# US 2020/0399646 A9 · Constructs and cells for enhanced protein expression

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- Number: US 2020/0399646 A9
- Document ID: US20200399646A9
- Status: Published application
- Kind: A9
- Application number: 16/080,844
- Filed: 2018-01-10
- Published: 2020-12-24
- Earliest claimed: 2017-01-10
- Assignees: Massachusetts Institute of Technology
- Applicants: Massachusetts Institute of Technology
- Inventors: Kerry R. Love; J. Christopher Love; Charles Whittaker; Joseph Brady; Catherine Bartlett Matthews; Noelle Colant; Neil C. Dalvie
- Web page: https://patentgrep.com/patent/US20200399646A9

## CPC

- C12N 15/81: for yeasts
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

Described are expression constructs, cells, and methods of producing proteins in Pichia pastoris.

## Claims

### Claim 1

An expression construct comprising an OLE1 promoter operably linked to a nucleic acid encoding a polypeptide comprising a signal sequence and a heterologous protein.

### Claim 2 (depends on 1)

The expression construct of claim 1, wherein the signal sequence is identical to the signal sequence of a naturally occurring yeast protein.

### Claim 3 (depends on 1, 2)

The expression construct of claim 1 or 2, wherein the signal sequence is the signal sequence of SCW11, MSC1, EXG1, 0841, 1286, BGL2, 2488, 2848, PRY2, 4355, or PIR1.

### Claim 4 (depends on 1, 2, 3)

The expression construct of any one of claims 1 to 3, wherein the OLE1 promoter has at least 95% homology with SEQ ID NO: 1 or a fragment thereof.

### Claim 5 (depends on 4)

The expression construct of claim 4, wherein the OLE1 promoter has the sequence SEQ ID NO: 1.

### Claim 6 (depends on 1, 2, 3, 4, 5)

The expression construct of any one of claims 1 to 5, wherein the expression construct is a plasmid or viral vector.

### Claim 7 (depends on 6)

The expression construct of claim 6, wherein the plasmid is an episomal plasmid or an integrative plasmid.

### Claim 8 (depends on 1, 2, 3, 4, 5, 6, 7)

The expression construct of any one of claims 1 to 7, wherein the expression construct is linearized.

### Claim 9 (depends on 1, 2, 3, 4, 5, 6, 7, 8)

The expression construct of any one of claims 1 to 8, wherein the heterologous protein is selected from the group consisting of enzymes, hormones, antibodies or antigen binding fragments thereof, vaccine components, blood factors, thrombolytic agents, cytokines, receptors, and fusion proteins.

### Claim 10

A methylotrophic cell expressing a heterologous protein, wherein the expression is under the control of an OLE1 promoter.

### Claim 11 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10)

The methylotrophic cell of claim 10, wherein the cell has been transformed by the expression construct of any of claims 1-9.

### Claim 12 (depends on 10, 11)

The methylotrophic cell of claim 10 or 11, wherein the OLE1 promoter is located at the OLE1, AOX1, GAPDH, DAS2, or PIF1 locus.

### Claim 13 (depends on 10, 11, 12)

The methylotrophic cell of any one of claims 10 to 12, wherein the methylotrophic cell is a yeast cell.

### Claim 14 (depends on 13)

The methylotrophic cell of claim 13, wherein the yeast cell is a Pichia pastoris cell.

### Claim 15 (depends on 14)

The methylotrophic cell of claim 14, wherein the Pichia pastoris cell is a Komagataella phaffii or Komagataella pastoris cell.

### Claim 16 (depends on 15)

The methylotrophic cell of claim 15, wherein the Komagataella phaffii cell is a Komagataella phaffii Y-11430, Y-7556, YB-4290, Y-12729, Y-17741, Y-48123, Y-48124, YB-378, YB-4289, GS115, KM71H, SMD1168, SMD11681H, or X-33 cell.

### Claim 17 (depends on 10, 11, 12, 13, 14, 15, 16)

The methylotrophic cell of any one of claims 10 to 16, wherein the OLE1 promoter has at least 95% homology with SEQ ID NO: 1 or a fragment thereof.

### Claim 18 (depends on 17)

The methylotrophic cell of claim 17, wherein the OLE1 promoter has the sequence SEQ ID NO: 1.

### Claim 19 (depends on 10, 11, 12, 13, 14, 15, 16, 17, 18)

The methylotrophic cell of any one of claims 10 to 18, wherein the heterologous protein is selected from the group consisting of enzymes, hormones, antibodies or antigen binding fragments thereof, vaccine components, blood factors, thrombolytic agents, cytokines, receptors, and fusion proteins.

### Claim 20 (depends on 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)

The methylotrophic cell of any one of claims 10 to 19, further comprising a signal sequence fused to the heterologous protein.

### Claim 21 (depends on 20)

The methylotrophic cell of claim 20, wherein the signal sequence is the signal sequence of SCW11, MSC1, EXG1, 0841, 1286, BGL2, 2488, 2848, PRY2, 4355, or PIR1.

### Claim 22

An expression construct comprising a DAS2 promoter operably linked to a nucleic acid encoding a polypeptide comprising a signal sequence and a heterologous protein and a targeting sequence for integration in a methylotrophic cell at a non-native locus.

### Claim 23 (depends on 22)

The expression construct of claim 22, wherein the signal sequence is identical to the signal sequence of a naturally occurring yeast protein.

### Claim 24 (depends on 22, 23)

The expression construct of claim 22 or 23, wherein the signal sequence is the signal sequence of SCW11, MSC1, EXG1, 0841, 1286, BGL2, 2488, 2848, PRY2, 4355, or PIR1.

### Claim 25 (depends on 22, 23, 24)

The expression construct of any one of claims 22-24, wherein the DAS2 promoter has at least 95% homology with SEQ ID NO: 2 or a fragment thereof.

### Claim 26 (depends on 25)

The expression construct of claim 25, wherein the DAS2 promoter has the sequence SEQ ID NO: 2.

### Claim 27 (depends on 22, 23, 24, 25, 26)

The expression construct of any one of claims 22-26, wherein the expression construct is a plasmid or viral vector.

### Claim 28 (depends on 27)

The expression construct of claim 27, wherein the plasmid is an episomal plasmid or an integrative plasmid.

### Claim 29 (depends on 22, 23, 24, 25, 26, 27, 28)

The expression construct of any one of claims 22 to 28, wherein the expression construct is linearized.

### Claim 30 (depends on 22, 23, 24, 25, 26, 27, 28, 29)

The expression construct of any one of claims 22-29, wherein the heterologous protein is selected from the group consisting of enzymes, hormones, antibodies or antigen binding fragments thereof, vaccine components, blood factors, thrombolytic agents, cytokines, receptors, and fusion proteins.

### Claim 31 (depends on 22, 23, 24, 25, 26, 27, 28, 29, 30)

The expression construct of any one of claims 22-30, wherein the expression construct further comprises a Kozak sequence beginning at the −3 position relative to the translation start site of the nucleic acid encoding the polypeptide.

### Claim 32 (depends on 31)

The expression construct of claim 31, wherein the Kozak sequence comprises:
(i) the sequence ANAATGNC, wherein N comprises A, T, G, or C; or
(ii) the sequence AMMATG, wherein M comprises A or C.

### Claim 33 (depends on 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)

The expression construct of any one of claims 22-32, wherein a mRNA secondary structure of the nucleic acid encoding a polypeptide has been reduced or eliminated relative to the endogenous nucleic acid encoding the polypeptide.

### Claim 34 (depends on 33)

The expression construct of claim 33, wherein the mRNA secondary structure is selected from a hairpin loop, a duplex, a single-stranded region, a hairpin, a bulge, an internal loop, or any other structure as predicted by likelihood of pairing and/or low free energy.

### Claim 35

A methylotrophic cell expressing a heterologous protein, wherein the expression is under the control of a DAS2 promoter integrated at a non-native locus.

### Claim 36 (depends on 35)

The methylotrophic cell of claim 35, wherein the non-native locus is an OLE1, AOX1, GAPDH, or PIF1 locus.

### Claim 37 (depends on 35, 36)

The methylotrophic cell of claim 35 or 36, wherein the methylotrophic cell is a yeast cell.

### Claim 38 (depends on 37)

The methylotrophic cell of claim 37, wherein the yeast cell is a Pichia pastoris cell.

### Claim 39 (depends on 38)

The methylotrophic cell of claim 38, wherein the Pichia pastoris cell is a Komagataella phaffii or Komagataella pastoris cell.

### Claim 40 (depends on 39)

The methylotrophic cell of claim 39, wherein the Komagataella phaffii cell is a Komagataella phaffii Y-11430, Y-7556, YB-4290, Y-12729, Y-17741, Y-48123, Y-48124, YB-378, YB-4289, GS115, KM71H, SMD1168, SMD1168H, or X-33 cell.

### Claim 41 (depends on 35, 36, 37, 38, 39, 40)

The methylotrophic cell of any one of claims 35 to 40, wherein the DAS2 promoter has at least 95% homology with SEQ ID NO: 2.

### Claim 42 (depends on 41)

The methylotrophic cell of claim 41, wherein the DAS2 promoter has the sequence SEQ ID NO: 2.

### Claim 43 (depends on 35, 36, 37, 38, 39, 40, 41, 42)

The methylotrophic cell of any one of claims 35 to 42, wherein the heterologous protein is selected from the group consisting of enzymes, hormones, antibodies or antigen binding fragments thereof, vaccine components, blood factors, thrombolytic agents, cytokines, receptors, and fusion proteins.

### Claim 44 (depends on 35, 36, 37, 38, 39, 40, 41, 42, 43)

The methylotrophic cell of any one of claims 35 to 43, further comprising a signal sequence fused to the heterologous protein.

### Claim 45 (depends on 44)

The methylotrophic cell of claim 44, wherein the signal sequence is the signal sequence of SCW11, MSC1, EXG1, 0841, 1286, BGL2, 2488, 2848, PRY2, 4355, or PIR1.

### Claim 46 (depends on 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45)

The methylotrophic cell of any one of claims 35-45, wherein the mRNA encoding the heterologous protein comprises a Kozak sequence beginning at the −3 position relative to the translation start site.

### Claim 47 (depends on 46)

The methylotrophic cell of claim 46, wherein the Kozak sequence comprises:
(i) the sequence ANAATGNC, wherein N comprises A, T, G, or C; or
(ii) the sequence AMMATG, wherein M comprises A or C.

### Claim 48 (depends on 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47)

The methylotrophic cell of any one of claims 35-47, wherein a mRNA secondary structure of the mRNA encoding the heterologous protein has been reduced or eliminated relative to the endogenous mRNA encoding the heterologous protein.

### Claim 49 (depends on 48)

The methylotrophic cell of claim 48, wherein the mRNA secondary structure is selected from a hairpin loop, a duplex, a single-stranded region, a hairpin, a bulge, an internal loop, or any other structure as predicted by likelihood of pairing and/or low free energy.

### Claim 50

An expression construct comprising an AOX1 promoter operably linked to a nucleic acid encoding a polypeptide comprising a signal sequence and a heterologous protein, the construct further comprising a targeting sequence for integration in a methylotrophic cell at a PIF1, OLE1, or DAS2 locus.

### Claim 51 (depends on 50)

The expression construct of claim 50, wherein the signal sequence is identical to the signal sequence of a naturally occurring yeast protein.

### Claim 52 (depends on 50, 51)

The expression construct of claim 50-51, wherein the signal sequence is the signal sequence of SCW11, MSC1, EXG1, 0841, 1286, BGL2, 2488, 2848, PRY2, 4355, or PIR1.

### Claim 53 (depends on 50, 51, 52)

The expression construct of any one of claims 50-52, wherein the AOX1 promoter has at least 95% homology with SEQ ID NO: 3 or a fragment thereof.

### Claim 54 (depends on 53)

The expression construct of claim 53, wherein the AOX1 promoter has the sequence SEQ ID NO: 3.

### Claim 55 (depends on 50, 51, 52, 53, 54)

The expression construct of any one of claims 50-54, wherein the expression construct is a plasmid or viral vector.

### Claim 56 (depends on 55)

The expression construct of claim 55, wherein the plasmid is an episomal plasmid or an integrative plasmid.

### Claim 57 (depends on 50, 51, 52, 53, 54, 55, 56)

The expression construct of any one of claims 50-56, wherein the expression construct is linearized.

### Claim 58 (depends on 50, 51, 52, 53, 54, 55, 56, 57)

The expression construct of any one of claims 50-57, wherein the heterologous protein is selected from the group consisting of enzymes, hormones, antibodies or antigen binding fragments thereof, vaccine components, blood factors, thrombolytic agents, cytokines, receptors, and fusion proteins.

### Claim 59 (depends on 50, 51, 52, 53, 54, 55, 56, 57, 58)

The expression construct of any one of claims 50-58, wherein the expression construct further comprises a Kozak sequence beginning at the −3 position relative to the translation start site of the nucleic acid encoding the polypeptide.

### Claim 60 (depends on 59)

The expression construct of claim 59, wherein the Kozak sequence comprises:
(i) the sequence ANAATGNC, wherein N comprises A, T, G, or C; or
(ii) the sequence AMMATG, wherein M comprises A or C.

### Claim 61 (depends on 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60)

The expression construct of any one of claims 50-60, wherein a mRNA secondary structure of the nucleic acid encoding a polypeptide has been reduced or eliminated relative to the endogenous nucleic acid encoding the polypeptide.

### Claim 62 (depends on 61)

The expression construct of claim 61, wherein the mRNA secondary structure is selected from a hairpin loop, a duplex, a single-stranded region, a hairpin, a bulge, an internal loop, or any other structure as predicted by likelihood of pairing and/or low free energy.

### Claim 63

A methylotrophic cell expressing a heterologous protein, wherein the expression is under the control of an AOX1 promoter integrated at a PIF1, OLE1, or DAS2 locus.

### Claim 64 (depends on 63)

The methylotrophic cell of claim 63, wherein the methylotrophic cell is a yeast cell.

### Claim 65 (depends on 64)

The methylotrophic cell of claim 64, wherein the yeast cell is a Pichia pastoris cell.

### Claim 66 (depends on 65)

The methylotrophic cell of claim 65, wherein the Pichia pastoris cell is a Komagataella phaffii or Komagataella pastoris cell.

### Claim 67 (depends on 66)

The methylotrophic cell of claim 66, wherein the Komagataella phaffii cell is a Komagataella phaffii
Y-11430, Y-7556, YB-4290, Y-12729, Y-17741, Y-48123, Y-48124, YB-378, YB-4289, GS115, KM71H, SMD1168, SMD1168H, or X-33 cell.

### Claim 68 (depends on 63, 64, 65, 66, 67)

The methylotrophic cell of any one of claims 63-67, wherein the AOX1 promoter has at least 95% homology with SEQ ID NO: 3.

### Claim 69 (depends on 68)

The methylotrophic cell of claim 68, wherein the AOX1 has the sequence SEQ ID NO: 3.

### Claim 70 (depends on 63, 64, 65, 66, 67, 68, 69)

The methylotrophic cell of any one of claims 63-69, wherein the heterologous protein is selected from the group consisting of enzymes, hormones, antibodies or antigen binding fragments thereof, vaccine components, blood factors, thrombolytic agents, cytokines, receptors, and fusion proteins.

### Claim 71 (depends on 63, 64, 65, 66, 67, 68, 69, 70)

The methylotrophic cell of any one of claims 63-70, further comprising a signal sequence fused to the heterologous protein.

### Claim 72 (depends on 71)

The methylotrophic cell of claim 71, wherein the signal sequence is the signal sequence of SCW11, MSC1, EXG1, 0841, 1286, BGL2, 2488, 2848, PRY2, 4355, or PIR1.

### Claim 73 (depends on 63, 64, 65, 66, 67, 68, 69, 70, 71, 72)

The methylotrophic cell of any one of claims 63-72, wherein the mRNA encoding the heterologous protein comprises a Kozak sequence beginning at the −3 position relative to the translation start site.

### Claim 74 (depends on 73)

The methylotrophic cell of claim 73, wherein the Kozak sequence comprises:
(i) the sequence ANAATGNC, wherein N comprises A, T, Ci, or C; or
(ii) the sequence AMMATG, wherein M comprises A or C.

### Claim 75 (depends on 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74)

The methylotrophic cell of any one of claims 63-74, wherein a mRNA secondary structure of the mRNA encoding the heterologous protein has been reduced or eliminated relative to the endogenous mRNA encoding the heterologous protein.

### Claim 76 (depends on 75)

The methylotrophic cell of claim 75, wherein the mRNA secondary structure is selected from a hairpin loop, a duplex, a single-stranded region, a hairpin, a bulge, an internal loop, or any other structure as predicted by likelihood of pairing and/or low free energy.

### Claim 77

An expression construct comprising a GAPDH promoter operably linked to a nucleic acid encoding a polypeptide comprising a signal sequence and a heterologous protein, the construct further comprising a targeting sequence for integration in a methylotrophic cell at an AOX1, PIF1, OLE1, or DAS2 locus.

### Claim 78 (depends on 77)

The expression construct of claim 77, wherein the signal sequence is identical to the signal sequence of a naturally occurring yeast protein.

### Claim 79 (depends on 77, 78)

The expression construct of claim 77 or 78, wherein the signal sequence is the signal sequence of SCW11, MSC1, EXG1, 0841, 1286, BGL2, 2488, 2848, PRY2, 4355, or PIR1.

### Claim 80 (depends on 77, 78, 79)

The expression construct of any one of claims 77-79, wherein the GAPDH promoter has at least 95% homology with SEQ ID NO: 4 or a fragment thereof.

### Claim 81 (depends on 80)

The expression construct of claim 80, wherein the GAPDH promoter has the sequence SEQ ID NO: 4.

### Claim 82 (depends on 77, 78, 79, 80, 81)

The expression construct of any one of claims 77-81, wherein the expression construct is a plasmid or viral vector.

### Claim 83 (depends on 82)

The expression construct of claim 82, wherein the plasmid is an episomal plasmid or an integrative plasmid.

### Claim 84 (depends on 77, 78, 79, 80, 81, 82, 83)

The expression construct of any one of claims 77-83, wherein the expression construct is linearized.

### Claim 85 (depends on 77, 78, 79, 80, 81, 82, 83, 84)

The expression construct of any one of claims 77-84, wherein the heterologous protein is selected from the group consisting of enzymes, hormones, antibodies or antigen binding fragments thereof, vaccine components, blood factors, thrombolytic agents, cytokines, receptors, and fusion proteins.

### Claim 86 (depends on 77, 78, 79, 80, 81, 82, 83, 84, 85)

The expression construct of any one of claims 77-85, wherein the expression construct further comprises a Kozak sequence beginning at the −3 position relative to the translation start site of the nucleic acid encoding the polypeptide.

### Claim 87 (depends on 86)

The expression construct of claim 86, wherein the Kozak sequence comprises:
(i) the sequence ANAATGNC, wherein N comprises A, T, G, or C; or
(ii) the sequence AMMATG, wherein M comprises A or C.

### Claim 88 (depends on 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87)

The expression construct of any one of claims 77-87, wherein a mRNA secondary structure of the nucleic acid encoding a polypeptide has been reduced or eliminated relative to the endogenous nucleic acid encoding the polypeptide.

### Claim 89 (depends on 88)

The expression construct of claim 88, wherein the mRNA secondary structure is selected from a hairpin loop, a duplex, a single-stranded region, a hairpin, a bulge, an internal loop, or any other structure as predicted by likelihood of pairing and/or low free energy.

### Claim 90

A methylotrophic cell expressing a heterologous protein, wherein the expression is under the control of a GAPDH promoter integrated at an AOX1, PIF1, OLE1, or DAS2 locus.

### Claim 91 (depends on 90)

The methylotrophic cell of claim 90, wherein the methylotrophic cell is a yeast cell.

### Claim 92 (depends on 91)

The methylotrophic cell of claim 91, wherein the yeast cell is a Pichia pastoris cell.

### Claim 93 (depends on 92)

The methylotrophic cell of claim 92, wherein the Pichia pastoris cell is a Komagataella phaffii or Komagataella pastoris cell.

### Claim 94 (depends on 93)

The methylotrophic cell of claim 93, wherein the Komagataella phaffii cell is a Komagataella phaffii
Y-11430, Y-7556, YB-4290, Y-12729, Y-17741, Y-48123, Y-48124, YB-378, YB-4289, GS115, KM71H, SMD1168, SMD1168H, or X-33 cell.

### Claim 95 (depends on 90, 91, 92, 93, 94)

The methylotrophic cell of any one of claims 90-94, wherein the GAPDH promoter has at least 95% homology with SEQ ID NO: 4.

### Claim 96 (depends on 95)

The methylotrophic cell of claim 95, wherein the GAPDH promoter has the sequence SEQ ID NO: 4.

### Claim 97 (depends on 90, 91, 92, 93, 94, 95, 96)

The methylotrophic cell of any one of claims 90-96, wherein the heterologous protein is selected from the group consisting of enzymes, hormones, antibodies or antigen binding fragments thereof, vaccine components, blood factors, thrombolytic agents, cytokines, receptors, and fusion proteins.

### Claim 98 (depends on 90, 91, 92, 93, 94, 95, 96, 97)

The methylotrophic cell of any one of claims 90-97, further comprising a signal sequence fused to the heterologous protein.

### Claim 99 (depends on 98)

The methylotrophic cell of claim 98, wherein the signal sequence is the signal sequence of SCW11, MSC1, EXG1, 0841, 1286, BGL2, 2488, 2848, PRY2, 4355, or PIR1.

### Claim 100 (depends on 90, 91, 92, 93, 94, 95, 96, 97, 98, 99)

The methylotrophic cell of any one of claims 90-99, wherein the mRNA encoding the heterologous protein comprises a Kozak sequence beginning at the −3 position relative to the translation start site.

### Claim 101 (depends on 100)

The methylotrophic cell of claim 100, wherein the Kozak sequence comprises:
(i) the sequence ANAATGNC, wherein N comprises A, T, G, or C; or
(ii) the sequence AMMATG, wherein M comprises A or C.

### Claim 102 (depends on 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101)

The methylotrophic cell of any one of claims 90-101, wherein a mRNA secondary structure of the mRNA encoding the heterologous protein has been reduced or eliminated relative to the endogenous mRNA encoding the heterologous protein.

### Claim 103 (depends on 102)

The methylotrophic cell of claim 102, wherein the mRNA secondary structure is selected from a hairpin loop, a duplex, a single-stranded region, a hairpin, a bulge, an internal loop, or any other structure as predicted by likelihood of pairing and/or low free energy.

### Claim 104

An expression construct comprising a promoter operably linked to a nucleic acid encoding a polypeptide comprising a signal sequence and a heterologous protein, wherein the signal sequence is a signal sequence of KAR2, MSC1, TOS1, 2241, LHS1, TIF1, CTS1, or 5326.

### Claim 105 (depends on 104)

The expression construct of claim 104, wherein the promoter in an OLE1, AOX1, DAS2, or GAPDH promoter.

### Claim 106 (depends on 104, 105)

The expression construct of any one of claims 104-105, wherein the expression construct is a plasmid or viral vector.

### Claim 107 (depends on 106)

The expression construct of claim 106, wherein the plasmid is an episomal plasmid or an integrative plasmid.

### Claim 108 (depends on 104, 105, 106, 107)

The expression construct of any one of claims 104-107, wherein the expression construct is linearized.

### Claim 109 (depends on 104, 105, 106, 107, 108)

The expression construct of any one of claims 104-108, wherein the heterologous protein is selected from the group consisting of enzymes, hormones, antibodies or antigen binding fragments thereof, vaccine components, blood factors, thrombolytic agents, cytokines, receptors, and fusion proteins.

### Claim 110 (depends on 104, 105, 106, 107, 108, 109)

The expression construct of any of claims 104-109, further comprising a targeting sequence for integration in a methylotrophic cell at an AOX1, PIF1, OLE1, GAPDH, or DAS2 locus.

### Claim 111

A methylotrophic cell expressing a heterologous protein fused to a signal sequence, wherein the signal sequence is a signal sequence of KAR2, MSC1, TOS1, 2241, LHS1, TIF1, CTS1, or 5326.

### Claim 112 (depends on 111)

The methylotrophic cell of claim 111, wherein the methylotrophic cell is a yeast cell.

### Claim 113 (depends on 112)

The methylotrophic cell of claim 112, wherein the yeast cell is a Pichia pastoris cell.

### Claim 114 (depends on 113)

The methylotrophic cell of claim 113, wherein the Pichia pastoris cell is a Komagataella phaffii or Komagataella pastoris cell.

### Claim 115 (depends on 114)

The methylotrophic cell of claim 114, wherein the Komagataella phaffii cell is a Komagataella phaffii
Y-11430, Y-7556, YB-4290, Y-12729, Y-17741, Y-48123, Y-48124, YB-378, YB-4289, GS115, KM71H, SMD1168, SMD1168H, or X-33 cell.

### Claim 116 (depends on 111, 112, 113, 114, 115)

The methylotrophic cell of any one of claims 111-115, wherein the expression is under the control of an OLE1, AOX1, DAS2, or GAPDH promoter.

### Claim 117 (depends on 111, 112, 113, 114, 115, 116)

The methylotrophic cell of any one of claims 111-116, wherein the heterologous protein is selected from the group consisting of enzymes, hormones, antibodies or antigen binding fragments thereof, vaccine components, blood factors, thrombolytic agents, cytokines, receptors, and fusion proteins.

### Claim 118 (depends on 111, 112, 113, 114, 115, 116, 117)

The methylotrophic cell of any of claims 111-117, wherein the heterologous protein is integrated at an AOX1, PIF1, OLE1, GAPDH, or DAS2 locus.

### Claim 119 (depends on 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99)

A method of producing a heterologous protein with the methylotrophic cell of any one of claims 10 to 21, 35-45, 63-72, 90-99, and 111-118, the method comprising culturing the cell under conditions suitable to express the heterologous protein.

### Claim 120 (depends on 119)

The method of claim 119, further comprising first culturing the cell with a first carbon source lacking methanol under conditions in which the heterologous protein is substantially not expressed, followed by switching the carbon source to a carbon source that includes methanol to express the heterologous protein.

### Claim 121 (depends on 119, 120)

The method of any one of claims 119-120, further comprising isolating the protein.

### Claim 122

A methylotrophic cell expressing a heterologous protein under the control of a promoter, wherein:
(i) the promoter is an AOX1 promoter or a DAS2 promoter and/or the promoter is located at an AOX1 or DAS2 locus;
(ii) mRNA encoding the heterologous protein comprises a Kozak sequence beginning at the −3 position relative to the translation start site; and/or
(iii) a mRNA secondary structure of the mRNA encoding the heterologous protein has been reduced or eliminated relative to the endogenous mRNA encoding the heterologous protein.

### Claim 123 (depends on 122)

The methylotrophic cell of claim 122, wherein the methylotrophic cell is a yeast cell.

### Claim 124 (depends on 123)

The methylotrophic cell of claim 123, wherein the yeast cell is a Pichia pastoris cell.

### Claim 125 (depends on 124)

The methylotrophic cell of claim 124, wherein the Pichia pastoris cell is a Komagataella phaffii or Komagataella pastoris cell.

### Claim 126 (depends on 125)

The methylotrophic cell of claim 125, wherein the Komagataella phaffii cell is a Komagataella phaffii Y-11430, Y-7556, YB-4290, Y-12729, Y-17741, Y-48123, Y-48124, YB-378, YB-4289, GS115, KM71H, SMD1168, SMD1168H, or X-33 cell.

### Claim 127 (depends on 122, 123, 124, 125, 126)

The methylotrophic cell of any one of claims 122-126, wherein the AOX1 promoter has at least 95% homology with SEQ ID NO: 3 or a fragment thereof.

### Claim 128 (depends on 127)

The methylotrophic cell of claim 127, wherein the AOX1 promoter has the sequence SEQ ID NO: 3.

### Claim 129 (depends on 122, 123, 124, 125, 126)

The methylotrophic cell of any one of claims 122-126, wherein the DAS2 promoter has at least 95% homology with SEQ ID NO: 2 or a fragment thereof.

### Claim 130 (depends on 127)

The methylotrophic cell of claim 127, wherein the DAS2 promoter has the sequence SEQ ID NO: 2.

### Claim 131 (depends on 122, 123, 124, 125, 126, 127, 128, 129, 130)

The methylotrophic cell of any one of claims 122-130, wherein the heterologous protein is selected from the group consisting of an enzyme, hormone, antibody or antigen-binding antibody fragments, vaccine component, blood factor, thrombolytic agent, cytokine, receptor, and fusion protein.

### Claim 132 (depends on 122, 123, 124, 125, 126, 127, 128, 129, 130, 131)

The methylotrophic cell of any one of claims 122-131, wherein the heterologous protein is fused to a signal sequence.

### Claim 133 (depends on 132)

The methylotrophic cell of claim 132, wherein the signal sequence is the signal sequence of SCW11, MSC1, EXG1, 0841, 1286, BGL2, 2488, 2848, PRY2, 4355, or PIR1.

### Claim 134 (depends on 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133)

The methylotrophic cell of any one of claims 122-133, wherein the Kozak sequence comprises:
(i) the sequence ANAATGNC, wherein N comprises A, T, G, or C; or
(ii) the sequence AMMATG, wherein M comprises A or C.

### Claim 135 (depends on 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134)

The methylotrophic cell of any one of claims 122-134, wherein the mRNA secondary structure is selected from a hairpin loop, a duplex, a single-stranded region, a hairpin, a bulge, an internal loop, or any other structure as predicted by likelihood of pairing and/or low free energy.

### Claim 136

An expression construct comprising a promoter operably linked to a nucleic acid encoding a polypeptide comprising a signal sequence and a heterologous protein, wherein:
(i) the promoter is an AOX1 or DAS2 promoter and/or the construct further comprises a targeting sequence for integration in a methylotrophic cell at an AOX1 or DAS2 locus;
(ii) the expression construct further comprises a Kozak sequence beginning at the −3 position relative to the translation start site of the nucleic acid encoding the polypeptide; and/or
(iii) a mRNA secondary structure of the nucleic acid encoding a polypeptide has been reduced or eliminated relative to the endogenous nucleic acid encoding the polypeptide.

### Claim 137 (depends on 136)

The expression construct of claim 136, wherein the signal sequence is identical to the signal sequence of a naturally occurring yeast protein.

### Claim 138 (depends on 136, 137)

The expression construct of claim 136 or 137, wherein the signal sequence is the signal sequence of SCW11, MSC1, EXG1, 0841, 1286, BGL2, 2488, 2848, PRY2, 4355, or PIR1.

### Claim 139 (depends on 136, 137, 138)

The expression construct of any one of claims 136 to 138, wherein the AOX1 promoter has at least 95% homology with SEQ ID NO: 3 or a fragment thereof.

### Claim 140 (depends on 139)

The expression construct of claim 139, wherein the AOX1 promoter has the sequence SEQ ID NO: 3.

### Claim 141 (depends on 136, 137, 138)

The expression construct of any one of claims 136 to 138, wherein the DAS2 promoter has at least 95% homology with SEQ ID NO: 2 or a fragment thereof.

### Claim 142 (depends on 141)

The expression construct of claim 141, wherein the DAS2 promoter has the sequence SEQ ID NO: 2.

### Claim 143 (depends on 136, 137, 138, 139, 140, 141, 142)

The expression construct of any one of claims 136 to 142, wherein the expression construct is a plasmid or viral vector.

### Claim 144 (depends on 143)

The expression construct of claim 143, wherein the plasmid is an episomal plasmid or an integrative plasmid.

### Claim 145 (depends on 136, 137, 138, 139, 140, 141, 142, 143, 144)

The expression construct of any one of claims 136 to 144, wherein the expression construct is linearized.

### Claim 146 (depends on 136, 137, 138, 139, 140, 141, 142, 143, 144, 145)

The expression construct of any one of claims 136 to 145, wherein the heterologous protein is selected from the group consisting of an enzyme, hormone, antibody or antigen-binding antibody fragment, vaccine component, blood factor, thrombolytic agent, cytokine, receptor, and fusion protein.

### Claim 147 (depends on 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146)

The expression construct of any one of claims 136 to 146, wherein the Kozak sequence comprises:
(i) the sequence ANAATGNC, wherein N comprises A, T, G, or C; or
(ii) the sequence AMMATG, wherein M comprises A or C.

### Claim 148 (depends on 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147)

The expression construct of any one of claims 136 to 147, wherein the mRNA secondary structure is selected from a hairpin loop, a duplex, a single-stranded region, a hairpin, a bulge, an internal loop, or any other structure as predicted by likelihood of pairing and/or low free energy.

### Claim 149

A method for preparing a transgene expression construct for expressing a heterologous protein in Pichia comprising:
providing a nucleic acid encoding a heterologous protein; and
(i) selecting a promoter that increases expression of genes of the Mut pathway upon integration; or
(ii) selecting a targeting sequence for guided recombination into a locus, wherein insertion of the heterologous protein into the locus increases expression of genes of the Mut pathway; or
(i) and (ii).

### Claim 150 (depends on 149)

The method of claim 149, further comprising selecting a Kozak sequence beginning at the −3 position relative to the translation start site of the nucleic acid encoding the heterologous protein.

### Claim 151 (depends on 149, 150)

The method of claim 149 or 150, further comprising reducing or eliminating a mRNA secondary structure of the nucleic acid encoding a polypeptide has been reduced or eliminated relative to the endogenous nucleic acid encoding the polypeptide.

### Claim 152 (depends on 149, 150, 151)

The method of any of claims 149-151, wherein the nucleic acid further encodes a signal sequence.

### Claim 153 (depends on 152)

The method of claim 152, wherein the signal sequence is identical to the signal sequence of a naturally occurring yeast protein.

### Claim 154 (depends on 152, 153)

The method of claim 152 or 153, wherein the signal sequence is the signal sequence of SCW11, MSC1, EXG1, 0841, 1286, BGL2, 2488, 2848, PRY2, 4355, or PIR1.

### Claim 155 (depends on 149, 150, 151, 152, 153, 154)

The method of any one of claims 149-154, wherein the promoter is DAS1, DAS2, AOX1, GAPDH, and ATG30.

### Claim 156 (depends on 149, 150, 151, 152, 153, 154, 155)

The method of any one of claims 149-155, wherein the locus is DAS1, DAS2, AOX1, GAPDH, and ATG30.

### Claim 157 (depends on 149, 150, 151, 152, 153, 154, 155, 156)

The expression construct of any one of claims 149-156, wherein the heterologous protein is selected from the group consisting of enzymes, hormones, antibodies or antigen binding fragments thereof, vaccine components, blood factors, thrombolytic agents, cytokines, receptors, and fusion proteins.

### Claim 158 (depends on 149, 150, 151, 152, 153, 154, 155, 156, 157)

The method of any one of claims 149-157, wherein the Kozak sequence comprises:
(i) the sequence ANAATGNC, wherein N comprises A, T, G, or C; or
(ii) the sequence AMMATG, wherein M comprises A or C.

### Claim 159 (depends on 149, 150, 151, 152, 153, 154, 155, 156, 157, 158)

The method of any one of claims 149-158, wherein the mRNA secondary structure is selected from a hairpin loop, a duplex, a single-stranded region, a hairpin, a bulge, an internal loop, or any other structure as predicted by likelihood of pairing and/or low free energy.