JP7246102B2 - Modified strain for producing recombinant silk - Google Patents

Description

FIELD OF THE INVENTION The present disclosure relates to methods of strain optimization for producing or enhancing production of proteins or metabolites from cells. The disclosure also relates to compositions resulting from those methods. In particular, the present disclosure relates to yeast cells selected or genetically engineered to inhibit degradation of recombinant proteins expressed by the yeast cells, and yeast cell culture methods for producing useful compounds.

BACKGROUND OF THE INVENTION The methylotrophic yeast Pichia pastoris is widely used for the production of recombinant proteins. P. pastoris grows to high cell densities, provides tightly regulated methanol-inducible transgene expression, and efficiently secretes heterologous proteins into defined media.

However, P.I. During cultivation of strains pastoris, the recombinantly expressed protein was sometimes degraded before it could be recovered, and included fragments of the recombinantly expressed protein and low yields of full-length recombinant protein. Form a protein mixture. Therefore, P.I. There is a need for means and engineered strains to attenuate proteolysis in S. pastoris.

In some embodiments, provided herein is a microorganism of Pichia pastoris having reduced or eliminated activity of YPS1-1 protease and YPS1-2 protease, wherein said microorganism expresses a recombinant polypeptide. do.

In some embodiments, the YPS1-1 protease comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:67. In some embodiments, the YPS1-1 protease comprises SEQ ID NO:67. 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 that is at least 95% identical to SEQ ID NO:1. In some embodiments, the YPS1-1 gene comprises at least 15, 20, 25, 30, 40, or 50 contiguous nucleotides of SEQ ID NO:1. In some embodiments, the YPS1-1 gene comprises SEQ ID NO:1. In some embodiments, the YPS1-1 gene is at locus PAS_chr4_0584 of said microorganism.

In some embodiments, the YPS1-2 protease comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:68. In some embodiments, the YPS1-2 protease comprises SEQ ID NO:68. 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 that is at least 95% identical to SEQ ID NO:2. In some embodiments, the YPS1-2 gene comprises at least 15, 20, 25, 30, 40, or 50 contiguous nucleotides of SEQ ID NO:2. In some embodiments, the YPS1-2 gene comprises SEQ ID NO:2. In some embodiments, the YPS1-2 gene is at locus PAS_chr3 — 1157 of said microorganism.

In some embodiments, the YPS1-1 gene or said YPS1-2 gene, or both, have been mutated or knocked out.

In some embodiments, the microorganism expresses the recombinant protein. In some embodiments, the recombinant protein comprises at least one block polypeptide sequence derived from 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 {GGY-[GPG-X ₁ ] _n1 -GPS-(A) _n2 } _n3 (SEQ ID NO: 514) , wherein , X ₁ = SGGQQ (SEQ ID NO: 515) or GAGQQ (SEQ ID NO: 516) or GQGPY (SEQ ID NO: 517) or AGQQ (SEQ ID NO: 518) or SQ and n1 is 4 to 8 Yes, n2 is 6-20 and n3 is 2-20. In some embodiments, the silk-like polypeptide comprises a polypeptide sequence encoded by SEQ ID NO:462.

In some embodiments, the activity of one or more additional proteases of the microorganism is attenuated or eliminated. In some embodiments, the one or more additional proteases comprise YPS1-5, MCK7, or YPS1-3.

In some embodiments, the YPS1-5 gene is at locus PAS_chr3_0688 of said microorganism.

In some embodiments, the MCK7 protease is encoded by a MCK7 gene comprising a polynucleotide sequence that is at least 95% identical to SEQ ID NO:7. In some embodiments, the MCK7 gene comprises at least 15, 20, 25, 30, 40, or 50 contiguous nucleotides of SEQ ID NO:7. In some embodiments, the MCK7 gene comprises SEQ ID NO:7. In some embodiments, the MCK7 gene is at locus PAS_chr1-1 — 0379 of said microorganism.

In some embodiments, the YPS1-3 protease is encoded by a YPS1-3 gene comprising a polynucleotide sequence that is at least 95% identical to SEQ ID NO:3. In some embodiments, the YPS1-3 gene comprises at least 15, 20, 25, 30, 40, or 50 contiguous nucleotides of SEQ ID NO:3. In some embodiments, the YPS1-3 gene comprises SEQ ID NO:3. In some embodiments, the YPS1-3 gene is at locus PAS_chr3_0299 of said microorganism.

In some embodiments, the one or more additional proteases comprise a polypeptide sequence that is at least 95% identical to a polypeptide sequence selected from the group consisting of SEQ ID NOs:68-130. In some embodiments, the one or more additional proteases comprise a polypeptide sequence selected from the group consisting of SEQ ID NOs:68-130. In some embodiments, one or more additional proteases are encoded by a polynucleotide sequence that is at least 95% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 3-66. In some embodiments, the one or more additional proteases comprise at least 15, 20, 25, 30, 40, or 50 contiguous nucleotides of a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 3-66. encoded by a polynucleotide sequence comprising

In some embodiments, the microorganism comprises a 3X, 4X or 5X protease knockout.

Also herein, according to some embodiments of the present invention, the YPS1-1 gene comprising SEQ ID NO: 1 and the YPS1-2 gene comprising SEQ ID NO:2 were reduced by mutation or deletion. Also provided is a modified microorganism of Pichia pastoris comprising the activity of YPS1-1 and YPS1-2, wherein said microorganism further comprises a recombinantly expressed polypeptide sequence encoded by SEQ ID NO:462. Contains protein.

Also provided herein, in some embodiments, is a cell culture comprising a protease-attenuated microorganism as described herein.

Also provided herein, according to some embodiments, is a cell culture comprising a microorganism having attenuated or abolished activity of YPS1-1 and YPS1-2 as described herein, wherein A cell culture comprising a Pichia pastoris microorganism identical except that said microorganism recombinantly expresses a protein, and said recombinantly expressed protein does not attenuate or eliminate the activity of YPS1-1 and YPS1-2. It decomposes less than material.

In some embodiments, provided herein are methods of producing a recombinant protein with reduced degradation, which attenuates the activity of YPS1-1 and YPS1-2 as described herein. or culturing the inactivated microorganism in culture medium under conditions suitable for expression of the recombinantly expressed protein and isolating the recombinant protein from the microorganism or culture medium.

In some embodiments, the recombinant protein is secreted from said microorganism, wherein isolating said recombinant protein comprises collecting medium containing said secreted recombinant protein. In some embodiments, the recombinant protein has a level of degradation compared to said recombinant protein produced by the same microorganism except that said YPS1-1 and said YPS1-2 protease activities are not attenuated or abolished. is low.

Also provided herein are methods of modifying Pichia pastoris to inhibit the degradation of recombinantly expressed proteins by knocking out or modifying the genes encoding the YPS1-1 and YPS1-2 proteins. Including mutating. In some embodiments, the method of modifying Pichia pastoris to inhibit degradation of a recombinantly expressed protein further comprises one or more Including knocking out or mutating additional genes. In some embodiments, the method of modifying Pichia pastoris to inhibit degradation of a recombinantly expressed protein further comprises a protein comprising a polypeptide selected from the group consisting of SEQ ID NOs: 68-130. Including knocking out one or more of the encoding genes.

In some embodiments, the recombinantly expressed protein comprises a poly A sequence (SEQ ID NO: 519) . 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-X ₁ ] _n1 -GPS-(A) _n2 } _n3 (SEQ ID NO: 514) , wherein , X ₁ = SGGQQ (SEQ ID NO: 515) or GAGQQ (SEQ ID NO: 516) or GQGPY (SEQ ID NO: 517) or AGQQ (SEQ ID NO: 518) or SQ and n1 is 4 to 8 Yes, n2 is 6-20 and n3 is 2-20. In some embodiments, the recombinantly expressed protein comprises a polypeptide sequence encoded by SEQ ID NO:462.
[Invention 1001]
A Pichia pastoris microorganism having reduced or eliminated activity of YPS1-1 protease and YPS1-2 protease, said microorganism expressing a recombinant polypeptide.
[Invention 1002]
1001. The microorganism of the invention 1001, wherein said YPS1-1 protease comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:67.
[Invention 1003]
1001. The microorganism of the invention 1001, wherein said YPS1-1 protease comprises SEQ ID NO:67.
[Invention 1004]
The microorganism of invention 1001 or invention 1002, wherein said YPS1-1 protease is encoded by the YPS1-1 gene.
[Invention 1005]
1004. The microorganism of the invention 1004, wherein said YPS1-1 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO:1.
[Invention 1006]
1004. The microorganism of the invention 1004, wherein said YPS1-1 gene comprises at least 15, 20, 25, 30, 40, or 50 contiguous nucleotides of SEQ ID NO:1.
[Invention 1007]
1004. The microorganism of the invention 1004, wherein said YPS1-1 gene comprises SEQ ID NO:1.
[Invention 1008]
1004. The microorganism of the invention 1004, wherein said YPS1-1 gene is at locus PAS_chr4 — 0584 of said microorganism.
[Invention 1009]
The microorganism of any preceding invention, wherein said YPS1-2 protease comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:68.
[Invention 1010]
1009. The microorganism of the invention 1009, wherein said YPS1-2 protease comprises SEQ ID NO:68.
[Invention 1011]
The microorganism of any preceding invention, wherein said YPS1-2 protease is encoded by the YPS1-2 gene.
[Invention 1012]
1011. The microorganism of the invention 1011, wherein said YPS1-2 gene comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO:2.
[Invention 1013]
1011. The microorganism of the invention 1011, wherein said YPS1-2 gene comprises at least 15, 20, 25, 30, 40, or 50 contiguous nucleotides of SEQ ID NO:2.
[Invention 1014]
1011. The microorganism of the present invention 1011, wherein said YPS1-2 gene comprises SEQ ID NO:2.
[Invention 1015]
1011. The microorganism of the invention 1011, wherein said YPS1-2 gene is at locus PAS_chr3 — 1157 of said microorganism.
[Invention 1016]
The microorganism of any preceding invention, wherein said YPS1-1 gene or said YPS1-2 gene or both have been mutated or knocked out.
[Invention 1017]
A microorganism of any preceding invention that expresses a recombinant protein.
[Invention 1018]
1017. The microorganism of the invention 1017, wherein said recombinant protein comprises at least one blocked polypeptide sequence derived from silk protein.
[Invention 1019]
1017. The microorganism of the invention 1017, wherein said recombinant protein comprises a silk-like polypeptide.
[Invention 1020]
The silk-like polypeptide comprises one or more repeat sequences {GGY-[GPG-X ₁ ] _n1 -GPS-(A) _n2 } _n3 , wherein
X = SGGQQ or GAGQQ or GQGPY or AGQQ or SQ,
n1 is 4 to 8;
n2 is 6 to 20, and
n is 2 to 20;
A microorganism of the invention 1019.
[Invention 1021]
1020. The microorganism of the invention 1019, wherein said silk-like polypeptide comprises a polypeptide sequence encoded by SEQ ID NO:462.
[Invention 1022]
The microorganism of any preceding invention, wherein the activity of one or more additional proteases has been reduced or eliminated.
[Invention 1023]
1022. The microorganism of the invention 1022, wherein said one or more additional proteases comprise YPS1-5, MCK7, or YPS1-3.
[Invention 1024]
1023. The microorganism of the invention 1023, wherein said YPS1-5 gene is at locus PAS_chr3 — 0688 of said microorganism.
[Invention 1025]
1023. The microorganism of the invention 1023, wherein said MCK7 protease is encoded by a MCK7 gene comprising a polynucleotide sequence that is at least 95% identical to SEQ ID NO:7.
[Invention 1026]
1023. The microorganism of the invention 1023, wherein said MCK7 gene comprises at least 15, 20, 25, 30, 40, or 50 contiguous nucleotides of SEQ ID NO:7.
[Invention 1027]
1023. The microorganism of the invention 1023, wherein said MCK7 gene comprises SEQ ID NO:7.
[Invention 1028]
1023. The microorganism of the invention 1023, wherein said MCK7 gene is at locus PAS_chr1-1 — 0379 of said microorganism.
[Invention 1029]
1023. The microorganism of the invention 1023, wherein said YPS1-3 protease is encoded by a YPS1-3 gene comprising a polynucleotide sequence that is at least 95% identical to SEQ ID NO:3.
[Invention 1030]
1023. The microorganism of the invention 1023, wherein said YPS1-3 gene comprises at least 15, 20, 25, 30, 40, or 50 contiguous nucleotides of SEQ ID NO:3.
[Invention 1031]
1023. The microorganism of the invention 1023, wherein said YPS1-3 gene comprises SEQ ID NO:3.
[Invention 1032]
1023. The microorganism of the invention 1023, wherein said YPS1-3 gene is at locus PAS_chr3 — 0299 of said microorganism.
[Invention 1033]
1023. The microorganism of the invention 1022, wherein said one or more additional proteases comprise a polypeptide sequence that is at least 95% identical to a polypeptide sequence selected from the group consisting of SEQ ID NOs: 68-130.
[Invention 1034]
1022. The microorganism of the invention 1022, wherein said one or more additional proteases comprise a polypeptide sequence selected from the group consisting of SEQ ID NOs: 68-130.
[Invention 1035]
1023. The microorganism of the invention 1022, wherein said one or more additional proteases are encoded by a polynucleotide sequence that is at least 95% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 3-66.
[Invention 1036]
The one or more additional proteases are encoded by a polynucleotide sequence comprising at least 15, 20, 25, 30, 40, or 50 contiguous nucleotides of a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 3-66 1022 of the present invention.
[Invention 1037]
The microorganism of any of Inventions 1022-1036, wherein said microorganism comprises a 3X, 4X or 5X protease knockout.
[Invention 1038]
Pichia comprising the activity of said YPS1-1 and said YPS1-2 reduced by mutation or deletion of the YPS1-1 gene comprising SEQ ID NO: 1 and the YPS1-2 gene comprising SEQ ID NO:2 A modified microorganism of pastoris,
Said microorganism, further comprising a recombinantly expressed protein comprising a polypeptide sequence encoded by SEQ ID NO:462.
[Invention 1039]
A cell culture comprising the microorganism of any of the inventions 1001-1038.
[Invention 1040]
A cell culture comprising the microorganism of any of the inventions 1017-1038,
Said cell culture, wherein said recombinantly expressed protein is less degraded than a cell culture containing the identical Pichia pastoris microorganism, except that it does not attenuate or eliminate the activity of YPS1-1 and YPS1-2.
[Invention 1041]
culturing the microorganism of any of the inventions 1017-1037 in a medium under conditions suitable for expression of said recombinantly expressed protein; and
isolating said recombinant protein from said microorganism or said medium;
A method for producing a recombinant protein with reduced degradation, comprising:
[Invention 1042]
1041. The method of invention 1041, wherein said recombinant protein is secreted from said microorganism, and said isolating said recombinant protein comprises recovering medium containing said secreted recombinant protein.
[Invention 1043]
The present invention, wherein said recombinant protein has a lower level of degradation compared to said recombinant protein produced by the same microorganism except that said YPS1-1 and said YPS1-2 protease activities are not attenuated or eliminated. 1041 ways.
[Invention 1044]
A method of modifying Pichia pastoris to inhibit degradation of a recombinantly expressed protein, comprising:
The above method, comprising knocking out or mutating the genes encoding the YPS1-1 protein and the YPS1-2 protein.
[Invention 1045]
The method of the invention 1044 further comprising knocking out or mutating one or more additional genes encoding YPS1-3 protein, YPS1-5 protein, or MCK7 protein.
[Invention 1046]
1044. The method of the invention 1044, further comprising knocking out one or more genes encoding a protein comprising a polypeptide selected from the group consisting of SEQ ID NO: 68-130.
[Invention 1047]
1044. The method of invention 1044, wherein said recombinantly expressed protein comprises a poly A sequence comprising at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive alanine residues.
[Invention 1048]
1044. The method of invention 1044, wherein said recombinantly expressed protein comprises a silk-like polypeptide.
[Invention 1049]
The silk-like polypeptide comprises one or more repeat sequences {GGY-[GPG-X ₁ ] _n1 -GPS-(A) _n2 } _n3 , wherein
X ₁ = SGGQQ or GAGQQ or GQGPY or AGQQ or SQ;
n1 is 4 to 8;
n2 is 6 to 20, and
n is 2 to 20;
The method of the invention 1048.
[Invention 1050]
1044. The method of invention 1044, wherein said recombinantly expressed protein comprises a polypeptide sequence encoded by SEQ ID NO:462.

The foregoing and other objects, features and advantages will become apparent from the following description of specific embodiments of the invention, illustrated in the accompanying drawings in which like reference numerals refer to the same parts throughout the different drawings. The drawings are not necessarily drawn to scale, emphasis instead being placed on illustrating the principles of various embodiments of the invention.

KU70 deletion plasmid map with zeocin resistance marker. Plasmid map of plasmids containing nourseothricin markers used with homology arms for targeted protease gene deletion. Figures 3A and 3B are protease knockout cassettes with homology arms pointing to the desired protease gene flanked by a nourseothricin resistance marker. Representative Western blots of proteins isolated from single KO strains, demonstrating proteolysis from these strains. Representative Western blots of proteins isolated from double KO strains showing proteolytic degradation from these strains. Representative Western blots of proteins isolated from strains with 2X, 3X, 4X, and 5X protease KO subcultured in BMGY or YPD, showing proteolysis in these strains.

DETAILED DESCRIPTION The details of various embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will become apparent from the description and drawings, and from the claims.

Definitions Unless otherwise specified herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The terms "a" and "an" include plural referents unless the context dictates otherwise. In general terms used in connection with biochemistry, enzymology, molecular and cell biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization as described herein and techniques relating thereto are It is a well-known and generally used one.

Unless otherwise specified, the following terms should be understood to have the following meanings.

The term "polynucleotide" or "nucleic acid molecule" refers to a polymeric form of nucleotides that is at least 10 bases long. Such terms include DNA molecules (eg, cDNA or genomic DNA or synthetic DNA) and RNA molecules (eg, mRNA or synthetic RNA), as well as non-naturally occurring nucleotide analogs, non-natural internucleoside linkages, or both. DNA or RNA analogues are included. Nucleic acids can be in any morphological conformation. For example, nucleic acids can be single-stranded, double-stranded, triple-stranded, quadruplex, partially double-stranded, branched, hairpinned, circular, or padlocked conformation.

Unless otherwise specified, as an example for any sequence set forth herein in the general form "SEQ ID NO", "a nucleic acid comprising SEQ ID NO: 1" means, at least in part, (i) SEQ ID NO: Refers to a nucleic acid having the sequence set forth in NO:1 or (ii) having a sequence complementary to SEQ ID NO:1. The alternative is determined by context. For example, when nucleic acids are used as probes, the alternative is determined by the requirement that the probes must be complementary to the desired target.

An "isolated" RNA, DNA or mixed polymer is free from other cellular components that naturally accompany the native polynucleotide in its natural host cell, such as ribosomes, polymerases and genomic sequences with which it is naturally associated. Means something that is substantially separated.

An "isolated" organic molecule (e.g., silk protein) is one that is substantially separated from the cellular components (membrane lipids, chromosomes, proteins) of the host cell from which it was derived, or from the medium in which the host cell was cultured. say. Although such terms do not necessarily require that the biomolecule be separated from all other chemicals, a particular isolated biomolecule can be purified to near homogeneity.

The term "recombinant" refers to a biomolecule, such as a gene or protein, that (1) has been removed from its naturally occurring environment, (2) the entire polynucleotide in 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" includes cloned DNA isolates, chemically synthesized polynucleotide analogues, or polynucleotide analogues synthesized biologically by heterologous systems, and any such nucleic acids encoded by such nucleic acids. can be used with respect to proteins and/or mRNAs.

An endogenous nucleic acid sequence of an organism's genome (or the protein product encoded by that sequence) is defined as the present invention if a heterologous sequence is placed adjacent to the endogenous nucleic acid sequence (or the protein product encoded by that sequence) so as to alter its expression. It is considered "recombinant" in the specification. In this case, the heterologous sequence is naturally endogenous nucleic acid, whether the heterologous sequence is itself endogenous (from the same host cell or progeny thereof) or exogenous (from a different host cell or progeny thereof). An array that is not adjacent to an array. By way of example, a promoter sequence can replace (eg, by homologous recombination) the native promoter of a gene in the genome of the host cell, such that the expression pattern of this gene is altered. Here, the gene will be considered "recombinant" because it is separated from at least some of the sequences that naturally flank it.

A nucleic acid is also considered "recombinant" if it contains some non-naturally occurring modification to the corresponding nucleic acid in the genome. For example, an endogenous coding sequence is considered "recombinant" if it contains insertions, deletions or point mutations introduced artificially, such as by human intervention. "Recombinant nucleic acid" also includes nucleic acids that are integrated at non-homologous sites within the host cell chromosome, and nucleic acid constructs that exist as episomes.

As used herein, the phrase "degenerate variant" of a reference nucleic acid sequence 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. It includes nucleic acid sequences. The terms "degenerate oligonucleotide" or "degenerate primer" are to mean oligonucleotides that are not necessarily identical in sequence but are capable of hybridizing to target nucleic acid sequences that are homologous to each other within one or more specified segments. used.

The term "percent sequence identity" or "identical" in nucleic acid sequences refers to those residues in two sequences that are the same when aligned for maximum correspondence. The length of sequence identity comparison is at least about 9 nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, preferably can span a region of 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, programs provided with the Wisconsin Package version 10.0 (Genetics Computer Group (GCG), Madison, Wis.). FASTA provides alignments and percent sequence identities of the most overlapping regions between the query and search sequences. Pearson, Methods Enzymol. 183:63-98 (1990) (incorporated herein by reference in its entirety). For example, the percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (string size is 6, and scoring matrix factor is NOPAM), or Gap can be determined using its default parameters. Alternatively, sequences can be analyzed 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.). 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, comparisons can be made using blastp or tblastn (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).

The terms "substantial homology" or "substantial similarity" when referring to a nucleic acid or fragment thereof refer to another nucleic acid (or its complementary strand) with appropriate insertions or deletions of nucleotides. When properly aligned, nucleotide sequence identity of at least about 75%, 80%, 85% of the nucleotide bases as measured by any of the well-known sequence identity algorithms such as FASTA, BLAST or Gap as described above , preferably at least about 90%, more preferably at least about 95%, 96%, 97%, 98% or 99%.

Alternatively, substantial homology or similarity exists when a nucleic acid or fragment thereof will hybridize under stringent hybridization conditions to another nucleic acid, another strand of nucleic acid, or its complementary strand. "Stringent hybridization conditions" and "stringent wash conditions" in nucleic acid hybridization experiments depend on a number of different physical parameters. Nucleic acid hybridization, as will be readily understood by those skilled in the art, is defined by the following conditions: salt concentration, temperature, solvent, base composition of the hybridizing species, length of the complementary region, and number of nucleotide base mismatches between the hybridizing nucleic acids. affected by conditions such as One skilled in the art knows how to vary these parameters to achieve a particular stringency of hybridization.

Generally, "stringent hybridization" is performed at about 25°C lower than the thermal melting point ( _Tm ) of an individual DNA hybrid under a specified set of conditions. "Stringent washing" is performed at temperatures about 5°C lower than the _Tm of individual DNA hybrids under a specified set of conditions. The _Tm is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe. Sambrook et al., incorporated herein by reference. , Molecular Cloning: A Laboratory Manual, 2d ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.M. Y. (1989), page 9.51. For the purposes of this specification, "stringent conditions" means solution phase hybridizations in 6x SSC (20x SSC contains 3.0 M NaCl and 0.3 M sodium citrate), 1% SDS at 65°C. Defined as aqueous hybridization (ie, no formamide) of 8-12 hours of hybridization followed by two 20 minute washes in 0.2×SSC, 0.1% SDS at 65°C. A skilled practitioner will readily appreciate that hybridization at 65° C. will occur at different rates depending on a number of factors, such as the length and percent identity of the hybridizing sequences.

Nucleic acids (also called polynucleotides) of the invention can include RNA, cDNA, both sense and antisense strands of 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 appreciated by those skilled in the art. Such modifications include, for example, labels, methylation, substitution of one or more naturally occurring nucleotides with analogues, 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., acridine, psoralen, etc.), chelating agents, alkyl agents, modified linkages (eg, alpha anomeric nucleic acids, etc.), and the like. Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which phosphate bonds in the molecular backbone are replaced with peptide bonds. Other modifications can include, for example, analogs in which the ribose ring contains bridging moieties or other structures, such as modifications found in "locked" nucleic acids.

The term "mutated" when applied to nucleic acid sequences means that nucleotides within a nucleic acid sequence have been inserted, deleted or altered when compared to a reference nucleic acid sequence. Single alterations (point mutations) may be made at a locus, and insertions, deletions or alterations of multiple nucleotides may be made at a single locus. Additionally, one or more alterations may be made at any number of loci within a given nucleic acid sequence. Nucleic acid sequences may be mutated by any method known in the art, including "error-prone PCR" (where the replication fidelity of the DNA polymerase is increased to yield a high rate of point mutations over the entire length of the PCR product). A method of performing PCR under reduced conditions, see, for example, 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 the generation of site-directed mutations in any cloned DNA segment of interest, see, for example, Reidhaar-Olson and Sauer, Science 241:53-57 (1988). )) and other mutagenesis techniques, but are not limited to these.

As used herein, the term "attenuating" generally refers to functional deletions, including mutations, partial or complete deletions, insertions, or gene sequences or transcription of gene sequences. Other alterations made to the regulatory sequences are included that reduce or inhibit production of the gene product or render the gene product non-functional. In some instances, functional deletions are expressed as knockout mutations. Attenuation may be by altering the nucleic acid sequence, placing the gene under the control of a less active promoter, down-regulating, expressing interfering RNA, ribozyme or antisense sequences that target the gene of interest. Alteration of the amino acid sequence, or alteration of the amino acid sequence through any other technique known in the art, is also included. In one example, the susceptibility of a particular enzyme to feedback inhibition or inhibition caused by a composition that is neither a product nor a reactant (pathway non-specific feedback) is rendered unaffected by the presence of the compound. In other cases, an enzyme that has been altered to have less activity may be referred to as attenuated.

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, the regions on either side of which are linked together. Point.

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

As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which generally refers to a circular double-stranded DNA loop into which additional DNA segments may be ligated, although linear double-stranded molecules such as polymerase chain Also included are those resulting from amplification by reaction (PCR), or from treatment of circular plasmids with restriction enzymes. Other vectors include cosmids, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC). Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome (detailed below). Certain vectors are capable of autonomous replication in a host cell into which they are introduced (eg, vectors having an origin of replication that is functional in the host cell). Other vectors, when introduced into a host cell, can integrate into the genome of the host cell and are thus replicated along with the host genome. Moreover, 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").

An “operably linked” or “operably linked” expression control sequence means that the expression control sequence flanks a gene of interest and a linkage that controls such gene of interest. , as well as expression control sequences that act in trans or at a distance to control a gene of interest.

The term "expression control sequence" refers to polynucleotide sequences necessary to affect the expression of coding sequences to which they are operably linked. Expression control sequences are sequences that control the transcription, post-transcriptional events and translation of nucleic acid sequences. Expression control sequences include appropriate transcription initiation, transcription termination, promoter and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency. (eg, ribosome binding sites); sequences that enhance protein stability; and, optionally, sequences that enhance secretion of the protein. The nature of such control sequences varies depending on the host organism, and in prokaryotes such control sequences generally include promoters, ribosome binding sites, and transcription termination sequences. The term "regulatory sequence" is intended to include, at a minimum, all components whose presence is essential for expression, including additional components that are conveniently present, such as leader sequences and fusions. Partner sequences and the like may also be included.

The term "regulatory element" refers to any element that affects the transcription or translation of a nucleic acid molecule. These include, by way of example and without limitation, regulatory proteins (eg, transcription factors), chaperones, signaling proteins, RNAi molecules, antisense RNA molecules, microRNAs and RNA aptamers. A regulatory element may be endogenous to the host organism. A regulatory element may be exogenous to the host organism. The regulatory element may be a synthetically generated regulatory element.

As used herein, the term "promoter," "promoter element," or "promoter sequence" refers to the ability to control the transcription of a nucleotide sequence of interest into mRNA when linked to that nucleotide sequence of interest. refers to a DNA sequence capable of A promoter is typically, but not necessarily, located 5' (i.e., upstream) to the nucleotide sequence of interest whose transcription into mRNA is controlled by the promoter and provides access to RNA polymerase and other media for transcription initiation. Provides sites for specific binding of transcription factors. The promoter may be endogenous to the host organism. The promoter may be exogenous to the host organism. A promoter may be a synthetically generated regulatory element.

Promoters useful for expressing the recombinant genes described herein include both constitutive promoters and inducible/repressible promoters. When expressing multiple recombinant genes in the artificially engineered organisms of the invention, such different genes can be controlled by different promoters or by the same promoter in separate operons, and two Expression of the above genes may be controlled by a single promoter as part of the operon.

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 parental cell, as certain modifications may occur with passage due to mutation or environmental influences, but still fall within the term "host cell" as used herein. include. A recombinant host cell may be an isolated cell, a cell line grown in culture, or a cell present in a living tissue or organism.

As used herein, the term "peptide" refers to short polypeptides, eg, typically less than about 50 amino acids in length, more typically less than about 30 amino acids in length. The term as used herein includes analogs as well as mimetics that mimic biological function by mimicking structural function.

The term "polypeptide" encompasses both naturally occurring and non-naturally occurring proteins, as well as fragments, variants, derivatives and analogs thereof. Polypeptides can be monomeric or polymeric. Moreover, a polypeptide may contain a number of different domains, each with one or more distinct activities.

The terms "isolated protein" or "isolated polypeptide", in terms of their origin or derivation source, are defined as follows: (1) a protein that in its natural state is unassociated with the naturally associated components that accompany it; or polypeptide, (2) a protein or polypeptide that exists in a purity not found in nature, where purity is determinable with respect to the presence of other cellular material (e.g., other proteins from the same species (3) a protein or polypeptide expressed by cells from a different species; or (4) a protein or polypeptide that does not occur in nature (e.g., is a fragment of a polypeptide found in nature, or amino acid analogs or derivatives that are not found in nature or that contain bonds other than standard peptide bonds). Thus, a polypeptide that is chemically synthesized, or that is synthesized in a cellular system other than the cell in which it naturally originates, 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. As so defined, "isolated" does not necessarily require that the protein, polypeptide, peptide or oligopeptide so described be physically removed from its natural environment.

The term "polypeptide fragment" refers to a polypeptide having deletions, eg, amino- and/or carboxy-terminal deletions, as compared to the full-length polypeptide. In preferred embodiments, a polypeptide fragment is a contiguous sequence that is identical to the corresponding position in the sequence in which the amino acid sequence of the fragment occurs in nature. Fragments are typically at least 5, 6, 7, 8, 9 or 10 amino acids long, preferably at least 12, 14, 16 or 18 amino acids long, more preferably at least 20 amino acids long, more preferably at least 25, 30, 35, 40 or 45 amino acids, even more preferably at least 50 or 60 amino acids long, even more preferably at least 70 amino acids long.

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 (particularly with regard to predicted structural similarity) is taken to mean similarity in function.

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 one amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of proteins. 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 substitutions. Means for making such modifications are well known to those skilled in the art. See, eg, Pearson, 1994, Methods Mol. Biol. 24:307-31 and 25:365-89, incorporated herein by reference.

The twenty conventional amino acids and their abbreviations follow convention. See Immunology-A Synthesis (Golub and Gren eds., Sinauer Associates, Sunderland, Mass., ^2nd ed. 1991), incorporated herein by reference. Stereoisomers of the 20 conventional amino acids (eg, D-amino acids), unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, etc., and other unconventional amino acids are also present in the present invention. can be a suitable component of a polypeptide of 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 (eg, 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.

The following six groups each contain amino acids that are conservative substitutions for each other: 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).

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

An algorithm useful in comparing a particular polypeptide sequence to databases containing large numbers 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. ), Zhang and Madden, Genome Res. 7:649-656 (1997)), especially blastp or tblastn (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).

Preferred parameters for BLASTp are: Expectation: 10 (default); Filter: seg (default); Cost to open gap: 11 (default); Cost to extend gap: 1 (default); Maximum alignment: 100 (default); String size: 11 (default); Display count: 100 (default); Penalty matrix: BLOWSUM62.

Preferred parameters for BLASTp are: Expectation: 10 (default); Filter: seg (default); Cost to open gap: 11 (default); Cost to extend gap: 1 (default); Maximum alignment: 100 (default); String size: 11 (default); Display count: 100 (default); Penalty matrix: BLOWSUM62. The length of the polypeptide sequences to be compared for homology is generally at least about 16 amino acid residues, usually at least about 20 residues, more generally at least about 24 residues, typically at least about 28 residues, preferably about 35 residues. more than residues. When searching a database containing sequences from many different organisms, it is preferable 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 the GCG version 6.1 program, FASTA. FASTA provides alignments and percent sequence identities of the most overlapping regions between the query and search sequences. Pearson, Methods Enzymol. 183:63-98 (1990) (incorporated herein by reference). For example, percent sequence identity between amino acid sequences can be determined using FASTA provided with GCG version 6.1, which is incorporated herein by reference, with its default parameters (string size is 2 , and the score matrix is PAM250).

Throughout this specification and claims, the word "comprises" or variations such as "comprises" or "comprising" include the stated integer or group of integers. and no other integers or groups of integers are excluded.

Although exemplary methods and materials are described below, methods and materials similar or equivalent to those described herein can be used in the practice of the invention and will be apparent to those skilled in the art. deaf. 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.

SUMMARY This document provides recombinant strains and methods of producing recombinant strains for increasing production of full-length desired products in target cells, such as by reducing degradation by proteases.

In some embodiments, to attenuate the protease activity of Pichia pastoris, the genes encoding these enzymes are inactivated or mutated to reduce or eliminate activity. This can be done through mutations or insertions into the gene itself, or through modification of the gene's regulatory elements. This is achievable by standard yeast genetics techniques. Examples of such techniques include gene replacement by double homologous recombination, in which regions of homology flanking the gene to be inactivated are cloned into a vector and a selectable marker gene (antibiotic resistance gene or yeast genes that complement the strain's auxotrophy).

Alternatively, the homologous region can be amplified by PCR and joined to the selectable marker gene by overlapping PCR. Such DNA fragments are then transformed into Pichia pastoris by methods known in the art, such as electroporation. Transformants grown under selective conditions are then analyzed for gene disruption events by standard techniques, such as PCR or Southern blot of genomic DNA. In an alternative experiment, gene inactivation can be achieved by single homologous recombination, where, for example, the 5' end of the gene's ORF is cloned onto a promoterless vector that also contains a selectable marker gene. do. Once such vectors are linearized by digestion with a restriction enzyme that cuts the vector only at the target gene homology fragment, such vectors are transformed into Pichia pastoris. Integration into the target gene site is confirmed by PCR or Southern blot of genomic DNA. In this way replication of the gene fragment cloned on the vector is achieved in the genome and two copies of the target locus, i.e. the ORF is truncated (if expressed) due to incompleteness. A first copy expressing an inactive protein and a second copy lacking a promoter to drive transcription are produced.

Alternatively, transposon mutagenesis is used to inactivate the target gene. Libraries of such mutants can be screened for insertion events within the target gene by PCR.

Functional phenotypes (ie, defects) of engineered/knockout strains can be assessed using techniques known in the art. For example, the lack of protease activity in engineered strains can be confirmed using any of a wide variety of methods known in the art, particularly assays for hydrolytic activity of chromogenic protease substrates, substrates for selected proteases. There is a protein band shift.

Attenuation of protease activity as described herein can be achieved by mechanisms other than knockout mutations. amino acid sequence by, for example, altering the nucleic acid sequence, placing the gene under the control of a less active promoter, down-regulating, expressing an interfering RNA, ribozyme or antisense sequence that targets the gene of interest. A desired protease can be attenuated by altering the amino acid sequence, such as by altering , or any other technique known in the art. In preferred strains, the protease activity of proteases encoded by PAS_chr4_0584 (YPS1-1) and PAS_chr3_1157 (YPS1-2) (e.g., polypeptides comprising SEQ ID NOs: 66 and 67) is attenuated by any of the methods described above. be done. In some aspects, the invention relates to methylotrophic yeast strains, particularly Pichia pastoris strains, wherein the YPS1-1 and YPS1-2 genes (eg, set forth in SEQ ID NO:1 and SEQ ID NO:2) are inactivated. In some embodiments, additional protease encoding genes 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.

Generating Recombinant Strains This specification describes the transformation of strains into activity by, for example, using vectors to deliver or knock out recombinant genes or other methods to attenuate the desired endogenous gene. provide a way to reduce These vectors are either vector backbones containing an origin of replication and a selectable marker (typically antibiotic resistance, but many others are possible), or linear fragments that allow integration into the chromosome of the target cell. can take the form of The vector should correspond to the chosen organism and method of insertion.

Once the vector elements have been selected, vector construction can be carried out in many different ways. In one embodiment, a DNA synthesis service may be utilized, or a method for making the entire vector individually may be used.

Once the DNA for each vector (including the additional elements necessary for insertion and manipulation) is obtained, it must be assembled. There are many possible assembly methods, including restriction enzyme cloning, blunt-end ligation, and overlap assembly [eg Gibson, D.; G. , 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)]. but not limited to). Overlap assembly provides a method to ensure that all of the elements are assembled in the correct position and do not introduce any undesired alignment.

The vectors generated above can be inserted into target cells using standard molecular biology techniques, such as molecular cloning. In one embodiment, the target cell is already engineered or selected to already contain the genes required for production of the desired product, but this is done during or after subsequent insertion of the vector. may

Depending on the organism and the type of library element (plasmid or genomic insert), several known methods of inserting vectors containing DNA for integration into cells may be used. These include, for example, transformation of microorganisms capable of taking up and replicating DNA from the local environment, transformation by electroporation or chemical means, transduction with viruses or phages, conjugation of two or more cells. mating, or conjugation from different cells.

Several methods of introducing recombinant DNA into bacterial cells are known in the art, including transformation, transduction, and electroporation (Sambrook, et al., Molecular Cloning: A Laboratory Manual). 1989), Second Edition, Cold Spring Harbor Press, Plainview, N.Y.). 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 ( Registered Trademark) BL21 (DE3)E. coli cells, One Shot® BL21(DE3) pLys E. coli cells. 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, Calif., USA), ElectroMAX™ A.I. tumefaciens LBA4404 cells (Invitrogen Corp., Carlsbad, Calif., USA).

Several methods are known in the art for introducing recombinant nucleic acids into eukaryotic cells. Exemplary methods include transfection, electroporation, nucleic acid delivery by liposomes, microinjection into host cells (Sambrook, et al., Molecular Cloning: A Laboratory Manual (1989), Second Edition, Cold Spring Harbor Press, Plainview, N.Y.). Non-limiting examples of commercially available kits and reagents for transfecting eukaryotic cells with recombinant nucleic acids 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 can be introduced into insect cells (eg, sf9, sf21, High Five™) through the use of baculovirus vectors.

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

Colonies are screened for desired production of proteins, metabolites, or other products, or for decreased protease activity. In one embodiment, the screening identifies the recombinant cells that produce the highest (or sufficiently high) yields or production efficiencies of the product. This includes decreasing the proportion of degradation products or increasing the total amount of full-length desired polypeptide recovered from cell culture.

This assay can be performed by growing individual clones in multiwell 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 culture is harvested by spinning at high speed in a centrifuge to pellet the cells and removing the supernatant. Each culture supernatant can then be tested for protease activity and/or proteolysis.

Silk Sequences In some embodiments, the low protease activity modified strains described herein recombinantly express silk-like polypeptide sequences. In some embodiments, the silk-like polypeptide sequence is 1) a block copolymer polypeptide composition generated by mixing and matching repeat domains from the silk polypeptide sequence, and/or 2) Recombinant expression of block copolymer polypeptides large enough in size (approximately 40 kDa) to be secreted from industrially scalable microorganisms to form useful fibers. Large (about 40 kDa to about 100 kDa) block copolymer polypeptides engineered from silk repeat domain fragments, including sequences derived from nearly all published spider silk polypeptide amino acid sequences, are described herein. It can be expressed in modified microorganisms described in the literature. In some embodiments, the silk polypeptide sequences are matched and designed to produce a highly expressed and highly secreted polypeptide that is capable of forming fibrils. In some embodiments, knocking out the protease gene or reducing protease activity in the host engineered strain inhibits degradation of the silk-like polypeptide.

Provided herein, in some embodiments, are compositions for expressing and secreting engineered block copolymers by exhaustively combining and mixing silk polypeptide domains throughout the silk polypeptide sequence space. , wherein such block copolymers undergo minimal degradation. Some embodiments herein provide methods for secreting block copolymers in scalable organisms with minimal degradation (eg, yeast, fungi, and Gram-positive bacteria). In some embodiments, the block copolymer polypeptide comprises zero or more N-terminal domains (NTD), one or more repeat domains (REP), and zero or more C-terminal domains (CTD )including. In some aspects of such embodiments, the block copolymer polypeptide is a single chain polypeptide of greater than 100 amino acids. In some embodiments, the block copolymer polypeptide is a block copolymer disclosed in International Publication No. WO/2015/042164, "Methods and Compositions for Synthesizing Improved Silk Fibers," which is incorporated by reference in its entirety. at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% of the sequence of the polypeptide , 95%, 96%, 97%, 98%, or 99% identical domains.

Several species of natural spider silk have been identified so far. Various mechanical properties of naturally spun spider silk are believed to be closely related to the molecular composition of the spider silk. For example, 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).

Exemplification Grape-like (AcSp) silk tends to be tough as a result of a combination of moderately high strength and moderately high stretchability. AcSp silks are characterized by large block (“aggregate repeat”) sizes that often incorporate polyserine and GPX motifs. Tubular (TuSp or cylindrical) silk threads tend to have large diameters, moderate strength and high stretchability. TuSp silk is characterized by its polyserine and polythreonine content and short polyalanine sequences. Large bottle (MaSp) silk tends to have high strength and moderate stretchability. MaSp silk can be either one of two subtypes, MaSp1 and MaSp2. MaSp1 silks are generally less stretchable than MaSp2 silks and feature polyalanine, GX, and GGX motifs. MaSp2 silk features polyalanine, GGX, and GPX motifs. Villial (MiSp) silk tends to have moderate strength and moderate stretchability. MiSp silks are characterized by GGX, GA, and polyA motifs and often contain spacer elements of about 100 amino acids. Flag silks tend to have very high stretchability and moderate strength. Flag silks are usually characterized by motifs of GPG, GGX, and short spacers.

The properties of each silk species can vary from species to species, and spiders with different lifestyles (e.g., stationary web-building spiders versus wandering and predatory spiders) or evolutionarily older spiders may produce different silk than described above (a description of spider diversity and taxonomy can be found in (Hormiga, G., and Griswold, C. E., Systematics, phylogeny, and evolution of orb-weaving spiders 59, pg.487-512 (2014), and Blackedge, TA et al., Reconstructing web evolution and spider diversification in the molecular era, Proc. S. A., 106:13, pg.5229-5234 (2009)), however, synthetic block copolymer poly(s) with sequence similarity and/or amino acid composition similarity to repeat domains of natural silk proteins. Using peptides, it is possible to produce consistent silk-like fibers on a commercial scale that reproduce properties corresponding to natural silk fibers.

In some embodiments, a list of putative silk sequences can be collected, e.g., by searching GenBank for related terms such as "spidroin", "fibroin", "MaSp", etc., and the sequences are collected separately. Can be pooled together with additional sequences obtained from sequencing efforts. The sequence is then translated into amino acids, duplicate entries are filtered, and manually divided into domains (NTD, REP, CTD). In some embodiments, the candidate amino acid sequence is back-translated into a DNA sequence optimized for expression in Pichia (Komagataella) pastoris. Each DNA sequence is cloned into an expression vector and transformed into Pichia (Komagataella) pastoris. In some embodiments, the various silk domains that have demonstrated successful expression and secretion are then assembled in a combinatorial fashion to construct a silk molecule capable of fiber formation.

Silk polypeptides are characteristically composed of repetitive domains (REPs) flanked by non-repetitive regions (eg, C-terminal and N-terminal domains). In one embodiment, both the C-terminal domain and the N-terminal domain are 75-350 amino acids long. Repeat domains exhibit a hierarchical structure as shown in FIG. A repeat domain comprises a series of blocks (also called repeat units). The blocks are sometimes completely and sometimes imperfectly repeated (constituting quasi-repetitive domains) throughout the silk repeat domain. The length and composition of the blocks vary between different silk types and between different species. Table 1 lists example block sequences for selected species and silk species; further examples are found 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. et al. , Extreme diversity, conservation, and convergence of spider silk fibroin sequences, Science, 291:5513, pg. 2603-2605 (2001). In some cases, blocks may be arranged in a regular pattern to form larger macrorepeats that occur multiple times (usually 2-8 times) in repeat domains of silk sequences. A spacing element may separate a repeated block within a repeat domain or macrorepeat and a repeated macrorepeat within the repeat domain. In some embodiments, the blocking sequence comprises a glycine-rich region followed by a poly A region. In some embodiments, short (about 1-10) amino acid motifs occur multiple times within a block. For the purposes of the present invention, blocks derived from different natural silk polypeptides can be selected regardless of circular permutation (i.e. circular permutation if identified blocks are otherwise similar among silk polypeptides). may not be sorted due to permutation). Thus, for example, a "block" of SGAGG (SEQ ID NO:494) is for the purposes of the present invention the same as GSGAG (SEQ ID NO:495) and the same as GGSGA (SEQ ID NO:496); They are all just circular permutations of each other. The particular permutation chosen for a given silk sequence may be determined by convenience (usually starting with G), among other things. Silk sequences obtained from the NCBI database can be divided into blocks and non-repetitive regions.

(Table 1) Block sequence sample

Fibre-forming block copolymer polypeptides derived from block and/or macrorepeat domains, according to certain embodiments of the present 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 from de novo sequencing are disrupted by domains (N-terminal domain, repeat domain, and C-terminal domain). The N-terminal and C-terminal domain sequences selected for post-synthetic assembly into fibers include the native amino acid sequence information and other modifications described herein. Repeat domains are broken down into repetitive sequences, which contain representative blocks (usually 1-8, depending on the type of silk) that capture important amino acid information, while the DNA encoding the amino acids Reduce the size to easily synthesizable fragments. In some embodiments, a suitably formed block copolymer polypeptide comprises at least one repeat domain comprising at least one repeat sequence, optionally flanked by an N-terminal domain and/or a C-terminal domain. Let

In some embodiments, the repeat domain comprises at least one repeat sequence. In some embodiments, the repeat sequence is 150-300 amino acid residues. In some embodiments, the repeat sequence comprises multiple blocks. In some embodiments, the repetitive sequence comprises multiple macrorepeats. In some embodiments, blocks or macrorepeats are split across multiple repeat sequences.

In some embodiments, the repeat sequence begins with a glycine and contains phenylalanine (F), tyrosine (Y), tryptophan (W), cysteine (C), histidine (H), to meet DNA assembly requirements. It cannot end with asparagine (N), methionine (M), or aspartic acid (D). In some embodiments, some repetitive sequences may be altered compared to native sequences. In some embodiments, repetitive sequences can be altered, such as by adding a serine to the C-terminus of the polypeptide (avoiding termination at F, Y, W, C, H, N, M, or D For). In some embodiments, repetitive sequences can be modified by filling incomplete blocks with homologous sequences from another block. In some embodiments, repeat sequences can be altered by rearranging the order of blocks or macrorepeats.

In some embodiments, non-repetitive N-terminal and C-terminal domains can be selected for synthesis. In some embodiments, the N-terminal domain may be due to the removal of a leading signal sequence, such as that identified by SignalP (Peterson, TN, et.Al., SignalP 4.0: Discriminating signal peptides from transmembrane regions, Nat. Methods, 8:10, pg.785-786 (2011).

In some embodiments, the N-terminal domain sequence, repeat sequence, or C-terminal domain sequence is derived from Agelenopsis aperta, Aliatypus gulosus, Aphonopelma seemanni, Aptostichus sp. sp.) AS217, Aptostichus sp. AS220, Araneus diadematus, Araneus gemmoides, Araneus ventricosus, Argiope amoena, Argiope amoena (Argoate amoena) 、アルギオペ・ブルエンニチ（Ａｒｇｉｏｐｅ ｂｒｕｅｎｎｉｃｈｉ）、アルギオペ・トリファスシアタ（Ａｒｇｉｏｐｅ ｔｒｉｆａｓｃｉａｔａ）、アチポイデス・リヴェルシ（Ａｔｙｐｏｉｄｅｓ ｒｉｖｅｒｓｉ）、アヴィキュラリア・ジュルエンシス（Ａｖｉｃｕｌａｒｉａ ｊｕｒｕｅｎｓｉｓ）、ボスリオシルツム・カリフォルニカム（Ｂｏｔｈｒｉｏｃｙｒｔｕｍ ｃａｌｉｆｏｒｎｉｃｕｍ）、デイノピス・スピノサ(Deinopis Spinosa), Diguetia canities, Dolomedes tenebrosus, Euagrus chisoseus, Euprosthenops australis, Gasteracantha mammosacan 、ヒポキルス・トレルリ（Ｈｙｐｏｃｈｉｌｕｓ ｔｈｏｒｅｌｌｉ）、ククルカニア・ヒベルナリス（Ｋｕｋｕｌｃａｎｉａ ｈｉｂｅｒｎａｌｉｓ）、ラトロデクツス・ヘスペルス（Ｌａｔｒｏｄｅｃｔｕｓ ｈｅｓｐｅｒｕｓ）、メガヘクスラ・フルヴァ（Ｍｅｇａｈｅｘｕｒａ ｆｕｌｖａ）、メテペイラ・グランディオサ（Ｍｅｔｅｐｅｉｒａ ｇｒａｎｄｉｏｓａ）、ネフィラ・アンチポディアナ（Ｎｅｐｈｉｌａ ａｎｔｉｐｏｄｉａｎａ ), Nephila clavat a), Nephila clavipes, Nephila madagascariensis, Nephila pilipes, Nephilengys cruentata, Parawixia bistriata, Parawixia vitriata (Peucetia viridans), Plectreurys tristis, Poecilotheria regalis, Tetragnatha kauaiensis, or Uloborus diversus.

In some embodiments, a silk polypeptide nucleotide coding sequence can be operably linked to an alpha mating factor nucleotide coding sequence. In some embodiments, a silk polypeptide nucleotide coding sequence can be operably linked to another endogenous or heterologous secretory signal coding sequence. In some embodiments, a silk polypeptide nucleotide coding sequence can be operably linked to a 3X FLAG nucleotide coding sequence. In some embodiments, the silk polypeptide nucleotide coding sequence is operably linked to other affinity tags such as 6-8 His residues (SEQ ID NO: 520) .

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

In some embodiments, each “repeat unit” of the silk-like polypeptide comprises 2-20 “quasi-repeat” units (ie, _n3 is 2-20). A quasi-repetition need not be an exact repetition. Each repeat may consist of concatenated quasi-repeats. Equation 1 shows the repeat unit composition according to this disclosure and WO2015/042164, which is incorporated by reference. Each silk-like polypeptide may have one or more repeating units defined by Equation 1.
{GGY-[GPG-X ₁ ] _n1 -GPS-(A) _n2 } _n3 (SEQ ID NO: 514) . (equation 1)

The compositional variable X ₁ (referred to as the “motif”) follows any one of the amino acid sequences shown in Equation 2 below, with X ₁ varying randomly within each quasi-repeat unit.
X ₁ = SGGQQ (SEQ ID NO: 515) or GAGQQ (SEQ ID NO: 516) or GQGPY (SEQ ID NO: 517) or AGQQ (SEQ ID NO: 518) or SQ
(equation 2)

Referring again to Equation 1, the constituent elements of the quasi-repeat unit represented in Equation 1 by "GGY-[GPG-X ₁ ] _n1 -GPS" (SEQ ID NO: 521) are referred to as the "first region." . A quasi-repeat unit is formed in part by 4 to 8 repetitions of the first region within the quasi-repeat unit. That is, the value of _n1 indicates the number of first region units repeated within a single quasi-repeated unit, such value of _n1 being any one of 4, 5, 6, 7 or 8. The building block (i.e., the polyA sequence) designated "(A) _n2 " (SEQ ID NO: 522 ) is referred to as the "second region" and contains the amino acid sequence "A" within each quasi-repeat unit. n is formed by repeating _twice (SEQ ID NO: 522) . That is, the value of _n2 indicates the number of second region units repeated within a single quasi-repeat unit, such values of _n2 being 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17, 18, 19, or 20. In some embodiments, the repeat units of the polypeptides of this disclosure have at least 95% sequence identity to the sequences containing the quasi-repeats set forth in Equations 1 and 2. In some embodiments, the repeat units of the polypeptides of the present disclosure have at least 80% sequence identity, or at least 90%, or at least 95% sequence identity to sequences containing quasi-repeats according to Equations 1 and 2, or At least 99%.

In a further embodiment, three "long" quasi-repeats are followed by three "short" quasi-repeat units. Short quasi-repeat units are those with n ₁ =4 or 5. Long quasi-repeat units are defined as those with n ₁ =6, 7 or 8. In some embodiments, both short quasi-repeats have the same X ₁ motif at the same position within each quasi-repeat of the repeat unit. In some embodiments, no more than 3 of the 6 quasi-repeat units share the same _X1 motif.

In a further embodiment, the repeating unit is composed of quasi-repeating units that do not use the same X ₁ three or more times in a row within the repeating unit. In a further embodiment, the repeat unit is composed of quasi-repeat units, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 of the quasi-repeats , 15, 16, 17, 18, 19 or 20 quasi-repeats do not use the same X ₁ more than 3 times consecutively within a single quasi-repeat unit of the repeating unit.

Thus, in some embodiments, the present disclosure recombinantly expresses a low-degradation silk-like polypeptide to increase the amount of full-length polypeptide present in products isolated from cell culture. Provided is a yeast strain that causes In some embodiments, the strain expressing the silk-like polypeptide is P. pastoris strain and contains the PAS_chr4_0584 knockout and the PAS_chr3_1157 knockout.

Equivalents and Ranges Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the invention described herein. would be possible. The scope of the invention is not intended to be limited to the above Detailed Description, but rather the scope is set forth in the appended claims.

In the claims, articles such as "a," "an," and "the" mean one or more than one unless stated otherwise or construed otherwise clearly from the context. obtain. A claim or statement that includes "or" between one or more members of a group indicates that one, more than one, or all of the members of that group are present in a given product or process. used, or otherwise related, shall be deemed to satisfy the requirement unless stated otherwise or construed otherwise clearly from the context. The invention includes embodiments in which exactly one member of the group is present, used, or otherwise related to a given product or process. The invention includes embodiments in which two or more or all of the group members are present, used, or otherwise associated with a given product or process.

It is also noted that the term "comprising" is intended to be open-ended and permissive, but need not include additional elements or steps. Where the term "comprising" is used herein, the term "consisting of" is so included and disclosed.

The endpoints are included if a range is given. Further, unless otherwise stated and the context and understanding of one of ordinary skill in the art make other senses apparent, values expressed as ranges may vary from the stated range in different embodiments of the invention, unless the context clearly dictates otherwise. It is to be understood that any particular value or subrange within can be taken to tenths of the lower limit of that range.

All sources, such as references, publications, databases, database entries, and art cited herein are incorporated by reference into this application even if not explicitly stated in the citation. If there is any discrepancy between the description of the cited source and this application, the description of this application shall prevail.

Subheadings and table titles are not intended to be limiting.

Below are specific example embodiments for carrying out the present invention. These examples are provided for illustration only and are not intended to limit the scope of the invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (eg amounts, temperature, etc.) but some experimental errors and deviations should, of course, be allowed for.

The practice of the present invention employs conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology within the skill of the art, unless otherwise indicated. Such techniques are well described in the literature. For example, T. E. Creighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); L. 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).

Example 1 Generation of Recombinant Yeast Expressing 18B Initially, to facilitate subsequent editing and engineering, P. KU70 function was abrogated by transforming strains of S.pastoris. GS115 (NRRL Y15851), a HIS+ derivative of Pichia pastoris (Komagataella phaffii) strain, was electroporated with a DNA cassette targeting the KU70 locus, consisting of homology arms flanked by zeocin resistance markers. . A map of the cassette is shown in FIG. 1 and the sequences are listed in Table 10. Transformants were plated on YPD agar plates supplemented with zeocin. As a result, KU70 function was inhibited.

This strain was then modified to express a recombinant gene encoding a silk-like polypeptide. GS115 (NRRL Y15851), a HIS+ derivative of Pichia pastoris (Komagataella phaffii) strain, was transformed into a recombinant vector (SEQ ID NO: 463) to effect expression and secretion of silk-like polypeptide (“18B”) (SEQ ID NO: 463). 462). Transformation was accomplished by electroporation as described in PMID 15679083, incorporated herein by reference.

Each vector contains an 18B expression cassette with a polynucleotide sequence encoding a silk-like protein in a recombinant vector, flanked by a promoter (pGCW14) and a terminator (tAOX1 pA signal). The recombinant vector further contained a dominant resistance marker to select for bacterial and yeast transformants, and a bacterial origin of replication. The first recombinant vector contained a directional region that directs the integration of the 18B polynucleotide sequence into the Pichia pastoris genome 3' adjacent to the AOX2 locus. A resistance marker in the first vector conferred resistance to G418 (aka geneticin). The second recombinant vector contained a directional region that directs the integration of the 18B polynucleotide sequence into the Pichia pastoris genome 3' adjacent to the TEF1 locus. A resistance marker in the second vector conferred resistance to hygromycin B.

Example 2: Generation of a library of single protease KO mutants After successful transformation and secretion of 18B in a recombinant Pichia pastoris strain, 65 open reading frames (ORFs) encoding proteases were individually deleted. targeted (Table 2). Cells were transformed with a vector containing a DNA cassette with approximately 1150 bp of homology arms flanked by a nourseothricin resistance marker. A plasmid map containing the nourseothricin resistance marker is shown in FIG. 2 and the sequences are listed in Table 11.

The homology arms used for each target were amplified with the primers listed in Table 7 and inserted into nourseothricin-resistant plasmids. The homology arms were inserted into the nourseothricin plasmid to create a cassette containing a nourseothricin resistance marker flanked by 3' and 5' homology arms to the target protease, as shown in Figures 3A and 3B. FIG. 3A shows the resistance cassette (Nour resistance cassette) flanked by homology arms (HA1 and HA2). FIG. 3B shows the details of the nourseothricin markers, the promoter from the ILV5 gene of Saccharomyces cerevisiae (pILV5), the promoter of the nourseothricin acetyltransferase from Streptomyces noursei (nat). genes and the polyA signal from the CYC1 gene of Saccharomyces cerevisiae.

The homology arms of each vector targeted one of the 65 desired protease loci listed in Table 2. Transformants were plated on YPD agar plates supplemented with nourseothricin and incubated at 30° C. for 48 hours.

(Table 2) P.I. Proteases targeted for deletion in Pastoris strains

Example 3: Testing of single protease knockout clones for low proteolytic degradation.
The resulting clones were inoculated into 400 μL buffered glycerol complex medium (BMGY) in a 96-well block and incubated at 30° C. for 48 hours with agitation at 1,000 rpm. After 48 hours of incubation, 4 μL of each culture was used to inoculate 400 μL of BMGY in a 96-well block, followed by incubation at 30° C. for 48 hours. To extract the recombinant protein, guanidine thiocyanate was added to the cell culture to a final concentration of 2.5M. After 5 minutes of incubation, the solution was centrifuged and the supernatant was collected and analyzed by Western blot.

Western blot data for representative clones of each protease knockout are shown in FIG. Single protease deletion had no discernible effect on the distribution of silk fragments detected by Western blot.

Example 4 Generation of Double Knockout Libraries of Proteases In addition to individual KOs, different combinations of proteases in different pairs were knocked out. These proteases were chosen in part because they were paralogs that could have compensatory functions for each other.

To generate a double knockout, nourseothricin resistance was eliminated from the single protease knockout strain generated in Example 2, and the second protease was deleted by transformation with the second nourseothricin resistance cassette described in Example 2. let me Transformants were plated on YPD agar plates supplemented with nourseothricin and incubated at 30° C. for 48 hours. The double protease knockouts tested are listed in Table 3.

(Table 3) P. cerevisiae expressing silk-like polypeptides. Protease double KO strain of Pastoris

Example 5 Testing Double Protease Knockout Clones for Low Proteolytic Degradability The resulting clones were inoculated into 96-well blocks of 400 μL buffered glycerol complex medium (BMGY) and stirred at 1,000 rpm at 30° C. for 48 hours. incubated. After 48 hours of incubation, 4 μL of each culture was used to inoculate 400 μL of BMGY in a 96-well block, followed by incubation at 30° C. for 48 hours. To extract the recombinant protein, guanidine thiocyanate was added to the cell culture to a final concentration of 2.5M. After 5 minutes of incubation, the solution was centrifuged and the supernatant was collected and analyzed by Western blot.

Figure 4 shows representative results from different protease double knockout strains. As shown, the combination of protease knockouts PAS_chr4_0584 and PAS_chr3_1157 (strain 3 in Table 3) resulted in almost complete loss of degradation products, despite the presence of proteolysis in all single knockout strains tested. brought None of the other combinations of proteases resulted in degradation products disappearance.

Example 6: Additional Protease Knockout Strains As shown in Examples 4 and 5, modified Pichia pastoris cells capable of producing a desired protein (e.g., 18B) were transfected with PAS_chr4_0584 and PAS_chr3_1157 to attenuate the degradation of the desired protein. was transformed to lack the protease of One or more additional proteases were additionally knocked out to enhance production of full-length product and minimize degradation.

For each additional knockout, the additional protease gene was converted to single protease KO (1X KO), double protease KO (2X KO) by transformation with a nourseothricin resistance cassette with homology arms pointing to the desired gene as described in Example 2. , triple protease KO (3X KO), or quadruple protease KO (4X KO). Table 4 shows the protease gene knocked out in each strain.

(Table 4) 2X to 5X KO strains

The resulting cells were isolated on selective media plates (by auxotrophic or antibiotic resistance markers) and individual clones were isolated for further testing. Individual clones were tested in a liquid culture assay under product protein production conditions as follows. Briefly, isolated colonies of each strain were inoculated into 400 μL of buffered glycerol complex medium (BMGY) in a 96-well block and incubated at 30° C. with agitation at 1,000 rpm for 48 hours. After 48 hours of incubation, 4 μL of each culture was used to inoculate 400 μL of BMGY or 400 μL of YPD (yeast extract-peptone-dextrose medium) in a 96-well block, followed by 30 μL with agitation at 1,000 rpm. °C for 48 hours.

Proteins expressed by the cells were isolated and analyzed for degradation as follows. Briefly, to extract the recombinant protein, guanidine thiocyanate was added to the cell culture to a final concentration of 2.5M. After 5 minutes of incubation, the solution was centrifuged and the supernatant was collected and analyzed by Western blot.

Figure 5 shows Western blot results of purified proteins from 2X KO, 3X KO, 4X KO and 5X KO strains inoculated in BMGY or YPD. As shown, deletion of additional protease genes from the strain with the PAS_chr4_0584+PAS_chr3_1157 protease knockout (strain 3 in Table 3) resulted in further loss of degradation products.

Other Embodiments The words that have been used are words of description rather than of limitation, and the broader scope of the invention is set forth in the appended claims without departing from the true scope and spirit of the invention. It should be understood that changes may be made in aspects.

Although the present invention has been described at considerable length and in considerable detail with respect to several described embodiments, it is limited to any such individual details or embodiments or to any specific embodiments. Rather, to obtain the broadest possible interpretation of the appended claims in light of the prior art, any interpretation of such claims may be made on the basis of such claims. and so the intended scope of the invention should be effectively covered.

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. Further, the section headings, materials, methods, and examples are intended to be illustrative only and not limiting.

Sequence Listing (Table 5) P.S. Nucleotide sequences of open reading frames of proteases targeted for deletion in pastoris

(Table 6) Polypeptide sequences of target proteases

(Table 7) Forward primer (F) and reverse primer (R) for both 5' and 3' homology arms (HA) targeting the protease ORF

(Table 8) Forward and reverse primers that amplify modified sequences

(Table 9) 18B vector

(Table 10) P.I. Zeocin cassette with HA arms for KU70 deletion in pastoris

(Table 11)P. Noseothricin cassette for protease deletion in pastoris

(Table 12) P.I. Exemplary nourseothricin cassettes with HA arms for protease deletion in pastoris

Claims (17)

A Pichia pastoris microorganism having attenuated or abolished YPS1-1 protease and YPS1-2 protease activity expressing a recombinant protein, said recombinant protein comprising at least one Said microorganism comprising a blocked polypeptide sequence or wherein said recombinant protein comprises a silk-like polypeptide . 2. The microorganism of claim 1, wherein said YPS1-1 protease comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:67. 2. The microorganism of claim 1, wherein said YPS1-1 protease comprises SEQ ID NO:67. Said YPS1-1 protease is encoded by the YPS1-1 gene, said YPS1-1 gene comprising a polynucleotide sequence at least 95% identical to SEQ ID NO: 1, or said YPS1-1 gene comprises SEQ ID NO: at least 15, 20, 25, 30, 40, or 50 contiguous nucleotides of: 1, or said YPS1-1 gene comprises SEQ ID NO: 1, or said YPS1-1 gene is a gene of said microorganism 3. A microorganism according to claim 1 or claim 2 at the locus PAS_chr4_0584. 5. The microorganism of any one of claims 1-4, wherein the YPS1-2 protease comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:68. 6. The microorganism of claim 5, wherein said YPS1-2 protease comprises SEQ ID NO:68. Said YPS1-2 protease is encoded by the YPS1-2 gene, wherein said YPS1-2 gene comprises a polynucleotide sequence at least 95% identical to SEQ ID NO:2, or said YPS1-2 gene comprises SEQ ID NO: at least 15, 20, 25, 30, 40, or 50 contiguous nucleotides of: 2, or said YPS1-2 gene comprises SEQ ID NO: 2, or said YPS1-2 gene is a gene of said microorganism A microorganism according to any one of claims 1 to 6, at the locus PAS_chr3_1157. The microorganism according to any one of claims 1 to 7, wherein said YPS1-1 gene or said YPS1-2 gene or both are mutated or knocked out. said silk-like polypeptide comprises one or more repeat sequences {GGY-[GPG-X ₁ ] _n1 -GPS-(A) _n2 } _n3 (SEQ ID NO: 514), wherein
X1 = SGGQQ (SEQ ID NO: 515) or GAGQQ (SEQ ID NO: 516) or GQGPY (SEQ ID NO: 517) or AGQQ (SEQ ID NO: 518) or SQ,
n1 is 4 to 8,
n2 is 6-20, and n3 is 2-20;
The microorganism according to claim 1 . 2. The microorganism of claim 1 , wherein said silk-like polypeptide comprises a polypeptide sequence encoded by SEQ ID NO:462. Microorganism according to any one of claims 1 to 10 , wherein the activity of one or more additional proteases is attenuated or eliminated. 12. The microorganism of claim 11 , wherein said one or more additional proteases comprise YPS1-5, MCK7, or YPS1-3. YPS1-1 gene comprising SEQ ID NO: 1 and YPS1-2 gene comprising SEQ ID NO: 2 are mutated or deleted to reduce the activity of said YPS1-1 and said YPS1-2, Pichia A modified microorganism of pastoris,
Said microorganism, further comprising a recombinantly expressed protein comprising a polypeptide sequence encoded by SEQ ID NO:462. A cell culture comprising a microorganism according to any one of claims 1-13 . culturing the microorganism of any one of claims 1 to 13 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. A method for producing a recombinant protein with reduced degradation, comprising: A method of modifying Pichia pastoris such that degradation of a recombinantly expressed silk-like polypeptide is inhibited, comprising:
The above method, comprising knocking out or mutating the genes encoding the YPS1-1 protein and the YPS1-2 protein. said silk-like polypeptide comprises one or more repeat sequences {GGY-[GPG-X ₁ ] _n1 -GPS-(A) _n2 } _n3 (SEQ ID NO: 514), wherein
X ₁ = SGGQQ (SEQ ID NO: 515) or GAGQQ (SEQ ID NO: 516) or GQGPY (SEQ ID NO: 517) or AGQQ (SEQ ID NO: 518) or SQ,
n1 is 4 to 8,
n2 is 6-20 and n3 is 2-20, or said silk-like polypeptide comprises a polypeptide sequence encoded by SEQ ID NO:462;
17. The method of claim 16 .

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