (default); maximum alignments: 100 (default); string size: 11 (default); number of items to display: 100 (default); penalty matrix: BLOWSUM62.

[0068] Preferred parameters for BLASTp are expectation: 10 (default); filter: seg (default); cost of opening a gap: 11 (default); cost of extending a gap: 1 (default); maximum alignment: 100 (default); string size: 11 (default); number of results displayed: 100 (default); penalty matrix: BLOWSUM62. The length of polypeptide sequences to be compared for homology will generally be at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, usually at least about 28 residues, and preferably more than about 35 residues. When searching a database containing sequences from many different organisms, it is preferred to compare amino acid sequences. Database searches using amino acid sequences can be measured by algorithms known in the art other than blastp. For example, polypeptide sequences can be compared using FASTA, a program in GCG version 6.1. FASTA provides alignments and percent sequence identity of the regions of best overlap between the query and search sequences. Pearson, Methods Enzymol. 183:63-98 (1990), which is incorporated herein by reference. For example, percent sequence identity between amino acid sequences can be determined using FASTA, provided in GCG version 6.1, which is incorporated herein by reference, with its default parameters (string size of 2 and scoring matrix PAM250).

[0069] Throughout this specification and the claims, the word "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0070] Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in practicing the present invention and will be apparent to those skilled in the art. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are intended to be illustrative only and not limiting.

[0071] overview Provided herein are recombinant strains and methods for improving secretion and productivity of recombinantly expressed proteins in yeast strains (eg, P. pastoris).

[0072] Proteins destined for secretion must first cross the membrane of the endoplasmic reticulum (ER translocation). Multiple targeting pathways recruit elongating ribosomes or fully translated proteins to the ER membrane, where their polypeptide chains enter through pore-forming protein complexes called translocons. Yeast, including S. cerevisiae and P. pastoris, express two translocons, the SEC61 complex and the SSH1 complex. Each complex is composed of a core trimeric complex, but the SEC61 translocon also associates with the tetrameric SEC63 complex. Figure 1 shows the protein subunits contained within each of these translocon complexes.

[0073] Although SEC72 encodes a non-essential component of the SEC63 complex, deletion of SEC72 (Δsec72) results in the accumulation of some secreted precursors. Surprisingly, we show here that Δsec72 strains have improved secretion of recombinant silk polypeptides.

[0074] In some embodiments, the strains are modified to delete the sec72 gene (SEQ ID NO: 1). As we describe and demonstrate herein, deletion of sec72, which expresses SEC72 (SEQ ID NO: 2), a cofactor for protein translocation into the ER for secretion, unexpectedly enhances silk secretion (up to +75%) in bench-scale block model assays. This is true across expression levels, signal sequences, and different silks.

[0075] In some embodiments, deletion of sec72 is achieved using a plasmid with a 5' homology arm to sec72 and a 3' homology arm to sec72 flanked by yeast selectable markers, for example as shown in Figure 2. Deleting Δsec72 induced a transcriptional adaptation that can indirectly promote secretion. The sequences of the sec72 gene and SEC72 protein are shown in Table 1.

[0076] Table 1. Sec72 open reading frame and SEC72 protein sequence TIFF0007623680000003.tif121149

[0077] In some cases, the deletion of sec72 slowed the growth of the strain, and fermentation production screening of Δsec72 strains struggled with glucose accumulation. Thus, in some embodiments, strains are provided herein that recombinantly overexpress proteins comprising the SSH1 translocon complex. As shown herein, overexpression of the SSH1 translocon complex in Δsec72 strains improved the growth rate and fermentation capacity of the strains while maintaining improved secretion. Testing of this combination deletion-overexpression strain with only four copies of the recombinant silk-like block polypeptide expressing gene (i.e., 18B) showed similar titer and specific productivity improvement (+18%) compared to strains with six copies of the silk-like block polypeptide expressing gene (i.e., 18B).

[0078] In some embodiments, the SSH1 translocon complex is overexpressed in the host cell by inserting a plasmid (FIG. 3) containing genes encoding recombinant SBH2, SSS1, and SSH1. The sequences of these genes and the expressed proteins are shown in Table 2.

[0079] Table 2. Open reading frames and protein sequences for the subunits of the SSH1 translocon complex. TIFF0007623680000004.tif50149TIFF0007623680000005.tif218149TIFF0007623680000006.tif76149

[0080] Protease knockout In some embodiments, to attenuate protease activity in Pichia pastoris, the genes encoding these enzymes are inactivated or mutated to reduce or eliminate activity. This can be done through mutations or insertions in the genes themselves, or through modification of the gene's regulatory elements. This can be achieved by standard yeast genetic techniques. An example of such a technique is gene replacement by double homologous recombination, in which homologous regions flanking the gene to be inactivated are cloned into a vector adjacent to a selectable marker gene, such as an antibiotic resistance gene or a gene that complements an auxotrophy of the yeast strain. In some embodiments, the nourseothricin selection plasmid shown in FIG. 4A can be used as the base plasmid, and homology arms (HA) flanking the resistance cassette (FIGS. 4B and 4C) can be used, which specifically target the desired protease to be knocked out. Descriptions of protease knockouts and methods for modifying host cells to inhibit degradation of recombinant proteins are provided in U.S. Application No. 15 / 724,196 and PCT Application No. PCT / US2017 / 054997, each of which is incorporated by reference in its entirety.

[0081] Alternatively, the homologous region can be PCR amplified and linked to the selection marker gene by overlap PCR. The DNA fragment is then transformed into Pichia pastoris by methods known in the art, for example, electroporation. The transformants grown under selective conditions are then analyzed for gene disruption events by standard techniques, for example, PCR or Southern blot of genomic DNA. In an alternative experiment, gene inactivation can be achieved by single homologous recombination, for example, by cloning the 5' end of the ORF of the gene onto a promoterless vector that also contains a selection marker gene. When the vector is linearized by digestion with a restriction enzyme that cuts the vector only at the target gene homologous fragment, the vector is transformed into Pichia pastoris. Integration into the target gene site is confirmed by PCR or Southern blot of genomic DNA. In this method, the gene fragment cloned onto the vector is replicated within the genome, resulting in two copies of the target locus: the first copy expressing a truncated, inactive protein (if expressed at all) due to an incomplete ORF, and the second copy lacking a promoter to drive transcription.

[0082] Alternatively, transposon mutagenesis is used to inactivate the target gene, and a library of such mutants can be screened for insertion events within the target gene by PCR.

[0083] The functional phenotype (i.e., deficiency) of the engineered / knocked out strain can be assessed using techniques known in the art. For example, the deficiency of protease activity in the engineered strain can be confirmed using any of a wide variety of methods known in the art, including hydrolytic activity assays of chromogenic protease substrates, band shifts of substrate proteins for the protease of choice, among others.

[0084] The attenuation of protease activity as described herein can be achieved by mechanisms other than knockout mutation. For example, the desired protease can be attenuated by changing the nucleic acid sequence, placing the gene under the control of a less active promoter, downregulation, modifying the amino acid sequence by expressing an interfering RNA, ribozyme or antisense sequence targeting the gene of interest, or any other technique known in the art. In a preferred strain, the protease activity of the proteases encoded by PAS_chr4_0584 (YPS1-1) and PAS_chr3_1157 (YPS1-2) (e.g., polypeptides comprising SEQ ID NOs: 10 and 12) is attenuated by any of the above methods. In some embodiments, the present invention is directed to methylotrophic yeast strains, particularly Pichia pastoris strains, in which the YPS1-1 and YPS1-2 genes (e.g., as set forth in SEQ ID NOs: 9 and 11) have been inactivated. In some embodiments, additional genes encoding proteases may be knocked out according to the methods provided herein to further reduce the protease activity of the desired protein product expressed by the strain.

[0085] Table 3. Open reading frame nucleotide and polypeptide sequences for proteases targeted for deletion in P. pastoris. TIFF0007623680000007.tif248153TIFF0007623680000008.tif147153

[0086] Production of recombinant strains The present specification provides methods for transforming strains to reduce activity, for example, using vectors that deliver recombinant genes or knock out or other methods to attenuate the desired endogenous gene. These vectors may take the form of a vector backbone containing an origin of replication and a selection marker (commonly antibiotic resistance, but many other methods are possible), or a linear fragment that allows integration into the chromosome of the target cell. The vector must be compatible with the organism and insertion method selected.

[0087] Once the vector elements have been selected, vector construction can be accomplished in a number of different ways. In one embodiment, DNA synthesis services can be utilized or methods can be used to create all vectors individually.

[0088] Once the DNA of each vector (including any additional elements required for insertion and manipulation) is obtained, it must be assembled. There are many possible assembly methods, including but not limited to restriction enzyme cloning, blunt end ligation, and overlap assembly [see, for example, Gibson, DG, et al., Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature methods, 6(5), 343-345(2009), and GeneArt Kit (http: / / tools.invitrogen.com / content / sfs / manuals / geneart_seamless_cloning_and_assembly_man.pdf)]. Overlap assembly provides a method to ensure that all of the elements are assembled in the correct location and that no undesired sequences are introduced.

[0089] The vectors generated above can be inserted into target cells using standard molecular biology techniques, such as molecular cloning, etc. In one embodiment, the target cells have already been genetically engineered or selected to already contain the genes required for production of the desired product, which may be done during or after the subsequent insertion of the vector.

[0090] Depending on the type of organism and library element (plasmid or genomic insertion), several known methods may be used to insert the vector containing DNA into the cell, including, for example, transformation of microorganisms that can take up and replicate DNA from the local environment, transformation by electroporation or chemical means, transduction with viruses or phages, mating of two or more cells, or conjugation from different cells.

[0091] Several methods of introducing recombinant DNA into bacterial cells are known in the art, including, but not limited to, transformation, transduction, and electroporation (see Sambrook, et al., Molecular Cloning: A Laboratory Manual (1989), Second Edition, Cold Spring Harbor Press, Plainview, NY). Non-limiting examples of commercially available kits and bacterial host cells for transformation include NovaBlue Singles™ (EMD Chemicals Inc., NJ, USA), Max Efficiency™ DH5α™, One Shot™ BL21(DE3) E. coli cells, One Shot™ BL21(DE3)pLys E. coli cells (Invitrogen Corp., Carlsbad, Calif., USA), XL1-Blue competent cells (Stratagene, CA, USA). Non-limiting examples of commercially available kits and bacterial host cells for electroporation include Zappers™ electrocompetent cells (EMD Chemicals Inc., NJ, USA), XL1-Blue electroporation-competent cells (Stratagene, CA, USA), ElectroMAX™ A. tumefaciens LBA4404 cells (Invitrogen Corp., Carlsbad, Calif., USA).

[0092] Several methods of introducing recombinant nucleic acids into eukaryotic cells are known in the art. Exemplary methods include transfection, electroporation, liposomal nucleic acid delivery, and microinjection into host cells (see Sambrook, et al., Molecular Cloning: A Laboratory Manual (1989), Second Edition, Cold Spring Harbor Press, Plainview, NY). Non-limiting examples of commercially available kits and reagents for transfecting recombinant nucleic acids into eukaryotic cells include Lipofectamine™ 2000, Optifect™ Reagent, Calcium Phosphate Transfection Kit (Invitrogen Corp., Carlsbad, Calif., USA), GeneJammer® Transfection Reagent, LipoTAXI® Transfection Reagent (Stratagene, CA, USA). Alternatively, recombinant nucleic acids may be introduced into insect cells (e.g., sf9, sf21, High Five™) by use of baculovirus vectors.

[0093] The transformed cells are isolated so that each clone can be tested separately. In one embodiment, this is done by plating on one or more plates of medium containing (or lacking) a selection agent that ensures that only transformed cells survive and reproduce. The specific agent may be an antibiotic (if the library contains an antibiotic resistance marker), a metabolite deprivation (for complementation of an auxotrophy), or other selection means. The cells grow into individual colonies, each of which contains a single clone.

[0094] Colonies are screened for the desired production of a protein, metabolite, or other product, for reduced protease activity, for growth, or for increased secretion activity. In one embodiment, recombinant cells with the highest (or sufficiently high) titer or efficiency of production of the product are identified by screening. This includes a reduced rate of degradation products or an increase in the total amount of the desired polypeptide secreted from the cells and recovered from the cell culture.

[0095] This assay can be performed by growing individual clones in multi-well culture plates, one clone per well. Once the cells reach an appropriate biomass density, they are induced with methanol. After a period of time, typically 24-72 hours of induction, the cultures are harvested by spinning in a centrifuge to pellet the cells and removing the supernatant. The supernatant from each culture may then be tested to determine if the desired secretion rate has been achieved.

[0096] Silk arrangement In some embodiments, the engineered strains described herein recombinantly express silk-like polypeptide sequences. In some embodiments, the silk-like polypeptide sequences are 1) block copolymer polypeptide compositions produced by mixing and matching repeat domains from silk polypeptide sequences, and / or 2) recombinantly expressed block copolymer polypeptides large enough (about 40 kDa) to form useful fibers upon secretion from industrially scalable microorganisms. Large (about 40 kDa to about 100 kDa) block copolymer polypeptides engineered with silk repeat domain fragments, including sequences from nearly all of the published amino acid sequences of spider silk polypeptides, can be expressed in the engineered microorganisms described herein. In some embodiments, the silk polypeptide sequences are matched and engineered to produce highly expressed and highly secreted polypeptides capable of fiber formation. In some embodiments, the silk-like polypeptide degradation is reduced by knocking out protease genes or reducing protease activity in the host engineered strain. In some embodiments, knockout of sec72 and overexpression of the SSH1 translocon complex improves secretion while mitigating the growth defect and maintaining or enhancing growth of the strain.

[0097] Provided herein in some embodiments are compositions for expressing and secreting engineered block copolymers by comprehensive combinatorial mixing of silk polypeptide domains spanning the entire silk polypeptide sequence space, wherein the block copolymers are minimally degraded. Provided herein in some embodiments are methods for secreting block copolymers in scalable organisms (e.g., yeast, fungi, and gram-positive bacteria) with minimally degraded. In some embodiments, the block copolymer polypeptide comprises zero or more N-terminal domains (NTDs), one or more repeat domains (REPs), and zero or more C-terminal domains (CTDs). In some aspects of the above embodiments, the block copolymer polypeptide is a single-chain polypeptide of more than 100 amino acids. In some embodiments, the block copolymer polypeptide comprises a domain that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of a block copolymer polypeptide disclosed in International Publication No. WO / 2015 / 042164, "Methods and Compositions for Synthesizing Improved Silk Fibers," which is incorporated by reference in its entirety.

[0098] Several types of natural spider silk have been identified so far. The various mechanical properties of naturally spun spider silk are thought to be closely related to the molecular composition of the silk. See, e.g., Garb, J. E., et al., Untangling spider silk evolution with spidroin terminal domains, BMC Evol. Biol., 10:243 (2010); Bittencourt, D., et al., Protein families, natural history and biotechnological aspects of spider silk, Genet. Mol. Res., 11:3 (2012); Rising, A., et al., Spider silk proteins: recent advances in recombinant production, structure-function relationships and biomedical applications, Cell. Mol. Life Sci., 68:2, pg. 169-184 (2011); and Humenik, M., et al., Spider silk: understanding the structure-function relationship of a natural fiber, Prog. Mol. Biol. Transl. Sci., 103, pg. 131-85 (2011). For example:

[0099] Grape-like (AcSp) silks tend to be tough, resulting from a combination of moderately high strength and moderately high elasticity. AcSp silks are characterized by large block ("repeat aggregate") sizes that often incorporate polyserine and GPX motifs. Tubular (TuSp or cylindrical) silks tend to be large in diameter, moderate in strength and high elasticity. TuSp silks are characterized by their polyserine and polythreonine content, and short polyalanine sequences. Macroampelar (MaSp) silks tend to be high in strength and moderate in elasticity. MaSp silks are one of two subtypes, MaSp1 and MaSp2. MaSp1 silks are generally less elastic than MaSp2 silks, and are characterized by polyalanine, GX, and GGX motifs. MaSp2 silks are characterized by polyalanine, GGX, and GPX motifs. Ampullate (MiSp) silks tend to have moderate strength and moderate elasticity. MiSp silks are characterized by GGX, GA, and polyA motifs and often contain a spacer element of about 100 amino acids. Flagelliform (Flag) silks tend to have very high elasticity and moderate strength. Flag silks are usually characterized by GPG, GGX, and a short spacer motif.

[0100] The properties of each silk species may vary from species to species, and spiders with different lifestyles (e.g. stationary web-weaving spiders vs. wandering feeding spiders) or evolutionarily older spiders may produce silks different from those described above (for a description of spider diversity and classification, see Hormiga, G., and Griswold, CE, Systematics, phylogeny, and evolution of orb-weaving spiders, Annu. Rev. Entomol. 59, pg.487-512(2014); and Blackedge, TA et al., Reconstructing web evolution and spider diversification in the molecular era, Proc. Natl. Acad. Sci. USA, 106:13, pg. 5229-5234(2009)). However, using synthetic block copolymer polypeptides with sequence similarity and / or amino acid composition similarity to the repeating domains of natural silk proteins, it is possible to produce consistent silk-like fibers on a commercial scale that recapitulate the properties corresponding to natural silk fibers.

[0101] In some embodiments, a list of putative silk sequences can be collected by searching GenBank for related terms such as "spidroin", "fibroin", "MaSp", etc., and these sequences can be pooled with additional sequences obtained from independent sequencing efforts. The sequences are then translated into amino acids, duplicate entries are filtered, and manually divided into each domain (NTD, REP, CTD). In some embodiments, the candidate amino acid sequences are reverse translated into DNA sequences optimized for expression in Pichia (Komagataella) pastoris. The DNA sequences are each cloned into an expression vector and transformed into Pichia (Komagataella) pastoris. In some embodiments, the various silk domains that show successful expression and secretion are then assembled in a combinatorial manner to construct fiber-forming silk molecules.

[0102] Silk polypeptides characteristically consist of repetitive domains (REPs) flanked by non-repetitive regions (e.g., C-terminal and N-terminal domains). In one embodiment, both the C-terminal and N-terminal domains are 75-350 amino acids in length. The repetitive domains exhibit a hierarchical structure. The repetitive domains contain a series of blocks (also called repeat units), which are repeated sometimes perfectly and sometimes imperfectly (forming quasi-repetitive domains) throughout the repetitive domain of the silk. The length and composition of the blocks vary between different silk types and between different species. Table 1 lists examples of block sequences for selected species and silk species, with further examples described in Rising, A. et al., Spider silk proteins: recent advances in recombinant production, structure-function relationships and biomedical applications, Cell Mol. Life Sci., 68:2, pg 169-184 (2011), and Gatesy, J. et al., Extreme diversity, conservation, and convergence of spider silk fibroin sequences, Science, 291:5513, pg.2603-2605 (2001). In some cases, the blocks may be arranged in a regular pattern to form larger macrorepeats that occur multiple times (usually 2-8 times) in the repeat domain of the silk sequence. Blocks that are repeated inside the repeat domain or macrorepeat may be separated by spacing elements from macrorepeats that are repeated within the repeat domain. In some embodiments, the block sequence comprises a glycine-rich region followed by a polyA region. In some embodiments, a short (about 1-10) amino acid motif occurs multiple times within a block. For purposes of the present invention, blocks derived from different native silk polypeptides can be selected without regard to circular permutation (i.e., identified blocks may not align due to circular permutation if they are otherwise similar between silk polypeptides).Thus, for example, a "block" of SGAGG (SEQ ID NO:23) is, for purposes of the present invention, identical to GSGAG (SEQ ID NO:24), which is also identical to GGSGA (SEQ ID NO:25), which are all merely circular permutations of each other. The particular permutation selected for a given silk sequence may be determined more by convenience than anything else (usually starting with G). Silk sequences obtained from the NCBI database can be divided into blocks and non-repetitive regions.

[0103] (Table 4) Block sequence samples TIFF0007623680000009.tif240148TIFF0007623680000010.tif181148

[0104] Fiber-forming block copolymer polypeptides derived from block and / or macro-repeat domains according to certain embodiments of the invention are described in International Publication No. WO / 2015 / 042164, which is incorporated by reference. Natural silk sequences obtained from protein databases such as GenBank or by de novo sequencing are broken down into domains (N-terminal, repeat, and C-terminal). The N-terminal and C-terminal domain sequences selected for post-synthetic assembly to construct fibers include the native amino acid sequence information and other modifications described herein. The repeat domains are broken down into repeat sequences that contain representative blocks (usually 1-8, depending on the silk type) that capture the key amino acid information while reducing the size of the DNA encoding the amino acids to a readily synthesizable fragment. In some embodiments, a properly formed block copolymer polypeptide comprises at least one repeat domain that includes at least one repeat sequence, optionally flanked by N-terminal and / or C-terminal domains.

[0105] In some embodiments, the repeat domain comprises at least one repeat sequence. In some embodiments, the repeat sequence is between 150 and 300 amino acid residues. In some embodiments, the repeat sequence comprises multiple blocks. In some embodiments, the repeat sequence comprises multiple macrorepeats. In some embodiments, the blocks or macrorepeats are divided throughout the multiple repeat sequences.

[0106] In some embodiments, the repeat sequence must start with glycine and cannot end with phenylalanine (F), tyrosine (Y), tryptophan (W), cysteine ​​(C), histidine (H), asparagine (N), methionine (M), or aspartic acid (D) to meet DNA assembly requirements. In some embodiments, some repeat sequences can be altered compared to the native sequence. In some embodiments, the repeat sequence can be altered, such as by adding a serine to the C-terminus of the polypeptide (to avoid ending at F, Y, W, C, H, N, M, or D). In some embodiments, the repeat sequence can be modified by filling in an incomplete block with a homologous sequence from another block. In some embodiments, the repeat sequence can be modified by rearranging the order of the blocks or macrorepeats.

[0107] In some embodiments, unique N-terminal and C-terminal domains can be selected for synthesis. In some embodiments, the N-terminal domain can be by removal of a leading signal sequence, such as those identified by SignalP (Peterson, TN, et. al., SignalP 4.0: discriminating signal peptides from transmembrane regions, Nat. Methods, 8:10, pg. 785-786 (2011)).

[0108] In some embodiments, the N-terminal domain sequence, the repeat sequence, or the C-terminal domain sequence is selected from the group consisting of Agelenopsis aperta, Aliatypus gulosus, Aphonopelma seemanni, Aptostichus sp. AS217, Aptostichus sp. AS220, Araneus diadematus, Araneus gemmoides, Araneus ventricosus, Argiope amoena, Argiope argentata, Argiope bruennichi, Argiope trifasciata, Argiope trifasciata, Atypoides riversi, Avicularia juruensis, Bothriocyrtum californicum, Deinopis spinosa, Diguetia canities, Dolomedes tenebrosus, Euagrus chisoseus, Euprosthenops australis, Gasteracantha mammosa, Hypochilus thorelli, Kukulcania hibernalis, Latrodectus hesperus hesperus, Megahexura fulva, Metepeira grandiosa, Nephila antipodiana, Nephila clavata, Nephila clavipesclavipes, Nephila madagascariensis, Nephila pilipes, Nephilengys cruentata, Parawixia bistriata, Peucetia viridans, Plectreurys tristis, Poecilotheria regalis, Tetragnatha kauaiensis, or Uloborus diversus.

[0109] In some embodiments, the silk polypeptide nucleotide coding sequence may be operably linked to an alpha mating factor nucleotide coding sequence. In some embodiments, the silk polypeptide nucleotide coding sequence may be operably linked to another endogenous or heterologous secretion signal coding sequence. In some embodiments, the silk polypeptide nucleotide coding sequence may be operably linked to a 3X FLAG nucleotide ... (SEQ ID NO:107) An example of a vector for delivering a coding sequence of a silk polypeptide nucleotide to a host cell is shown in FIG. 5, which shows a plasmid map of a vector containing an open reading frame (ORF) for expressing a silk-like polypeptide, including a secretion signal and a FLAG tag. The ORF is operably linked to a pGCW14 promoter and a tAOX1 terminator. The vector also contains a selection marker for selecting successfully transformed cells.

[0110] In some embodiments, the silk polypeptide comprises a full length spider silk polypeptide. In some embodiments, the full length spider silk polypeptide is major ampullate spidron 1 (MaSpl) or major ampullate spidroin 2 (MaSp2).

[0111] Silk-like Polypeptides In some embodiments, the P. pastoris strains disclosed herein are modified to express silk-like polypeptides. Methods for producing silk-like polypeptides of preferred embodiments are provided in WO2015 / 042164, particularly paragraphs 114-134, which are incorporated herein by reference. WO2015 / 042164 discloses synthetic proteinaceous copolymers based on recombinant spider silk protein fragment sequences from MaSp2, such as from the species Argiope bruennichi. Silk-like polypeptides are described that include 2-20 repeat units, in which each repeat unit has a molecular weight greater than about 20 kDa. Within each repeat unit of the copolymer are more than about 60 amino acid residues that are organized into multiple "quasi-repeat units." In some embodiments, the sequence identity of the repeat units of the polypeptides described in this disclosure to the sequence of the MaSp2 dragline silk protein is at least 95%.

[0112] In some embodiments, each "repeat unit" of the silk-like polypeptide comprises 2-20 "quasi-repeat" units (i.e., n3 is 2-20). The quasi-repeats do not have to be exact repeats. Each repeat may be composed of a concatenation of quasi-repeats. Formula 1 shows the composition of the repeat unit according to this disclosure and WO2015 / 042164, which is incorporated by reference. Each silk-like polypeptide may have one or more repeat units defined by Formula 1. TIFF0007623680000011.tif4128

[0113] The compositional variable element X1 (referred to as a "motif") follows any one of the amino acid sequences shown in Formula 2 below, with X1 varying randomly within each quasi-repeat unit. TIFF0007623680000012.tif11161

[0114] Referring again to Equation 1, in Equation 1, The compositional element of the quasi-repeated unit represented by TIFF0007623680000013.tif4128 is called the "first region." Some quasi-repeated units are formed by repeating the first region 4 to 8 times within the quasi-repeated unit. That is, the value of n1 indicates the number of first region units repeated within a single quasi-repeated unit, and the value of n1 is any one of 4, 5, 6, 7, or 8. "(A) n2 " (SEQ ID NO: 19) is called the "second region" and is formed by repeating the amino acid sequence "A" n2 times within each quasi-repeat unit (SEQ ID NO: 19). That is, the value of n2 indicates the number of units of the second region that are repeated within a single quasi-repeat unit, and the value of n2 is any one of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the sequence identity of the repeat unit of a polypeptide of the present disclosure to a sequence containing quasi-repeats according to Formulas 1 and 2 is at least 95%. In some embodiments, the sequence identity of the repeat unit of a polypeptide of the present disclosure to a sequence containing quasi-repeats according to Formulas 1 and 2 is at least 80%, or at least 90%, or at least 95%, or at least 99%.

[0115] In further embodiments, three "long" quasi-repeats are followed by three "short" quasi-repeat units. Short quasi-repeat units are those where n1=4 or 5. Long quasi-repeat units are defined as those where n1=6, 7 or 8. In some embodiments, all of the short quasi-repeats have the same X1 motif at the same position within each quasi-repeat unit of the repeat unit. In some embodiments, no more than three of the six quasi-repeat units share the same X1 motif.

[0116] In further embodiments, the repeat unit is comprised of quasi-repeat units that do not use the same X1 more than two consecutive occurrences within the repeat unit. In further embodiments, the repeat unit is comprised of quasi-repeat units, where at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the quasi-repeats do not use the same X1 more than two consecutive occurrences within a single quasi-repeat unit of the repeat unit.

[0117] Thus, in some embodiments, provided herein is a yeast strain that recombinantly expresses a silk-like polypeptide with reduced degradation to increase the amount of full-length polypeptide present in the product isolated from a cell culture. In some embodiments, the silk-like polypeptide expressing strain is a P. pastoris strain that comprises a PAS_chr4_0584 knockout and a PAS_chr3_1157 knockout. In some embodiments, the silk-like polypeptide expressing strain is a P. pastoris strain that comprises a sec72 knockout and / or an overexpressed SSH1 translocon complex.

[0118] Table 5. 18B vectors TIFF0007623680000014.tif194139TIFF0007623680000015.tif194144

[0119] Equivalence and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments according to the invention described herein. The scope of the invention is not intended to be limited to the above Detailed Description, but rather is set forth in the appended claims.

[0120] In the claims, articles such as "a", "an" and "the" may mean one or more, unless indicated to the contrary or the context makes clear otherwise. A claim or description containing "or" between one or more members of a group is deemed to satisfy the requirement if one, more than one or all of the members of the group are present in, used in, or otherwise related to a given product or process, unless indicated to the contrary or the context makes clear otherwise. The invention includes embodiments in which exactly one member of the group is present in, used in, or otherwise related to a given product or process. The invention includes embodiments in which one or more or all of the members of the group are present in, used in, or otherwise related to a given product or process.

[0121] It should also be noted that the term "comprising" is intended to be open-ended and permissive, but does not require the inclusion of additional elements or steps. Thus, when the term "comprising" is used herein, the term "consisting of" is also included and disclosed.

[0122] When ranges are given, the endpoints are included. Additionally, unless otherwise indicated or clear from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges are to be understood as including any particular value or subrange within the ranges set forth in various embodiments of the invention, to the nearest tenth of the lower limit of that range, unless the context clearly dictates otherwise.

[0123] All cited sources, e.g., references, publications, databases, database entries, and art cited herein, are incorporated by reference into this application, even if not explicitly stated in the citation. In the event of a discrepancy between the statement in the cited source and this application, the statement in this application shall control.

[0124] Subheadings and table titles are not intended to be limiting. EXAMPLES

[0125] Below are examples of specific embodiments for carrying out the present invention. These examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0126] The practice of the present invention employs, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology within the skill of the art. Such techniques are fully explained in the literature. See, for example, TE Creighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); AL Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Ed. (Plenum Press) Vols A and B (1992).

[0127] Example 1: Production of recombinant yeast expressing 18B A strain of P. pastoris was engineered to recombinantly express the 18B silk-like polypeptide as follows.

[0128] First, a strain of P. pastoris was transformed to silence KU70 function to facilitate subsequent editing and engineering. A HIS+ derivative of Pichia pastoris (Komagataella phaffii) strain, GS115 (NRRL Y15851), was electroporated with a DNA cassette consisting of homology arms flanking a Zeocin resistance marker and targeting the KU70 locus. The sequence is listed in Table 12. Transformants were plated on YPD agar plates supplemented with Zeocin, resulting in silenced KU70 function.

[0129] This strain was then engineered to express a recombinant gene encoding a silk-like polypeptide. A HIS+ derivative of Pichia pastoris (Komagataella phaffii) strain, GS115 (NRRL Y15851), was transformed with the recombinant vector (SEQ ID NO:20) to result in expression and secretion of the silk-like polypeptide ("18B") (SEQ ID NO:21). Transformation was achieved by electroporation as described in PMID 15679083, which is incorporated herein by reference.

[0130] Each vector contained an 18B expression cassette with a polynucleotide sequence encoding a silk-like protein flanked by a promoter (pGCW14) and a terminator (tAOX1 pA signal) in the recombinant vector. The recombinant vectors further contained a dominant resistance marker for selection of bacterial and yeast transformants, and a bacterial origin of replication. The first recombinant vector contained a targeting region that directed integration of the 18B polynucleotide sequence adjacent to the 3' of the AOX2 locus in the Pichia pastoris genome. The resistance marker of the first vector conferred resistance to G418 (also known as geneticin). The second recombinant vector contained a targeting region that directed integration of the 18B polynucleotide sequence adjacent to the 3' of the TEF1 locus in the Pichia pastoris genome. The resistance marker of the second vector conferred resistance to hygromycin B.

[0131] Example 2: Generation of recombinant Δsec72 strains Cells engineered to express 18B were transformed with a vector containing a DNA cassette with 5' and 3' homology arms targeted to sec72 (SEQ ID NO: 1). As shown in the plasmid map in Figure 2, the homology arms flank a yeast selectable marker and, when inserted into the sec72 gene, knock out the sec72 gene. Transformants were plated on YPD agar plates supplemented with yeast selection medium and incubated at 30°C for 48 hours.

[0132] Example 3: Generation of recombinant Δsec72 strains overexpressing the SSH1 translocon complex P. pastoris (Δsec72) cells engineered to express 18B (SEQ ID NO: 21) were transformed with a vector for overexpressing the SSH1 translocon complex. The plasmid map of the vector is shown in FIG. 3. The vector contains open reading frames for SSH1 (SEQ ID NO: 3), SSS1 (SEQ ID NO: 5), and SBH2 (SEQ ID NO: 7). Each open reading frame is operably linked to a promoter and a terminator. Transformants were plated on YPD agar plates supplemented with yeast selection medium and incubated at 30° C. for 48 hours.

[0133] Example 4: Production of a protease double knockout strain To generate the ΔΔ protease strain (i.e., double protease knockout), the selected yeast strain was transformed with a vector containing a DNA cassette with approximately 1150 bp homology arms flanking a nourseothricin resistance marker. The plasmid map containing the nourseothricin resistance marker is shown in FIG. 4A and the sequence is listed in Table 13. The homology arms used for each target were amplified with primers listed in Table 8 and inserted into the nourseothricin resistance plasmid. The homology arms were inserted into the nourseothricin plasmid to generate a cassette containing the nourseothricin resistance marker flanked by 3' and 5' homology arms to the target protease, as shown in FIG. 4B and FIG. 4C. The resistance cassette (Nour resistance cassette) flanked by homology arms (HA1 and HA2) is shown in FIG. 4B. Figure 4C shows details of the nourseothricin marker, which includes the promoter from the ILV5 gene from Saccharomyces cerevisiae (pILV5), the nourseothricin acetyltransferase gene from Streptomyces noursei (nat), and the polyA signal from the CYC1 gene from Saccharomyces cerevisiae.

[0134] The engineered yeast strain was transformed with a vector carrying homology arms targeting YPS1-1 (SEQ ID NO: 77). The transformants were plated on YPD agar plates supplemented with nourseothricin and incubated at 30° C. for 48 hours.

[0135] Table 6. Proteases targeted for deletion in P. pastoris strains TIFF0007623680000016.tif40128

[0136] To generate a double knockout, the nourseothricin resistance was eliminated from the single protease knockout strain prepared above. The single protease knockout strain was transformed with a vector carrying a homology arm targeting YPS1-2 (SEQ ID NO: 80). The transformant was plated on YPD agar plates supplemented with nourseothricin and incubated at 30°C for 48 hours.

[0137] Example 5: Δsec72 improves MFα-18B secretion across integration copy numbers Pichia pastoris strains engineered to express two, four, or six copies of 18B (AbMaSp2 79 kDa) containing the MFα1(sc) prepro leader sequence were prepared using the techniques described in Example 1.

[0138] Each of the above Δsec72 strains was prepared using the technique described in Example 2. Δsec72 and WT strains were prepared for each of the 2X, 4X, and 6X 18B expression strains. For the 2X, 4X, and 6X 18B expression strains, secretion of MFα-18B from the WT and Δsec72 strains (i.e., SEC72KO strains) was measured by ELISA, and the results are shown in FIG.

[0139] Referring to FIG. 6, "Silk copies" is the number of expression cassettes for 18B (Ab MaSp2 79 kDa) with MFα1(sc) prepro leader sequence in each strain. 18B is C-terminally tagged with 3xFLAG epitope for ELISA detection. Error bars indicate standard error of the mean among n≧4 biological replicates. The results show that deleting the sec72 gene improves secretion of MFα-18B across integrated copy numbers (i.e., 2X, 4X, and 6X silk copies).

[0140] Example 6: Δsec72 improves secretion from non-MFα signals P. pastoris strains were modified according to Example 1 to express four different silk polypeptides, each containing a different secretion signal, i.e., PEP4(sc), CPY+4(sc), DAP2(sc), and MFα1(sc). Each complete leader sequence is a hybrid consisting of the indicated signal peptide and the MFα1(sc) propeptide (systematic name of S. cerevisiae orthologue: YPL187W). (sc) indicates that the signal peptide is derived from S. cerevisiae. Strains recombinantly expressing polypeptides using PEP4(sc), CPY+4(sc), and DAP2(sc) each contain a single expression cassette (i.e., one silk copy). Strains recombinantly expressing MFα1(sc) contain two expression cassettes (i.e., two silk copies). To compare secretion from the WT and Δsec72 strains, each of the Δsec72 strains listed above was prepared using the techniques described in Example 2. For the Δsec72 strain containing DAP2(sc), the strain was further modified to knock out the YPS1-1 and YPS1-2 proteases as described in Example 4. Secretion of polypeptides by each secretion signal from the WT and Δsec72 strains was measured by ELISA, and the results are shown in Figure 7.

[0141] The above results indicate that deletion of the sec72 gene improves secretion of polypeptides containing signal peptides other than MFα1(sc). Error bars indicate the standard error of the mean among n≧4 biological replicates.

[0142] Table 7A: MFα1(sc) peptide sequence TIFF0007623680000017.tif66132

[0143] Table 7B Silk secretion signals TIFF0007623680000018.tif205132

[0144] Example 7: Δsec72 improves secretion of longer silks and distinct silk sequences P. pastoris strains were engineered according to Example 1 to express the long silk polypeptides Argioppe bruennichi (Ab) MaSp2 (106 kDa) (SEQ ID NO: 43) and Latrodectus hesperus (Lh) MaSp1 (55 kDa) (SEQ ID NO: 44). Ab MaSp2 106 kDa (also known as 24B) is a longer concatamer of Ab MaSp2 79 kDa (18B). Lh MaSp1 55 kDa is a distinct sequence from other spidroins of the same family.

[0145] To compare secretion from the WT and Δsec72 strains, each Δsec72 strain was prepared using the techniques described in Example 2. The strain containing Lh MaSpl (55 kDa) was further modified to knock out the YPS1-1 and YPS1-2 proteases as described in Example 4. Secretion of long silk polypeptides from the WT and Δsec72 strains was measured by ELISA and the results are shown in Figure 8. Error bars indicate the standard error of the mean among n>4 biological replicates.

[0146] Argioppe bruennichi MaSp2 protein amino acid sequence (SEQ ID NO:43): TIFF0007623680000019.tif210120

[0147] Latrodectus hesperus MaSp1 protein amino acid sequence (SEQ ID NO:44): TIFF0007623680000020.tif198120

[0148] Example 8: Deletion of SEC72 improves secretion, but neither knockdown nor overexpression improves P. pastoris strains were modified according to Example 1 to contain four copies of a DNA cassette containing a recombinant gene expressing the 18B silk-like polypeptide. The Δsec72 strains were prepared using the techniques described in Example 2. These strains were further modified to knock out the YPS1-1 and YPS1-2 proteases as described in Example 4.

[0149] From this strain, a strain overexpressing sec72 was prepared by transformation with a vector containing the recombinant sec72 gene operably linked to the THI11 promoter (pTHI11). pTHI11 is derepressed in minimal medium without the vitamin thiamine. From previous RNAseq and promoter fusion studies, pTHI11 is one of the strongest promoters in block and tank minimal medium without thiamine.

[0150] Furthermore, instead of knocking out sec72, we knocked down SEC72 expression from a P. pastoris strain engineered to express four copies of a recombinant gene expressing the 18B silk-like polypeptide using DAmP (decreased abundance by mRNA perturbation), a transcriptional knockdown method that disrupts the 3'UTR of a gene with a marker cassette.

[0151] Secretion of 18B silk-like polypeptide from the WT strain, the Δsec72 strain, the Δsec72 strain with recombinantly overexpressed sec72 (operably linked to pTHI11), and the sec72 knockdown strain was measured by ELISA, and the results are shown in Figure 9A.

[0152] The Δsec72 phenotype was reversed by complementation with an inducible allele of sec72. Similarly, knockdown of SEC72 had no measurable effect on silk secretion compared to WT. Thus, secretion is improved by deleting sec72 but not by knockdown or overexpression of sec72.

[0153] Example 9: Other perturbations to the stoichiometry of the SEC63 complex do not affect silk secretion A P. pastoris strain was modified to contain four copies of a DNA cassette containing a recombinant gene expressing the 18B silk-like polypeptide according to Example 1. The Δsec72 strain was prepared using the techniques described in Example 2.

[0154] To test whether the Δsec72 phenotype was a direct effect of depleting SEC72 or an indirect effect of perturbing the stoichiometry of the SEC63 complex, we simultaneously overexpressed three non-SEC72 complex members (SEC62, SEC63, and SEC66) using pTHI11 (by transforming the strain with a vector containing a triple-linked expression cassette with sec62, sec63, and sec66). We also overexpressed SEC72 using pTHI11. We measured the secretion of 18B from each strain using ELISA and compared it to the Δsec72 strain, and the results are shown in Figure 9B.

[0155] Neither stoichiometric change (overexpression of SEC62, SEC63, and SEC66, nor overexpression of SEC72) had any measurable effect on silk secretion. Error bars indicate the standard error of the mean among n>3 biological replicates.

[0156] Example 10: Demonstration of transcriptional adaptation to Δsec72, including at the translocon level, by RTqPCR. A Δsec72 strain was prepared using the techniques described in Example 2. After growth, mRNA was isolated and RTqPCR was performed for selected markers to analyze transcriptional adaptation to Δsec72.

[0157] Expression normalized to reference (ALG9 or ACT1) as fold change from "WT" strain RMs71 in minimal medium is shown in Figure 10A. Shading indicates range, and the middle line represents the median across n=3 biological replicate experiments (except n=2 for Δsec72). SSH1 transcript levels increase 4-8 fold across all three perturbations. Δsec72 reduces expression of SEC61 translocon components by 2-4 fold.

[0158] Figure 10B shows the projection of centered, log-scaled, reference-normalized expression data onto the principal component space (left panel = PC1 vs. PC3, right panel = PC2 vs. PC3). Replicate data are color-coded by strain genotype, showing low intra-genotype variation compared to inter-genotype variation. Examination of the loading coefficients reveals that most of the variation along PC1 is driven by strong SSA3 repression, and most of the variation along PC3 is driven by repression of the SEC61 translocon complex. These 3 PCs reconstruct 99% of the variation from the original 20 dimensions.

[0159] Example 11: Overexpression of SSH1 improves the growth rate of Δsec72 silk-secreting strains SSH1 also functions in ER translocation but differs from SEC61 in its dispensability for S. cerevisiae growth and its lack of interaction with the SEC63 complex. SSH1 can also display preferences for translocation substrates, but these preferences overlap extensively with those of SEC61.

[0160] To test whether overexpression of SSH1 can promote the growth of the Δsec72 strain, P. pastoris Δsec72 strain was prepared using the technique described in Example 2. The control and coding sequences of SEC61 and SSH1 complex (SSS1, SBH2, and one of SEC61 or SSH1) were assembled on an integration plasmid by PCR. The resulting integration duplicates the gene copy number of SEC61 or one of SSH1 complex. P. pastoris(Δsec72) cells were transformed with a vector for overexpression of SSH1 translocon complex as described in Example 3. Similarly, P. pastoris(Δsec72) cells were transformed with a vector for overexpression of SEC61. The selected strain (indicated by "ΔΔ" in FIG. 11) was further modified to knock out YPS1-1 and YPS1-2 proteases as described in Example 4.

[0161] Precultures in YPD were diluted 1:1600 in minimal medium (RMml7). OD600 was recorded at six time points over an 8-h exponential growth period starting 19 hours after dilution. A linear model fits log(OD600) versus time to estimate growth rate and standard error of slope between n=6 biological replicates. Growth measured for each strain is shown in FIG. 11. Doubling times are expressed as the reciprocal of growth rate. Error bars are asymmetric due to reciprocal transformation of estimates.

[0162] Duplication of SEC61 and SSH1 promoted faster doubling of the SEC72 silk-producing strain (Figure 11, left). This effect may be due to a decrease in the silk load accumulated in the cells. However, Δsec72 exhibited a significant growth defect that was partially rescued by duplication of SSH1 but not by duplication of SEC61 (Figure 11, right).

[0163] Example 12: Duplication of SSH1 improves the fermentation ability of the Δsec72 strain According to Example 1, P. pastoris strains were modified to contain 4 or 6 copies of a DNA cassette containing a recombinant gene expressing an 18B silk-like polypeptide. The Δsec72 strain was prepared from recombinant cells expressing 4 copies of the DNA cassette using the technique described in Example 2. A portion of the resulting P. pastoris(Δsec72) cells was also transformed with a vector for overexpression of the SSH1 translocon complex as described in Example 3. Secretion of 18B from each strain was measured using ELISA, and the results are shown in Figures 12A and 12B.

[0164] In FIG. 12A, each curve represents an independent fed-batch fermentation. The marked points are samples taken for analysis, including the concentration of silk protein secreted into the broth ("18B titer", y-axis). The x-axis is the cumulative glucose feed, since the feeding program is time-varying. The shading indicates the range of all experiments for the same strain. The Δsec72 deletion strain shows highly variable performance. When SSH1 is duplicated in this strain ("2N SSH1"), the performance is improved and the variability is reduced. The silk secretion of the Δsec72 2N SSH1 strain, which has only four silk expression cassettes, approaches the range of secretion of the reference strain with six silk expression cassettes.

[0165] Specific productivity (ratio of product to biomass) was measured for cultures of each strain and is shown in FIG. 12B. Δsec72 background growth is slower and produces less biomass, and these metrics rise above those of the higher expressing, highest titer strains. Due to high variability in Δsec72 strains, only the top n=2 experiments are shown in the pooled data. Error bars are standard error of the mean.

[0166] Other embodiments The words which have been used are words of description rather than of limitation, and it is to be understood that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the invention in its broader aspects.

[0167] While the present invention has been described at some length and in some detail with respect to certain illustrated embodiments, it is not intended that the present invention should be limited to any of such individual details or embodiments, or to any particular embodiment, but rather should be interpreted with reference to the appended claims in order to obtain the broadest possible interpretation of such claims in light of the prior art, so as to effectively encompass the intended scope of the invention.

[0168] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, the section headings, materials, methods, and examples are illustrative only and are intended to be non-limiting.

[0169] array Table 8: Forward (F) and reverse (R) primers with 5' and 3' homology arms (HA) targeting the protease ORF TIFF0007623680000021.tif53156

[0170] Table 9. Forward and reverse primers for amplifying modified sequences TIFF0007623680000022.tif28135

[0171] Table 10. sec72KO vector TIFF0007623680000023.tif226129TIFF0007623680000024.tif226147TIFF0007623680000025.tif226147TIFF0007623680000026.tif22681

[0172] Table 11. SSH1 complex overexpression vector TIFF0007623680000027.tif22643TIFF0007623680000028.tif226149TIFF0007623680000029.tif226149TIFF00076236800 00030.tif226149TIFF0007623680000031.tif226148TIFF0007623680000032.tif226138TIFF0007623680000033.tif226142

[0173] Table 12. Zeocin cassette with HA arms for KU70 deletion in P. pastoris TIFF0007623680000034.tif219130TIFF0007623680000035.tif219146TIFF0007623680000036.tif219113

[0174] Table 13. Template nooseothricin cassette for protease deletion in P. pastoris TIFF0007623680000037.tif217132TIFF0007623680000038.tif21757

[0175] Table 14. Exemplary nourseothricin cassettes with HA arms for deletion of YPS1-1 and YPS1-2 proteases in P. pastoris TIFF0007623680000039.tif21966TIFF0007623680000040.tif219148TIFF00076236800 00041.tif219148TIFF0007623680000042.tif219149TIFF0007623680000043.tif219114

[0176] Table 15. Gene and sequence information disclosed herein TIFF0007623680000044.tif94156

[0177] Table 16. Promoter and terminator information for SSH1 complex genes TIFF0007623680000045.tif88135

Claims

1. A microorganism called Pichia pastoris, in which the sec72 gene is deleted, The microorganism expresses a recombinant protein comprising a secretory signal sequence derived from yeast, The recombinant protein comprises at least one block polypeptide sequence derived from a silk protein or is a silk-like protein; and The secretory signal sequence is selected from the group consisting of a PEP4 signal sequence, a CPY+4 signal sequence, a DAP2 signal sequence, and an MFα1 signal sequence. The microorganism.

2. The microorganism of claim 1 , wherein the sec72 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO:

1.

3. The microorganism of claim 1 , wherein the sec72 gene comprises SEQ ID NO:

1.

4. 2. The microorganism of claim 1, wherein the sec72 gene is present at locus PAS_chr2-1_0448 of the microorganism.

5. The microorganism according to any one of claims 1 to 4, further comprising a recombinantly expressed SSH1 translocon complex.

6. The microorganism of claim 5, wherein the SSH1 translocon complex comprises a first polypeptide sequence at least 95% identical to SEQ ID NO:4, a second polypeptide sequence at least 95% identical to SEQ ID NO:6, and a third polypeptide sequence at least 95% identical to SEQ ID NO:

8.

7. The microorganism of claim 5, wherein the SSH1 translocon complex comprises a first polypeptide comprising SEQ ID NO:4, a second polypeptide comprising SEQ ID NO:6, and a third polypeptide comprising SEQ ID NO:

8.

8. The microorganism according to any one of claims 1 to 7, further comprising a recombinantly expressed translocon complex.

9. The microorganism of claim 8 , wherein the translocon complex is expressed from a recombinant SSH1 gene, a recombinant SSS1 gene, and a recombinant SBH2 gene.

10. The microorganism of claim 9, wherein the SSH1 gene comprises SEQ ID NO:

3.

11. The microorganism of claim 9, wherein the SSH1 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO:

3.

12. The microorganism of claim 9, wherein the SSH1 gene comprises SEQ ID NO:

5.

13. The microorganism of claim 9, wherein the SSH1 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO:

5.

14. The microorganism of claim 9, wherein the SBH2 gene comprises SEQ ID NO:

7.

15. The microorganism of claim 9, wherein the SBH2 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO:

7.

16. The microorganism described in claim 9, wherein expression of the recombinant SSH1 gene to increase the level of the SSH1 translocon in the microorganism beyond the level of the SSH1 translocon expressed by the natural organism improves the growth rate and / or fermentation capacity of the microorganism.

17. The microorganism of claim 8 , wherein the translocon complex comprises an SSH1 protein, an SSS1 protein, and an SBH2 protein.

18. The microorganism of claim 17, wherein the SSH1 protein comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:

4.

19. The microorganism of claim 17, wherein the SSH1 protein comprises SEQ ID NO:

4.

20. The microorganism described in claim 17, wherein the SSS1 protein comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:

6.

21. The microorganism described in claim 17, wherein the SSS1 protein comprises SEQ ID NO:

6.

22. 18. The microorganism of claim 17, wherein the SBH2 protein comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:

8.

23. The microorganism of claim 17 , wherein the SBH2 protein comprises SEQ ID NO:

8.

24. The microorganism according to any one of claims 1 to 23, wherein the activities of YPS1-1 protease and YPS1-2 protease are weakened or eliminated.

25. 25. The microorganism of claim 24, wherein the YPS1-1 protease comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:

10.

26. 25. The microorganism of claim 24, wherein the YPS1-1 protease comprises SEQ ID NO:

10.

27. The microorganism of claim 24, wherein the YPS1-1 protease is encoded by the YPS1-1 gene.

28. The microorganism of claim 27, wherein the YPS1-1 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO:

9.

29. The microorganism of claim 27, wherein the YPS1-1 gene comprises SEQ ID NO:

9.

30. 28. The microorganism of claim 27, wherein the YPS1-1 gene is present at locus PAS_chr4_0584 of the microorganism.

31. The microorganism of any one of claims 24 to 30, wherein the YPS1-2 protease comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:

12.

32. 25. The microorganism of claim 24, wherein the YPS1-2 protease comprises SEQ ID NO:

12.

33. The microorganism of claim 24, wherein the YPS1-2 protease is encoded by the YPS1-2 gene.

34. The microorganism of claim 33, wherein the YPS1-2 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO:

11.

35. The microorganism of claim 33, wherein the YPS1-2 gene comprises SEQ ID NO:

11.

36. The microorganism of claim 33, wherein the YPS1-2 gene is present at locus PAS_chr3_1157 of the microorganism.

37. 37. The microorganism according to any one of claims 24 to 36, wherein the YPS1-1 gene or the YPS1-2 gene, or both, are mutated or knocked out.

38. The microorganism according to any one of claims 24 to 37, wherein the activity of one or more further proteases is reduced or eliminated.

39. The silk-like polypeptide comprises one or more repeat sequences (SEQ ID NO: 13), During the ceremony, X1=SGGQQ (SEQ ID NO:14) or GAGQQ (SEQ ID NO:15) or GQGPY (SEQ ID NO:16) or AGQQ (SEQ ID NO:17) or SQ; n1 is 4 to 8; n2 is 6 to 20, and n3 is 2 to 20; The microorganism described in claim 1.

40. The microorganism of claim 1 , wherein the silk-like polypeptide comprises a polypeptide sequence encoded by SEQ ID NO:

21.

41. The microorganism according to any one of claims 1 to 40, wherein the secretory signal sequence is selected from the group consisting of SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, and SEQ ID NO:

90.

42. A microorganism called Pichia pastoris, The sec72 gene comprising SEQ ID NO:1 of the microorganism is knocked out, a recombinantly expressed SSH1 gene comprising SEQ ID NO:3, a recombinantly expressed SSS1 gene comprising SEQ ID NO:5, and a recombinantly expressed SBH2 gene comprising SEQ ID NO:7; a recombinantly expressed protein comprising a silk-like polypeptide comprising a yeast-derived secretory signal sequence and a polypeptide encoded by SEQ ID NO:21; and The secretory signal sequence is selected from the group consisting of a PEP4 signal sequence, a CPY+4 signal sequence, a DAP2 signal sequence, and an MFα1 signal sequence. The microorganism.

43. A cell culture comprising a microorganism according to any one of claims 1 to 42.

44. 43. A cell culture comprising a microorganism according to any one of claims 5 to 42, wherein the growth rate and fermentation capacity of the strain is improved under standard cell culture conditions compared to a cell culture not comprising a recombinantly expressed SSH1 translocon complex.

45. A cell culture comprising the microorganism according to any one of claims 5 to 42, Under standard cell culture conditions, microorganisms which are otherwise identical except that they contain a functional sec72 gene and do not contain a recombinantly expressed SSH1 translocon complex, each of which has the same number of copies of a recombinant silk polypeptide gene. the yield or specific productivity of the recombinant protein is increased compared to a cell culture of The cell culture.

46. Cultivating the microorganism according to any one of claims 1 to 42 in a medium under conditions suitable for the expression of the recombinantly expressed protein; isolating said recombinant protein from said microorganism or from said culture medium. A method for producing a recombinant protein comprising the steps of:

47. 47. The method of claim 46, wherein the recombinant protein is secreted from the microorganism and the step of isolating the recombinant protein comprises recovering the medium containing the secreted recombinant protein.

48. 47. The method of claim 46, wherein the yield or specific productivity of the recombinant protein is increased in the microorganism compared to an otherwise identical microorganism in which the sec72 gene has not been deleted.

49. an identical microorganism except that it does not contain the recombinantly expressed SSH1 translocon complex and the sec72 gene is not deleted.

47. The method of claim 46, wherein the yield or specific productivity of the recombinant protein in the microorganism is increased compared to

50. 1. A method for modifying Pichia pastoris to improve secretion of a recombinantly expressed protein that contains a yeast-derived secretory signal sequence, comprising: the method comprising knocking out a gene encoding a SEC72 protein, the recombinantly expressed protein comprises at least one blocked polypeptide sequence derived from a silk protein or comprises a silk-like protein; and The secretory signal sequence is selected from the group consisting of a PEP4 signal sequence, a CPY+4 signal sequence, a DAP2 signal sequence, and an MFα1 signal sequence. The method.

51. 51. The method of claim 50, further comprising transforming the Pichia pastoris with a vector comprising a gene encoding a recombinantly expressed SSH1 translocon complex.

52. The silk-like polypeptide has one or more repeat sequences {GGY-[GPG-X １ ] ｎ１ -GPS-(A) ｎ２ } ｎ３ (SEQ ID NO: 13), During the ceremony, X １ = SGGQQ (SEQ ID NO: 14) or GAGQQ (SEQ ID NO: 15) or GQGPY (SEQ ID NO: 16) or AGQQ (SEQ ID NO: 17) or SQ, n1 is 4 to 8; n2 is 6 to 20, and n3 is 2 to 20; 51. The method of claim 50.

53. 51. The method of claim 50, wherein the recombinantly expressed protein comprises a polypeptide sequence encoded by SEQ ID NO:21.

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