Materials and methods for protein production

Abstract

To provide constructs that can be used to efficiently express one or more products (e.g., proteins) in cells such as yeast cells or methylotrophic yeast cells.SOLUTION: The present invention provides a nucleic acid construct including a first alcohol oxidase promoter element, wherein the first alcohol oxidase promoter element includes a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 668-734 relative to a specific sequence.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority to U.S. Provisional Application No. 62 / 835,338, filed on April 17, 2019, which is incorporated herein by reference in its entirety.

[0002] Description of electronically submitted text files A text file submitted electronically with this specification: The file name is 38767 - 0193WO1_Se quenceListing.txt, the recording date is April 17, 2020, and the file size is approximately 53 kilobytes. A copy of the sequence listing in a computer - readable format is incorporated herein by reference in its entirety.

[0003] Technical Field The present disclosure generally relates to DNA constructs and methods of genetically engineering cells such as yeast cells or methanol - assimilating yeast cells using such DNA constructs.

Background Art

[0004] Recombinant expression of products is a common method for producing said products. In some cases, proteins can be produced by recombinant production. Herein, constructs are provided that can be used to efficiently express one or more products (e.g., proteins) intracellularly in cells such as yeast cells or methanol - assimilating yeast cells. (e.g., proteins) within cells such as yeast cells or methanol - assimilating yeast cells.

Summary of the Invention

[0005] This document provides an AOX1 promoter capable of conferring an increase in the expression of linked coding sequences. ​​​​​is at least partially based on the identification of point mutations. Mutation A described herein The OX1 promoter can be used, for example, for the efficient expression of an operably linked coding sequence in Pichia. sequence.

[0006] In one aspect of the present specification, a nucleic acid construct is provided that includes a first alcohol oxidase promoter element, wherein the first alcohol oxidase promoter element contains a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 668 to 734 relative to SEQ ID NO: 28. A nucleic acid construct is provided.

[0007] The implementation may have one or more of the following features. The first alcohol oxidase promoter element may contain a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 673 to 729 relative to SEQ ID NO: 28. The first alcohol oxidase promoter element may contain a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 678 to 724 relative to SEQ ID NO: 28. The first alcohol oxidase promoter element may contain a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 683 to 719 relative to SEQ ID NO: 28. The first alcohol oxidase promoter element may contain a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 688 to 714 relative to SEQ ID NO: 28. The first alcohol oxidase promoter element may contain a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 683 to 719 relative to SEQ ID NO: 28. The first alcohol oxidase promoter element may contain a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 688 to 714 relative to SEQ ID NO: 28. The first alcohol oxidase promoter element may contain a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 688 to 714 relative to SEQ ID NO: 28. The first alcohol oxidase promoter element may contain a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 688 to 714 relative to SEQ ID NO: 28. The first The first alcohol oxidase promoter element may contain a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 688 to 714 relative to SEQ ID NO: 28. The first alcohol oxidase promoter element ​​​It may contain two or more mutations at nucleotide positions corresponding to any of nucleotide positions 668 to 734 of the oligonucleotide. The first alcohol oxidase promoter element may contain three or more mutations at nucleotide positions corresponding to any of nucleotide positions 668 to 734 relative to SEQ ID NO: 28. The first alcohol o xidase promoter element may contain four or more mutations at nucleotide positions corresponding to any of nucleotide positions 668 to 734 relative to SEQ ID NO: 28. The first alcohol o xidase promoter element may contain five or more mutations at nucleotide positions corresponding to any of nucleotide positions 668 to 734 relative to SEQ ID NO: 28. In another aspect of the present specification, a nucleic acid construct comprising the first alcohol oxidase promoter element wherein the first alcohol oxidase promoter element may contain one or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to T146, C154, T303, T426, A433, A435, T 530, C572, T596, T617, T688, A696, T702, A709, A 712, T714, A790, A841, and T862 relative to SEQ ID NO: 28 is provided.

[0008] The implementation may include one or more of the following features. The first alcohol oxidase promoter element, relative to SEQ ID NO: 28, has T146, C154, T303, T426, A433, A435, T530, C572, T596, T617, T688,

[0009] The implementation may include one or more of the following features. The first alcohol oxidase promoter element, relative to SEQ ID NO: 28, has T146, C154, T303, T426, A433, A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790, A841, and T8 It may contain two or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to 62. The first alcohol oxidase promoter element is, with respect to SEQ ID NO: 28, T146, C154, T303, T426, A433, It may contain mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to 62. The first alcohol oxidase promoter element is, with respect to SEQ ID NO: 28, T146, C154, T303, T426, A433, A435, T530, It may contain three or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to 62. The first alcohol oxidase promoter element is, with respect to SEQ ID NO: 28, T146, C154, T303, T426, A433, A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790, A841, and T862. It may contain four or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to 62. The first alcohol oxidase promoter element is, with respect to SEQ ID NO: 28, T146, C154, T303, T426, A433, A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790, A841, and T862. It may contain five or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to 62. The first alcohol oxidase promoter element is, with respect to SEQ ID NO: 28, T146, C154, T303, T426, A433, A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790, A841, and T862. The first alcohol oxidase promoter element may contain mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to 62. The first alcohol oxidase promoter element is, with respect to SEQ ID NO: 28, T688, A696, T702, It may contain mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to 62. The first alcohol oxidase promoter element is, with respect to SEQ ID NO: 28, T146, C154, T303, T426, A433, A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790, A841, and T862. It may contain mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to 62. The first alcohol oxidase promoter element is, with respect to SEQ ID NO: 28, T146, C154, T303, T426, A433, A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790, A841, and T862. It may contain mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to 62. The first alcohol oxidase promoter element is, with respect to SEQ ID NO: 28, T146, C154, T303, T426, A433, A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790, A841, and T862. It may contain mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to 62. The first alcohol oxidase promoter element is, with respect to SEQ ID NO: 28, T146, C154, T303, T426, A433, A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790, A841, and T862. It may contain mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to 62. The first alcohol oxidase promoter element is, with respect to SEQ ID NO: 28, T146, C154, T303, T426, A433, A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790, A841, and T862. It may contain mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to 62. The first alcohol oxidase promoter element is, with respect to SEQ ID NO: 28, T146, C154, T303, T426, A433, A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790, A841, and T862. It may contain mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to 62. The first alcohol oxidase promoter element is, with respect to SEQ ID NO: 28, T146, C154, T303, T426, A433, A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790, A841, and T862. It may contain mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to 62. The first alcohol oxidase promoter element is, with respect to SEQ ID NO: 28, T146, C154, T303, T426, A433, A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790, A841, and T862. The first alcohol oxidase promoter element may contain mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to 62. The first alcohol oxidase promoter element is, with respect to SEQ ID NO: 28, T688, A696, T702, It may contain one or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to A712 and T714. The first alcohol oxidase promoter element may contain two or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to T688, A696, T702, A71 2, and T714 with respect to SEQ ID NO: 28. The first alcohol oxidase promoter element may contain three or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to T688, A696, T702, A71 2, and T714 with respect to SEQ ID NO: 28. The first alcohol oxidase promoter element may contain four or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to T688, A696, T702, A71 2, and T714 with respect to SEQ ID NO: 28. The first alcohol oxidase promoter element may contain mutations at nucleotide positions corresponding to T688, A696, T702, A71 2, and T714. In another aspect of the present specification, a nucleic acid construct comprising a first alcohol oxidase promoter element, wherein the first alcohol oxidase promoter element has a sequence number 28, T146C, C154T, T303C, T426A, A433T, A 435G, T530A, C572T, T596C, T617C, T688C, A696T with respect to, T702C, A709G, A712G, T714G, A790G, A841T, and

[0010] In another aspect of the present disclosure, a nucleic acid construct comprising a first alcohol oxidase promoter element, wherein the first alcohol oxidase promoter element has a sequence number 28, T146C, C154T, T303C, T426A, A433T, A 435G, T530A, C572T, T596C, T617C, T688C, A696T with respect to, T702C, A709G, A712G, T714G, A790G, A841T, and comprising one or more mutations selected from the group consisting of mutations corresponding to T862A a nucleic acid construct is provided that may be included

[0011] The implementation may include one or more of the following features. The first alcohol oxidase promoter element may include two or more mutations selected from the group consisting of mutations corresponding to T146C, C154T, T303C, T426A, A433T, A435G, T530A, C572T, T596C, T617C, T688C, A696T, T702C, A709G, A712G, T714G, A790G, A841T, and T862A with respect to SEQ ID NO: 28 3C, T426A, A433T, A435G, T530A, C572T, T596C, T 617C, T688C, A696T, T702C, A709G, A712G, T714G with respect to SEQ ID NO: 28. The first alcohol oxidase promoter element may include two or more mutations selected from the group consisting of mutations corresponding to T146C, C154T, T303C, T426A, A433T, A435G, T530A, C572T, T596C, T617C T688C, A696T, T702C, A709G, A712G, T714G, A790G, A841T, and T862A with respect to SEQ ID NO: 28 The first alcohol oxidase promoter element may include three or more mutations selected from the group consisting of mutations corresponding to T146C, C154T, T303C, T 426A, A433T, A435G, T530A, C572T, T596C, T617C T688C, A696T, T702C, A709G, A712G, T714G, A79 0G, A841T, and T862A with respect to SEQ ID NO: 28 The first alcohol oxidase promoter element may include three or more mutations selected from the group consisting of mutations corresponding to T146C, C154T, T303C, T426A A433T, A435G, T530A, C572T, T596C, T617C, T68 8C, A696T, T702C, A709G, A712G, T714G, A790G, A 841T, and T862A with respect to SEQ ID NO: 28 The first alcohol oxidase promoter element may include four or more mutations selected from the group consisting of mutations corresponding to T146C, C154T, T303C, T426A, A433T, A435G, T530A, C572T, T596C, T617C, T688C, A696T, T702C, A709G, A712G, T714G, A790G, A841T, and T862A with respect to SEQ ID NO: 28 The first alcohol oxidase promoter element may include four or more mutations selected from the group consisting of mutations corresponding to T146C, C154T, T303C, T426A, A43 3T, A435G, T530A, C572T, T596C, T617C, T688C, A696T, T702C, A709G, A712G, T714G, A790G, A841T, and T862A with respect to SEQ ID NO: 28 3T, A435G, T530A, C572T, T596C, T617C, T688C, A 696T, T702C, A709G, A712G, T714G, A790G, A841T 、and 5 or more mutations selected from the group consisting of mutations corresponding to T862A. The first alcohol oxidase promoter element is capable of containing a mutation. With respect to SEQ ID NO: 28, the first alcohol oxidase promoter element can contain one or more mutations selected from the group consisting of T688C, A696T, T702C, A712G, and T714 G. With respect to SEQ ID NO: 28, the first alcohol oxidase promoter element can contain two or more mutations selected from the group consisting of T688C, A696T , T702C, A712G, and T714G. With respect to SEQ ID NO: 28, the first alcohol oxidase promoter element can contain three or more mutations selected from the group consisting of T688C, A696T , T702C, A712G, and T714G. With respect to SEQ ID NO: 28, the first alcohol oxidase promoter element can contain four or more mutations selected from the group consisting of T688C, A696T , T702C, A712G, and T714G. With respect to SEQ ID NO: 28, the first alcohol oxidase promoter element can contain the mutations T688C, A696T, T702C, A712G, and T714G. Any implementation of the nucleic acid constructs described herein can have one or more of the following features. The first alcohol oxidase promoter element is capable of containing the mutations T688C, A696T, T702C, A712G, and T714G.

[0012] Any implementation of the nucleic acid constructs described herein can have one or more of the following features. The first alcohol oxidase promoter element is capable of having one or more of the following characteristics. The first alcohol oxidase promoter element is It may be a cytochrome oxidase 1 promoter element. The first alcohol oxidase promoter element may have at least 90% sequence identity to SEQ ID NO: 28 The first alcohol oxidase promoter element may have at least 95% sequence identity to SEQ ID NO: 28 The nucleic acid construct may further contain a nucleotide sequence encoding a first protein. In this case, the nucleotide sequence encoding the first protein is operably linked to the first alcohol oxidase promoter element. The first protein may be exogenous to the methanol-assimilating yeast cell The first protein may be heterologous to the methanol-assimilating yeast cell. The first protein may be selected from the group consisting of an antibody or a fragment thereof, an enzyme, a regulatory protein, a peptide hormone, a blood coagulation protein, a cytokine, a cytokine inhibitor, and a heme-binding protein. The first protein may be a heme-binding protein. The he me-binding protein may be selected from the group consisting of globin, cytochrome, cytochrome c oxidase, lignin ase, catalase, and peroxidase. The heme-binding protein may be selected from the group consisting of androglobin, chlorocruorin, cytoglobin, erythrocru lorin, flavohemoglobin, globin E, globin X, globin Y, hemoglobin, his toglobin, leghemoglobin, myoglobin, neuroglobin, non-symbiotic hemoglobin protein, protoglobin, and truncated hemoglobin. The heme-binding protein may be non-symbiotic hemoglobin. The heme-binding protein may be leghemoglobin The heme-binding protein may be selected from the group consisting of androglobin, chlorocruorin, cytoglobin, erythrocruorin, flavohemoglobin, globin E, globin X, globin Y, hemoglobin, histoglobin, leghemoglobin, myoglobin, neuroglobin, non-symbiotic hemoglobin, protoglobin, and truncated hemoglobin. The heme-binding protein may be selected from the group consisting of non-symbiotic hemoglobin. The heme-binding protein may be leghemoglobin The heme-binding protein may be non-symbiotic hemoglobin. The heme-binding protein may be leghemoglobin The heme-binding protein may be any one of SEQ ID NOs: 1-27 It may include an amino acid sequence having at least 90% sequence identity to the amino acid sequence. The first alcohol oxidase promoter element may include a recognition sequence for a transcription factor. It may include.

[0013] In another aspect of the present specification, there is also a methanol-assimilating yeast cell containing a first nucleic acid construct, wherein the first nucleic acid construct is any nucleic acid construct described herein, and a methanol-assimilating yeast cell is also provided.

[0014] The implementation may have one or more of the following features. The methanol-assimilating yeast cell is a Pichia cell, a Candida cell, a Hansenula cell or a Torulopsis cell. The methanol-assimilating yeast cell is a P ichia methanolica cell, a Pichia pastoris ) cell, a Candida boidinii cell, or a Hansenula polymor pha (Hansenula polymorpha) cell. The methanol-assimilating yeast cell is a Pichia pa storis (Pichia pastoris) cell. The methanol-assimilating yeast cell may further include a second nucleic acid construct containing a nucleotide sequence encoding a second protein , in which case the nucleotide sequence encoding the second protein is operably linked to the first alcohol oxidase promoter element or to a second promoter element. The nucleotide sequence encoding the second protein has the same sequence as the first alcohol oxidase promoter element, a second promoter element linked thereto. element. It can be operably linked to the to. The second protein can be a transcription factor. The second tanpa The nucleotide sequence encoding the protein can include a recognition sequence for the transcription factor, the second pu It can be operably linked to the romoter element. The first alcohol oxidase promoter The ter element can include a recognition sequence for the transcription factor. The second protein can be a heme A protein involved in biosynthesis. Proteins involved in heme biosynthesis include am Nolevulinic acid synthase (ALAS), δ-aminolevulinic acid dehydratase (ALAD ), porphobilinogen deaminase (PBGD), uroporphyrinogen III syn Tase (UPG3S), uroporphyrinogen III decarboxylase (UPG3D ), coproporphyrinogen oxidase (COPROX), protoporphyrinogen Gen IX oxidase (PROTOX), and ferrochelatase (FC). It can be selected from the group consisting of selected.

[0015] In another aspect of the present specification, a method for producing a protein in a methanol-assimilating yeast cell is That includes expressing a nucleic acid construct containing a nucleotide sequence encoding a first protein operably linked to a first alcohol oxidase promoter element Including steps, the first alcohol oxidase promoter element is relative to SEQ ID NO: 28 One or more nucleotides corresponding to any of nucleotide positions 668 to 734 A method is provided that includes a mutation at the nucleotide position. The implementation can include one or more of the following features. The first alcohol oxidase

[0016] It can include one or more of the following characteristics. The first alcohol oxidase The zep promoter element may contain a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 673 to 729 relative to SEQ ID NO: 28. The first alcohol oxidase promoter element may contain a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 678 to 724 relative to SEQ ID NO: 28. The first alcohol oxidase promoter element may contain a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 683 to 719 relative to SEQ ID NO: 28. The first alcohol oxidase promoter element may contain a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 688 to 714 relative to SEQ ID NO: 28. The first alcohol oxidase promoter element may contain a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 688 to 714 relative to SEQ ID NO: 28. The first alcohol oxidase promoter element may contain two or more mutations at nucleotide positions corresponding to any of nucleotide positions 668 to 734 relative to SEQ ID NO: 28. The first alcohol oxidase promoter element may contain three or more mutations at nucleotide positions corresponding to any of nucleotide positions 668 to 734 relative to SEQ ID NO: 28. The first alcohol oxidase promoter element may contain four or more mutations at nucleotide positions corresponding to any of nucleotide positions 668 to 734 relative to SEQ ID NO: 28. The first alcohol oxidase promoter element may contain five or more mutations at nucleotide positions corresponding to any of nucleotide positions 668 to 734 relative to SEQ ID NO: 28.

[0017] ​​​​​​​​​​ In another aspect of the present specification, there is also provided a method for producing a protein in methanol-assimilating yeast cells comprising expressing a nucleic acid construct comprising a nucleotide sequence encoding a first protein, operably linked to a first alcohol oxidase promoter element wherein the first alcohol oxidase promoter element comprises one or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to T146, C154, T303, T426, A433, A435, T530, C 572, T596, T617, T688, A696, T702, A709, A712, T 714, A790, A841, and T862 relative to SEQ ID NO: 28 A method is also provided. The implementation may include one or more of the following features. The first alcohol oxidase promoter element may comprise two or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to T146, C154, T303,

[0018] T426, A433, A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790, A841, and T8 62 relative to SEQ ID NO: 28. The first alcohol oxidase promoter element may comprise three or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to T146, C154, T303, T426, A433, A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790, A841, and T862 relative to SEQ ID NO: 28. The first alcohol oxidase promoter element may comprise three or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to T146, C154, T303, T426, A433, A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790, A841, and T862 relative to SEQ ID NO: 28. otide positions. ations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to T146, C154, T303, T426, A433, may contain differences. The first alcohol oxidase promoter element has nucleotide positions corresponding to T146, C154, T303, T426, A433, A435, T530, with respect to SEQ ID NO: 28, and may contain 4 or more mutations at nucleotide positions selected from the group consisting of C572, T596, T617, T688, A696, T702, A709, A712, T714, A790, A841, and T862. The first alcohol oxidase promoter element may contain 5 or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to T146, C154, T303, T426, A433, A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790, A841, and T862 with respect to SEQ ID NO: 28. The first alcohol oxidase promoter element may contain 1 or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to T688, A696, T702, A712, and T714 with respect to SEQ ID NO: 28. The first alcohol oxidase pro moter element may contain 2 or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to T688, A696, T702, A712, and T714 with respect to SEQ ID NO: 28. The first alcohol oxidase pro moter element may contain 3 or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to T688, A696, T702, A712, and T714 with respect to SEQ ID NO: 28. The first alcohol oxidase pro moter element may contain 2 or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to T688, A696, T702, A712, and T714 with respect to SEQ ID NO: 28. The first alcohol oxidase pro moter element may contain 3 or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to T688, A696, T702, A712, and T714 with respect to SEQ ID NO: 28. The first alcohol oxidase pro moter element may contain 3 or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to T688, The motor element may contain four or more mutations at nucleotide positions corresponding to T688, A696, T702, A712, and T714, when compared to SEQ ID NO: 28. The first alcohol oxidase promoter element may contain a mutation at the nucleotide position corresponding to T688, A696, T702, A712, and T714, when compared to SEQ ID NO: 28. from the group consisting of nucleotide positions corresponding to T688, A696, T702, A712, and T714. In another aspect of the present specification, a method for producing a protein in a methanol-assimilating yeast cell, comprising expressing a nucleic acid construct containing a nucleotide sequence encoding a first protein operably linked to a first alcohol oxidase promoter element, wherein the first alcohol oxidase promoter element contains one or more mutations selected from the group consisting of mutations corresponding to T146C, C154T, T303C, T426A, A433T, A435G, T530A, C572T, T596C, T617C, T688C, A696T, T702C, A709G, A712G, T714G, A790G, A841T, and T862A, when compared to SEQ ID NO: 28, is provided.

[0019] The implementation may include one or more of the following features. The first alcohol oxidase promoter element contains one or more mutations selected from the group consisting of mutations corresponding to T146C, C154T, T303C, T426A, A433T, A435G, T530A, C572T, T596C, T617C, T688C, A696T, T702C, A709G, A712G, T714G, when compared to SEQ ID NO: 28. including expressing a nucleic acid construct containing a nucleotide sequence encoding a first protein operably linked to a first alcohol oxidase promoter element; wherein the first alcohol oxidase promoter element contains one or more mutations selected from the group consisting of mutations corresponding to T146C, C154T, T303C, T426A, A433T, A435G, T530A, C572T, T596C, T617C, T688C, A696T, T702C, A709G, A712G, T714G, A790G, A841T, and T862A, when compared to SEQ ID NO: 28. A709G, A712G, T714G, A790G, A841T, and T862A, when compared to SEQ ID NO: 28. is provided. The implementation may include one or more of the following features. The first alcohol oxidase promoter element contains one or more mutations selected from the group consisting of mutations corresponding to T146C, C154T, T303C, T426A, A433T, A435G, T530A, C572T, T596C, T617C, T688C, A696T, T702C, A709G, A712G, T714G, when compared to SEQ ID NO: 28.

[0020] The implementation may include one or more of the following features. The first alcohol oxidase promoter element contains one or more mutations selected from the group consisting of mutations corresponding to T146C, C154T, T303C, T426A, A433T, A435G, T530A, C572T, T596C, T617C, T688C, A696T, T702C, A709G, A712G, T714G, when compared to SEQ ID NO: 28. A709G, A712G, T714G, A790G, A841T, and T862A, when compared to SEQ ID NO: 28. A709G, A712G, T714G, A790G, A841T, and T862A, when compared to SEQ ID NO: 28. , two or more mutations selected from the group consisting of mutations corresponding to A790G, A841T, and T862A may be included. The first alcohol oxidase promoter element may include three or more mutations selected from the group consisting of mutations corresponding to T146C, C154T, T303C, T426A, A433T, A435G, T530A, C572T, T596C, T617C, T688C, A696T, T702C, A709G, A712G, T714G, A790G, A841T, and T862A with respect to SEQ ID NO: 28. The first alcohol oxidase promoter element may include four or more mutations selected from the group consisting of mutations corresponding to T146C, C154T, T303C, T426A, A433T, A435G, T530A, C572T, T596C, T617C, T688C, A696T, T702C, A709G, A712G, T714G, A790G, A841T, and T862A with respect to SEQ ID NO: 28. The first alcohol oxidase promoter element may include five or more mutations selected from the group consisting of mutations corresponding to T146C, C154T, T303C, T426A, A433T, A435G, T530A, C572T, T596C, T617C, T688C, A696T, T702C, A709G, A712G, T714G, A790G, A841T, and T862A with respect to SEQ ID NO: 28. The first alcohol oxidase promoter element may include two or more mutations selected from the group consisting of T688C, A696T, T702C, A712G, and T714G with respect to SEQ ID NO: 28. The first alcohol can include two or more mutations selected from the group consisting of mutations corresponding to A790G, A841T, and T862A. The first alcohol oxidase promoter element can include three or more mutations selected from the group consisting of mutations corresponding to T146C, C154T, T303C, T 426A, A433T, A435G, T530A, C572T, T596C, T617C , T688C, A696T, T702C, A709G, A712G, T714G, A79 0G, A841T, and T862A with respect to SEQ ID NO: 28. The first alcohol oxidase promoter element can include four or more mutations selected from the group consisting of mutations corresponding to T146C, C154T, T303C, T426A , A433T, A435G, T530A, C572T, T596C, T617C , T688C, A696T, T702C, A709G, A712G, T714G, A790G, A 841T, and T862A with respect to SEQ ID NO: 28. The first alcohol oxidase promoter element can include five or more mutations selected from the group consisting of mutations corresponding to T146C, C154T, T303C, T426A, A43 3T, A435G, T530A, C572T, T596C, T617C, T688C, A 696T, T702C, A709G, A712G, T714G, A790G, A841T , and T862A with respect to SEQ ID NO: 28. The first alcohol oxidase promoter element can include two or more mutations selected from the group consisting of T688C, A696T, T702C, A712G, and T714 G with respect to SEQ ID NO: 28. The first alcohol oxidase promoter element may include two or more mutations selected from the group consisting of mutations corresponding to T688C, A696T, T702C, A712G, and T714 G with respect to SEQ ID NO: 28. The first alcohol oxidase promoter element may include two or more mutations selected from the group consisting of mutations corresponding to T688C, A696T, T702C, A712G, and T714 G with respect to SEQ ID NO: 28. The first alcohol oxidase promoter element may include two or more mutations selected from the group consisting of T688C, A696T, T702C, A712G, and T714 G with respect to SEQ ID NO: 28. The first alcohol The alcohol oxidase promoter element may contain three or more mutations selected from the group consisting of T688C, A6 96T, T702C, A712G, and T714G with respect to SEQ ID NO: 28. The first alcohol oxidase promoter element may contain four or more mutations selected from the group consisting of T688C, A696T, T702C, A712G, and T714G with respect to SEQ ID NO: 28. The first alcohol oxidase promoter element may contain the mutations T688C, A696T, T702C, A712G, and T714G with respect to SEQ ID NO: 28. Any implementation of the methods described herein may have one or more of the following features. The first alcohol oxidase promoter element may be an alcohol

[0021] oxidase 1 promoter element. The first alcohol oxidase promoter element may have at least 90% sequence identity with respect to SEQ ID NO: 28 . The first alcohol oxidase promoter element may have at least 95% sequence identity with respect to SEQ ID NO: 28. The first protein may be exogenous to the methanol-assimilating yeast cell . The first protein may be heterologous to the methanol-assimilating yeast cell . The first protein may be selected from the group consisting of an antibody or a fragment thereof, an enzyme, a regulatory protein, a peptide hormone, a blood coagulation protein, a cytokine, and a heme-binding protein. The first protein may be a heme-binding protein. The heme-binding protein may be a globin, a cytochrome, a cytochrome c oxidase, a ligninase, . The first protein may be heterologous to the methanol-assimilating yeast cell. The first protein may be selected from the group consisting of an antibody or a fragment thereof, an enzyme, a regulatory protein, a peptide hormone, a blood coagulation protein, a cytokine, and a heme-binding protein. The first protein may be a heme-binding protein. The heme-binding protein may be a globin, a cytochrome, a cytochrome c oxidase, a ligninase, It may be selected from the group consisting of catalase and peroxidase. The heme-binding protein may be selected from the group consisting of androglobin, chlorocruorin, cytoglobin, erythrocruorin, f labohemoglobin, globin E, globin X, globin Y, hemoglobin, histoglobin , leghemoglobin, myoglobin, neuroglobin, non-symbiotic hemoglobin, pro toglobin, and truncated hemoglobin. The heme-binding protein may be non-symbiotic hemoglobin. The heme-binding protein may be leghemoglobin . The heme-binding protein may contain an amino acid sequence having at least 90% sequence identity to any of the amino acid sequences of SEQ ID NOs: 1-27. The first alcohol oxidase promoter element may contain one or more recognition sequences for a transcription factor. The method may further include the step of expressing a second nucleic acid construct comprising a nucleotide sequence encoding a second protein, wherein the nucleotide sequence encoding the second protein is operably linked to the first alcohol oxidase promoter element or to a promoter element of the second . The nucleotide sequence encoding the second protein may be operably linked to a second promoter element having the same sequence as the first alcohol oxidase promoter element . The second protein may be a transcription factor. The nucleotide sequence encoding the second protein may be operably linked to a second promoter element that may contain a recognition sequence for a transcription factor . The first alcohol oxidase promoter element may contain a recognition sequence for a transcription factor. The second protein may be involved in the biosynthesis of heme . The nucleotide sequence encoding the second protein may be operably linked to a second promoter element having the same sequence as the first alcohol oxidase promoter er element. The second protein may be a transcription factor. The nucleotide sequence encoding the second protein may be operably linked to a second promoter element that may contain a recognition sequence for a transcription factor . The first alcohol oxidase promoter element may contain a recognition sequence for a transcription factor. The second protein may be involved in the biosynthesis of heme . ​ It can be a protein. Proteins involved in heme biosynthesis include ALAS, ALAD, P BGD, UPG3S, UPG3D, COPROX, PROTOX, and FC. It can be selected from the group. The method can be carried out in the absence of the addition of methanol.

[0022] In another aspect of the present specification, a nucleic acid construct comprising a nucleotide sequence encoding a first alcohol oxidase promoter element, wherein the first alcohol oxidase pro moter element comprises one or more mutations selected from the group consisting of mutations corresponding to T146C, C154T, T303C, T426A, A433T, A435G, T530A, C572T, T596C, T61 7C, T688C, A696T, T702C, A709G, A712G, T714G, A 790G, A841T, and T862A with respect to SEQ ID NO: 28 is provided. In some embodiments, the one or more mutations are selected from the group consisting of mutations corresponding to T688C, A696T, T702C, A712G, and T714G with respect to SEQ ID NO: 28. Pichia pastoris cells comprising the nucleic acid construct are provided. In some embodiments, the one or more mutations can be selected from the group consisting of mutations corresponding to SEQ ID NO: 28. T688C, A696T, T702C, A712G, and T714G

[0023] In another aspect of the present specification, a method for producing leghemoglobin is also provided, which comprises the step of expressing a nucleic acid construct comprising a nucleotide sequence encoding leghemoglobin operably linked to a first alcohol oxidase promoter element, wherein the first alcohol oxidase promoter element comprises one or more mutations selected from the group consisting of mutations corresponding to T146C, C154T, T303C, T426A, A433T, A435G, T530A, C572 T, T596C, T617C, T688C, A696T, T702C, A709G, A712G, T714G, A790G, A841T, and T862A with respect to SEQ ID NO: 28. T, T596C, T617C, T688C, A696T, T702C, A709G, A7 12G, T714G, A790G, A841T, and mutations corresponding to T862A One or more mutations selected from the group consisting of are also provided. In some imp lementations, the method can be performed in the absence of the addition of methanol. In some implementations , one or more mutations can be selected from the group consisting of mutations corresponding to T688C, A696T, T7 02C, A712G, and T714G relative to SEQ ID NO: 28 .

[0024] In another aspect of the present specification, a first nucleic acid construct comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 28 is included in Pichia pastoris cells, wherein the first nucleic acid construct is selected from the group consisting of mutations corresponding to T146C, C154T , T303C, T426A, A433T, A435G, T530A, C572T, T59 6C, T617C, T688C, A696T, T702C, A709G, A712G, T 714G, A790G, A841T, and T862A relative to SEQ ID NO: 28 One or more mutations selected from the group consisting of are included in Pichia pastoris cells. In some implementations, one or more mutations can be selected from the group consisting of mutations corresponding to T688C, A696T, T702C, A712G, and T714G relative to SEQ ID NO: 28 . Unless otherwise specified, all technical terms

[0025] and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present invention pertains ​ has the meaning. In the implementation of the present invention, it is the same as the methods and materials described in this specification or methods and materials equivalent thereto may be used, but the appropriate methods and materials are described below. All publications, patent applications, patents, and other references mentioned in this specification are incorporated by reference in their entirety. In case of conflict, follow this specification including the definitions. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0026] Details of one or more embodiments of the present invention are set forth in the accompanying drawings and the following description will be apparent. Other features, objects, and advantages of the present invention will be apparent from the description and drawings, as well as from the claims. The phrase "comprising" in the claims, in accordance with standard practice in patent law, may be replaced by "consisting essentially of" or "consisting of" ".

Brief Description of the Drawings

[0027]

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Modes for Carrying Out the Invention

[0028] This document relates to materials and methods for protein production. For example, in one aspect, the present The literature relates to materials and methods for producing products (e.g., proteins (e.g., plant proteins)) in cells (e.g., yeast (e.g., methanol-utilizing yeast)) using an inducible promoter. Methanol-utilizing yeasts such as Pichia pastoris are commonly used to produce recombinant products (e.g., proteins). Pichia strains are typically capable of growing on methanol as the sole carbon source. The term "Pichia pastoris" is still used and may refer to any suitable Komagataella species, although Pichia pastoris has been reclassified as a Komagataella species such as Komagataella phaffii, Komagataella pastoris, or Komagataella pseudopastoris. Generally, laboratory strains of P. pastoris are Komagataella phaffii. The utilization of methanol can be induced by the conversion of methanol to formaldehyde via the action of alcohol oxidase. P. pastoris contains two genes for alcohol oxidase, AOX1 and AOX2. Strains with reduced alcohol oxidase activity ( "slow methanol-utilizing" strains or MutS strains)

[0029]

[0030] ​​​​​​​​​​​​​​Typically, recombinant products (e.g., proteins) expressed from the AOX1 promoter can produce more than strains with reduced alcohol oxidase activity. The Pichia pastoris promoter for the alcohol oxidase 1 (AOX1) gene, designated pAOX1, can be used for the production of heterologous products (e.g., proteins (e.g., industrially relevant proteins)). Expression from this promoter can be induced in the presence of methanol, a flammable and toxic compound. In some embodiments, the materials and methods described herein can enable high-level expression of recombinant products (e.g., proteins) from this promoter or promoter elements derived therefrom in the absence of methanol. In some embodiments, the materials and methods described herein can enable high-level expression of recombinant products (e.g., proteins) from this promoter or promoter elements derived therefrom in the absence of the addition of methanol. Expression from pAOX1 is typically absent or very poor in the presence of non-inducing carbon sources such as glucose or glycerol. Mutations within pAOX1 that enable significant expression from pAOX1 in the absence of methanol are described herein. Mutations within pAOX1 that enable significant expression from pAOX1 in the absence of the addition of methanol are described herein. The reference pAOX1 sequence is presented in SEQ ID NO: 28 (Figure 2). The pAOX1 mutations described herein are within Pichia pastoris which can be used for the production of heterologous products (e.g., proteins (e.g., industrially relevant proteins)). Expression from this promoter can be induced in the presence of methanol, a flammable and toxic compound. In some embodiments, the materials and methods described herein can enable high-level expression of recombinant products (e.g., proteins) from this promoter or promoter elements derived therefrom in the absence of methanol. In some embodiments, the materials and methods described herein can enable high-level expression of recombinant products (e.g., proteins) from this promoter or promoter elements derived therefrom in the absence of the addition of methanol. which can be used for the production of heterologous products (e.g., proteins (e.g., industrially relevant proteins)). Expression from this promoter can be induced in the presence of methanol, a flammable and toxic compound. In some embodiments, the materials and methods described herein can enable high-level expression of recombinant products (e.g., proteins) from this promoter or promoter elements derived therefrom in the absence of methanol. In some embodiments, the materials and methods described herein can enable high-level expression of recombinant products (e.g., proteins) from this promoter or promoter elements derived therefrom in the absence of the addition of methanol. which can be induced in the presence of methanol, a flammable and toxic compound. In some embodiments, the materials and methods described herein can enable high-level expression of recombinant products (e.g., proteins) from this promoter or promoter elements derived therefrom in the absence of methanol. In some embodiments, the materials and methods described herein can enable high-level expression of recombinant products (e.g., proteins) from this promoter or promoter elements derived therefrom in the absence of the addition of methanol. In some embodiments, the materials and methods described herein can enable high-level expression of recombinant products (e.g., proteins) from this promoter or promoter elements derived therefrom in the absence of methanol. (e.g., proteins) from this promoter or promoter elements derived therefrom. In some embodiments, the materials and methods described herein can enable high-level expression of recombinant products (e.g., proteins) from this promoter or promoter elements derived therefrom in the absence of the addition of methanol. In the absence of the addition of methanol, recombinant products (e.g., proteins) from this promoter or promoter elements derived therefrom. In the absence of the addition of methanol, recombinant products (e.g., proteins)

[0031] Typically, expression from pAOX1 is absent or very poor in the presence of non-inducing carbon sources such as glucose or glycerol. Mutations within pAOX1 that enable significant expression from pAOX1 in the absence of methanol are described herein. Mutations within pAOX1 that enable significant expression from pAOX1 in the absence of the addition of methanol are described herein. The reference pAOX1 sequence is presented in SEQ ID NO: 28 (Figure 2). The pAOX1 mutations described herein are within Typically, expression from pAOX1 is absent or very poor in the presence of non-inducing carbon sources such as glucose or glycerol. Mutations within pAOX1 that enable significant expression from pAOX1 in the absence of methanol are described herein. Mutations within pAOX1 that enable significant expression from pAOX1 in the absence of the addition of methanol are described herein. The reference pAOX1 sequence is presented in SEQ ID NO: 28 (Figure 2). The pAOX1 mutations described herein are within Mutations within pAOX1 that enable significant expression from pAOX1 in the absence of methanol are described herein. Mutations within pAOX1 that enable significant expression from pAOX1 in the absence of methanol are described herein. Mutations within pAOX1 that enable significant expression from pAOX1 in the absence of the addition of methanol are described herein. The reference pAOX1 sequence is presented in SEQ ID NO: 28 (Figure 2). The pAOX1 mutations described herein are within , Exemplary mutations are presented in SEQ ID NO: 29 (Figure 2). These mutations may exist individually or in any combination. These mutations may also result in a further increase in expression from pAOX1 when methanol is present.

[0032] Accordingly, provided herein is a nucleic acid construct (also sometimes referred to as a nucleic acid molecule) comprising a promoter element having a sequence that includes one or more mutations as compared to a reference promoter sequence. In some embodiments, the promoter element can be an alcohol oxidase promoter element. In some embodiments, the promoter element can have at least 70% (e.g., at least 75 %, 80%, 85%, 90%, 95%, 97%, 98%, or 99%) sequence identity to an alcohol oxidase promoter element (e.g., SEQ ID NO: 28 or SEQ ID NO: 29). In some embodiments, the promoter element can have the sequence of SEQ ID NO: 29. In some embodiments, a single mutation can be present within the promoter element. For example, in some embodiments, a single mutation corresponding to a mutation at one of nucleotide positions 668 - 734 relative to SEQ ID NO: 28 ( e.g., nucleotide positions 673 - 729, nucleotide positions 678 - 724, nucleotide positions 683 - 719, or nucleotide positions 688 - 714) can be present within the promoter element. For example, in some embodiments, the following mutations relative to SEQ ID NO: 28: T146C; C154 T; T303C; T426A; A433T; A435G; T530A; C572T; T5 ... ... ... can be present within the promoter element. For example, in some embodiments, the following mutations relative to SEQ ID NO: 28: T146C; C154 T; T303C; T426A; A433T; A435G; T530A; C572T; T5 96C; T617C; T688C; A696T; T702C; A709G; A712G; A single mutation corresponding to one of T714G; A790G; A841T; or T862A may be present within the promoter element. For example, in some embodiments, the following mutations relative to SEQ ID NO: 28: 146C; 154T; 303C; 426A; 433 T; 435G; 530A; 572T; 596C; 617C; 688C; 696T; 702 C; 709G; 712G; 714G; 790G; 841T; or 862A A single mutation corresponding to one of may be present within the promoter element provided that the indicated nucleobase is not the same as the corresponding naturally occurring nucleobase. For example, in some embodiments, a single mutation at a position corresponding to one of the following positions relative to SEQ ID NO: 28: T146; C154; T303; T4 26; A433; A435; T530; C572; T596; T617; T688; A6 96; T702; A709; A712; T714; A790; A841; or T862 may be present within the promoter element. For example, in some embodiments, a single mutation at a position corresponding to one of the following positions relative to SEQ ID NO: 28: 146; 1 54; 303; 426; 433; 435; 530; 572; 596; 617; 688; 6 96; 702; 709; 712; 714; 790; 841; or 862 may be present within the promoter element. For example, in some embodiments, a single mutation corresponding to one of the following mutations relative to SEQ ID NO: 28: T688C; A696 T; T702C; A712G; or T714G may be present within the promoter element. For example, in some embodiments, the following mutations relative to SEQ ID NO: 2 A single mutation corresponding to one of may be present within the promoter element. For example, in some embodiments, a single mutation corresponding to one of the following mutations relative to SEQ ID NO: 28: T688C; A696 T; T702C; A712G; or T714G may be present within the promoter element. For example, in some embodiments, the following mutations relative to SEQ ID NO: 2 The following mutations to 8: 688C; 696T; 702C; 712G; or 714G A single mutation corresponding to one of these may be present within the promoter element, provided that the indicated nucleotide is not the same as the corresponding naturally occurring nucleotide. For example, in some embodiments, a single mutation at a position corresponding to one of the following positions relative to SEQ ID NO: 28: T688; A696; T 702; A712; or T714 may be present within the promoter element. For example, in some embodiments, a single mutation at a position corresponding to one of the following positions relative to SEQ ID NO: 2 8: 688; 696; 702; 712; or 714 may be present within the promoter element. Also provided herein are nucleic acid constructs comprising a promoter element having a sequence that includes a plurality (e.g., 2, 3, 4 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 18, 19, 20, or more) of mutations as compared to a reference promoter sequence. For example, in some embodiments, at least two (e.g., at least three

[0033] 4, at least five, at least 10, at least 15, 2-5, 2-1 0, 2-15, 2-20, 5-10, 5-15, 5-20, 10-15, 10-20, or 15-20) mutations corresponding to mutations at nucleotide positions 668-734 (e.g., nucleotide positions 673-729, nucleotide positions 678-724, nucleotide positions 683-719, or nucleotide positions 688-714) relative to SEQ ID NO: 28 may be present within the promoter element. For example, in some embodiments ​​​​​In an embodiment, the following mutations for SEQ ID NO: 28: T146C; C154T; T3 03C; T426A; A433T; A435G; T530A; C572T; T596C; T617C; T688C; A696T; T702C; A709G; A712G; T714 G; A790G; A841T; or at least two (e.g., at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twelve, at least fourteen, at least sixteen, at least eighteen, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 1 4, 15, 16, 17, 18, or 19) mutations may be present within the promoter element. For example, in some embodiments, the following mutations for SEQ ID NO: 28: 14 6C; 154T; 303C; 426A; 433T; 435G; 530A; 572T; 59 6C; 617C; 688C; 696T; 702C; 709G; 712G; 714G; 79 0G; 841T; or 862A corresponding to at least two (e.g., at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twelve, at least fourteen, at least sixteen, at least eighteen, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, or 19) mutations may be present within the promoter element provided that the indicated nucleobases are not the same as the corresponding naturally occurring nucleobases. For example, in some embodiments, the following positions for SEQ ID NO: 28: T146; C154; T 303; T426; A433; A435; T530; C572; T596; T617; T 303; T426; A433; A435; T530; C572; T596; T617; T 303; T426; A433; A435; T530; C572; T596; T617; T 688; A696; T702; A709; A712; T714; A790; A841; or at least two (e.g., at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine , at least ten, at least twelve, at least fourteen, at least sixteen, at least eighteen , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, or 19) mutations may be present within the promoter element. For example, in some embodiments, the following positions relative to SEQ ID NO: 28: 146; 154; 303; 42 6; 433; 435; 530; 572; 596; 617; 688; 696; 702; 70 9; 712; 714; 790; 841; or at least two corresponding to 862 (e.g., at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twelve, at least fourteen, at least sixteen, at least eighteen, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, or 19) mutations may be present within the promoter element. For example, in some embodiments, the following mutations relative to SEQ ID NO: 28 : T688C; A696T; T702C; A712G; or at least two corresponding to T714G (e.g., at least three, at least four, 2, 3, 4, or five) mutations may be present within the promoter element. For example, in some embodiments, the following mutations relative to SEQ ID NO: 28 : 688C; 696T; 702C; 7 ; or at least two corresponding to 714G (e.g., at least three, at least four, 2, 3, 4, 12G; or at least two (e.g., at least three, at least four, two, three, four, or five) mutations corresponding to 714G can be present within the promoter element, provided that the indicated nucleobases are not the same as the corresponding naturally occurring nucleobases. For example, in some embodiments, at least two (e.g., at least three, at least four, two, three, four, or five) mutations corresponding to one of the following positions relative to SEQ ID NO: 28: T688; A696; T702; A712; or T714 can be present within the promoter element. For example, in some embodiments, at least two (e.g., at least three, at least four, two, three, four, or five) mutations corresponding to one of the following positions relative to SEQ ID NO: 28: 688; 696; 702; 712; or 714 can be present within the promoter element. (e.g., at least three, at least four, two, three, four, or five) mutations can be present within the promoter element. In some embodiments, the mutation in the nucleic acid can be an insertion, deletion, or substitution. In some embodiments, the mutation in the nucleic acid can be a substitution (e.g., a guanosine to cytosine mutation). In some embodiments, the mutation in the nucleic acid can be a mutation within a non-coding sequence. In some embodiments, a substitution within a coding sequence (e.g., encoding a protein) can be a silent mutation (e.g., encoding the same amino acid). In some embodiments, a substitution within a coding sequence can be a non-synonymous mutation (e.g., a missense mutation or a nonsense mutation). In some embodiments, a substitution within a coding sequence can be a missense mutation (e.g., encoding a different amino acid). In some embodiments,

[0034] In some embodiments, the mutation in the nucleic acid can be an insertion, deletion, or substitution. In some embodiments, the mutation in the nucleic acid can be a substitution (e.g., a guanosine to cytosine mutation). In some embodiments, the mutation in the nucleic acid can be a mutation within a non-coding sequence. In some embodiments, a substitution within a coding sequence (e.g., encoding a protein) can be a silent mutation (e.g., encoding the same amino acid). In some embodiments, a substitution within a coding sequence can be a non-synonymous mutation (e.g., a missense mutation or a nonsense mutation). In some embodiments, a substitution within a coding sequence can be a missense mutation (e.g., encoding a different amino acid). In some embodiments, substitutions within a coding sequence can be silent mutations (e.g., encoding the same amino acid). In some embodiments, substitutions within a coding sequence can be non-synonymous mutations (e.g., missense mutations or nonsense mutations). In some embodiments, substitutions within a coding sequence can be missense mutations (e.g., encoding different amino acids). In some embodiments, substitutions within a coding sequence can be, missense mutations (e.g., encoding different amino acids). In some embodiments, substitutions within a coding sequence can be, missense mutations (e.g., encoding different amino acids). In some embodiments, In some embodiments, substitutions within the coding sequence can be nonsense mutations (e.g., a premature stop codon is encoded). Mutations can be used, for example, with CRISPR, TALEN, and / or zinc finger nucleases to alter endogenous nucleic acids, as will be understood. In some embodiments, mutations within the protein sequence can be insertions, deletions, or substitutions. It will be understood that mutations within the nucleic acid encoding the protein can cause mutations within the protein sequence. In some embodiments, mutations within the protein sequence are substitutions (e.g., a mutation from cysteine to serine, or a mutation from cysteine to alanine). The "corresponding" nucleic acid position (or substitution) within a nucleic acid sequence that is different from a reference nucleic acid sequence (e.g., the nucleic acid sequence of the pAOX1 promoter that is cleaved, extended, or mutated compared to the reference pAOX nucleic acid sequence such as SEQ ID NO: 28) used herein can be identified by performing a sequence alignment between the nucleic acid sequences of interest. It will be understood that gaps can be present in the nucleic acid alignment in some cases. Similarly, the "corresponding" amino acid position (or substitution) within a protein sequence that is different from a reference protein sequence (e.g., the myoglobin protein sequence of a different organism compared to the reference myoglobin protein sequence such as SEQ ID NO: 18) can be identified by performing a sequence alignment between the protein sequences of interest. It will be understood that gaps can be present in the protein alignment in some cases. As used herein, "compared to" the reference sequence

[0035]

[0036] ​​​​​​​​​​​​​​​​"Beta", the nucleotide position or amino acid position, can be the corresponding nucleotide position or amino acid position within the reference sequence.

[0037] In some embodiments, the reference sequence can be from the same taxonomic class as the comparison sequence. In some embodiments, the reference sequence can be from the same domain as the comparison sequence. For example, in some embodiments, both the reference sequence and the comparison sequence can be from the Eukarya domain. In some embodiments, the reference sequence can be from the same kingdom as the comparison sequence. For example, in some embodiments, both the reference sequence and the comparison sequence can be from the Fungi kingdom. In some embodiments, the reference sequence can be from the same phylum as the comparison sequence. For example, in some embodiments, both the reference sequence and the comparison sequence can be from the Ascomycota phylum. In some embodiments, the reference sequence can be from the same class as the comparison sequence. For example, in some embodiments, both the reference sequence and the comparison sequence can be from the Saccharomycetes class. In some embodiments, the reference sequence can be from the same order as the comparison sequence. For example, in some embodiments, both the reference sequence and the comparison sequence can be from the Saccharomycetales order. In some embodiments, the reference sequence can be from the same family as the comparison sequence. For example, in some embodiments, both the reference sequence and the comparison sequence can be from the Saccharomycetaceae family. In some embodiments, the reference sequence can be from the same genus as the comparison sequence. For example, in some embodiments, both the reference sequence and the comparison sequence can be from the Pichia genus. In some embodiments, the reference sequence can be from the same species as the comparison sequence.

[0038] In some embodiments, both the reference array and the comparison array can be derived from yeast. In some embodiments, both the reference array and the comparison array can be derived from methanol-utilizing yeast.

[0039] In some embodiments, the reference array and the comparison array can have at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 99% ) sequence identity.

[0040] In some embodiments, the nucleotide sequence of the promoter element provided herein can contain two mutations when compared to the nucleotide sequence of the reference promoter element. For example, in some embodiments, the following mutations relative to SEQ ID NO: 28: T146C and C154T; T146C and T303C; T146C and T426A; T146C and A433T; T146C and A435G; T146C and T530A; T146C and C572T; T146C and T596C; T146C and T617C; T146C and T688C; T146C and A696T; T146C and T702C; T146C and A709G; T146C and A712G; T146C and T714G; T146C and A790G; T146C and A841T; T146C and T862A; C154T and T303C; C154T and T426A; C154T and A433T; C154T and A435G; C154T and T530A; C154T and C572T; C154T and T596C; C154T and T617C; ​​​C154T and T688C; C154T and A696T; C154T and T702 C; C154T and A709G; C154T and A712G; C154T and T7 14G; C154T and A790G; C154T and A841T; C154T and T862A; T303C and T426A; T303C and A433T; T303C and A435G; T303C and T530A; T303C and C572T; T303 C and T596C; T303C and T617C; T303C and T688C; T3 03C and A696T; T303C and T702C; T303C and A709G; T303C and A712G; T303C and T714G; T303C and A790 G; T303C and A841T; T303C and T862A; T426A and A4 33T; T426A and A435G; T426A and T530A; T426A and C572T; T426A and T596C; T426A and T617C; T426A and T688C; T426A and A696T; T426A and T702C; T426 A and A709G; T426A and A712G; T426A and T714G; T4 26A and A790G; T426A and A841T; T426A and T862A; A433T and A435G; A433T and T530A; A433T and C572 T; A433T and T596C; A433T and T617C; A433T and T6 88C; A433T and A696T; A433T and T702C; A433T and A709G; A433T and A712G; A433T and T714G; A433T and A790G; A433T and A841T; A433T and T862A; A435 G and T530A; A435G and C572T; A435G and T596C; A4 35G and T617C; A435G and T688C; A435G and A696T; A435G and T702C; A435G and A709G; A435G and A712 G; A435G and T714G; A435G and A790G; A435G and A8 41T; A435G and T862A; T530A and C572T; T530A and T596C; T530A and T617C; T530A and T688C; T530A and T596C; T530A and T617C; T530A and T688C; T530A and A696T; T530A and T702C; T530A and A709G; T530 A and A712G; T530A and T714G; T530A and A790G; T5 30A and A841T; T530A and T862A; C572T and T596C; C572T and T617C; C572T and T688C; C572T and A696 T; C572T and T702C; C572T and A709G; C572T and A7 12G; C572T and T714G; C572T and A790G; C572T and A841T; C572T and T862A; T596C and T617C; T596C and T688C; T596C and A696T; T596C and T702C; T596 C and A709G; T596C and A712G; T596C and T714G; T5 96C and A790G; T596C and A841T; T596C and T862A; T617C and T688C; T617C and A696T; T617C and T702 14G; T617C and A790G; T617C and A841T; T617C and T862A; T688C and A696T; T688C and T702C; T688C and A709G; T688C and A712G; T688C and T714G; T688 C and A790G; T688C and A841T; T688C and T862A; A6 96T and T702C; A696T and A709G; A696T and A712G; A696T and T714G; A696T and A790G; A696T and A841 T; A696T and T862A; T702C and A709G; T702C and A7 12G; T702C and T714G; T702C and A790G; T702C and A841T; T702C and T862A; A709G and A712G; A709G and T714G; A709G and A790G; A709G and A841T; A709 G and T862A; A712G and T714G; A712G and A790G; A7 12G and A841T; A712G and T862A; T714G and A790G; T714G and A841T; T714G and T862A; A790G and A841 T; A790G and T862A; or two mutations corresponding to A841T and T862A may be present within the promoter sequence.

[0041] In some embodiments, the nucleotide sequence of the promoter element provided herein may contain three mutations as compared to the nucleotide sequence of the reference promoter element. For example, in some embodiments, the following mutations relative to SEQ ID NO: 28: T146 C, C154T, and T303C; T146C, C154T, and T426A; T1 46C, C154T, and A433T; T146C, C154T, and A435G; T146C, C154T, and T530A; T146C, C154T, and C572 T; T146C, C154T, and T596C; T146C, C154T, and T6 17C; T146C, C154T, and T688C; T146C, C154T, and A696T; T146C, C154T, and T702C; T146C, C154T, and A709G; T146C, C154T, and A712G; T146C, C154T and T714G; T146C, C154T, and A790G; T146C, C15 4T, and A841T; T146C, C154T, and T862A; T146C, T 303C, and T426A; T146C, T303C, and A433T; T146C , T303C, and A435G; T146C, T303C, and T530A; T14 6C, T303C, and C572T; T146C, T303C, and T596C; T 146C, T303C, and T617C; T146C, T303C, and T688C ; T146C, T303C, and A696T; T146C, T303C, and T70 2C; T146C, T303C, and A709G; T146C, T303C, and A 712G; T146C, T303C, and T714G; T146C, T303C, and A790G; T146C, T303C, and A841T; T146C, T303C, and T862A; T146C, T426A, and A433T; T146C, T426 A, and A435G; T146C, T426A, and T530A; T146C, T4 26A, and C572T; T146C, T426A, and T596C; T146C, T426A, and T617C; T146C, T426A, and T688C; T146 C, T426A, and A696T; T146C, T426A, and T702C; T1 46C, T426A, and A709G; T146C, T426A, and A712G; T146C, T426A, and T714G; T146C, T426A, and A790 G; T146C, T426A, and A841T; T146C, T426A, and T8 62A; T146C, A433T, and A435G; T146C, A433T, and T530A; T146C, A433T, and C572T; T146C, A433T, and T596C; T146C, A433T, and T617C; T146C, A433T and T688C; T146C, A433T, and A696T; T146C, A43 3T, and T702C; T146C, A433T, and A709G; T146C, A 433T, and A712G; T146C, A433T, and T714G; T146C A433T, and A790G; T146C, A433T, and A841T; T14 6C, A433T, and T862A; T146C, A435G, and T530A; T 146C, A435G, and C572T; T146C, A435G, and T596C ; T146C, A435G, and T617C; T146C, A435G, and T68 8C; T146C, A435G, and A696T; T146C, A435G, and T 702C; T146C, A435G, and A709G; T146C, A435G, and A712G; T146C, A435G, and T714G; T146C, A435G, and A790G; T146C, A435G, and A841T; T146C, A435 G, and T862A; T146C, T530A, and C572T; T146C, T5 30A, and T596C; T146C, T530A, and T617C; T146C, T530A, and T688C; T146C, T530A, and A696T; T146 C, T530A, and T702C; T146C, T530A, and A709G; T1 46C, T530A, and A712G; T146C, T530A, and T714G; T146C, T530A, and A790G; T146C, T530A, and A841 T; T146C, T530A, and T862A; T146C, C572T, and T5 96C; T146C, C572T, and T617C; T146C, C572T, and T688C; T146C, C572T, and A696T; T146C, C572T, and よびT702C; T146C, C572T, and A709G; T146C, C572T 、and A712G; T146C, C572T, and T714G; T146C, C57 2T, and A790G; T146C, C572T, and A841T; T146C, C 572T, and T862A; T146C, T596C, and T617C; T146C 、T596C, and T688C; T146C, T596C, and A696T; T14 6C, T596C, and T702C; T146C, T596C, and A709G; T 146C, T596C, and A712G; T146C, T596C, and T714G ; T146C, T596C, and A790G; T146C, T596C, and A84 1T; T146C, T596C, and T862A; T146C, T617C, and T 688C; T146C, T617C, and A696T; T146C, T617C, and o y T702C; T146C, T617C, and A709G; T146C, T617C, and A712G; T146C, T617C, and T714G; T146C, T617 C, and A790G; T146C, T617C, and A841T; T146C, T6 17C, and T862A; T146C, T688C, and A696T; T146C, T688C, and T702C; T146C, T688C, and A709G; T146 C, T688C, and A712G; T146C, T688C, and T714G; T1 46C, T688C, and A790G; T146C, T688C, and A841T; T146C, T688C, and T862A; T146C, A696T, and T702 C; T146C, A696T, and A709G; T146C, A696T, and A7 12G; T146C, A696T, and T714G; T146C, A696T, and A790G; T146C, A696T, and A841T; T146C, A696T, and y T862A; T146C, T702C, and A709G; T146C, T702C and A712G; T146C, T702C, and T714G; T146C, T70 2C, and A790G; T146C, T702C, and A841T; T146C, T 702C, and T862A; T146C, A709G, and A712G; T146C A709G, and T714G; T146C, A709G, and A790G; T14 6C, A709G, and A841T; T146C, A709G, and T862A; T 146C, A712G, and T714G; T146C, A712G, and A790G ; T146C, A712G, and A841T; T146C, A712G, and T86 2A; T146C, T714G, and A790G; T146C, T714G, and A 841T; T146C, T714G, and T862A; T146C, A790G, and also A841T; T146C, A790G, and T862A; T146C, A841T, and T862A; C154T, T303C, and T426A; C154T, T303 C, and A433T; C154T, T303C, and A435G; C154T, T3 03C, and T530A; C154T, T303C, and C572T; C154T, T303C, and T596C; C154T, T303C, and T617C; C154 T, T303C, and T688C; C154T, T303C, and A696T; C1 54T, T303C, and T702C; C154T, T303C, and A709G; C154T, T303C, and A712G; C154T, T303C, and T714 G; C154T, T303C, and A790G; C154T, T303C, and A8 41T; C154T, T303C, and T862A; C154T, T426A, and A433T; C154T, T426A, and A435G; C154T, T426A, and also T530A; C154T, T426A, and C572T; C154T, T426A 、and T596C; C154T, T426A, and T617C; C154T, T42 6A, and T688C; C154T, T426A, and A696T; C154T, T 426A, and T702C; C154T, T426A, and A709G; C154T , T426A, and A712G; C154T, T426A, and T714G; C15 4T, T426A, and A790G; C154T, T426A, and A841T; C 154T, T426A, and T862A; C154T, A433T, and A435G ; C154T, A433T, and T530A; C154T, A433T, and C57 2T; C154T, A433T, and T596C; C154T, A433T, and T 617C; C154T, A433T, and T688C; C154T, A433T, and O bviously A696T; C154T, A433T, and T702C; C154T, A433T, and A709G; C154T, A433T, and A712G; C154T, A433 T, and T714G; C154T, A433T, and A790G; C154T, A4 33T, and A841T; C154T, A433T, and T862A; C154T, A435G, and T530A; C154T, A435G, and C572T; C154 T, A435G, and T596C; C154T, A435G, and T617C; C1 54T, A435G, and T688C; C154T, A435G, and A696T; C154T, A435G, and T702C; C154T, A435G, and A709 G; C154T, A435G, and A712G; C154T, A435G, and T7 14G; C154T, A435G, and A790G; C154T, A435G, and A841T; C154T, A435G, and T862A; C154T, T530A, and also C572T; C154T, T530A, and T596C; C154T, T530A and T617C; C154T, T530A, and T688C; C154T, T53 0A, and A696T; C154T, T530A, and T702C; C154T, T 530A, and A709G; C154T, T530A, and A712G; C154T , T530A, and T714G; C154T, T530A, and A790G; C15 4T, T530A, and A841T; C154T, T530A, and T862A; C 154T, C572T, and T596C; C154T, C572T, and T617C ; C154T, C572T, and T688C; C154T, C572T, and A69 6T; C154T, C572T, and T702C; C154T, C572T, and A 709G; C154T, C572T, and A712G; C154T, C572T, and o r T714G; C154T, C572T, and A790G; C154T, C572T, and A841T; C154T, C572T, and T862A; C154T, T596 C, and T617C; C154T, T596C, and T688C; C154T, T5 96C, and A696T; C154T, T596C, and T702C; C154T, T596C, and A709G; C154T, T596C, and A712G; C154 T, T596C, and T714G; C154T, T596C, and A790G; C1 54T, T596C, and A841T; C154T, T596C, and T862A; C154T, T617C, and T688C; C154T, T617C, and A696 T; C154T, T617C, and T702C; C154T, T617C, and A7 09G; C154T, T617C, and A712G; C154T, T617C, and T714G; C154T, T617C, and A790G; C154T, T617C, and A841T; C154T, T617C, and T862A; C154T, T688C , and A696T; C154T, T688C, and T702C; C154T, T68 8C, and A709G; C154T, T688C, and A712G; C154T, T 688C, and T714G; C154T, T688C, and A790G; C154T , T688C, and A841T; C154T, T688C, and T862A; C15 4T, A696T, and T702C; C154T, A696T, and A709G; C 154T, A696T, and A712G; C154T, A696T, and T714G ; C154T, A696T, and A790G; C154T, A696T, and A84 1T; C154T, A696T, and T862A; C154T, T702C, and A 709G; C154T, T702C, and A712G; C154T, T702C, and T714G; C154T, T702C, and A790G; C154T, T702C, and A841T; C154T, T702C, and T862A; C154T, A709 G, and A712G; C154T, A709G, and T714G; C154T, A7 09G, and A790G; C154T, A709G, and A841T; C154T, A709G, and T862A; C154T, A712G, and T714G; C154 T, A712G, and A790G; C154T, A712G, and A841T; C1 54T, A712G, and T862A; C154T, T714G, and A790G; C154T, T714G, and A841T; C154T, T714G, and T862 A; C154T, A790G, and A841T; C154T, A790G, and T8 62A; C154T, A841T, and T862A; T303C, T426A, and A433T; T303C, T426A, and A435G; T303C, T426A, and T530A; T303C, T426A, and C572T; T303C, T426A , and T596C; T303C, T426A, and T617C; T303C, T42 6A, and T688C; T303C, T426A, and A696T; T303C, T 426A, and T702C; T303C, T426A, and A709G; T303C , T426A, and A712G; T303C, T426A, and T714G; T30 3C, T426A, and A790G; T303C, T426A, and A841T; T 303C, T426A, and T862A; T303C, A433T, and A435G ; T303C, A433T, and T530A; T303C, A433T, and C57 2T; T303C, A433T, and T596C; T303C, A433T, and T 617C; T303C, A433T, and T688C; T303C, A433T, and A696T; T303C, A433T, and T702C; T303C, A433T, and A709G; T303C, A433T, and A712G; T303C, A433 T, and T714G; T303C, A433T, and A790G; T303C, A4 33T, and A841T; T303C, A433T, and T862A; T303C, A435G, and T530A; T303C, A435G, and C572T; T303 C, A435G, and T596C; T303C, A435G, and T617C; T3 03C, A435G, and T688C; T303C, A435G, and A696T; T303C, A435G, and T702C; T303C, A435G, and A709 G; T303C, A435G, and A712G; T303C, A435G, and T7 14G; T303C, A435G, and A790G; T303C, A435G, and A841T; T303C, A435G, and T862A; T303C, T530A, and C572T; T303C, T530A, and T596C; T303C, T530A , and T617C; T303C, T530A, and T688C; T303C, T53 0A, and A696T; T303C, T530A, and T702C; T303C, T 530A, and A709G; T303C, T530A, and A712G; T303C , T530A, and T714G; T303C, T530A, and A790G; T30 3C, T530A, and A841T; T303C, T530A, and T862A; T 303C, C572T, and T596C; T303C, C572T, and T617C ; T303C, C572T, and T688C; T303C, C572T, and A69 6T; T303C, C572T, and T702C; T303C, C572T, and A 709G; T303C, C572T, and A712G; T303C, C572T, and T714G; T303C, C572T, and A790G; T303C, C572T, and A841T; T303C, C572T, and T862A; T303C, T596 C, and T617C; T303C, T596C, and T688C; T303C, T5 96C, and A696T; T303C, T596C, and T702C; T303C, T596C, and A709G; T303C, T596C, and A712G; T303 C, T596C, and T714G; T303C, T596C, and A790G; T3 03C, T596C, and A841T; T303C, T596C, and T862A; T303C, T617C, and T688C; T303C, T617C, and A696 T; T303C, T617C, and T702C; T303C, T617C, and A7 09G; T303C, T617C, and A712G; T303C, T617C, and T714G; T303C, T617C, and A790G; T303C, T617C, and A841T; T303C, T617C, and T862A; T303C, T688C and A696T; T303C, T688C, and T702C; T303C, T68 8C, and A709G; T303C, T688C, and A712G; T303C, T 688C, and T714G; T303C, T688C, and A790G; T303C , T688C, and A841T; T303C, T688C, and T862A; T30 3C, A696T, and T702C; T303C, A696T, and A709G; T 303C, A696T, and A712G; T303C, A696T, and T714G ; T303C, A696T, and A790G; T303C, A696T, and A84 1T; T303C, A696T, and T862A; T303C, T702C, and A 709G; T303C, T702C, and A712G; T303C, T702C, and T714G; T303C, T702C, and A790G; T303C, T702C, and A841T; T303C, T702C, and T862A; T303C, A709 G, and A712G; T303C, A709G, and T714G; T303C, A7 09G, and A790G; T303C, A709G, and A841T; T303C, A709G, and T862A; T303C, A712G, and T714G; T303 C, A712G, and A790G; T303C, A712G, and A841T; T3 03C, A712G, and T862A; T303C, T714G, and A790G; T303C, T714G, and A841T; T303C, T714G, and T862 A; T303C, A790G, and A841T; T303C, A790G, and T8 62A; T303C, A841T, and T862A; T426A, A433T, and A435G; T426A, A433T, and T530A; T426A, A433T, and C572T; T426A, A433T, and T596C; T426A, A433T , and T617C; T426A, A433T, and T688C; T426A, A43 3T, and A696T; T426A, A433T, and T702C; T426A, A 433T, and A709G; T426A, A433T, and A712G; T426A , A433T, and T714G; T426A, A433T, and A790G; T42 6A, A433T, and A841T; T426A, A433T, and T862A; T 426A, A435G, and T530A; T426A, A435G, and C572T ; T426A, A435G, and T596C; T426A, A435G, and T61 7C; T426A, A435G, and T688C; T426A, A435G, and A 696T; T426A, A435G, and T702C; T426A, A435G, and o r A709G; T426A, A435G, and A712G; T426A, A435G, and T714G; T426A, A435G, and A790G; T426A, A435 G, and A841T; T426A, A435G, and T862A; T426A, T5 30A, and C572T; T426A, T530A, and T596C; T426A, T530A, and T617C; T426A, T530A, and T688C; T426 A, T530A, and A696T; T426A, T530A, and T702C; T4 26A, T530A, and A709G; T426A, T530A, and A712G; T426A, T530A, and T714G; T426A, T530A, and A790 G; T426A, T530A, and A841T; T426A, T530A, and T8 62A; T426A, C572T, and T596C; T426A, C572T, and T617C; T426A, C572T, and T688C; T426A, C572T, a nd A696T; T426A, C572T, and T702C; T426A, C572T , and A709G; T426A, C572T, and A712G; T426A, C57 2T, and T714G; T426A, C572T, and A790G; T426A, C 572T, and A841T; T426A, C572T, and T862A; T426A , T596C, and T617C; T426A, T596C, and T688C; T42 6A, T596C, and A696T; T426A, T596C, and T702C; T 426A, T596C, and A709G; T426A, T596C, and A712G ; T426A, T596C, and T714G; T426A, T596C, and A79 0G; T426A, T596C, and A841T; T426A, T596C, and T 862A; T426A, T617C, and T688C; T426A, T617C, and oyo bi A696T; T426A, T617C, and T702C; T426A, T617C, and A709G; T426A, T617C, and A712G; T426A, T617 C, and T714G; T426A, T617C, and A790G; T426A, T6 17C, and A841T; T426A, T617C, and T862A; T426A, T688C, and A696T; T426A, T688C, and T702C; T426 A, T688C, and A709G; T426A, T688C, and A712G; T426A, T688C, and T714G; T426A, T688C, and A790 G; T426A, T688C, and A841T; T426A, T688C, and T8 62A; T426A, A696T, and T702C; T426A, A696T, and A709G; T426A, A696T, and A712G; T426A, A696T, o yobi T714G; T426A, A696T, and A790G; T426A, A696T , and A841T; T426A, A696T, and T862A; T426A, T70 2C, and A709G; T426A, T702C, and A712G; T426A, T 702C, and T714G; T426A, T702C, and A790G; T426A , T702C, and A841T; T426A, T702C, and T862A; T42 6A, A709G, and A712G; T426A, A709G, and T714G; T 426A, A709G, and A790G; T426A, A709G, and A841T ; T426A, A709G, and T862A; T426A, A712G, and T71 4G; T426A, A712G, and A790G; T426A, A712G, and A 841T; T426A, A712G, and T862A; T426A, T714G, and oyo bi A790G; T426A, T714G, and A841T; T426A, T714G, and T862A; T426A, A790G, and A841T; T426A, A790 G, and T862A; T426A, A841T, and T862A; A433T, A4 35G, and T530A; A433T, A435G, and C572T; A433T, A435G, and T596C; A433T, A435G, and T617C; A433 T, A435G, and T688C; A433T, A435G, and A696T; A4 33T, A435G, and T702C; A433T, A435G, and A709G; A433T, A435G, and A712G; A433T, A435G, and T714 G; A433T, A435G, and A790G; A433T, A435G, and A8 41T; A433T, A435G, and T862A; A433T, T530A, and C572T; A433T, T530A, and T596C; A433T, T530A, and o Call T617C; A433T, T530A, and T688C; A433T, T530A , and A696T; A433T, T530A, and T702C; A433T, T53 0A, and A709G; A433T, T530A, and A712G; A433T, T 530A, and T714G; A433T, T530A, and A790G; A433T , T530A, and A841T; A433T, T530A, and T862A; A43 3T, C572T, and T596C; A433T, C572T, and T617C; A 433T, C572T, and T688C; A433T, C572T, and A696T ; A433T, C572T, and T702C; A433T, C572T, and A70 9G; A433T, C572T, and A712G; A433T, C572T, and T 714G; A433T, C572T, and A790G; A433T, C572T, and O r A841T; A433T, C572T, and T862A; A433T, T596C, and T617C; A433T, T596C, and T688C; A433T, T596 C, and A696T; A433T, T596C, and T702C; A433T, T5 96C, and A709G; A433T, T596C, and A712G; A433T, T596C, and T714G; A433T, T596C, and A790G; A433 T, T596C, and A841T; A433T, T596C, and T862A; A4 33T, T617C, and T688C; A433T, T617C, and A696T; A433T, T617C, and T702C; A433T, T617C, and A709 G; A433T, T617C, and A712G; A433T, T617C, and T7 14G; A433T, T617C, and A790G; A433T, T617C, and A841T; A433T, T617C, and T862A; A433T, T688C, and A696T; A433T, T688C, and T702C; A433T, T688C and A709G; A433T, T688C, and A712G; A433T, T68 8C, and T714G; A433T, T688C, and A790G; A433T, T 688C, and A841T; A433T, T688C, and T862A; A433T , A696T, and T702C; A433T, A696T, and A709G; A43 3T, A696T, and A712G; A433T, A696T, and T714G; A 433T, A696T, and A790G; A433T, A696T, and A841T ; A433T, A696T, and T862A; A433T, T702C, and A70 9G; A433T, T702C, and A712G; A433T, T702C, and T 714G; A433T, T702C, and A790G; A433T, T702C, and o r A841T; A433T, T702C, and T862A; A433T, A709G, and A712G; A433T, A709G, and T714G; A433T, A709 G, and A790G; A433T, A709G, and A841T; A433T, A7 09G, and T862A; A433T, A712G, and T714G; A433T, A712G, and A790G; A433T, A712G, and A841T; A433 T, A712G, and T862A; A433T, T714G, and A790G; A4 33T, T714G, and A841T; A433T, T714G, and T862A; A433T, A790G, and A841T; A433T, A790G, and T862 A; A433T, A841T, and T862A; A435G, T530A, and C5 72T; A435G, T530A, and T596C; A435G, T530A, and T617C; A435G, T530A, and T688C; A435G, T530A, and A696T; A435G, T530A, and T702C; A435G, T530A , and A709G; A435G, T530A, and A712G; A435G, T53 0A, and T714G; A435G, T530A, and A790G; A435G, T 530A, and A841T; A435G, T530A, and T862A; A435G , C572T, and T596C; A435G, C572T, and T617C; A43 5G, C572T, and T688C; A435G, C572T, and A696T; A 435G, C572T, and T702C; A435G, C572T, and A709G ; A435G, C572T, and A712G; A435G, C572T, and T71 4G; A435G, C572T, and A790G; A435G, C572T, and A 841T; A435G, C572T, and T862A; A435G, T596C, and T617C; A435G, T596C, and T688C; A435G, T596C, and A696T; A435G, T596C, and T702C; A435G, T596 C, and A709G; A435G, T596C, and A712G; A435G, T5 96C, and T714G; A435G, T596C, and A790G; A435G, T596C, and A841T; A435G, T596C, and T862A; A435 G, T617C, and T688C; A435G, T617C, and A696T; A4 35G, T617C, and T702C; A435G, T617C, and A709G; A435G, T617C, and A712G; A435G, T617C, and T714 G; A435G, T617C, and A790G; A435G, T617C, and A8 41T; A435G, T617C, and T862A; A435G, T688C, and A696T; A435G, T688C, and T702C; A435G, T688C, and A709G; A435G, T688C, and A712G; A435G, T688C and T714G; A435G, T688C, and A790G; A435G, T68 8C, and A841T; A435G, T688C, and T862A; A435G, A 696T, and T702C; A435G, A696T, and A709G; A435G A696T, and A712G; A435G, A696T, and T714G; A43 5G, A696T, and A790G; A435G, A696T, and A841T; A 435G, A696T, and T862A; A435G, T702C, and A709G ; A435G, T702C, and A712G; A435G, T702C, and T71 4G; A435G, T702C, and A790G; A435G, T702C, and A 841T; A435G, T702C, and T862A; A435G, A709G, and A712G; A435G, A709G, and T714G; A435G, A709G, and A790G; A435G, A709G, and A841T; A435G, A709 G, and T862A; A435G, A712G, and T714G; A435G, A7 12G, and A790G; A435G, A712G, and A841T; A435G, A712G, and T862A; A435G, T714G, and A790G; A435 G, T714G, and A841T; A435G, T714G, and T862A; A4 35G, A790G, and A841T; A435G, A790G, and T862A; A435G, A841T, and T862A; T530A, C572T, and T596 C; T530A, C572T, and T617C; T530A, C572T, and T6 88C; T530A, C572T, and A696T; T530A, C572T, and T702C; T530A, C572T, and A709G; T530A, C572T, and A712G; T530A, C572T, and T714G; T530A, C572T , and A790G; T530A, C572T, and A841T; T530A, C57 2T, and T862A; T530A, T596C, and T617C; T530A, T 596C, and T688C; T530A, T596C, and A696T; T530A , T596C, and T702C; T530A, T596C, and A709G; T53 0A, T596C, and A712G; T530A, T596C, and T714G; T 530A, T596C, and A790G; T530A, T596C, and A841T ; T530A, T596C, and T862A; T530A, T617C, and T68 8C; T530A, T617C, and A696T; T530A, T617C, and T 702C; T530A, T617C, and A709G; T530A, T617C, and A712G; T530A, T617C, and T714G; T530A, T617C, and A790G; T530A, T617C, and A841T; T530A, T617 C, and T862A; T530A, T688C, and A696T; T530A, T6 88C, and T702C; T530A, T688C, and A709G; T530A, T688C, and A712G; T530A, T688C, and T714G; T530 A, T688C, and A790G; T530A, T688C, and A841T; T5 30A, T688C, and T862A; T530A, A696T, and T702C; T530A, A696T, and A709G; T530A, A696T, and A712 G; T530A, A696T, and T714G; T530A, A696T, and A790G; T530A, A696T, and A841T; T530A, A696 T, and T862A; T530A, T702C, and A709G; T530A, T7 02C, and A712G; T530A, T702C, and T714G; T530A, T702C, and A790G; T530A, T702C, and A841T; T530 A, T702C, and T862A; T530A, A709G, and A712G; T5 30A, A709G, and T714G; T530A, A709G, and A790G; T530A, A709G, and A841T; T530A, A709G, and T862 A; T530A, A712G, and T714G; T530A, A712G, and A7 90G; T530A, A712G, and A841T; T530A, A712G, and T862A; T530A, T714G, and A790G; T530A, T714G, and A841T; T530A, T714G, and T862A; T530A, A790G and A841T; T530A, A790G, and T862A; T530A, A84 1T, and T862A; C572T, T596C, and T617C; C572T, T 596C, and T688C; C572T, T596C, and A696T; C572T T596C, and T702C; C572T, T596C, and A709G; C57 2T, T596C, and A712G; C572T, T596C, and T714G; C 572T, T596C, and A790G; C572T, T596C, and A841T ; C572T, T596C, and T862A; C572T, T617C, and T68 8C; C572T, T617C, and A696T; C572T, T617C, and T 702C; C572T, T617C, and A709G; C572T, T617C, and O A712G; C572T, T617C, and T714G; C572T, T617C, and A790G; C572T, T617C, and A841T; C572T, T617 C, and T862A; C572T, T688C, and A696T; C572T, T6 88C, and T702C; C572T, T688C, and A709G; C572T, T688C, and A712G; C572T, T688C, and T714G; C572 T, T688C, and A790G; C572T, T688C, and A841T; C5 72T, T688C, and T862A; C572T, A696T, and T702C; C572T, A696T, and A709G; C572T, A696T, and A712 G; C572T, A696T, and T714G; C572T, A696T, and A7 90G; C572T, A696T, and A841T; C572T, A696T, and T862A; C572T, T702C, and A709G; C572T, T702C, and A712G; C572T, T702C, and T714G; C572T, T702C , and A790G; C572T, T702C, and A841T; C572T, T70 2C, and T862A; C572T, A709G, and A712G; C572T, A 709G, and T714G; C572T, A709G, and A790G; C572T , A709G, and A841T; C572T, A709G, and T862A; C57 2T, A712G, and T714G; C572T, A712G, and A790G; C 572T, A712G, and A841T; C572T, A712G, and T862A ; C572T, T714G, and A790G; C572T, T714G, and A84 1T; C572T, T714G, and T862A; C572T, A790G, and A 841T; C572T, A790G, and T862A; C572T, A841T, and T862A; T596C, T617C, and T688C; T596C, T617C, and A696T; T596C, T617C, and T702C; T596C, T617 C, and A709G; T596C, T617C, and A712G; T596C, T6 17C, and T714G; T596C, T617C, and A790G; T596C, T617C, and A841T; T596C, T617C, and T862A; T596 C, T688C, and A696T; T596C, T688C, and T702C; T5 96C, T688C, and A709G; T596C, T688C, and A712G; T596C, T688C, and T714G; T596C, T688C, and A790 G; T596C, T688C, and A841T; T596C, T688C, and T8 62A; T596C, A696T, and T702C; T596C, A696T, and A709G; T596C, A696T, and A712G; T596C, A696T, and o y and T714G; T596C, A696T, and A790G; T596C, A696T , and A841T; T596C, A696T, and T862A; T596C, T70 2C, and A709G; T596C, T702C, and A712G; T596C, T 702C, and T714G; T596C, T702C, and A790G; T596C , T702C, and A841T; T596C, T702C, and T862A; T59 6C, A709G, and A712G; T596C, A709G, and T714G; T 596C, A709G, and A790G; T596C, A709G, and A841T ; T596C, A709G, and T862A; T596C, A712G, and T71 4G; T596C, A712G, and A790G; T596C, A712G, and A 841T; T596C, A712G, and T862A; T596C, T714G, and o y and A790G; T596C, T714G, and A841T; T596C, T714G, and T862A; T596C, A790G, and A841T; T596C, A790 G, and T862A; T596C, A841T, and T862A; T617C, T6 88C, and A696T; T617C, T688C, and T702C; T617C, T688C, and A709G; T617C, T688C, and A712G; T617 C, T688C, and T714G; T617C, T688C, and A790G; T6 17C, T688C, and A841T; T617C, T688C, and T862A; T617C, A696T, and T702C; T617C, A696T, and A709 G; T617C, A696T, and A712G; T617C, A696T, and T7 14G; T617C, A696T, and A790G; T617C, A696T, and A841T; T617C, A696T, and T862A; T617C, T702C, and and A709G; T617C, T702C, and A712G; T617C, T702C and T714G; T617C, T702C, and A790G; T617C, T70 2C, and A841T; T617C, T702C, and T862A; T617C, A 709G, and A712G; T617C, A709G, and T714G; T617C , A709G, and A790G; T617C, A709G, and A841T; T61 7C, A709G, and T862A; T617C, A712G, and T714G; T 617C, A712G, and A790G; T617C, A712G, and A841T ; T617C, A712G, and T862A; T617C, T714G, and A79 0G; T617C, T714G, and A841T; T617C, T714G, and T 862A; T617C, A790G, and A841T; T617C, A790G, and i.e., T862A; T617C, A841T, and T862A; T688C, A696T, and T702C; T688C, A696T, and A709G; T688C, A696 T, and A712G; T688C, A696T, and T714G; T688C, A6 96T, and A790G; T688C, A696T, and A841T; T688C, A696T, and T862A; T688C, T702C, and A709G; T688 C, T702C, and A712G; T688C, T702C, and T714G; T6 88C, T702C, and A790G; T688C, T702C, and A841T; T688C, T702C, and T862A; T688C, A709G, and A712 G; T688C, A709G, and T714G; T688C, A709G, and A7 90G; T688C, A709G, and A841T; T688C, A709G, and T862A; T688C, A712G, and T714G; T688C, A712G, and also A790G; T688C, A712G, and A841T; T688C, A712G , and T862A; T688C, T714G, and A790G; T688C, T71 4G, and A841T; T688C, T714G, and T862A; T688C, A 790G, and A841T; T688C, A790G, and T862A; T688C , A841T, and T862A; A696T, T702C, and A709G; A69 6T, T702C, and A712G; A696T, T702C, and T714G; A 696T, T702C, and A790G; A696T, T702C, and A841T ; A696T, T702C, and T862A; A696T, A709G, and A71 2G; A696T, A709G, and T714G; A696T, A709G, and A 790G; A696T, A709G, and A841T; A696T, A709G, and T862A; A696T, A712G, and T714G; A696T, A712G, and A790G; A696T, A712G, and A841T; A696T, A712 G, and T862A; A696T, T714G, and A790G; A696T, T7 14G, and A841T; A696T, T714G, and T862A; A696T, A790G, and A841T; A696T, A790G, and T862A; A696 T, A841T, and T862A; T702C, A709G, and A712G; T7 02C, A709G, and T714G; T702C, A709G, and A790G; T702C, A709G, and A841T; T702C, A709G, and T862 A; T702C, A712G, and T714G; T702C, A712G, and A7 90G; T702C, A712G, and A841T; T702C, A712G, and T862A; T702C, T714G, and A790G; T702C, T714G, and A841T; T702C, T714G, and T862A; T702C, A790G , and A841T; T702C, A790G, and T862A; T702C, A84 1T, and T862A; A709G, A712G, and T714G; A709G, A 712G, and A790G; A709G, A712G, and A841T; A709G , A712G, and T862A; A709G, T714G, and A790G; A70 9G, T714G, and A841T; A709G, T714G, and T862A; A 709G, A790G, and A841T; A709G, A790G, and T862A ; A709G, A841T, and T862A; A712G, T714G, and A79 0G; A712G, T714G, and A841T; A712G, T714G, and T 862A; A712G, A790G, and A841T; A712G, A790G, and oyo bi T862A; A712G, A841T, and T862A; T714G, A790G, and A841T; T714G, A790G, and T862A; T714G, A841 T, and T862A; or A790G, A841T, and T862A may be present within the promoter sequence.

[0042] In some embodiments, the nucleotide sequence of the promoter element provided herein may contain two mutations as compared to the nucleotide sequence of the reference promoter element. For example, in some embodiments, the following mutations relative to SEQ ID NO: 28: T688 C and A696T; T688C and T702C; T688C and A712G; T6 88C and T714G; A696T and T702C; A696T and A712G; A696T and T714G; T702C and A712G; T702C and T714 G; or two mutations corresponding to A712G and T714G may be present within the promoter sequence.

[0043] In some embodiments, the nucleotide sequence of the promoter element provided herein ​​The column contains three mutations as compared to the nucleotide sequence of the reference promoter element. It is possible. For example, in some embodiments, the following mutations relative to SEQ ID NO: 28: T688 C, A696T, and T702C; T688C, A696T, and A712G; T6 88C, A696T, and T714G; T688C, T702C, and A712G; T688C, T702C, and T714G; T688C, A712G, and T714 G; A696T, T702C, and A712G; A696T, T702C, and T7 14G; A696T, A712G, and T714G; or the three mutations corresponding to T702C, A712G, and T714G may be present within the promoter sequence.

[0044] In some embodiments, the nucleotide sequence of the promoter element provided herein contains four mutations as compared to the nucleotide sequence of the reference promoter element. It is possible. For example, in some embodiments, the following mutations relative to SEQ ID NO: 28: T688 C, A696T, T702C, and A712G; T688C, A696T, T702C and T714G; T688C, A696T, A712G, and T714G; T68 8C, T702C, A712G, and T714G; or the four mutations corresponding to A696T, T702C, A 712G, and T714G may be present within the promoter sequence. It is possible.

[0045] In some embodiments, the nucleotide sequence of the promoter element provided herein contains five mutations as compared to the nucleotide sequence of the reference promoter element. may be present. For example, in some embodiments, the following mutations for SEQ ID NO: 28: T688 C, five mutations corresponding to A696T, T702C, A712G, and T714G may be present within the promoter sequence.

[0046] In some embodiments, the nucleotide sequence of the promoter element provided herein may contain two mutations compared to the nucleotide sequence of the reference promoter element. may be present. For example, in some embodiments, the following mutations for SEQ ID NO: 28: 146C and 154T; 146C and 303C; 146C and 426A; 146C and 4 33T; 146C and 435G; 146C and 530A; 146C and 572T; 146C and 596C; 146C and 617C; 146C and 688C; 146C and 696T; 146C and 702C; 146C and 709G; 146C and 7 12G; 146C and 714G; 146C and 790G; 146C and A841T ; 146C and 862A; 154T and 303C; 154T and 426A; 154 T and 433T; 154T and 435G; 154T and 530A; 154T and 572T; 154T and 596C; 154T and 617C; 154T and 688C ; 154T and 696T; 154T and 702C; 154T and 709G; 154 T and 712G; 154T and 714G; 154T and 790G; 154T and A841T; 154T and 862A; 303C and 426A; 303C and 433 T; 303C and 435G; 303C and 530A; 303C and 572T; 30 3C and 596C; 303C and 617C; 303C and 688C; 303C and and 696T;303C and 702C;303C and 709G;303C and 712 G;303C and 714G;303C and 790G;303C and A841T;3 03C and 862A;426A and 433T;426A and 435G;426A and 530A;426A and 572T;426A and 596C;426A and 61 7C;426A and 688C;426A and 696T;426A and 702C;4 26A and 709G;426A and 712G;426A and 714G;426A and 790G;426A and A841T;426A and 862A;433T and 4 35G;433T and 530A;433T and 572T;433T and 596C; 433T and 617C;433T and 688C;433T and 696T;433T and 702C;433T and 709G;433T and 712G;433T and 7 14G;433T and 790G;433T and A841T;433T and 862A ;435G and 530A;435G and 572T;435G and 596C;435 G and 617C;435G and 688C;435G and 696T;435G and 702C;435G and 709G;435G and 712G;435G and 714G ;435G and 790G;435G and A841T;435G and 862A;53 0A and 572T;530A and 596C;530A and 617C;530A and and 688C;530A and 696T;530A and 702C;530A and 709 G;530A and 712G;530A and 714G;530A and 790G;53 0A and A841T; 530A and 862A; 572T and 596C; 572T and 617C; 572T and 688C; 572T and 696T; 572T and 70 2C; 572T and 709G; 572T and 712G; 572T and 714G; 5 72T and 790G; 572T and A841T; 572T and 862A; 596C and 617C; 596C and 688C; 596C and 696T; 596C and 7 02C; 596C and 709G; 596C and 712G; 596C and 714G; 596C and 790G; 596C and A841T; 596C and 862A; 617 C and 688C; 617C and 696T; 617C and 702C; 617C and 709G; 617C and 712G; 617C and 714G; 617C and 790G ; 617C and A841T; 617C and 862A; 688C and 696T; 68 8C and 702C; 688C and 709G; 688C and 712G; 688C and 714G; 688C and 790G; 688C and A841T; 688C and 86 2A; 696T and 702C; 696T and 709G; 696T and 712G; 6 96T and 714G; 696T and 790G; 696T and A841T; 696T and 862A; 702C and 709G; 702C and 712G; 702C and 7 14G; 702C and 790G; 702C and A841T; 702C and 862A ; 709G and 712G; 709G and 714G; 709G and 790G; 709 G and A841T; 709G and 862A; 712G and 714G; 712G and 790G; 712G and A841T; 712G and 862A; 714G and 79 0G; 714G and A841T; 714G and 862A; 790G and A841T ; two mutations corresponding to 790G and 862A; or A841T and 862A can be present within the promoter sequence, provided that the nucleotide of the display is not the same as the corresponding naturally occurring nucleotide thereof.

[0047] In some embodiments, the nucleotide sequence of the promoter element provided herein can contain three mutations compared to the nucleotide sequence of the reference promoter element . For example, in some embodiments, the following mutations relative to SEQ ID NO: 28: 146C , 154T, and 303C; 146C, 154T, and 426A; 146C, 154 T, and 433T; 146C, 154T, and 435G; 146C, 154T, and 530A; 146C, 154T, and 572T; 146C, 154T, and 596 C; 146C, 154T, and 617C; 146C, 154T, and 688C; 14 6C, 154T, and 696T; 146C, 154T, and 702C; 146C, 1 54T, and 709G; 146C, 154T, and 712G; 146C, 154T, and 714G; 146C, 154T, and 790G; 146C, 154T, and A 841T; 146C, 154T, and 862A; 146C, 303C, and 426A ; 146C, 303C, and 433T; 146C, 303C, and 435G; 146 C, 303C, and 530A; 146C, 303C, and 572T; 146C, 30 3C, and 596C; 146C, 303C, and 617C; 146C, 303C, and 688C; Call 688C; 146C, 303C, and 696T; 146C, 303C, and 70 2C; 146C, 303C, and 709G; 146C, 303C, and 712G; 1 46C, 303C, and 714G; 146C, 303C, and 790G; 146C, 303C, and A841T; 146C, 303C, and 862A; 146C, 426 A, and 433T; 146C, 426A, and 435G; 146C, 426A, and so on 530A; 146C, 426A, and 572T; 146C, 426A, and 596 C; 146C, 426A, and 617C; 146C, 426A, and 688C; 14 6C, 426A, and 696T; 146C, 426A, and 702C; 146C, 4 26A, and 709G; 146C, 426A, and 712G; 146C, 426A, and 714G; 146C, 426A, and 790G; 146C, 426A, and A 841T; 146C, 426A, and 862A; 146C, 433T, and 435G ; 146C, 433T, and 530A; 146C, 433T, and 572T; 146 C, 433T, and 596C; 146C, 433T, and 617C; 146C, 43 3T, and 688C; 146C, 433T, and 696T; 146C, 433T, and o yon 702C; 146C, 433T, and 709G; 146C, 433T, and 71 2G; 146C, 433T, and 714G; 146C, 433T, and 790G; 1 46C, 433T, and A841T; 146C, 433T, and 862A; 146C , 435G, and 530A; 146C, 435G, and 572T; 146C, 435 G, and 596C; 146C, 435G, and 617C; 146C, 435G, and so and 688C; 146C, 435G, and 696T; 146C, 435G, and 702 C; 146C, 435G, and 709G; 146C, 435G, and 712G; 14 6C, 435G, and 714G; 146C, 435G, and 790G; 146C, 4 35G, and A841T; 146C, 435G, and 862A; 146C, 530A and 572T; 146C, 530A, and 596C; 146C, 530A, and 617C; 146C, 530A, and 688C; 146C, 530A, and 696T ; 146C, 530A, and 702C; 146C, 530A, and 709G; 146 C, 530A, and 712G; 146C, 530A, and 714G; 146C, 53 0A, and 790G; 146C, 530A, and A841T; 146C, 530A, and 862A; 146C, 572T, and 596C; 146C, 572T, and 6 17C; 146C, 572T, and 688C; 146C, 572T, and 696T; 146C, 572T, and 702C; 146C, 572T, and 709G; 146C 572T, and 712G; 146C, 572T, and 714G; 146C, 572 T, and 790G; 146C, 572T, and A841T; 146C, 572T, o and 862A; 146C, 596C, and 617C; 146C, 596C, and 68 8C; 146C, 596C, and 696T; 146C, 596C, and 702C; 1 46C, 596C, and 709G; 146C, 596C, and 712G; 146C, 596C, and 714G; 146C, 596C, and 790G; 146C, 596C , and A841T; 146C, 596C, and 862A; 146C, 617C, and 688C; 146C, 617C, and 696T; 146C, 617C, and 702 C; 146C, 617C, and 709G; 146C, 617C, and 712G; 14 6C, 617C, and 714G; 146C, 617C, and 790G; 146C, 6 17C, and A841T; 146C, 617C, and 862A; 146C, 688C , and 696T; 146C, 688C, and 702C; 146C, 688C, and 709G; 146C, 688C, and 712G; 146C, 688C, and 714G ; 146C, 688C, and 790G; 146C, 688C, and A841T; 14 6C, 688C, and 862A; 146C, 696T, and 702C; 146C, 6 96T, and 709G; 146C, 696T, and 712G; 146C, 696T, and 714G; 146C, 696T, and 790G; 146C, 696T, and A 841T; 146C, 696T, and 862A; 146C, 702C, and 709G ; 146C, 702C, and 712G; 146C, 702C, and 714G; 146 C, 702C, and 790G; 146C, 702C, and A841T; 146C, 7 02C, and 862A; 146C, 709G, and 712G; 146C, 709G, and 714G; 146C, 709G, and 790G; 146C, 709G, and A 841T; 146C, 709G, and 862A; 146C, 712G, and 714G ; 146C, 712G, and 790G; 146C, 712G, and A841T; 14 6C, 712G, and 862A; 146C, 714G, and 790G; 146C, 7 14G, and A841T; 146C, 714G, and 862A; 146C, 790G , and A841T; 146C, 790G, and 862A; 146C, A841T, and 862A; 154T, 303C, and 426A; 154T, 303C, and 43 3T; 154T, 303C, and 435G; 154T, 303C, and 530A; 1 54T, 303C, and 572T; 154T, 303C, and 596C; 154T, 303C, and 617C; 154T, 303C, and 688C; 154T, 303C , and 696T; 154T, 303C, and 702C; 154T, 303C, and 709G; 154T, 303C, and 712G; 154T, 303C, and 714G ; 154T, 303C, and 790G; 154T, 303C, and A841T; 15 4T, 303C, and 862A; 154T, 426A, and 433T; 154T, 4 26A, and 435G; 154T, 426A, and 530A; 154T, 426A, and 572T; 154T, 426A, and 596C; 154T, 426A, and 6 17C; 154T, 426A, and 688C; 154T, 426A, and 696T; 154T, 426A, and 702C; 154T, 426A, and 709G; 154T , 426A, and 712G; 154T, 426A, and 714G; 154T, 426 A, and 790G; 154T, 426A, and A841T; 154T, 426A, and 862A; 154T, 433T, and 435G; 154T, 433T, and 53 0A; 154T, 433T, and 572T; 154T, 433T, and 596C; 1 54T, 433T, and 617C; 154T, 433T, and 688C; 154T, 433T, and 696T; 154T, 433T, and 702C; 154T, 433T , and 709G; 154T, 433T, and 712G; 154T, 433T, and 714G; 154T, 433T, and 790G; 154T, 433T, and A841 T; 154T, 433T, and 862A; 154T, 435G, and 530A; 15 4T, 435G, and 572T; 154T, 435G, and 596C; 154T, 4 35G, and 617C; 154T, 435G, and 688C; 154T, 435G, and 696T; 154T, 435G, and 702C; 154T, 435G, and 7 09G; 154T, 435G, and 712G; 154T, 435G, and 714G; 154T, 435G, and 790G; 154T, 435G, and A841T; 154 T, 435G, and 862A; 154T, 530A, and 572T; 154T, 53 0A, and 596C; 154T, 530A, and 617C; 154T, 530A, and 688C; 154T, 530A, and 696T; 154T, 530A, and 70 2C; 154T, 530A, and 709G; 154T, 530A, and 712G; 1 54T, 530A, and 714G; 154T, 530A, and 790G; 154T, 530A, and A841T; 154T, 530A, and 862A; 154T, 572 T, and 596C; 154T, 572T, and 617C; 154T, 572T, and 688C; 154T, 572T, and 696T; 154T, 572T, and 702 C; 154T, 572T, and 709G; 154T, 572T, and 712G; 15 4T, 572T, and 714G; 154T, 572T, and 790G; 154T, 5 72T, and A841T; 154T, 572T, and 862A; 154T, 596C , and 617C; 154T, 596C, and 688C; 154T, 596C, and 696T; 154T, 596C, and 702C; 154T, 596C, and 709G ; 154T, 596C, and 712G; 154T, 596C, and 714G; 154 T, 596C, and 790G; 154T, 596C, and A841T; 154T, 5 96C, and 862A; 154T, 617C, and 688C; 154T, 617C, , and 696T; 154T, 617C, and 702C; 154T, 617C, and 7 09G; 154T, 617C, and 712G; 154T, 617C, and 714G; 154T, 617C, and 790G; 154T, 617C, and A841T; 154 T, 617C, and 862A; 154T, 688C, and 696T; 154T, 68 8C, and 702C; 154T, 688C, and 709G; 154T, 688C, and 712G; 154T, 688C, and 714G; 154T, 688C, and 79 0G; 154T, 688C, and A841T; 154T, 688C, and 862A; 154T, 696T, and 702C; 154T, 696T, and 709G; 154T , 696T, and 712G; 154T, 696T, and 714G; 154T, 696 T, and 790G; 154T, 696T, and A841T; 154T, 696T, and 862A; 154T, 702C, and 709G; 154T, 702C, and 71 2G; 154T, 702C, and 714G; 154T, 702C, and 790G; 1 54T, 702C, and A841T; 154T, 702C, and 862A; 154T , 709G, and 712G; 154T, 709G, and 714G; 154T, 709G, and 790G; 154T, 709G, and A841T; 154 T, 709G, and 862A; 154T, 712G, and 714G; 154T, 71 2G, and 790G; 154T, 712G, and A841T; 154T, 712G, and 862A; 154T, 714G, and 790G; 154T, 714G, and A 841T; 154T, 714G, and 862A; 154T, 790G, and A841 T; 154T, 790G, and 862A; 154T, A841T, and 862A; 3 03C, 426A, and 433T; 303C, 426A, and 435G; 303C, 426A, and 530A; 303C, 426A, and 572T; 303C, 426A , and 596C; 303C, 426A, and 617C; 303C, 426A, and 688C; 303C, 426A, and 696T; 303C, 426A, and 702C ; 303C, 426A, and 709G; 303C, 426A, and 712G; 303 C, 426A, and 714G; 303C, 426A, and 790G; 303C, 42 6A, and A841T; 303C, 426A, and 862A; 303C, 433T, and 435G; 303C, 433T, and 530A; 303C, 433T, and 5 72T; 303C, 433T, and 596C; 303C, 433T, and 617C; 303C, 433T, and 688C; 303C, 433T, and 696T; 303C , 433T, and 702C; 303C, 433T, and 709G; 303C, 433 T, and 712G; 303C, 433T, and 714G; 303C, 433T, and also 790G; 303C, 433T, and A841T; 303C, 433T, and 86 2A; 303C, 435G, and 530A; 303C, 435G, and 572T; 3 03C, 435G, and 596C; 303C, 435G, and 617C; 303C, 435G, and 688C; 303C, 435G, and 696T; 303C, 435G , and 702C; 303C, 435G, and 709G; 303C, 435G, and 712G; 303C, 435G, and 714G; 303C, 435G, and 790G ; 303C, 435G, and A841T; 303C, 435G, and 862A; 30 3C, 530A, and 572T; 303C, 530A, and 596C; 303C, 5 30A, and 617C; 303C, 530A, and 688C; 303C, 530A, and 696T; 303C, 530A, and 702C; 303C, 530A, and 7 09G; 303C, 530A, and 712G; 303C, 530A, and 714G; 303C, 530A, and 790G; 303C, 530A, and A841T; 303 C, 530A, and 862A; 303C, 572T, and 596C; 303C, 57 2T, and 617C; 303C, 572T, and 688C; 303C, 572T, and also 696T; 303C, 572T, and 702C; 303C, 572T, and 70 9G; 303C, 572T, and 712G; 303C, 572T, and 714G; 3 03C, 572T, and 790G; 303C, 572T, and A841T; 303C 、572T, and 862A; 303C, 596C, and 617C; 303C, 596 C, and 688C; 303C, 596C, and 696T; 303C, 596C, and 702C; 303C, 596C, and 709G; 303C, 596C, and 712 G; 303C, 596C, and 714G; 303C, 596C, and 790G; 30 3C, 596C, and A841T; 303C, 596C, and 862A; 303C, 617C, and 688C; 303C, 617C, and 696T; 303C, 617C , and 702C; 303C, 617C, and 709G; 303C, 617C, and 712G; 303C, 617C, and 714G; 303C, 617C, and 790G ; 303C, 617C, and A841T; 303C, 617C, and 862A; 30 3C, 688C, and 696T; 303C, 688C, and 702C; 303C, 6 88C, and 709G; 303C, 688C, and 712G; 303C, 688C, and 714G; 303C, 688C, and 790G; 303C, 688C, and A 841T; 303C, 688C, and 862A; 303C, 696T, and 702C ; 303C, 696T, and 709G; 303C, 696T, and 712G; 303 C, 696T, and 714G; 303C, 696T, and 790G; 303C, 69 6T, and A841T; 303C, 696T, and 862A; 303C, 702C, and 709G; 303C, 702C, and 712G; 303C, 702C, and 7 14G; 303C, 702C, and 790G; 303C, 702C, and A841T ; 303C, 702C, and 862A; 303C, 709G, and 712G; 303 C, 709G, and 714G; 303C, 709G, and 790G; 303C, 70 9G, and A841T; 303C, 709G, and 862A; 303C, 712G, and 714G; 303C, 712G, and 790G; 303C, 712G, and A 841T; 303C, 712G, and 862A; 303C, 714G, and 790G ; 303C, 714G, and A841T; 303C, 714G, and 862A; 30 3C, 790G, and A841T; 303C, 790G, and 862A; 303C, A841T, and 862A; 426A, 433T, and 435G; 426A, 433 T, and 530A; 426A, 433T, and 572T; 426A, 433T, and o b 596C; 426A, 433T, and 617C; 426A, 433T, and 688 C; 426A, 433T, and 696T; 426A, 433T, and 702C; 42 6A, 433T, and 709G; 426A, 433T, and 712G; 426A, 4 33T, and 714G; 426A, 433T, and 790G; 426A, 433T, and A841T; 426A, 433T, and 862A; 426A, 435G, and 530A; 426A, 435G, and 572T; 426A, 435G, and 596C ; 426A, 435G, and 617C; 426A, 435G, and 688C; 426 A, 435G, and 696T; 426A, 435G, and 702C; 426A, 43 5G, and 709G; 426A, 435G, and 712G; 426A, 435G, and y 714G; 426A, 435G, and 790G; 426A, 435G, and A8 41T; 426A, 435G, and 862A; 426A, 530A, and 572T; 426A, 530A, and 596C; 426A, 530A, and 617C; 426A 530A, and 688C; 426A, 530A, and 696T; 426A, 530 A, and 702C; 426A, 530A, and 709G; 426A, 530A, and 712G; 426A, 530A, and 714G; 426A, 530A, and 790 G; 426A, 530A, and A841T; 426A, 530A, and 862A; 4 26A, 572T, and 596C; 426A, 572T, and 617C; 426A, 572T, and 688C; 426A, 572T, and 696T; 426A, 572T and 702C; 426A, 572T, and 709G; 426A, 572T, and 712G; 426A, 572T, and 714G; 426A, 572T, and 790G ; 426A, 572T, and A841T; 426A, 572T, and 862A; 42 6A, 596C, and 617C; 426A, 596C, and 688C; 426A, 5 96C, and 696T; 426A, 596C, and 702C; 426A, 596C, and 709G; 426A, 596C, and 712G; 426A, 596C, and 7 14G; 426A, 596C, and 790G; 426A, 596C, and A841T ; 426A, 596C, and 862A; 426A, 617C, and 688C; 426 A, 617C, and 696T; 426A, 617C, and 702C; 426A, 61 7C, and 709G; 426A, 617C, and 712G; 426A, 617C, and Call 714G;426A, 617C, and 790G;426A, 617C, and A8 41T;426A, 617C, and 862A;426A, 688C, and 696T; 426A, 688C, and 702C;426A, 688C, and 709G;426A , 688C, and 712G;426A, 688C, and 714G;426A, 688 C, and 790G;426A, 688C, and A841T;426A, 688C, and Call 862A;426A, 696T, and 702C;426A, 696T, and 70 9G;426A, 696T, and 712G;426A, 696T, and 714G;4 26A, 696T, and 790G;426A, 696T, and A841T;426A , 696T, and 862A;426A, 702C, and 709G;426A, 702 C, and 712G;426A, 702C, and 714G;426A, 702C, and Call 790G;426A, 702C, and A841T;426A, 702C, and 86 2A;426A, 709G, and 712G;426A, 709G, and 714G;4 26A, 709G, and 790G;426A, 709G, and A841T;426A , 709G, and 862A;426A, 712G, and 714G;426A, 712 G, and 790G;426A, 712G, and A841T;426A, 712G, and Call 790G;426A, 714G, and A8 41T;426A, 714G, and 862A;426A, 790G, and A841T ;426A, 790G, and 862A;426A, A841T, and 862A;43 3T, 435G, and 530A;433T, 435G, and 572T;433T, 4 35G, and 596C; 433T, 435G, and 617C; 433T, 435G, and 688C; 433T, 435G, and 696T; 433T, 435G, and 7 02C; 433T, 435G, and 709G; 433T, 435G, and 712G; 433T, 435G, and 714G; 433T, 435G, and 790G; 433T , 435G, and A841T; 433T, 435G, and 862A; 433T, 53 0A, and 572T; 433T, 530A, and 596C; 433T, 530A, and 617C; 433T, 530A, and 688C; 433T, 530A, and 69 6T; 433T, 530A, and 702C; 433T, 530A, and 709G; 4 33T, 530A, and 712G; 433T, 530A, and 714G; 433T, 530A, and 790G; 433T, 530A, and A841T; 433T, 530 A, and 862A; 433T, 572T, and 596C; 433T, 572T, and 617C; 433T, 572T, and 688C; 433T, 572T, and 696 T; 433T, 572T, and 702C; 433T, 572T, and 709G; 43 3T, 572T, and 712G; 433T, 572T, and 714G; 433T, 5 72T, and 790G; 433T, 572T, and A841T; 433T, 572T , and 862A; 433T, 596C, and 617C; 433T, 596C, and 688C; 433T, 596C, and 696T; 433T, 596C, and 702C ; 433T, 596C, and 709G; 433T, 596C, and 712G; 433 T, 596C, and 714G; 433T, 596C, and 790G; 433T, 596C, and A841T; 433T, 596C, and 862A; 433T, 617C, and 688C; 433T, 617C, and 696T ; 433T, 617C, and 702C; 433T, 617C, and 709G; 433 T, 617C, and 712G; 433T, 617C, and 714G; 433T, 61 7C, and 790G; 433T, 617C, and A841T; 433T, 617C, and 862A; 433T, 688C, and 696T; 433T, 688C, and 7 02C; 433T, 688C, and 709G; 433T, 688C, and 712G; 433T, 688C, and 714G; 433T, 688C, and 790G; 433T 、688C, and A841T; 433T, 688C, and 862A; 433T, 69 6T, and 702C; 433T, 696T, and 709G; 433T, 696T, and yobi 712G; 433T, 696T, and 714G; 433T, 696T, and 79 0G; 433T, 696T, and A841T; 433T, 696T, and 862A; 433T, 702C, and 709G; 433T, 702C, and 712G; 433T 、702C, and 714G; 433T, 702C, and 790G; 433T, 702 C, and A841T; 433T, 702C, and 862A; 433T, 709G, and yobi 712G; 433T, 709G, and 714G; 433T, 709G, and 79 0G; 433T, 709G, and A841T; 433T, 709G, and 862A; 433T, 712G, and 714G; 433T, 712G, and 790G; 433T 、712G, and A841T; 433T, 712G, and 862A; 433T, 71 4G, and 790G; 433T, 714G, and A841T; 433T, 714G, and 862A; 433T, 790G, and A841T; 433T, 790G, and 862A; 433T, A841T, and 862A; 435G, 530A, and 572 T; 435G, 530A, and 596C; 435G, 530A, and 617C; 43 5G, 530A, and 688C; 435G, 530A, and 696T; 435G, 5 30A, and 702C; 435G, 530A, and 709G; 435G, 530A, and 712G; 435G, 530A, and 714G; 435G, 530A, and 7 90G; 435G, 530A, and A841T; 435G, 530A, and 862A ; 435G, 572T, and 596C; 435G, 572T, and 617C; 435 G, 572T, and 688C; 435G, 572T, and 696T; 435G, 57 2T, and 702C; 435G, 572T, and 709G; 435G, 572T, and 712G; 435G, 572T, and 714G; 435G, 572T, and 79 0G; 435G, 572T, and A841T; 435G, 572T, and 862A; 435G, 596C, and 617C; 435G, 596C, and 688C; 435G , 596C, and 696T; 435G, 596C, and 702C; 435G, 596 C, and 709G; 435G, 596C, and 712G; 435G, 596C, and 714G; 435G, 596C, and 790G; 435G, 596C, and A84 1T; 435G, 596C, and 862A; 435G, 617C, and 688C; 4 35G, 617C, and 696T; 435G, 617C, and 702C; 435G, 617C, and 709G; 435G, 617C, and 712G; 435G, 617C , and 714G; 435G, 617C, and 790G; 435G, 617C, and A841T; 435G, 617C, and 862A; 435G, 688C, and 696 T; 435G, 688C, and 702C; 435G, 688C, and 709G; 43 5G, 688C, and 712G; 435G, 688C, and 714G; 435G, 6 88C, and 790G; 435G, 688C, and A841T; 435G, 688C , and 862A; 435G, 696T, and 702C; 435G, 696T, and 709G; 435G, 696T, and 712G; 435G, 696T, and 714G ; 435G, 696T, and 790G; 435G, 696T, and A841T; 43 5G, 696T, and 862A; 435G, 702C, and 709G; 435G, 7 02C, and 712G; 435G, 702C, and 714G; 435G, 702C, and 790G; 435G, 702C, and A841T; 435G, 702C, and 862A; 435G, 709G, and 712G; 435G, 709G, and 714G ; 435G, 709G, and 790G; 435G, 709G, and A841T; 43 5G, 709G, and 862A; 435G, 712G, and 714G; 435G, 7 12G, and 790G; 435G, 712G, and A841T; 435G, 712G , and 862A; 435G, 714G, and 790G; 435G, 714G, and A841T; 435G, 714G, and 862A; 435G, 790G, and A84 1T; 435G, 790G, and 862A; 435G, A841T, and 862A; 530A, 572T, and 596C; 530A, 572T, and 617C; 530A , 572T, and 688C; 530A, 572T, and 696T; 530A, 572 T, and 702C; 530A, 572T, and 709G; 530A, 572T, and 712G; 530A, 572T, and 714G; 530A, 572T, and 790 G; 530A, 572T, and A841T; 530A, 572T, and 862A; 5 30A, 596C, and 617C; 530A, 596C, and 688C; 530A, 596C, and 696T; 530A, 596C, and 702C; 530A, 596C , and 709G; 530A, 596C, and 712G; 530A, 596C, and 714G; 530A, 596C, and 790G; 530A, 596C, and A841 T; 530A, 596C, and 862A; 530A, 617C, and 688C; 53 0A, 617C, and 696T; 530A, 617C, and 702C; 530A, 6 17C, and 709G; 530A, 617C, and 712G; 530A, 617C, , and 714G; 530A, 617C, and 790G; 530A, 617C, and A 841T; 530A, 617C, and 862A; 530A, 688C, and 696T ; 530A, 688C, and 702C; 530A, 688C, and 709G; 530 A, 688C, and 712G; 530A, 688C, and 714G; 530A, 68 8C, and 790G; 530A, 688C, and A841T; 530A, 688C, and 862A; 530A, 696T, and 702C; 530A, 696T, and 7 09G; 530A, 696T, and 712G; 530A, 696T, and 714G; 530A, 696T, and 790G; 530A, 696T, and A841T; 530 A, 696T, and 862A; 530A, 702C, and 709G; 530A, 70 2C, and 712G; 530A, 702C, and 714G; 530A, 702C, and 790G; 530A, 702C, and A841T; 530A, 702C, and 8 62A; 530A, 709G, and 712G; 530A, 709G, and 714G; 530A, 709G, and 790G; 530A, 709G, and A841T; 530 A, 709G, and 862A; 530A, 712G, and 714G; 530A, 71 2G, and 790G; 530A, 712G, and A841T; 530A, 712G, and 862A; 530A, 714G, and 790G; 530A, 714G, and A 841T; 530A, 714G, and 862A; 530A, 790G, and A841 T; 530A, 790G, and 862A; 530A, A841T, and 862A; 5 72T, 596C, and 617C; 572T, 596C, and 688C; 572T, 596C, and 696T; 572T, 596C, and 702C; 572T, 596C , and 709G; 572T, 596C, and 712G; 572T, 596C, and 714G; 572T, 596C, and 790G; 572T, 596C, and A841 T; 572T, 596C, and 862A; 572T, 617C, and 688C; 57 2T, 617C, and 696T; 572T, 617C, and 702C; 572T, 6 17C, and 709G; 572T, 617C, and 712G; 572T, 617C, and 714G; 572T, 617C, and 790G; 572T, 617C, and A 841T; 572T, 617C, and 862A; 572T, 688C, and 696T ; 572T, 688C, and 702C; 572T, 688C, and 709G; 572 T, 688C, and 712G; 572T, 688C, and 714G; 572T, 68 8C, and 790G; 572T, 688C, and A841T; 572T, 688C, and 862A; 572T, 696T, and 702C; 572T, 696T, and 7 09G; 572T, 696T, and 712G; 572T, 696T, and 714G; 572T, 696T, and 790G; 572T, 696T, and A841T; 572 T, 696T, and 862A; 572T, 702C, and 709G; 572T, 70 2C, and 712G; 572T, 702C, and 714G; 572T, 702C, and 790G; 572T, 702C, and A841T; 572T, 702C, and 8 62A; 572T, 709G, and 712G; 572T, 709G, and 714G; 572T, 709G, and 790G; 572T, 709G, and A841T; 572 T, 709G, and 862A; 572T, 712G, and 714G; 572T, 71 2G, and 790G; 572T, 712G, and A841T; 572T, 712G, and 862A; 572T, 714G, and 790G; 572T, 714G, and A 841T; 572T, 714G, and 862A; 572T, 790G, and A841 T; 572T, 790G, and 862A; 572T, A841T, and 862A; 5 96C, 617C, and 688C; 596C, 617C, and 696T; 596C, 617C, and 702C; 596C, 617C, and 709G; 596C, 617C , and 712G; 596C, 617C, and 714G; 596C, 617C, and 790G; 596C, 617C, and A841T; 596C, 617C, and 862 A; 596C, 688C, and 696T; 596C, 688C, and 702C; 59 6C, 688C, and 709G; 596C, 688C, and 712G; 596C, 6 88C, and 714G; 596C, 688C, and 790G; 596C, 688C, and A841T; 596C, 688C, and 862A; 596C, 696T, and 702C; 596C, 696T, and 709G; 596C, 696T, and 712G ; 596C, 696T, and 714G; 596C, 696T, and 790G; 596 C, 696T, and A841T; 596C, 696T, and 862A; 596C, 7 02C, and 709G; 596C, 702C, and 712G; 596C, 702C, and 714G; 596C, 702C, and 790G; 596C, 702C, and A 841T; 596C, 702C, and 862A; 596C, 709G, and 712G ; 596C, 709G, and 714G; 596C, 709G, and 790G; 596 C, 709G, and A841T; 596C, 709G, and 862A; 596C, 712G, and 714G; 596C, 712G, and 7 90G; 596C, 712G, and A841T; 596C, 712G, and 862A ;596C, 714G, and 790G;596C, 714G, and A841T;59 6C, 714G, and 862A;596C, 790G, and A841T;596C, 790G, and 862A;596C, A841T, and 862A;617C, 688 C, and 696T;617C, 688C, and 702C;617C, 688C, and 709G;617C, 688C, and 712G;617C, 688C, and 714 G;617C, 688C, and 790G;617C, 688C, and A841T;6 17C, 688C, and 862A;617C, 696T, and 702C;617C, 696T, and 709G;617C, 696T, and 712G;617C, 696T , and 714G;617C, 696T, and 790G;617C, 696T, and A841T;617C, 696T, and 862A;617C, 702C, and 709 G;617C, 702C, and 712G;617C, 702C, and 714G;61 7C, 702C, and 790G;617C, 702C, and A841T;617C, 702C, and 862A;617C, 709G, and 712G;617C, 709G , and 714G;617C, 709G, and 790G;617C, 709G, and A841T;617C, 709G, and 862A;617C, 712G, and 714 G;617C, 712G, and 790G;617C, 712G, and A841T;6 17C, 712G, and 862A;617C, 714G, and 790G;617C, 714G, and A841T;617C, 714G, and 862A;617C, 790 G, and A841T;617C, 790G, and 862A;617C, A841T, and 862A; 688C, 696T, and 702C; 688C, 696T, and 7 09G; 688C, 696T, and 712G; 688C, 696T, and 714G; 688C, 696T, and 790G; 688C, 696T, and A841T; 688 C, 696T, and 862A; 688C, 702C, and 709G; 688C, 70 2C, and 712G; 688C, 702C, and 714G; 688C, 702C, and 862A; 688C, 702C, and 790G; 688C, 702C, and A841T; 688C, 702C, and 8 62A; 688C, 709G, and 712G; 688C, 709G, and 714G; 688C, 709G, and 790G; 688C, 709G, and A841T; 688 C, 709G, and 862A; 688C, 712G, and 714G; 688C, 71 2G, and 790G; 688C, 712G, and A841T; 688C, 712G, and 862A; 688C, 714G, and 790G; 688C, 714G, and A 841T; 688C, 714G, and 862A; 688C, 790G, and A841 T; 688C, 790G, and 862A; 688C, A841T, and 862A; 6 96T, 702C, and 709G; 696T, 702C, and 712G; 696T, 702C, and 714G; 696T, 702C, and 790G; 696T, 702C , and A841T; 696T, 702C, and 862A; 696T, 709G, and 712G; 696T, 709G, and 714G; 696T, 709G, and 790 G; 696T, 709G, and A841T; 696T, 709G, and 862A; 6 96T, 712G, and 714G; 696T, 712G, and 790G; 696T, 712G, and A841T; 696T, 712G, and 862A; 696T, 714 G, and 790G; 696T, 714G, and A841T; 696T, 714G, and 862A; 696T, 790G, and A841T; 696T, 790G, and 8 62A; 696T, A841T, and 862A; 702C, 709G, and 712G ; 702C, 709G, and 714G; 702C, 709G, and 790G; 702 C, 709G, and A841T; 702C, 709G, and 862A; 702C, 7 12G, and 714G; 702C, 712G, and 790G; 702C, 712G, and A841T; 702C, 712G, and 862A; 702C, 714G, and 790G; 702C, 714G, and A841T; 702C, 714G, and 862 A; 702C, 790G, and A841T; 702C, 790G, and 862A; 7 02C, A841T, and 862A; 709G, 712G, and 714G; 709G 、712G, and 790G; 709G, 712G, and A841T; 709G, 71 2G, and 862A; 709G, 714G, and 790G; 709G, 714G, and 862A; 709G, 790G, and A841T; 709G, 790G, and 862A; 709G, A841T, and 862 A; 712G, 714G, and 790G; 712G, 714G, and A841T; 7 12G, 714G, and 862A; 712G, 790G, and A841T; 712G 、790G, and 862A; 712G, A841T, and 862A; 714G, 79 、790G, and 862A; 714G, A841T, and 862A; 790G, A841T, and 862A 0G, and 862A; 714G, A841T, and 862A; or 790G, A8 Three mutations corresponding to 41T and 862A, where the indicated nucleobases can be present within the promoter sequence, provided that they are not the same as the corresponding naturally occurring nucleobases. .

[0048] In some embodiments, the nucleotide sequence of the promoter element provided herein can contain two mutations as compared to the nucleotide sequence of a reference promoter element. For example, in some embodiments, the following mutations relative to SEQ ID NO: 28: 688C and 696T; 688C and 702C; 688C and 712G; 688C and 7 14G; 696T and 702C; 696T and 712G; 696T and 714G; 702C and 712G; 702C and 714G; or 712G and 714G, where the indicated nucleobases can be present within the promoter sequence, provided that they are not the same as the corresponding naturally occurring nucleobases.

[0049] In some embodiments, the nucleotide sequence of the promoter element provided herein can contain three mutations as compared to the nucleotide sequence of a reference promoter element. For example, in some embodiments, the following mutations relative to SEQ ID NO: 28: 688C , 696T, and 702C; 688C, 696T, and 712G; 688C, 696 T, and 714G; 688C, 702C, and 712G; 688C, 702C, and also 714G; 688C, 712G, and 714G; 696T, 702C, and 712 G; 696T, 702C, and 714G; 696T, 712G, and 714G; also three mutations corresponding to 702C, 712G, and 714G may be present in the promoter sequence as long as the indicated nucleotide is not the same as the corresponding naturally occurring nucleotide.

[0050] In some embodiments, the nucleotide sequence of the promoter element provided herein may contain four mutations when compared to the nucleotide sequence of a reference promoter element. For example, in some embodiments, the following mutations relative to SEQ ID NO: 28: 688C , 696T, 702C, and 712G; 688C, 696T, 702C, and 714 G; 688C, 696T, 712G, and 714G; 688C, 702C, 712G, and 714G; or four mutations corresponding to 696T, 702C, 712G, and 714G may be present in the promoter sequence as long as the indicated nucleotide is not the same as the corresponding naturally occurring nucleotide.

[0051] In some embodiments, the nucleotide sequence of the promoter element provided herein may contain five mutations when compared to the nucleotide sequence of a reference promoter element. For example, in some embodiments, the following mutations relative to SEQ ID NO: 28: 688C , 696T, 702C, 712G, and 714G; five mutations corresponding to the indicated nucleotides may be present in the promoter sequence as long as the indicated nucleotide is not the same as the corresponding naturally occurring nucleotide.

[0052] In some embodiments, the nucleotide sequence of the promoter element provided herein​​​​​​​​ The column contains two mutations as compared to the nucleotide sequence of the reference promoter element and may. For example, in some embodiments, the following positions relative to SEQ ID NO: 28: T146 and C154; T146 and T303; T146 and T426; T146 and A43 3; T146 and A435; T146 and T530; T146 and C572; T1 46 and T596; T146 and T617; T146 and T688; T146 and A696; T146 and T702; T146 and A709; T146 and A71 2; T146 and T714; T146 and A790; T146 and A841; T1 46 and T862; C154 and T303; C154 and T426; C154 and A433; C154 and A435; C154 and T530; C154 and C57 2; C154 and T596; C154 and T617; C154 and T688; C1 54 and A696; C154 and T702; C154 and A709; C154 and A712; C154 and T714; C154 and A790; C154 and A84 1; C154 and T862; T303 and T426; T303 and A433; T3 03 and A435; T303 and T530; T303 and C572; T303 and T596; T303 and T617; T303 and T688; T303 and A69 6; T303 and T702; T303 and A709; T303 and A712; T3 03 and T714; T303 and A790; T303 and A841; T303 and T862; T426 and A433; T426 and A435; T426 and T53 0; T426 and C572; T426 and T596; T426 and T617; T4 26 and T688; T426 and A696; T426 and T702; T426 and A709; T426 and A712; T426 and T714; T426 and A79 0; T426 and A841; T426 and T862; A433 and A435; A4 33 and T530; A433 and C572; A433 and T596; A433 and T617; A433 and T688; A433 and A696; A433 and T70 2; A433 and A709; A433 and A712; A433 and T714; A4 33 and A790; A433 and A841; A433 and T862; A435 and T530; A435 and C572; A435 and T596; A435 and T61 7; A435 and T688; A435 and A696; A435 and T702; A4 35 and A709; A435 and A712; A435 and T714; A435 and A790; A435 and A841; A435 and T862; T530 and C57 2; T530 and T596; T530 and T617; T530 and T688; T5 30 and A696; T530 and T702; T530 and A709; T530 and A712; T530 and T714; T530 and A790; T530 and A84 1; T530 and T862; C572 and T596; C572 and T617; C5 72 and T688; C572 and A696; C572 and T702; C572 and A709; C572 and A712; C572 and T714; C572 and A79 0; C572 and A841; C572 and T862; T596 and T617; T5 96 and T688; T596 and A696; T596 and T702; T596 and A709; T596 and A712; T596 and T714; T596 and A79 0; T596 and A841; T596 and T862; T617 and T688; T6 17 and A696; T617 and T702; T617 and A709; T617 and A712; T617 and T714; T617 and A790; T617 and A84 1; T617 and T862; T688 and A696; T688 and T702; T6 88 and A709; T688 and A712; T688 and T714; T688 and A790; T688 and A841; T688 and T862; A696 and T70 2; A696 and A709; A696 and A712; A696 and T714; A6 96 and A790; A696 and A841; A696 and T862; T702 and A709; T702 and A712; T702 and T714; T702 and A79 0; T702 and A841; T702 and T862; A709 and A712; A7 09 and T714; A709 and A790; A709 and A841; A709 and A841; A709 and T862; A712 and T714; A712 and A790; A712 and A84 1; A712 and T862; T714 and A790; T714 and A841; T7 14 and T862; A790 and A841; A790 and T862; or A84 1 and T862 may be present within the promoter sequence at positions corresponding to two mutations that can occur.

[0053] In some embodiments, the nucleotide sequences of the promoter elements provided herein The column may contain three mutations as compared to the nucleotide sequence of the reference promoter element. For example, in some embodiments, the following positions relative to SEQ ID NO: 28: T146, C 154, and T303; T146, C154, and T426; T146, C154, and A433, T146, C154, and A435; T146, C154, and T 530; T146, C154, and C572; T146, C154, and T596; T146, C154, and T617; T146, C154, and T688; T146 , C154, and A696; T146, C154, and T702; T146, C15 4, and A709; T146, C154, and A712; T146, C154, and o f T714; T146, C154, and A790; T146, C154, and A84 1; T146, C154, and T862; T146, T303, and T426; T1 46, T303, and A433; T146, T303, and A435; T146, T 303, and T530; T146, T303, and C572; T146, T303, and T596; T146, T303, and T617; T146, T303, and T 688; T146, T303, and A696; T146, T303, and T702; T146, T303, and A709; T146, T303, and A712; T146 , T303, and T714; T146, T303, and A790; T146, T30 3, and A841; T146, T303, and T862; T146, T426, and o f A433; T146, T426, and A435; T146, T426, and T53 0; T146, T426, and C572; T146, T426, and T596; T1 46, T426, and T617; T146, T426, and T688; T146, T 426, and A696; T146, T426, and T702; T146, T426, and A709; T146, T426, and A712; T146, T426, and T 714; T146, T426, and A790; T146, T426, and A841; T146, T426, and T862; T146, A433, and A435; T146 , A433, and T530; T146, A433, and C572; T146, A43 3, and T596; T146, A433, and T617; T146, A433, and o r T688; T146, A433, and A696; T146, A433, and T70 2; T146, A433, and A709; T146, A433, and A712; T1 46, A433, and T714; T146, A433, and A790; T146, A 433, and A841; T146, A433, and T862; T146, A435, and T530; T146, A435, and C572; T146, A435, and T 596; T146, A435, and T617; T146, A435, and T688; T146, A435, and A696; T146, A435, and T702; T146 , A435, and A709; T146, A435, and A712; T146, A43 5, and T714; T146, A435, and A790; T146, A435, and o r A841; T146, A435, and T862; T146, T530, and C57 2; T146, T530, and T596; T146, T530, and T617; T1 46, T530, and T688; T146, T530, and A696; T146, T 530, and T702; T146, T530, and A709; T146, T530, and A712; T146, T530, and T714; T146, T530, and A 790; T146, T530, and A841; T146, T530, and T862; T146, C572, and T596; T146, C572, and T617; T146 , C572, and T688; T146, C572, and A696; T146, C57 2, and T702; T146, C572, and A709; T146, C572, and o ther A712; T146, C572, and T714; T146, C572, and A79 0; T146, C572, and A841; T146, C572, and T862; T1 46, T596, and T617; T146, T596, and T688; T146, T 596, and A696; T146, T596, and T702; T146, T596, and A709; T146, T596, and A712; T146, T596, and T 714; T146, T596, and A790; T146, T596, and A841; T146, T596, and T862; T146, T617, and T688; T146 , T617, and A696; T146, T617, and T702; T146, T61 7, and A709; T146, T617, and A712; T146, T617, and o ther T714; T146, T617, and A790; T146, T617, and A84 1; T146, T617, and T862; T146, T688, and A696; T1 46, T688, and T702; T146, T688, and A709; T146, T 688, and A712; T146, T688, and T714; T146, T688, and A790; T146, T688, and A841; T146, T688, and T 862; T146, A696, and T702; T146, A696, and A709; T146, A696, and A712; T146, A696, and T714; T146 , A696, and A790; T146, A696, and A841; T146, A69 6, and T862; T146, T702, and A709; T146, T702, and A712; T146, T702, and T714; T146, T702, and A79 0; T146, T702, and A841; T146, T702, and T862; T1 46, A709, and A712; T146, A709, and T714; T146, A 709, and A790; T146, A709, and A841; T146, A709, and T862; T146, A712, and T714; T146, A712, and A 790; T146, A712, and A841; T146, A712, and T862; T146, T714, and A790; T146, T714, and A841; T146 , T714, and T862; T146, A790, and A841; T146, A79 0, and T862; T146, A841, and T862; C154, T303, and T426; C154, T303, and A433; C154, T303, and A43 5; C154, T303, and T530; C154, T303, and C572; C1 54, T303, and T596; C154, T303, and T617; C154, T 303, and T688; C154, T303, and A696; C154, T303, and T702; C154, T303, and A709; C154, T303, and A 712; C154, T303, and T714; C154, T303, and A790; C154, T303, and A841; C154, T303, and T862; C154 , T426, and A433; C154, T426, and A435; C154, T42 6, and T530; C154, T426, and C572; C154, T426, and o r T596; C154, T426, and T617; C154, T426, and T68 8; C154, T426, and A696; C154, T426, and T702; C1 54, T426, and A709; C154, T426, and A712; C154, T 426, and T714; C154, T426, and A790; C154, T426, and A841; C154, T426, and T862; C154, A433, and A 435; C154, A433, and T530; C154, A433, and C572; C154, A433, and T596; C154, A433, and T617; C154 , A433, and T688; C154, A433, and A696; C154, A43 3, and T702; C154, A433, and A709; C154, A433, and o r A712; C154, A433, and T714; C154, A433, and A79 0; C154, A433, and A841; C154, A433, and T862; C1 54, A435, and T530; C154, A435, and C572; C154, A 435, and T596; C154, A435, and T617; C154, A435, and T688; C154, A435, and A696; C154, A435, and T 702; C154, A435, and A709; C154, A435, and A712; C154, A435, and T714; C154, A435, and A790; C154 , A435, and A841; C154, A435, and T862; C154, T53 0, and C572; C154, T530, and T596; C154, T530, and oyo bi T617; C154, T530, and T688; C154, T530, and oyo bi A69 6; C154, T530, and T702; C154, T530, and A709; C1 54, T530, and A712; C154, T530, and T714; C154, T 530, and A790; C154, T530, and A841; C154, T530, and T862; C154, C572, and T596; C154, C572, and T 617; C154, C572, and T688; C154, C572, and A696; C154, C572, and T702; C154, C572, and A709; C154 , C572, and A712; C154, C572, and T714; C154, C57 bi T862; C154, T596, and T617; C154, T596, and T68 8; C154, T596, and A696; C154, T596, and T702; C1 54, T596, and A709; C154, T596, and A712; C154, T 596, and T714; C154, T596, and A790; C154, T596, and A841; C154, T596, and T862; C154, T617, and T 688; C154, T617, and A696; C154, T617, and T702; C154, T617, and A709; C154, T617, and A712; C154 , T617, and T714; C154, T617, and A790; C154, T61 7, and A841; C154, T617, and T862; C154, T688, and o r A696; C154, T688, and T702; C154, T688, and A70 9; C154, T688, and A712; C154, T688, and T714; C1 54, T688, and A790; C154, T688, and A841; C154, T 688, and T862; C154, A696, and T702; C154, A696, and A709; C154, A696, and A712; C154, A696, and T 714; C154, A696, and A790; C154, A696, and A841; C154, A696, and T862; C154, T702, and A709; C154 , T702, and A712; C154, T702, and T714; C154, T70 2, and A790; C154, T702, and A841; C154, T702, and o r T862; C154, A709, and A712; C154, A709, and T71 4; C154, A709, and A790; C154, A709, and A841; C154, A709, and T862; C154, A712, and T714; C154 , A712, and A790; C154, A712, and A841; C154, A71 2, and T862; C154, T714, and A790; C154, T714, and A841; C154, T714, and T862; C154, A790, and A84 1; C154, A790, and T862; C154, A841, and T862; T3 03, T426, and A433; T303, T426, and A435; T303, T 426, and T530; T303, T426, and C572; T303, T426, and T596; T303, T426, and T617; T303, T426, and T 688; T303, T426, and A696; T303, T426, and T702; T303, T426, and A709; T303, T426, and A712; T303 , T426, and T714; T303, T426, and A790; T303, T42 6, and A841; T303, T426, and T862; T303, A433, and A435; T303, A433, and T530; T303, A433, and C57 2; T303, A433, and T596; T303, A433, and T617; T3 03, A433, and T688; T303, A433, and A696; T303, A 433, and T702; T303, A433, and A709; T303, A433, and A712; T303, A433, and T714; T303, A433, and A 790; T303, A433, and A841; T303, A433, and T862; T303, A435, and T530; T303, A435, and C572; T303 , A435, and T596; T303, A435, and T617; T303, A43 5, and T688; T303, A435, and A696; T303, A435, and and T702; T303, A435, and A709; T303, A435, and A71 2; T303, A435, and T714; T303, A435, and A790; T3 03, A435, and A841; T303, A435, and T862; T303, T 530, and C572; T303, T530, and T596; T303, T530, and T617; T303, T530, and T688; T303, T530, and A 696; T303, T530, and T702; T303, T530, and A709; T303, T530, and A712; T303, T530, and T714; T303 , T530, and A790; T303, T530, and A841; T303, T53 0, and T862; T303, C572, and T596; T303, C572, and o r T617; T303, C572, and T688; T303, C572, and A69 6; T303, C572, and T702; T303, C572, and A709; T3 03, C572, and A712; T303, C572, and T714; T303, C 572, and A790; T303, C572, and A841; T303, C572, and T862; T303, T596, and T617; T303, T596, and T 688; T303, T596, and A696; T303, T596, and T702; T303, T596, and A709; T303, T596, and A712; T303 , T596, and T714; T303, T596, and A790; T303, T59 6, and A841; T303, T596, and T862; T303, T617, and o and T688; T303, T617, and A696; T303, T617, and T70 2; T303, T617, and A709; T303, T617, and A712; T3 03, T617, and T714; T303, T617, and A790; T303, T 617, and A841; T303, T617, and T862; T303, T688, and A696; T303, T688, and T702; T303, T688, and A 709; T303, T688, and A712; T303, T688, and T714; T303, T688, and A790; T303, T688, and A841; T303 , T688, and T862; T303, A696, and T702; T303, A69 6, and A709; T303, A696, and A712; T303, A696, and o and T714; T303, A696, and A790; T303, A696, and A84 1; T303, A696, and T862; T303, T702, and A709; T3 03, T702, and A712; T303, T702, and T714; T303, T 702, and A790; T303, T702, and A841; T303, T702, and T862; T303, A709, and A712; T303, A709, and T 714; T303, A709, and A790; T303, A709, and A841; T303, A709, and T862; T303, A712, and T714; T303 , A712, and A790; T303, A712, and A841; T303, A71 2, and T862; T303, T714, and A790; T303, T714, and o and A841; T303, T714, and T862; T303, A790, and A84 1; T303, A790, and T862; T303, A841, and T862; T4 26, A433, and A435; T426, A433, and T530; T426, A 433, and C572; T426, A433, and T596; T426, A433, and T617; T426, A433, and T688; T426, A433, and A 696; T426, A433, and T702; T426, A433, and A709; T426, A433, and A712; T426, A433, and T714; T426 , A433, and A790; T426, A433, and A841; T426, A43 3, and T862; T426, A435, and T530; T426, A435, and C572; T426, A435, and T596; T426, A435, and T61 7; T426, A435, and T688; T426, A435, and A696; T4 26, A435, and T702; T426, A435, and A709; T426, A 435, and A712; T426, A435, and T714; T426, A435, and A790; T426, A435, and A841; T426, A435, and T 862; T426, T530, and C572; T426, T530, and T596; T426, T530, and T617; T426, T530, and T688; T426 , T530, and A696; T426, T530, and T702; T426, T53 0, and A709; T426, T530, and A712; T426, T530, and T714; T426, T530, and A790; T426, T530, and A84 1; T426, T530, and T862; T426, C572, and T596; T4 26, C572, and T617; T426, C572, and T688; T426, C 572, and A696; T426, C572, and T702; T426, C572, and A709; T426, C572, and A712; T426, C572, and T 714; T426, C572, and A790; T426, C572, and A841; T426, C572, and T862; T426, T596, and T617; T426 , T596, and T688; T426, T596, and A696; T426, T59 6, and T702; T426, T596, and A709; T426, T596, and oyo bi A712; T426, T596, and T714; T426, T596, and A79 0; T426, T596, and A841; T426, T596, and T862; T4 26, T617, and T688; T426, T617, and A696; T426, T 617, and T702; T426, T617, and A709; T426, T617, and A712; T426, T617, and T714; T426, T617, and A 790; T426, T617, and A841; T426, T617, and T862; T426, T688, and A696; T426, T688, and T702; T426 , T688, and A709; T426, T688, and A712; T426, T68 8, and T714; T426, T688, and A790; T426, T688, and oyo bi A841; T426, T688, and T862; T426, A696, and T70 2; T426, A696, and A709; T426, A696, and A712; T4 26, A696, and T714; T426, A696, and A790; T426, A 696, and A841; T426, A696, and T862; T426, T702, and A709; T426, T702, and A712; T426, T702, and T 714; T426, T702, and A790; T426, T702, and A841; T426, T702, and T862; T426, A709, and A712; T426 , A709, and T714; T426, A709, and A790; T426, A70 9, and A841; T426, A709, and T862; T426, A712, and T714; T426, A712, and A790; T426, A712, and A84 1; T426, A712, and T862; T426, T714, and A790; T4 26, T714, and A841; T426, T714, and T862; T426, A 790, and A841; T426, A790, and T862; T426, A841, and T862; A433, A435, and T530; A433, A435, and C 572; A433, A435, and T596; A433, A435, and T617; A433, A435, and T688; A433, A435, and A696; A433 , A435, and T702; A433, A435, and A709; A433, A43 5, and A712; A433, A435, and T714; A433, A435, and A790; A433, A435, and A841; A433, A435, and T86 2; A433, T530, and C572; A433, T530, and T596; A4 33, T530, and T617; A433, T530, and T688; A433, T 530, and A696; A433, T530, and T702; A433, T530, and A709; A433, T530, and A712; A433, T530, and T 714; A433, T530, and A790; A433, T530, and A841; A433, T530, and T862; A433, C572, and T596; A433 、C572, and T617; A433, C572, and T688; A433, C57 2, and A696; A433, C572, and T702; A433, C572, and o nd A709; A433, C572, and A712; A433, C572, and T71 4; A433, C572, and A790; A433, C572, and A841; A4 33, C572, and T862; A433, T596, and T617; A433, T 596, and T688; A433, T596, and A696; A433, T596, and T702; A433, T596, and A709; A433, T596, and A 712; A433, T596, and T714; A433, T596, and A790; A433, T596, and A841; A433, T596, and T862; A433 、T617, and T688; A433, T617, and A696; A433, T617, and T702; A433, T617, and A 709; A433, T617, and A712; A433, T617, and T714; A433, T617, and A790; A433, T617, and A841; A433 、T617, and T862; A433, T688, and A696; A433, T68 8, and T702; A433, T688, and A709; A433, T688, and also A712; A433, T688, and T714; A433, T688, and A79 0; A433, T688, and A841; A433, T688, and T862; A4 33, A696, and T702; A433, A696, and A709; A433, A 696, and A712; A433, A696, and T714; A433, A696, and A790; A433, A696, and A841; A433, A696, and T 862; A433, T702, and A709; A433, T702, and A712; A433, T702, and T714; A433, T702, and A790; A433 , T702, and A841; A433, T702, and T862; A433, A70 9, and A712; A433, A709, and T714; A433, A709, and also A790; A433, A709, and A841; A433, A709, and T86 2; A433, A712, and T714; A433, A712, and A790; A4 33, A712, and A841; A433, A712, and T862; A433, T 714, and A790; A433, T714, and A841; A433, T714, and T862; A433, A790, and A841; A433, A790, and T 862; A433, A841, and T862; A435, T530, and C572; A435, T530, and T596; A435, T530, and T617; A435 , T530, and T688; A435, T530, and A696; A435, T53 0, and T702; A435, T530, and A709; A435, T530, and and A712; A435, T530, and T714; A435, T530, and A79 0; A435, T530, and A841; A435, T530, and T862; A4 35, C572, and T596; A435, C572, and T617; A435, C 572, and T688; A435, C572, and A696; A435, C572, and T702; A435, C572, and A709; A435, C572, and A 712; A435, C572, and T714; A435, C572, and A790; A435, C572, and A841; A435, C572, and T862; A435 , T596, and T617; A435, T596, and T688; A435, T59 6, and A696; A435, T596, and T702; A435, T596, and o and A709; A435, T596, and A712; A435, T596, and T71 4; A435, T596, and A790; A435, T596, and A841; A4 35, T596, and T862; A435, T617, and T688; A435, T 617, and A696; A435, T617, and T702; A435, T617, and A709; A435, T617, and A712; A435, T617, and T 714; A435, T617, and A790; A435, T617, and A841; A435, T617, and T862; A435, T688, and A696; A435 , T688, and T702; A435, T688, and A709; A435, T68 8, and A712; A435, T688, and T714; A435, T688, and o and A790;A435, T688, and A841;A435, T688, and T86 2;A435, A696, and T702;A435, A696, and A709;A4 35, A696, and A712;A435, A696, and T714;A435, A 696, and A790;A435, A696, and A841;A435, A696, and T862;A435, T702, and A709;A435, T702, and A 712;A435, T702, and T714;A435, T702, and A790; A435, T702, and A841;A435, T702, and T862;A435 , A709, and A712;A435, A709, and T714;A435, A70 9, and A790;A435, A709, and A841;A435, A709, and o and T862;A435, A712, and T714;A435, A712, and A79 0;A435, A712, and A841;A435, A712, and T862;A4 35, T714, and A790;A435, T714, and A841;A435, T 714, and T862;A435, A790, and A841;A435, A790, and T862;A435, A841, and T862;T530, C572, and T 596;T530, C572, and T617;T530, C572, and T688; T530, C572, and A696;T530, C572, and T702;T530 , C572, and A709;T530, C572, and A712;T530, C57 2, and T714;T530, C572, and A790;T530, C572, and o and A841;T530, C572, and T862;T530, T596, and T61 7; T530, T596, and T688; T530, T596, and A696; T5 30, T596, and T702; T530, T596, and A709; T530, T 596, and A712; T530, T596, and T714; T530, T596, and A790; T530, T596, and A841; T530, T596, and T 862; T530, T617, and T688; T530, T617, and A696; T530, T617, and T702; T530, T617, and A709; T530 , T617, and A712; T530, T617, and T714; T530, T61 7, and A790; T530, T617, and A841; T530, T617, and T862; T530, T688, and A696; T530, T688, and T70 2; T530, T688, and A709; T530, T688, and A712; T5 30, T688, and T714; T530, T688, and A790; T530, T 688, and A841; T530, T688, and T862; T530, A696, and T702; T530, A696, and A709; T530, A696, and A 712; T530, A696, and T714; T530, A696, and A790; T530, A696, and A841; T530, A696, and T862; T530 , T702, and A709; T530, T702, and A712; T530, T70 2, and T714; T530, T702, and A790; T530, T702, and T862; T530, A709, and A71 2; T530, A709, and T714; T530, A709, and A790; T5 30, A709, and A841; T530, A709, and T862; T530, A 712, and T714; T530, A712, and A790; T530, A712, and A841; T530, A712, and T862; T530, T714, and A 790; T530, T714, and A841; T530, T714, and T862; T530, A790, and A841; T530, A790, and T862; T530 , A841, and T862; C572, T596, and T617; C572, T59 6, and T688; C572, T596, and A696; C572, T596, and o f T702; C572, T596, and A709; C572, T596, and A71 2; C572, T596, and T714; C572, T596, and A790; C5 72, T596, and A841; C572, T596, and T862; C572, T 617, and T688; C572, T617, and A696; C572, T617, and T702; C572, T617, and A709; C572, T617, and A 712; C572, T617, and T714; C572, T617, and A790; C572, T617, and A841; C572, T617, and T862; C572 , T688, and A696; C572, T688, and T702; C572, T68 8, and A709; C572, T688, and A712; C572, T688, and o f T714; C572, T688, and A790; C572, T688, and A84 1; C572, T688, and T862; C572, A696, and T702; C5 72, A696, and A709; C572, A696, and A712; C572, A 696, and T714; C572, A696, and A790; C572, A696, and A841; C572, A696, and T862; C572, T702, and A 709; C572, T702, and A712; C572, T702, and T714; C572, T702, and A790; C572, T702, and A841; C572 , T702, and T862; C572, A709, and A712; C572, A70 9, and T714; C572, A709, and A790; C572, A709, and o r A841; C572, A709, and T862; C572, A712, and T71 4; C572, A712, and A790; C572, A712, and A841; C5 72, A712, and T862; C572, T714, and A790; C572, T 714, and A841; C572, T714, and T862; C572, A790, and A841; C572, A790, and T862; C572, A841, and T 862; T596, T617, and T688; T596, T617, and A696; T596, T617, and T702; T596, T617, and A709; T596 , T617, and A712; T596, T617, and T714; T596, T61 7, and A790; T596, T617, and A841; T596, T617, and o r T862; T596, T688, and A696; T596, T688, and T70 2; T596, T688, and A709; T596, T688, and A712; T5 96, T688, and T714; T596, T688, and A790; T596, T 688, and A841; T596, T688, and T862; T596, A696, and T702; T596, A696, and A709; T596, A696, and A 712; T596, A696, and T714; T596, A696, and A790; T596, A696, and A841; T596, A696, and T862; T596 , T702, and A709; T596, T702, and A712; T596, T70 2, and T714; T596, T702, and A790; T596, T702, and o r A841; T596, T702, and T862; T596, A709, and A71 2; T596, A709, and T714; T596, A709, and A790; T5 96, A709, and A841; T596, A709, and T862; T596, A 712, and T714; T596, A712, and A790; T596, A712, and A841; T596, A712, and T862; T596, T714, and A 790; T596, T714, and A841; T596, T714, and T862; T596, A790, and A841; T596, A790 , and T862; T596, A841, and T862; T617, T688, and A696; T617, T688, and T702; T617, T688, and A709 ; T617, T688, and A712; T617, T688, and T714; T61 7, T688, and A790; T617, T688, and A841; T617, T6 88, and T862; T617, A696, and T702; T617, A696, and o Call A709; T617, A696, and A712; T617, A696, and T7 14; T617, A696, and A790; T617, A696, and A841; T 617, A696, and T862; T617, T702, and A709; T617, T702, and A712; T617, T702, and T714; T617, T702 , and A790; T617, T702, and A841; T617, T702, and T862; T617, A709, and A712; T617, A709, and T714 ; T617, A709, and A790; T617, A709, and A841; T61 7, A709, and T862; T617, A712, and T714; T617, A7 12, and A790; T617, A712, and A841; T617, A712, and and T862; T617, T714, and A790; T617, T714, and A8 41; T617, T714, and T862; T617, A790, and A841; T 617, A790, and T862; T617, A841, and T862; T688, A696, and T702; T688, A696, and A709; T688, A696 , and A712; T688, A696, and T714; T688, A696, and A790; T688, A696, and A841; T688, A696, and T862 ; T688, T702, and A709; T688, T702, and A712; T68 8, T702, and T714; T688, T702, and A790; T688, T7 02, and A841; T688, T702, and T862; T688, A709, and and A712; T688, A709, and T714; T688, A709, and A7 90; T688, A709, and A841; T688, A709, and T862; T 688, A712, and T714; T688, A712, and A790; T688, A712, and A841; T688, A712, and T862; T688, T714 , and A790; T688, T714, and A841; T688, T714, and T862; T688, A790, and A841; T688, A790, and T862 ; T688, A841, and T862; A696, T702, and A709; A69 6, T702, and A712; A696, T702, and T714; A696, T7 02, and A790; A696, T702, and A841; A696, T702, and T862; A696, A709, and A712; A696, A709, and T7 14; A696, A709, and A790; A696, A709, and A841; A 696, A709, and T862; A696, A712, and T714; A696, A712, and A790; A696, A712, and A841; A696, A712 , and T862; A696, T714, and A790; A696, T714, and A841; A696, T714, and T862; A696, A790, and A841 ; A696, A790, and T862; A696, A841, and T862; T70 2, A709, and A712; T702, A709, and T714; T702, A7 09, and A790; T702, A709, and A841; T702, A709, and T862; T702, A712, and T714; T702, A712, and A7 90; T702, A712, and A841; T702, A712, and T862; T 702, T714, and A790; T702, T714, and A841; T702, T714, and T862; T702, A790, and A841; T702, A790 , and T862; T702, A841, and T862; A709, A712, and T714; A709, A712, and A790; A709, A712, and A841 ; A709, A712, and T862; A709, T714, and A790; A70 9, T714, and A841; A709, T714, and T862; A709, A7 90, and A841; A709, A790, and T862; A709, A841, and T862; A712, T714, and A790; A712, T714, and A8 41; A712, T714, and T862; A712, A790, and A841; A 712, A790, and T862; A712, A841, and T862; T714, A790, and A841; T714, A790, and T862; T714, A841 , and T862; or three mutations at positions corresponding to A790, A841, and T862 may be present within the promoter sequence.

[0054] In some embodiments, the nucleotide sequence of the promoter element provided herein may contain two mutations as compared to the nucleotide sequence of the reference promoter element. For example, in some embodiments, the following positions relative to SEQ ID NO: 28: T688 and A696; T688 and T702; T688 and A712; T688 and T71 4; T688 and T862; A696 and A712; A696 and T714; A696 4; A696 and T702; A696 and A712; A696 and T714; T7 02 and A712; T702 and T714; or two mutations at positions corresponding to A712 and T714 may be present within the promoter sequence.

[0055] In some embodiments, the nucleotide sequence of the promoter element provided herein may contain three mutations compared to the nucleotide sequence of the reference promoter element. For example, in some embodiments, at the following positions relative to SEQ ID NO: 28: T688, A 696, and T702; T688, A696, and A712; T688, A696, and T714; T688, T702, and A712; T688, T702, and T 714; T688, A712, and T714; A696, T702, and A712; A696, T702, and T714; A696, A712, and T714; or T 702, A712, and T714, three mutations at positions corresponding thereto may be present within the promoter sequence.

[0056] In some embodiments, the nucleotide sequence of the promoter element provided herein may contain four mutations compared to the nucleotide sequence of the reference promoter element. For example, in some embodiments, at the following positions relative to SEQ ID NO: 28: T688, A 696, T702, and A712; T688, A696, T702, and T714; T688, A696, A712, and T714; T688, T702, A712, and T714; or four mutations at positions corresponding to A696, T702, A712, and T714 may be present within the promoter sequence. ​

[0057] In some embodiments, the nucleotide sequence of the promoter element provided herein may contain five mutations compared to the nucleotide sequence of the reference promoter element. For example, in some embodiments, five mutations may be present at positions corresponding to the following positions relative to SEQ ID NO: 28: T688, A 696, T702, A712, and T714 within the promoter sequence.

[0058] In some embodiments, the nucleotide sequence of the promoter element provided herein may contain two mutations compared to the nucleotide sequence of the reference promoter element. For example, in some embodiments, the following positions relative to SEQ ID NO: 28: 146 and 154; 146 and 303; 146 and 426; 146 and 433; 146 and 435; 146 and 530; 146 and 572; 146 and 596; 146 and 617; 146 and 688; 146 and 696; 146 and 702; 146 and 617; 146 and 688; 146 and 696; 146 and 702; 146 and 709; 146 and A712; 146 and 714; 146 and 790; 146 and 841; 146 and 862; 154 and 303; 154 and 426; 154 and 841; 146 and 862; 154 and 303; 154 and 426; 154 and 433; 154 and 435; 154 and 530; 154 and 572; 154 and 596; 154 and 617; 154 and 688; 154 and 696; 154 and 702; 154 and 709; 154 and A712; 154 and 714; 154 and 790; 154 and 841; 154 and 862; 303 and 426; 303 and and 596;303 and 617;303 and 688;303 and 696;303 and and 702;303 and 709;303 and A712;303 and 714;303 and 790;303 and 841;303 and 862;426 and 433;426 and 435;426 and 530;426 and 572;426 and 596;426 and 617;426 and 688;426 and 696;426 and 702;426 and 709;426 and A712;426 and 714;426 and 790;42 6 and 841;426 and 862;433 and 435;433 and 530;43 3 and 572;433 and 596;433 and 617;433 and 688;43 3 and 696;433 and 702;433 and 709;433 and A712;4 33 and 714;433 and 790;433 and 841;433 and 862;4 35 and 530;435 and 572;435 and 596;435 and 617;4 35 and 688;435 and 696;435 and 702;435 and 709;4 35 and A712;435 and 714;435 and 790;435 and 841; 435 and 862;530 and 572;530 and 596;530 and 617; 530 and 688;530 and 696;530 and 702;530 and 709; 530 and A712;530 and 714;530 and 790;530 and 841 ;530 and 862;572 and 596;572 and 617;572 and 688 ;572 and 696;572 and 702;572 and 709;572 and A71 2;572 and 714;572 and 790;572 and 841;572 and 86 2,596 and 617,596 and 688,596 and 696,596 and 70 2,596 and 709,596 and A712,596 and 714,596 and 7 90,596 and 841,596 and 862,617 and 688,617 and 6 96,617 and 702,617 and 709,617 and A712,617 and 714,617 and 790,617 and 841,617 and 862,688 and 696,688 and 702,688 and 709,688 and A712,688 and 714,688 and 790,688 and 841,688 and 862,696 and 702,696 and 709,696 and A712,696 and 714,696 and 790,696 and 841,696 and 862,702 and 709,702 and A712,702 and 714,702 and 790,702 and 841,702 and 862,709 and A712,709 and 714,709 and 790,70 9 and 841,709 and 862,A712 and 714,A712 and 790, A712 and 841,A712 and 862,714 and 790,714 and 84 1,714 and 862,790 and 841,790 and 862; or two mutations at positions corresponding to 841 and 862 may be present within the promoter sequence.

[0059] In some embodiments, the nucleotide sequence of the promoter element provided herein may contain three mutations as compared to the nucleotide sequence of a reference promoter element. For example, in some embodiments, the following positions relative to SEQ ID NO: 28: 146, 15 ​​4, and 303; 146, 154, and 426; 146, 154, and 433; 1 46, 154, and 435; 146, 154, and 530; 146, 154, and 572; 146, 154, and 596; 146, 154, and 617; 146, 15 4, and 688; 146, 154, and 696; 146, 154, and 702; 1 46, 154, and 709; 146, 154, and A712; 146, 154, and 714; 146, 154, and 790; 146, 154, and 841; 146, 1 54, and 862; 146, 303, and 426; 146, 303, and 433; 146, 303, and 435; 146, 303, and 530; 146, 303, and 714; 146, 303, and 790; 146, 303, and 841; 146, 303, and 688; 146, 303, and 696; 146, 303, and 702; 146, 303, and 709; 146, 303, and A712; 146, 303, and 714; 146, 303, and 790; 146, 303, and 841; 146, 303, and 862; 146, 426, and 433; 146, 426, and 435 ; 146, 426, and 530; 146, 426, and 572; 146, 426, and 714; 146, 426, and 790; 146, 426, and 841; 146, 426, and 696; 146, 426, and 702; 146, 426, and 709 ; 146, 426, and A712; 146, 426, and 714; 146, 426, and 790; 146, 426, and 841; 146, 426, and 862; 146 433, and 435; 146, 433, and 530; 146, 433, and 57 2; 146, 433, and 596; 146, 433, and 617; 146, 433, and 688; 146, 433, and 696; 146, 433, and 702; 146 , 433, and 709; 146, 433, and A712; 146, 433, and 7 14; 146, 433, and 790; 146, 433, and 841; 146, 433 , and 862; 146, 435, and 530; 146, 435, and 572; 14 6, 435, and 596; 146, 435, and 617; 146, 435, and 6 88; 146, 435, and 696; 146, 435, and 702; 146, 435 , and 709; 146, 435, and A712; 146, 435, and 714; 1 46, 435, and 790; 146, 435, and 841; 146, 435, and 862; 146, 530, and 572; 146, 530, and 596; 146, 53 0, and 617; 146, 530, and 688; 146, 530, and 696; 1 46, 530, and 702; 146, 530, and 709; 146, 530, and A712; 146, 530, and 714; 146, 530, and 790; 146, 5 30, and 841; 146, 530, and 862; 146, 572, and 596; 146, 572, and 617; 146, 572, and 688; 146, 572, and by 696; 146, 572, and 702; 146, 572, and 709; 146, 5 72, and A712; 146, 572, and 714; 146, 572, and 790 ; 146, 572, and 841; 146, 572, and 862; 146, 596, and Call 617; 146, 596, and 688; 146, 596, and 696; 146, 596, and 702; 146, 596, and 709; 146, 596, and A71 2; 146, 596, and 714; 146, 596, and 790; 146, 596, and 841; 146, 596, and 862; 146, 617, and 688; 146 , 617, and 696; 146, 617, and 702; 146, 617, and 70 9; 146, 617, and A712; 146, 617, and 714; 146, 617 , and 790; 146, 617, and 841; 146, 617, and 862; 14 6, 688, and 696; 146, 688, and 702; 146, 688, and 7 09; 146, 688, and A712; 146, 688, and 714; 146, 68 8, and 790; 146, 688, and 841; 146, 688, and 862; 1 46, 696, and 702; 146, 696, and 709; 146, 696, and A712; 146, 696, and 714; 146, 696, and 790; 146, 6 96, and 841; 146, 696, and 862; 146, 702, and 709; 146, 702, and A712; 146, 702, and 714; 146, 702, and and 790; 146, 702, and 841; 146, 702, and 862; 146, 709, and A712; 146, 709, and 714; 146, 709, and 79 0; 146, 709, and 841; 146, 709, and 862; 146, A712 , and 714; 146, A712, and 790; 146, A712, and 841; 146, A712, and 862; 146, 714, and 790; 146, 714, and and 841;146, 714, and 862;146, 790, and 841;146, 790, and 862;146, 841, and 862;154, 303, and 426 ;154, 303, and 433;154, 303, and 435;154, 303, and 530;154, 303, and 572;154, 303, and 596;154, 303, and 617;154, 303, and 688;154, 303, and 696 ;154, 303, and 702;154, 303, and 709;154, 303, and A712;154, 303, and 714;154, 303, and 790;154 , 303, and 841;154, 303, and 862;154, 426, and 43 3;154, 426, and 435;154, 426, and 530;154, 426, and 572;154, 426, and 596;154, 426, and 617;154 , 426, and 688;154, 426, and 696;154, 426, and 70 2;154, 426, and 709;154, 426, and A712;154, 426 , and 714;154, 426, and 790;154, 426, and 841;15 4, 426, and 862;154, 433, and 435;154, 433, and 5 30;154, 433, and 572;154, 433, and 596;154, 433 , and 617;154, 433, and 688;154, 433, and 696;15 4, 433, and 702;154, 433, and 709;154, 433, and A 712;154, 433, and 714;154, 433, and 790;154, 43 3, and 841; 154, 433, and 862; 154, 435, and 530; 1 54, 435, and 572; 154, 435, and 596; 154, 435, and 617; 154, 435, and 688; 154, 435, and 696; 154, 43 5, and 702; 154, 435, and 709; 154, 435, and A712; 154, 435, and 714; 154, 435, and 790; 154, 435, and 841; 154, 435, and 862; 154, 530, and 572; 154, 5 30, and 596; 154, 530, and 617; 154, 530, and 688; 154, 530, and 696; 154, 530, and 702; 154, 530, and 709; 154, 530, and A712; 154, 530, and 714; 154, 530, and 790; 154, 530, and 841; 154, 530, and 862 ; 154, 572, and 596; 154, 572, and 617; 154, 572, and 688; 154, 572, and 696; 154, 572, and 702; 154, 572, and 709; 154, 572, and A712; 154, 572, and 71 4; 154, 572, and 790; 154, 572, and 841; 154, 572, and 862; 154, 596, and 617; 154, 596, and 688; 154 , 596, and 696; 154, 596, and 702; 154, 596, and 70 9; 154, 596, and A712; 154, 596, and 714; 154, 596 , and 790; 154, 596, and 841; 154, 596, and 862; 15 4, 617, and 688; 154, 617, and 696; 154, 617, and 7 02; 154, 617, and 709; 154, 617, and A712; 154, 61 7, and 714; 154, 617, and 790; 154, 617, and 841; 1 54, 617, and 862; 154, 688, and 696; 154, 688, and 702; 154, 688, and 709; 154, 688, and A712; 154, 6 88, and 714; 154, 688, and 790; 154, 688, and 841; 154, 688, and 862; 154, 696, and 702; 154, 696, and 709; 154, 696, and A712; 154, 696, and 714; 154, 696, and 790; 154, 696, and 841; 154, 696, and 862 ; 154, 702, and 709; 154, 702, and A712; 154, 702, and 714; 154, 702, and 790; 154, 702, and 841; 154 , 702, and 862; 154, 709, and A712; 154, 709, and 7 14; 154, 709, and 790; 154, 709, and 841; 154, 709 , and 862; 154, A712, and 714; 154, A712, and 790; 154, A712, and 841; 154, A712, and 862; 154, 714, and 790; 154, 714, and 841; 154, 714, and 862; 154 , 790, and 841; 154, 790, and 862; 154, 841, and 86 2; 303, 426, and 433; 303, 426, and 435; 303, 426, and 530; 303, 426, and 572; 303, 426, and 596; 303 , 426, and 617; 303, 426, and 688; 303, 426, and 69 6; 303, 426, and 702; 303, 426, and 709; 303, 426, and A712; 303, 426, and 714; 303, 426, and 790; 30 3, 426, and 841; 303, 426, and 862; 303, 433, and 4 35; 303, 433, and 530; 303, 433, and 572; 303, 433 , and 596; 303, 433, and 617; 303, 433, and 688; 30 3, 433, and 696; 303, 433, and 702; 303, 433, and 7 09; 303, 433, and A712; 303, 433, and 714; 303, 43 3, and 790; 303, 433, and 841; 303, 433, and 862; 3 03, 435, and 530; 303, 435, and 572; 303, 435, and 596; 303, 435, and 617; 303, 435, and 688; 303, 43 5, and 696; 303, 435, and 702; 303, 435, and 709; 3 03, 435, and A712; 303, 435, and 714; 303, 435, and by 790; 303, 435, and 841; 303, 435, and 862; 303, 530, and 572; 303, 530, and 596; 303, 530, and 617 ; 303, 530, and 688; 303, 530, and 696; 303, 530, and by 702; 303, 530, and 709; 303, 530, and A712; 303 , 530, and 714; 303, 530, and 790; 303, 530, and 84 1; 303, 530, and 862; 303, 572, and 596; 303, 572, and 617; 303, 572, and 688; 303, 572, and 696; 303 , 572, and 702; 303, 572, and 709; 303, 572, and A7 12; 303, 572, and 714; 303, 572, and 790; 303, 572 , and 841; 303, 572, and 862; 303, 596, and 617; 30 3, 596, and 688; 303, 596, and 696; 303, 596, and 7 02; 303, 596, and 709; 303, 596, and A712; 303, 59 6, and 714; 303, 596, and 790; 303, 596, and 841; 3 03, 596, and 862; 303, 617, and 688; 303, 617, and 696; 303, 617, and 702; 303, 617, and 709; 303, 61 7, and A712; 303, 617, and 714; 303, 617, and 790; 303, 617, and 841; 303, 617, and 862; 303, 688, and by 696; 303, 688, and 702; 303, 688, and 709; 303, 6 88, and A712; 303, 688, and 714; 303, 688, and 790 ; 303, 688, and 841; 303, 688, and 862; 303, 696, and by 702; 303, 696, and 709; 303, 696, and A712; 303 , 696, and 714; 303, 696, and 790; 303, 696, and 84 1; 303, 696, and 862; 303, 702, and 709; 303, 702, and A712; 303, 702, and 714; 303, 702, and 790; 30 3, 702, and 841; 303, 702, and 862; 303, 709, and A 712; 303, 709, and 714; 303, 709, and 790; 303, 70 9, and 841; 303, 709, and 862; 303, A712, and 714; 303, A712, and 790; 303, A712, and 841; 303, A712 , and 862; 303, 714, and 790; 303, 714, and 841; 30 3, 714, and 862; 303, 790, and 841; 303, 790, and 8 62; 303, 841, and 862; 426, 433, and 435; 426, 433 , and 530; 426, 433, and 572; 426, 433, and 596; 42 6, 433, and 617; 426, 433, and 688; 426, 433, and 6 96; 426, 433, and 702; 426, 433, and 709; 426, 433 , and A712; 426, 433, and 714; 426, 433, and 790; 4 26, 433, and 841; 426, 433, and 862; 426, 435, and 530; 426, 435, and 572; 426, 435, and 596; 426, 43 5, and 617; 426, 435, and 688; 426, 435, and 696; 4 26, 435, and 702; 426, 435, and 709; 426, 435, and A712; 426, 435, and 714; 426, 435, and 790; 426, 4 35, and 841; 426, 435, and 862; 426, 530, and 572; 426, 530, and 596; 426, 530, and 617; 426, 530, and 688; 426, 530, and 696; 426, 530, and 702; 426, 5 30, and 709; 426, 530, and A712; 426, 530, and 714 ; 426, 530, and 790; 426, 530, and 841; 426, 530, and 862; 426, 572, and 596; 426, 572, and 617; 426, 572, and 688; 426, 572, and 696; 426, 572, and 702 ; 426, 572, and 709; 426, 572, and A712; 426, 572, and 714; 426, 572, and 790; 426, 572, and 841; 426 、572, and 862; 426, 596, and 617; 426, 596, and 68 8; 426, 596, and 696; 426, 596, and 702; 426, 596, and 709; 426, 596, and A712; 426, 596, and 714; 42 6, 596, and 790; 426, 596, and 841; 426, 596, and 8 62; 426, 617, and 688; 426, 617, and 696; 426, 617 、and 702; 426, 617, and 709; 426, 617, and A712; 4 26, 617, and 714; 426, 617, and 790; 426, 617, and 841; 426, 617, and 862; 426, 688, and 696; 426, 68 8, and 702; 426, 688, and 709; 426, 688, and A712; 426, 688, and 714; 426, 688, and 790; 426, 688, and 841; 426, 688, and 862; 426, 696, and 702; 426, 6 96, and 709; 426, 696, and A712; 426, 696, and 714 ; 426, 696, and 790; 426, 696, and 841; 426, 696, and 862; 426, 702, and 709; 426, 702, and A712; 426 , 702, and 714; 426, 702, and 790; 426, 702, and 84 1; 426, 702, and 862; 426, 709, and A712; 426, 709 , and 714; 426, 709, and 790; 426, 709, and 841; 42 6, 709, and 862; 426, A712, and 714; 426, A712, and 790; 426, A712, and 841; 426, A712, and 862; 426 , 714, and 790; 426, 714, and 841; 426, 714, and 86 2; 426, 790, and 841; 426, 790, and 862; 426, 841, , and 862; 433, 435, and 530; 433, 435, and 572; 433 , 435, and 596; 433, 435, and 617; 433, 435, and 68 8; 433, 435, and 696; 433, 435, and 702; 433, 435, , and 709; 433, 435, and A712; 433, 435, and 714; 43 3, 435, and 790; 433, 435, and 841; 433, 435, and 8 62; 433, 530, and 572; 433, 530, and 596; 433, 530 , and 617; 433, 530, and 688; 433, 530, and 696; 43 3, 530, and 702; 433, 530, and 709; 433, 530, and A 712; 433, 530, and 714; 433, 530, and 790; 433, 53 0, and 841; 433, 530, and 862; 433, 572, and 596; 4 33, 572, and 617; 433, 572, and 688; 433, 572, and 696; 433, 572, and 702; 433, 572, and 709; 433, 57 2, and A712; 433, 572, and 714; 433, 572, and 790; 433, 572, and 841; 433, 572, and 862; 433, 596, and 617; 433, 596, and 688; 433, 596, and 696; 433, 5 96, and 702; 433, 596, and 709; 433, 596, and A712 ; 433, 596, and 714; 433, 596, and 790; 433, 596, and 841; 433, 596, and 862; 433, 617, and 688; 433, 617, and 696; 433, 617, and 702; 433, 617, and 709 ; 433, 617, and A712; 433, 617, and 714; 433, 617, and 790; 433, 617, and 841; 433, 617, and 862; 433 , 688, and 696; 433, 688, and 702; 433, 688, and 70 9; 433, 688, and A712; 433, 688, and 714; 433, 688 , and 790; 433, 688, and 841; 433, 688, and 862; 43 3, 696, and 702; 433, 696, and 709; 433, 696, and A 712; 433, 696, and 714; 433, 696, and 790; 433, 69 6, and 841; 433, 696, and 862; 433, 702, and 709; 4 33, 702, and A712; 433, 702, and 714; 433, 702, and 790;433, 702, and 841;433, 702, and 862;433, 7 09, and A712;433, 709, and 714;433, 709, and 790 ;433, 709, and 841;433, 709, and 862;433, A712, and 714;433, A712, and 790;433, A712, and 841;4 33, A712, and 862;433, 714, and 790;433, 714, and 790 and 841;433, 714, and 862;433, 790, and 841;433, 7 90, and 862;433, 841, and 862;435, 530, and 572; 435, 530, and 596;435, 530, and 617;435, 530, and 617;435, 530, and 688;435, 530, and 696;435, 530, and 702;435, 5 30, and 709;435, 530, and A712;435, 530, and 714 ;435, 530, and 790;435, 530, and 841;435, 530, and 841;435, 530, and 862;435, 572, and 596;435, 572, and 617;435, 572, and 688;435, 572, and 696;435, 572, and 702 ;435, 572, and 709;435, 572, and A712;435, 572, and 714;435, 572, and 790;435, 572, and 841;435 572, and 862;435, 596, and 617;435, 596, and 688;435, 596, and 696;435, 596, and 702;435, 596, and 709;435, 596, and A712;435, 596, and 714;435, 596, and 790;435, 596, and 841;435, 596, and 8 33, A712, and 862;433, 714, and 790;433, 714, and 790;433, 714, and 841;433, 714, and 862;433, 790, and 841;433, 7 90, and 862;433, 841, and 862;435, 530, and 572;435, 530, and 596;435, 530, and 617;435, 530, and 688;435, 530, and 696;435, 530, and 702;435, 5 30, and 709;435, 530, and A712;435, 530, and 714;435, 530, and 790;435, 530, and 841;435, 530, and 862;435, 572, and 596;435, 572, and 617;435, 572, and 688;435, 572, and 696;435, 572, and 702;435, 572, and 709;435, 572, and A712;435, 572, and 714;435, 572, and 790;435, 572, and 841;435, 572, and 862;435, 596, and 617;435, 596, and 688;435, 596, and 696;435, 596, and 702;435, 596, 5, 596, and 790;435, 596, and 841;435, 596, and 8 62; 435, 617, and 688; 435, 617, and 696; 435, 617 , and 702; 435, 617, and 709; 435, 617, and A712; 4 35, 617, and 714; 435, 617, and 790; 435, 617, and 841; 435, 617, and 862; 435, 688, and 696; 435, 68 8, and 702; 435, 688, and 709; 435, 688, and A712; 435, 688, and 714; 435, 688, and 790; 435, 688, and 841; 435, 688, and 862; 435, 696, and 702; 435, 6 96, and 709; 435, 696, and A712; 435, 696, and 714 ; 435, 696, and 790; 435, 696, and 841; 435, 696, and 862; 435, 702, and 709; 435, 702, and A712; 435 , 702, and 714; 435, 702, and 790; 435, 702, and 84 1; 435, 702, and 862; 435, 709, and A712; 435, 709, and 714; 435, 709, and 790; 43 5, 709, and 841; 435, 709, and 862; 435, A712, and 714; 435, A712, and 790; 435, A712, and 841; 435, A712, and 862; 435, 714, and 790; 435, 714, and 84 1; 435, 714, and 862; 435, 790, and 841; 435, 790, and 862; 435, 841, and 862; 530, 572, and 596; 530 , 572, and 617; 530, 572, and 688; 530, 572, and 69 6; 530, 572, and 702; 530, 572, and 709; 530, 572, and A712; 530, 572, and 714; 530, 572, and 790; 53 0, 572, and 841; 530, 572, and 862; 530, 596, and 6 17; 530, 596, and 688; 530, 596, and 696; 530, 596 , and 702; 530, 596, and 709; 530, 596, and A712; 5 30, 596, and 714; 530, 596, and 790; 530, 596, and 841; 530, 596, and 862; 530, 617, and 688; 530, 61 7, and 696; 530, 617, and 702; 530, 617, and 709; 5 30, 617, and A712; 530, 617, and 714; 530, 617, and by 790; 530, 617, and 841; 530, 617, and 862; 530, 6 88, and 696; 530, 688, and 702; 530, 688, and 709; 530, 688, and A712; 530, 688, and 714; 530, 688, and by 790; 530, 688, and 841; 530, 688, and 862; 530, 696, and 702; 530, 696, and 709; 530, 696, and A71 2; 530, 696, and 714; 530, 696, and 790; 530, 696, and 841; 530, 696, and 862; 530, 702, and 709; 530 , 702, and A712; 530, 702, and 714; 530, 702, and 7 90; 530, 702, and 841; 530, 702, and 862; 530, 709 , and A712; 530, 709, and 714; 530, 709, and 790; 5 30, 709, and 841; 530, 709, and 862; 530, A712, and 714; 530, A712, and 790; 530, A712, and 841; 530 , A712, and 862; 530, 714, and 790; 530, 714, and 8 41; 530, 714, and 862; 530, 790, and 841; 530, 790 , and 862; 572, 596, and 617; 57 2, 596, and 688; 572, 596, and 696; 572, 596, and 7 02; 572, 596, and 709; 572, 596, and A712; 572, 59 6, and 714; 572, 596, and 790; 572, 596, and 841; 5 72, 596, and 862; 572, 617, and 688; 572, 617, and 696; 572, 617, and 702; 572, 617, and 709; 572, 61 7, and A712; 572, 617, and 714; 572, 617, and 790; 572, 617, and 841; 572, 617, and 862; 572, 688, and 696; 572, 688, and 702; 572, 688, and 709; 572, 6 88, and A712; 572, 688, and 714; 572, 688, and 790 ; 572, 688, and 841; 572, 688, and 862; 572, 696, and 702; 572, 696, and 709; 572, 696, and A712; 572 , 696, and 714; 572, 696, and 790; 572, 696, and 84 1; 572, 696, and 862; 572, 702, and 709; 572, 702, and A712; 572, 702, and 714; 572, 702, and 790; 57 2, 702, and 841; 572, 702, and 862; 572, 709, and A 712; 572, 709, and 714; 572, 709, and 790; 572, 70 9, and 841; 572, 709, and 862; 572, A712, and 714; 572, A712, and 790; 572, A712, and 841; 572, A712 , and 862; 572, 714, and 790; 572, 714, and 841; 57 2, 714, and 862; 572, 790, and 841; 572, 790, and 8 62; 572, 841, and 862; 596, 617, and 688; 596, 617 , and 696; 596, 617, and 702; 596, 617, and 709; 59 6, 617, and A712; 596, 617, and 714; 596, 617, and 790; 596, 617, and 841; 596, 617, and 862; 596, 68 8, and 696; 596, 688, and 702; 596, 688, and 709; 5 96, 688, and A712; 596, 688, and 714; 596, 688, and by 790; 596, 688, and 841; 596, 688, and 862; 596, 6 96, and 702; 596, 696, and 709; 596, 696, and A712 ; 596, 696, and 714; 596, 696, and 790; 596, 696, and by 841; 596, 696, and 862; 596, 702, and 709; 596, 702, and A712; 596, 702, and 714; 596, 702, and 79 0; 596, 702, and 841; 596, 702, and 862; 596, 709, and A712; 596, 709, and 714; 596, 709, and 790; 59 6, 709, and 841; 596, 709, and 862; 596, A712, and 714; 596, A712, and 790; 596, A712, and 841; 596, A712, and 862; 596, 714, and 790; 596, 714, and 84 1; 596, 714, and 862; 596, 790, and 841; 596, 790, and 862; 617, 688, and 696; 617 , 688, and 702; 617, 688, and 709; 617, 688, and A7 12; 617, 688, and 714; 617, 688, and 790; 617, 688 , and 841; 617, 688, and 862; 617, 696, and 702; 61 7, 696, and 709; 617, 696, and A712; 617, 696, and 714; 617, 696, and 790; 617, 696, and 841; 617, 69 6, and 862; 617, 702, and 709; 617, 702, and A712; 617, 702, and 714; 617, 702, and 790; 617, 702, and 841; 617, 702, and 862; 617, 709, and A712; 617, 709, and 714; 617, 709, and 790; 617, 709, and 841 ; 617, 709, and 862; 617, A712, and 714; 617, A712 , and 790; 617, A712, and 841; 617, A712, and 862; 617, 714, and 790; 617, 714, and 841; 617, 714, and 862, 617, 790, and 841, 617, 790, and 862, 617, 8 41, and 862, 688, 696, and 702, 688, 696, and 709; 688, 696, and A712, 688, 696, and 714, 688, 696, and 790, 688, 696, and 841, 688, 696, and 862, 688, 702, and 709, 688, 702, and A712, 688, 702, and 71 4, 688, 702, and 790, 688, 702, and 841, 688, 702, and 862, 688, 709, and A712, 688, 709, and 714, 68 8, 709, and 790, 688, 709, and 841, 688, 709, and 8 62, 688, A712, and 714, 688, A712, and 790, 688, A 712, and 841, 688, A712, and 862, 688, 714, and 79 0, 688, 714, and 841, 688, 714, and 862, 688, 790, and 841, 688, 790, and 862, 688, 841, and 862, 696 , 702, and 709, 696, 702, and A712, 696, 702, and 7 14, 696, 702, and 790, 696, 702, and 841, 696, 702 , and 862, 696, 709, and A712, 696, 709, and 714, 6 96, 709, and 790, 696, 709, and 841, 696, 709, and 862, 696, A712, and 714, 696, A712, and 790, 696, A712, and 841, 696, A712, and 862, 696, 714, and 7 90, 696, 714, and 841, 696, 714, and 862, 696, 790 and 841; 696, 790, and 862; 696, 841, and 862; 70 2, 709, and A712; 702, 709, and 714; 702, 709, and 790; 702, 709, and 841; 702, 709, and 862; 702, A7 12, and 714; 702, A712, and 790; 702, A712, and 84 1; 702, A712, and 862; 702, 714, and 790; 702, 714 and 841; 702, 714, and 862; 702, 790, and 841; 70 2, 790, and 862; 702, 841, and 862; 709, A712, and 714; 709, A712, and 790; 709, A712, and 841; 709, A712, and 862; 709, 714, and 790; 709, 714, and 84 1; 709, 714, and 862; 709, 790, and 841; 709, 790, and 862; 709, 841, and 862; A712, 714, and 790; A7 12, 714, and 841; A712, 714, and 862; A712, 790, and also 841; A712, 790, and 862; A712, 841, and 862; 71 4, 790, and 841; 714, 790, and 862; 714, 841, and 8 62; or three mutations at positions corresponding to 790, 841, and 862 may be present within the promoter sequence.

[0060] In some embodiments, the nucleotide sequence of the promoter element provided herein contains two mutations as compared to the nucleotide sequence of a reference promoter element can be found. For example, in some embodiments, the following positions for SEQ ID NO: 28: 688 and 696; 688 and 702; 688 and A712; 688 and 714; 696 and 702; 696 and A712; 696 and 714; 702 and A712; 702 and 714; or two mutations at positions corresponding to A712 and 714 may be present within the promoter sequence.

[0061] In some embodiments, the nucleotide sequence of the promoter element provided herein may contain three mutations compared to the nucleotide sequence of the reference promoter element. For example, in some embodiments, the following positions for SEQ ID NO: 28: 688, 69 6, and 702; 688, 696, and A712; 688, 696, and 714; 688, 702, and A712; 688, 702, and 714; 688, A712, and 714; 696, 702, and A712; 696, 702, and 714; 69 6, A712, and 714; or three mutations at positions corresponding to 702, A712, and 714 may be present within the promoter sequence. In some embodiments, the nucleotide sequence of the promoter element provided herein may contain four mutations compared to the nucleotide sequence of the reference promoter element.

[0062] For example, in some embodiments, the following positions for SEQ ID NO: 28: 688, 69 6, 702, and A712; 688, 696, 702, and 714; 688, 696 A712, and 714; 688, 702, A712, and 714; or 696, 688, 702, A712, and 714; 688, 696, 702, and 714; 688, 696 A712, and 714; 688, 702, A712, and 714; or 696, Four mutations at positions corresponding to 702, A712, and 714 may be present within the promoter -sequence.

[0063] In some embodiments, the nucleotide sequence of the promoter element provided herein may contain five mutations as compared to the nucleotide sequence of the reference promoter element. For example, in some embodiments, five mutations at the following positions relative to SEQ ID NO: 28: 688, 696, 702, A712, and 714 may be present within the promoter -sequence. In some embodiments, the nucleic acid molecules used in the methods described herein are typically DNA, but RNA molecules may also be used under appropriate circumstances. As used herein, "exogenous" refers to any nucleic acid sequence, or nucleic acid made by synthesis (e.g., a codon-optimized nucleic acid sequence), that has been introduced into a cell, for example, from the same organism or a different organism. For example, an exogenous nucleic acid can be a nucleic acid from one microorganism (e.g., one genus or species of methanol-assimilating yeast) that has been introduced into a different genus or species of methanol-assimilating yeast; an exogenous nucleic acid can also be a nucleic acid from methanol-assimilating yeast that has been recombinantly introduced into methanol-assimilating yeast as an additional copy, despite the presence of the corresponding native nucleic acid sequence, and can be a nucleic acid from methanol-assimilating yeast that has been recombinantly introduced into methanol-assimilating yeast and contains one or more

[0064] mutations, insertions, or deletions as compared to the sequence native to methanol-assimilating yeast. For example, P. pastoris contains an endogenous nucleic acid encoding ALAS; P. pastoris (P (P. pastoris) contains an endogenous nucleic acid encoding ALAS; P. pastoris (P​​​​​​​​​ . additional copies of the ALAS nucleic acid of P. pastoris (e.g., recombinantly introduced into P. pastoris (P. pastoris)) are considered to be exogenous. Similarly, an "exogenous" protein is a protein encoded by an exogenous nucleic acid.

[0065] In some cases, the exogenous nucleic acid can be a heterologous nucleic acid. As used herein, "heterologous" nucleic acid refers to any nucleic acid sequence that is not native to an organism (e.g., a heterologous nucleic acid can be a nucleic acid derived from one microorganism (e.g., a codon-optimized or non-codon-optimized nucleic acid) that has been introduced into a different genus or species of methanol-utilizing yeast such as P. pastoris). Similarly, a "heterologous" protein is a protein encoded by a heterologous nucleic acid.

[0066] A nucleic acid molecule is considered to be exogenous to a host organism if any part thereof (e.g., a promoter sequence or the sequence of the encoded protein) is exogenous to the host organism. A nucleic acid molecule is considered to be heterologous to a host organism if any part thereof (e.g., a promoter sequence or the sequence of the encoded protein) is heterologous to the host organism.

[0067] Provided herein are nucleic acid constructs that enable genetic manipulation of cells (e.g., yeast cells such as methanol-utilizing yeast cells). In some embodiments, provided herein are nucleic acid constructs that enable genetic manipulation of cells (e.g., yeast cells such as methanol-utilizing yeast cells) to produce RNA. Recombinant RNA can be, for example, RN (e.g., yeast cells such as methanol-utilizing yeast cells) to produce RNA. It may also be used for modifying the function of cells by interference and may be used as a guide for DNA editing. In some embodiments, as described herein, cells (e.g., yeast cells (e.g., methanol-assimilating yeast cells)) are genetically engineered to provide nucleic acid constructs that enable the production of products (e.g., proteins). In some embodiments, as described herein, cells (e.g., yeast cells (e.g., methanol-assimilating yeast cells)) are genetically engineered to provide nucleic acid constructs that enable the production of exogenous products (e.g., proteins). In some embodiments, as described herein, cells (e.g., yeast cells (e.g., methanol-assimilating yeast cells)) are genetically engineered to provide nucleic acid constructs that enable the production of heterologous products (e.g., proteins). In some embodiments, as described herein, cells ( e.g., yeast cells (e.g., methanol-assimilating yeast cells)) are genetically engineered to provide nucleic acid constructs that enable the production of products (e.g., proteins) in the absence of methanol. In addition, as described herein, cells (e.g., yeast cells (e.g., methanol-assimilating yeast cells)) are genetically engineered to provide nucleic acid constructs that enable the increased expression of heme-binding proteins. As described herein, cells containing any of the promoter elements described herein are also provided. The cells can be any suitable cells. For example, the cells can be bacterial cells (e.g., E. coli cells, B. subtilis cells, or Lactococcus lactis cells), fungal cells, algal cells, plant cells, insect cells ). For example, yeast cells (e.g., methanol-assimilating yeast cells)) are genetically engineered to provide nucleic acid constructs that enable the production of products (e.g., proteins) in the absence of methanol. In addition, as described herein, cells (e.g., yeast cells (e.g., methanol-assimilating yeast cells)) are genetically engineered to provide nucleic acid constructs that enable the increased expression of heme-binding proteins. In addition, as described herein, cells (e.g., yeast cells (e.g., methanol-assimilating yeast cells)) are genetically engineered to provide nucleic acid constructs that enable the increased expression of heme-binding proteins. In addition, as described herein, cells (e.g., yeast cells (e.g., methanol-assimilating yeast cells)) are genetically engineered to provide nucleic acid constructs that enable the increased expression of heme-binding proteins. are provided.

[0068] As described herein, cells containing any of the promoter elements described herein are also provided. The cells can be any suitable cells. For example, the cells can be bacterial cells (e.g., E. coli cells, B. subtilis cells, or Lactococcus lactis cells), fungal cells, algal cells, plant cells, insect cells For example, bacterial cells (e.g., E. coli cells, B. subtilis cells, or Lactococcus lactis cells), fungal cells, algal cells, plant cells, insect cells For example, bacterial cells (e.g., E. coli cells, B. subtilis cells, or Lactococcus lactis cells), fungal cells, algal cells, plant cells, insect cells or can be a mammalian cell. In some embodiments, the cell can be a yeast cell. Non-limiting examples of yeast cells include cells of the genus Pichia (e.g., Pichia methanolica, Pichia pastoris), Candida (e.g., Candida boidinii), Hansenula (e.g., Hansenula polymorpha), Torulopsis, and Saccharomyces (e.g., Saccharomyces cerevisiae). In some embodiments, the cell can be a methanol-assimilating yeast cell. Non-limiting examples of methanol-assimilating yeast cells include cells of the genus Pichia, Candida, Hansenula, and Torulopsis. In some embodiments, the cell can be a cell of the genus Pichia or a cell of the genus Saccharomyces. hanolica), Pichia pastoris), Candida (e.g., Candida boidinii)), Hansenula (e.g., Hansenula polymorpha)), Torulopsis cells, and Saccharomyces (e.g., Saccharomyces cerevisiae) cells. In some embodiments, the cell can be , a methanol-assimilating yeast cell. Non-limiting examples of methanol-assimilating yeast cells include cells of the genus Pichia (Pichia), Candida, Hansenula, and Torulopsis. In some embodiments, the cell can be a cell of the genus Pichia (Pichia) or a cell of the genus Saccharomyces.

[0069] In some embodiments, the present document provides a cell containing a nucleic acid construct (e.g., a first nucleic acid construct, a second nucleic acid construct, etc.) comprising a nucleotide sequence operably linked to a promoter element described herein. The nucleic acid construct comprising the nucleotide sequence can comprise any suitable nucleotide sequence. As used herein, "operably linked" means that a promoter, or other transcriptional regulatory element, is ligated to a nucleotide sequence such that the transcription of the nucleotide sequence is regulated by the promoter or other transcriptional regulatory element.

[0070] regulatory element, is ligated to a nucleotide sequence such that the transcription of the nucleotide sequence is The present element(s) is / are arranged to direct or regulate the expression (e.g., of an influenza -like) of the coding array with respect to the coding array.

[0071] A nucleic acid construct comprising a nucleotide sequence operably linked to any of the promoter elements described herein may be perceived to contain the nucleotide sequence of interest. In some embodiments, transcription and / or translation of the nucleotide sequence may result in the production of the product of interest (e.g., a protein, DNA, RNA, or small molecule). For example, in some embodiments, a nucleic acid construct comprising a nucleotide sequence may be a nucleic acid construct encoding a protein. For example, in some embodiments, a nucleic acid construct comprising a nucleotide sequence may be a nucleic acid construct encoding an RNA (e.g., mRNA, tRNA, ribozyme, siRNA, miRNA, or shRNA). For example, in some embodiments, a nucleic acid construct comprising a nucleotide sequence may be a nucleic acid construct encoding a DNA. For example, in some embodiments, a nucleic acid construct comprising a nucleotide sequence may be a nucleic acid construct whose transcription results in or contributes to the production of a small molecule (e.g., heme, ethanol, or a pharmaceutically active agent). In some embodiments, a nucleic acid construct comprising a nucleotide sequence (e.g., a first nucleic acid construct, a second nucleic acid construct, etc.) may be a nucleic acid construct encoding a protein (e.g., a first protein, a second protein, etc.). Recombinant expression proteins have many applications, such as for food, research, and drug applications.

[0072] In some embodiments, a nucleic acid construct comprising a nucleotide sequence (e.g., a first nucleic acid construct, a second nucleic acid construct, etc.) may be a nucleic acid construct encoding a protein (e.g., a first protein, a second protein, etc.).

[0073] Recombinant expression proteins have many applications, such as for food, research, and drug applications. In some embodiments, it can be widely used. The nucleic acid construct containing a nucleotide sequence operably linked to any of the promoter elements described herein The protein encoded thereby can be dehydrin, phytase, protease, catalase, lipase, peroxidase, amylase, transglutaminase, oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase. In some embodiments, the protein encoded by a nucleic acid operably linked to any of the promoter elements described herein can be an antibody or a fragment thereof (e.g., adalimumab, rituximab, trastuzumab, bevacizumab, infli ximab, or ranibizumab), an enzyme (e.g., alpha-galactosidase A, al pha-L-iduronidase, N-acetylgalactosamine-4-sulfatase, dol nase alpha, glucocerebrosidase, tissue plasminogen activator, lasbu case and other therapeutic enzymes, industrial enzymes (e.g., catalase, cellulase, laccase, glutaminase, or glycosidase), or a biocatalyst (e.g., transaminase, cytochrome P450, kinase, phosphorylase, or isomerase)), a regulatory protein (e.g., a transcription factor (e.g., Mxr1, Adr1)), a peptide hormone ( e.g., a growth hormone such as insulin, insulin-like growth factor 1, granulocyte colony-stimulating factor, follicle-stimulating hormone, or human growth hormone), a blood coagulation protein (e.g., factor VII), a cytokine (e.g., interferon or erythropoietin), or a cytokine inhibitor (e.g., etanercept). For example, insulin, insulin-like growth factor 1, granulocyte colony-stimulating factor, follicle-stimulating hormone, or a growth hormone such as human growth hormone), a blood coagulation protein (e.g., factor VII), a cytokine (e.g., interferon or erythropoietin), or a cytokine inhibitor (e.g., etanercept). It can be, for example, insulin, insulin-like growth factor 1, granulocyte colony-stimulating factor, follicle-stimulating hormone,

[0074] In some embodiments, the protein can be a heme-binding protein (e.g., an exogenous or heterologous heme-binding protein). In some embodiments, the heme-binding protein can be selected from the group consisting of globin (PF00042 in the Pfam database), cytochrome (e.g., cytochrome P450, cytochrome a, cytochrome b, cytochrome c), cytochrome c oxidase, ligninase, catalase, and peroxidase. In some embodiments, the globin can be selected from the group consisting of androglobin, chlorocruorin, cytoglobin, erythrocruorin, flavohemoglobin, globin E, globin X, globin Y, hemoglobin (e.g., beta hemoglobin, alpha hemoglobin), histoglobin, leghemoglobin, myoglobin, neuroglobin, non-symbiotic hemoglobin, protohemoglobin, and truncated hemoglobin (e.g., HbN, HbO, Glb3, cyanoglobin). In some embodiments, the heme-binding protein can be non-symbiotic hemoglobin. In some embodiments, the heme-binding protein can be leghemoglobin. In some embodiments, the heme-binding protein can be leghemoglobin from soybean (LegH). The reference amino acid sequence for LegH is presented as SEQ ID NO: 4 in FIG. 1. LegH is a protein that binds to heme, resulting in characteristic absorption at 415 nm and a distinct red color. The LegH protein (also known as LGB2) is naturally found in soybean root nodules (see, e.g., UniprotKB accession number: P02236). In some embodiments, the heme-binding protein can be selected from the group consisting of globin (PF00042 in the Pfam database), cytochrome (e.g., cytochrome P450, cytochrome a, cytochrome b, cytochrome c), cytochrome c oxidase, ligninase, catalase, and peroxidase. In some embodiments, the globin can be selected from the group consisting of androglobin, chlorocruorin, cytoglobin, erythrocruorin, flavohemoglobin, globin E, globin X, globin Y, hemoglobin (e.g., beta hemoglobin, alpha hemoglobin), histoglobin, leghemoglobin, myoglobin, neuroglobin, non-symbiotic hemoglobin, protohemoglobin, and truncated hemoglobin (e.g., HbN, HbO, Glb3, cyanoglobin). In some embodiments, the heme-binding protein can be non-symbiotic hemoglobin. In some embodiments, the heme-binding protein can be leghemoglobin. In some embodiments, the heme-binding protein can be leghemoglobin from soybean (LegH). The reference amino acid sequence for LegH is presented as SEQ ID NO: 4 in FIG. 1. LegH is a protein that binds to heme, resulting in characteristic absorption at 415 nm and a distinct red color. The LegH protein (also known as LGB2) is naturally found in soybean root nodules (see, e.g., UniprotKB accession number: P02236). In some embodiments, the heme-binding protein can be selected from the group consisting of globin (PF00042 in the Pfam database), cytochrome (e.g., cytochrome P450, cytochrome a, cytochrome b, cytochrome c), cytochrome c oxidase, ligninase, catalase, and peroxidase. In some embodiments, the globin can be selected from the group consisting of androglobin, chlorocruorin, cytoglobin, erythrocruorin, flavohemoglobin, globin E, globin X, globin Y, hemoglobin (e.g., beta hemoglobin, alpha hemoglobin), histoglobin, leghemoglobin, myoglobin, neuroglobin, non-symbiotic hemoglobin, protohemoglobin, and truncated hemoglobin (e.g., HbN, HbO, Glb3, cyanoglobin). In some embodiments, the heme-binding protein can be non-symbiotic hemoglobin. In some embodiments, the heme-binding protein can be leghemoglobin. In some embodiments, the heme-binding protein can be leghemoglobin from soybean (LegH). The reference amino acid sequence for LegH is presented as SEQ ID NO: 4 in FIG. 1. LegH is a protein that binds to heme, resulting in characteristic absorption at 415 nm and a distinct red color. The LegH protein (also known as LGB2) is naturally found in soybean root nodules (see, e.g., UniprotKB accession number: P02236). In some embodiments, the heme-binding protein can be leghemoglobin from soybean (LegH). The reference amino acid sequence for LegH is presented as SEQ ID NO: 4 in FIG. 1. LegH is a protein that binds to heme, resulting in characteristic absorption at 415 nm and a distinct red color. The LegH protein (also known as LGB2) is naturally found in soybean root nodules (see, e.g., UniprotKB accession number: P02236). In some embodiments, the heme-binding protein can be selected from the group consisting of globin (PF00042 in the Pfam database), cytochrome (e.g., cytochrome P450, cytochrome a, cytochrome b, cytochrome c), cytochrome c oxidase, ligninase, catalase, and peroxidase. In some embodiments, the globin can be selected from the group consisting of androglobin, chlorocruorin, cytoglobin, erythrocruorin, flavohemoglobin, globin E, globin X, globin Y, hemoglobin (e.g., beta hemoglobin, alpha hemoglobin), histoglobin, leghemoglobin, myoglobin, neuroglobin, non-symbiotic hemoglobin, protohemoglobin, and truncated hemoglobin (e.g., HbN, HbO, Glb3, cyanoglobin). In some embodiments, the heme-binding protein can be non-symbiotic hemoglobin. and International Publication No. 2005 / 013366, each of which is incorporated herein by reference in its entirety. International Publication No. 2014 / 110539 and International Publication No. 2014 / 110532 See also the brochure. In some embodiments, the heme binding protein is 1 to 27 (FIG. 1), % (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 9 4%, 95%, 96%, 97%, 98%, or 99%) identical amino acid sequence In some embodiments, the heme binding protein is any of SEQ ID NOs: 1-27 (FIG. 1). The amino acid sequence is one specified in any one of the above.

[0075] As used herein, Pichia sp. (P. pastoris) Materials and methods using the alcohol oxidase promoter element are exemplified. However, other organisms may be used, for example other species of the genus Pichia, or Candida. (Candida), Hansenula, Pichia, and Torulopsis The species may be derived from different yeasts, such as those from any of the Torulopsis species. An alcohol oxidase promoter element such as that described in Non-limiting examples of species include Pichia methanolica, Pichia pastoris, chia pastoris, Candida boidinii, and Hansenula porphyra Hansenula polymorpha (also known as Pichia angusta) also referred to) is included. In some embodiments, the promoter element is from Candida ndida), Hansenula, Pichia, and Torulopsis any of which may be an alcohol oxidase promoter element derived from . In some embodiments, the promoter element may have at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 9 7%, 98%, or 99%) sequence identity to an alcohol oxidase promoter element derived from any of Candida , Hansenula, Pichia, and Torulopsis . In some embodiments, the promoter element may be an alcohol oxidase promoter element derived from any of Candida , Hansenula, Pichia, and Torulopsis. In some embodiments, the promoter element is an alcohol oxidase promoter element derived from Pichia methanolica, Pichia pastoris, Candida boidinii, or Hansenula polymorpha . In some embodiments, the promoter element is an alcohol oxidase promoter element derived from Pichia methanolica, Pichia pastoris, Candida boidinii, or Hansenula polymorpha . In some embodiments, the promoter element may be an alcohol oxidase promoter element derived from Pichia methanolica, Pichia pastoris, Candida boidinii, or Hansenula polymorpha . In some embodiments, the promoter element is an alcohol oxidase promoter element derived from Pichia methanolica, Pichia pastoris, Candida boidinii, or Hansenula polymorpha . In some embodiments, the promoter element is an alcohol oxidase promoter element derived from Pichia methanolica, Pichia pastoris, Candida boidinii, or Hansenula polymorpha . In some embodiments, the promoter element is an alcohol oxidase promoter element derived from Pichia methanolica, Pichia pastoris, Candida boidinii, or Hansenula polymorpha . In some embodiments, the promoter element is an alcohol oxidase promoter element derived from Pichia methanolica, Pichia pastoris, Candida boidinii, or Hansenula polymorpha . In some embodiments, the promoter element is an alcohol oxidase promoter element derived from Pichia methanolica, Pichia pastoris, Candida boidinii, or Hansenula polymorpha . In some embodiments, the promoter element is an alcohol oxidase promoter element derived from Pichia methanolica, Pichia pastoris, Candida boidinii, or Hansenula polymorpha -(Candida boidinii), or Hansenula polymorpha at least 70% to an alcohol oxidase promoter element derived from (e.g., for example, at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 9 9%) sequence identity. In some embodiments, the promoter element is Pichia methanolica, Pichia pastoris, Candida boidinii, or an alcohol oxidase promoter element derived from Hansenula polymorpha a polymorpha. Non-limiting examples of other alcohol oxidase promoters are the AOX2 promoter derived from Pichia pastoris (e.g., see Ohi, Hideyuki, et al. Molecular and General Genetics MGG 243 .5 (1994): 489-499, which is incorporated herein by reference in its entirety), the alcohol oxidase (AOD1) promoter derived from Candida boidinii (e.g., see GenBank accession number: YSAAOD1A), the alcohol oxidase (MOX) promoter derived from Hansenula polymorpha (e.g., see GenB ank accession number: X02425), or the MOD1 or MOD2 promoter derived from Pichia methanolica (e.g., see, for example, ), or the MOD1 or MOD2 promoter derived from Pichia methanolica (e.g., ank accession number: X02425), or the MOD1 or MOD2 promoter derived from Pichia methanolica (e.g., ethanolica) (e.g., Raymond et al., 1998, Yeast, 14:11-23; and Nakagawa et al., 1999, Yeast, 15:12 (see for 23-30) and includes. In some embodiments, the alcohol oxidase promoter element can be selected from the group consisting of promoter elements derived from AOX1, AOX2, AOD1, MOX, MOD1, and MOD2 . In some embodiments, the alcohol oxidase promoter element can be a promoter element derived from AOX1. In some embodiments, the alcohol oxidase promoter element can be a promoter element derived from AOX2. In some embodiments, the alcohol oxidase promoter element can be a promoter element derived from AOD1. In some embodiments, the alcohol oxidase promoter element can be a promoter element derived from MOX. In some embodiments, the alcohol oxidase promoter element can be a promoter element derived from MOD1. In some embodiments, the alcohol oxidase promoter element can be a promoter element derived from MOD2.

[0076] In some embodiments, any of the cells described herein (e.g., yeast cells (e.g., methanol utilizing yeast cells)) can contain a second nucleic acid construct comprising a nucleotide sequence that can result in the production of a second product (e.g., a protein, RNA, DNA, or small molecule) whose transcription and / or translation is operably linked to the promoter element. In some embodiments, the nucleotide sequence of the second nucleic acid construct is made ​ The promoter element that is movably linked is the same as the promoter element operably linked to the nucleotide sequence of the first nucleic acid construct. In some embodiments, the promoter element operably linked to the nucleotide sequence of the second nucleic acid construct is the second promoter element. In some embodiments, the second promoter element can be any of the promoter elements described herein. In some embodiments, the second promoter element can have the same sequence as the first promoter element. In some embodiments, the second promoter element has one or more mutations corresponding to nucleotide positions 668-734 (e.g., nucleotide positions 673-72 9, nucleotide positions 678-724, nucleotide positions 683-719, or nucle otide positions 688-714) relative to SEQ ID NO: 2 8. In some embodiments, the second promoter element has one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) mutations selected from the group consisting of mutations corresponding to T146C, C 154T, T303C, T426A, A433T, A435G, T530A, C572T T596C, T617C, T688C, A696T, T702C, A709G, A71 2G, T714G, A790G, A841T, and T862A relative to SEQ ID NO: 28. In some embodiments, the second promoter element has a nucleotide base that is the same as the corresponding naturally occurring nucleotide base, except to the extent that it is not the same, SEQ ID NO: 36 ​For 28, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) mutations selected from the group consisting of mutations corresponding to A, 572T, 596C, 617C, 688C, 696T, 702C, 709G, 712 G, 714G, 790G, 841T, and 862A may be included. In some embodiments, the following positions relative to SEQ ID NO: 28: T146; C154 ; T303; T426; A433; A435; T530; C572; T596; T617 ; T688; A696; T702; A709; A712; T714; A790; A841 ; or one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 1 8, or 19) mutations at positions corresponding to T862 may be present within the second promoter element. In some embodiments, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 1 8, or 19) mutations at positions corresponding to the following positions relative to SEQ ID NO: 28: 146; 154; 303; 426; 4 33; 435; 530; 572; 596; 617; 688; 696; 702; 709; 7 12; 714; 790; 841; or 862 may be present within the second promoter element. In some embodiments, the second promoter element consists of one or more (e.g., 2, 3, 4 selected from the group consisting of mutations corresponding to T688C, A696T, T702C, A712G, or T714G relative to SEQ ID NO: 28 ), 3, 4 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) mutations may be present within the second promoter element. In some embodiments, the second promoter element is one or more (e.g., 2, 3, 4 selected from the group consisting of mutations corresponding to T688C, A696T, T702C, A712G, or T714G relative to SEQ ID NO: 28 ), 3, 4 or may contain one or more (e.g., two, three, four, or five) mutations. In some embodiments, the second promoter element is such that the indicated nucleobases are the same as the corresponding naturally occurring nucleobases only to the extent that with respect to SEQ ID NO: 28, consists of one or more (e.g., two, three, four, or five) mutations selected from the group consisting of the mutations corresponding to 688C, 696T, 702C, 712G, or 71 4G. In some embodiments, one or more (e.g., two, three, four, or five) mutations corresponding to the following positions with respect to SEQ ID NO: 28: T688; A696; T702; A712; or T714 may be present within the second promoter element. In some embodiments, one or more (e.g., two, three, four, or five) mutations corresponding to the following positions with respect to SEQ ID NO: 28: 688; 696; 702; 712; or 714 may be present within the second promoter element. In some embodiments, the second promoter element is, or may be, an inducible promoter element (e.g., a methanol-inducible promoter element ). When genetically engineering cells (e.g., yeast (e.g., methanol-assimilating yeast)), any of several inducible promoters may generally be used. For example, a methanol-inducible promoter, or a promoter element derived therefrom, may be used. Suitable methanol-inducible promoters include, in addition to pAOX1 described herein, other methanol-inducible promoters, or promoter elements derived therefrom. In some embodiments, the second promoter element is, or may be, a constitutive promoter element.

[0077] When genetically engineering cells (e.g., yeast (e.g., methanol-assimilating yeast)), any of several inducible promoters may generally be used. For example, a methanol-inducible promoter, or a promoter element derived therefrom, may be used. For example, a methanol-inducible promoter, or a promoter element derived therefrom, may be used. Suitable methanol-inducible promoters include, in addition to pAOX1 described herein, other methanol-inducible promoters, or promoter elements derived therefrom. These include, without limitation, p derived from Pichia pastoris AOX2 promoter, alcohol oxidase (AOD1) promoter derived from Candida boidinii (e.g., see GenBank accession number: YSAA OD1A), alcohol oxidase (MOX) promoter derived from Hansenula polymorpha (e.g., see GenBank accession number :X02425), MOD1 promoter or MOD2 promoter derived from Pichia methanolica (e.g., see Raymond et al., 1998 , Yeast, 14:11-23; and Nakagawa et al., 1999, Yeast, 15:1223-30 for reference), DHAS promoter derived from P. pastoris (e.g., see Gen Bank accession number: FJ752551) or a promoter element derived therefrom, formaldehyde dehydrogenase (FLD1) promoter derived from P. pastoris (e.g., see GenBank accession number: AF06605 4 for reference), or PEX8 promoter derived from P. pastoris (e.g., see Kranthi et al., 2010, Yeast, 27:705-11 for reference). All of these promoters are known to be induced by methanol. Suitable constitutive promoters, and constitutive promoter elements, include, without limitation, the gene for EF-1α (TEF1), a transcription elongation factor, which is constitutively transcribed strongly (e.g., see GenBank accession number: FJ752551) or a promoter element derived therefrom, formaldehyde dehydrogenase (FLD1) promoter derived from P. pastoris (e.g., see GenBank accession number: AF06605 4 for reference), or PEX8 promoter derived from P. pastoris (e.g., see Kranthi et al., 2010, Yeast, 27:705-11 for reference). All of these promoters are known to be induced by methanol. Suitable constitutive promoters, and constitutive promoter elements, include, without limitation, the gene for EF-1α (TEF1), a transcription elongation factor, which is constitutively transcribed strongly (e.g., see GenBank accession number: FJ752551) or a promoter element derived therefrom, formaldehyde dehydrogenase (FLD1) promoter derived from P. pastoris (e.g., see GenBank accession number: AF06605 4 for reference), or PEX8 promoter derived from P. pastoris (e.g., see Kranthi et al., 2010, Yeast, 27:705-11 for reference). All of these promoters are known to be induced by methanol. Suitable constitutive promoters, and constitutive promoter elements, include, without limitation, the gene for EF-1α (TEF1), a transcription elongation factor, which is constitutively transcribed strongly (e.g., see GenBank accession number: FJ752551) or a promoter element derived therefrom, formaldehyde dehydrogenase comprises a P. pastoris promoter (or a portion thereof). Without limitation, it may be mentioned that the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter from P. pastoris (e.g., see GenBank accession number: U 62648.1), the promoter from P. pastoris for the potential glycosylphospho inositol (GPI) anchor protein GCW14p (PAS_chr1-4_0586) (e.g., see GenB ank accession number: XM_002490678), and the promoter from P. pastoris for the 3-phosphoglycerate kinase gene (PGK1) (e.g., see GenBank accession number: AY288296). Other suitable constitutive promoters (or promoter elements derived therefrom) may also be used. Furthermore, it is noted that an inducible (e.g., methanol-inducible) promoter and a constitutive promoter (or promoter elements derived therefrom) can be combined to further increase the expression of any of the nucleic acids operably linked thereto.

[0078] In some embodiments, the second protein can be any of the proteins described above. In some embodiments, the second protein can be a transcription factor (e.g., Mxr 1). In some embodiments, any of the promoter elements herein (e.g., the first promoter element or the second promoter element (T) may contain one or more recognition sequences for a transcription factor. Thus, in some embodiments, the feedback loop can be constructed such that the transcription factor drives the expression of the protein of interest and also drives the expression of additional copies of the transcription factor. In some embodiments , the transcription factor can be Mxr1. In some embodiments, the second protein is a protein involved in the biosynthesis of heme (e.g., aminolevulinate synthase (ALAS ), δ-aminolevulinate dehydratase (ALAD), porphobilinogen deaminase (PBGD), uroporphyrinogen III synthase (UPG3S), uroporph yrinogen III decarboxylase (UPG3D), coproporphyrinogen oxidase (COPROX), protoporphyrinogen IX oxidase (PROTO X), and / or ferrochelatase (FC)) and can be selected from the group consisting of proteins .

[0079] A nucleic acid encoding one or more of eight different enzymes involved in the biosynthesis of heme (determined and annotated from the sequence of the Pichia pastoris genome ) can be expressed as described herein. For example, heterologous nucleic acid molecules encoding ALA synthase , ALA dehydratase, porphobilinogen deaminase, UPG III synthase , UPG III decarboxylase, CPG oxidase, PPG oxidase, and ferrochelatase can be expressed in the strains described herein (e.g., yeast strains (e.g., methylotrophic yeast strains)). Cells (e.g., yeast ( e.g., methylotrophic yeast)) can be transformed with more than one heterologous nucleic acid (e.g., a transgene) For genetic manipulation to contain, a methanol-inducible promoter and a constitutive promoter, or elements derived therefrom, can be combined to further increase the expression of such nucleic acids.

[0080] Any of the cells described herein (e.g., yeast cells (e.g., methanol-utilizing yeast cells)) can contain additional nucleic acid constructs such as third, fourth, fifth nucleic acid constructs, etc., and in some embodiments, it will be understood that such constructs are as previously described for the second nucleic acid construct.

[0081] Previous studies in Saccharomyces cerevisiae identified ALAD and porphobilinogen deaminase as rate-limiting enzymes in heme biosynthesis (see, e.g., Hoffman et al., 2003, Biochem. Biophys. Res. Commun., 310(4) :1247-53). However, heterologous expression of individual heme enzymes from the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter in P. pastoris failed to overcome the limitations associated with the expression of heme-containing recombinant proteins (see Krainer et al., 2015, Microb. Cell Fact., 13;14:4). One or more of the genes involved in the heme biosynthetic pathway can be expressed from one or more constitutive promoters (see, e.g., U.S. Patent No. 9,938, 327, incorporated by reference in its entirety), but P. pastoris (P. pastoris) The expression of recombinant heme-containing proteins can be achieved by co-expression of the entire heme biosynthesis pathway from the methanol -inducible promoter.

[0082] Also provided herein is a method of making a product (e.g., a protein) using any of the nucleic acid constructs and / or cells described herein. In some embodiments the method provided herein may include expressing a nucleic acid construct comprising a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter element. In some embodiments, the first promoter element can be any promoter element described herein. In some embodiments the first promoter element comprises one or more mutations corresponding to nucleotide positions 668 to 734 (e.g., nucleotide positions 673 to 729, nucleotide positions 678 to 724 nucleotide positions 683 to 719, or nucleotide positions 688 to 714) relative to SEQ ID NO: 28. In some embodiments, the method provided herein is a step of expressing a nucleic acid construct comprising a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter element, wherein the first promoter element is selected from the group consisting of mutations corresponding to T146C, C154T, T30 3C, T426A, A433T, A435G, T530A, C572T, T596C, T 617C, T688C, A696T, T702C, A709G, A712G, T714G A790G, A841T, and T862A relative to SEQ ID NO: 28. one or more (e.g., two, three, four, five, six, seven, eight, nine, ten , eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or nineteen) mutations, which may include steps. In some embodiments, the methods provided herein comprise expressing a nucleic acid construct comprising a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter element, wherein the first promoter element, except where the indicated nucleobase is the same as the corresponding naturally occurring nucleobase, relative to SEQ ID NO: 28, has one or more (e.g., two, three, four, five, six , seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or nineteen) mutations selected from the group consisting of mutations corresponding to 146C, 154T, 303C, 426A, 433 T, 435G, 530A, 572T, 596C, 617C, 688C, 696T, 702 C, 709G, 712G, 714G, 790G, 841T, and 862A. In some embodiments, the methods provided herein comprise expressing a nucleic acid construct comprising a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter element, wherein the promoter element has one or more (e.g., two, three, four, five, six , seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or nineteen) mutations at positions corresponding to: T146; C1 54; T303; T426; A433; A435; T530; C572; T596; T6 , 17; T688; A696; T702; A709; A712; T714; A790; A8 , 41; or T862 relative to SEQ ID NO: 28. (e.g., encoding a first protein), wherein the promoter element has one or more (e.g., two, three, four, five , six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen , nineteen) mutations at the positions corresponding to: T146; C154; T303; T426; A433; A435; T530; C572; T596; T617; T688; A696; T702; A709; A712; T714; A790; A841; or T862 relative to SEQ ID NO: 28. In some embodiments, the methods provided herein comprise expressing a nucleic acid construct comprising a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter element, wherein the promoter element has one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen and may include steps that include a mutation of 19). In some embodiments, the methods provided herein comprise expressing a nucleic acid construct comprising a nucleotide sequence operably linked to a first promoter element (e.g., encoding a first protein), wherein the promoter element has the following positions relative to SEQ ID NO: 28: 146; 154; 303; 426; 433; 435; 530; 572; 596; 617; 688; 696; 702; 709; 712; 714; 790; 841; or 862, and comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, or 19) mutations, and may include steps. In some embodiments, the methods provided herein comprise expressing a nucleic acid construct comprising a nucleotide sequence operably linked to a first promoter element (e.g., encoding a first protein), wherein the first promoter element comprises one or more (e.g., 2, 3, 4, or 5) mutations selected from the group consisting of mutations corresponding to T688C, A696T, T702C, A712G and T714G relative to SEQ ID NO: 28, and may include steps. In some embodiments, the methods provided herein comprise expressing a nucleic acid construct comprising a nucleotide sequence operably linked to a first promoter element (e.g., encoding a first protein), wherein the first promoter element has a nucleotide that is not the same as the corresponding naturally occurring nucleotide, relative to SEQ ID NO: 28, and may include steps. In some embodiments, the methods provided herein comprise expressing a nucleic acid construct comprising a nucleotide sequence operably linked to a first promoter element, wherein the first promoter element is operably linked to a nucleotide sequence (e.g., encoding a first protein), and the first promoter element has a nucleotide that is not the same as the corresponding naturally occurring nucleotide, relative to SEQ ID NO: 28, resulting in one or more (e.g., two, three, four, or five) mutations selected from the group consisting of 688C, 696T, 702C, 712G, and 714G and may include steps. In some embodiments, the methods provided herein comprise expressing a nucleic acid construct comprising a nucleotide sequence operably linked to a first promoter element (e.g., encoding a first protein), wherein the promoter element comprises one or more (e.g., two, three, four, or five) mutations at positions corresponding to T688; A696; T702; A712; or T714 with respect to SEQ ID NO: 28, and may include steps. In some embodiments, the methods provided herein comprise expressing a nucleic acid construct comprising a nucleotide sequence operably linked to a first promoter element (e.g., encoding a first protein), wherein the promoter element comprises one or more (e.g., two, three, four, or five) mutations at positions corresponding to 688; 696; 702; 712; or 714 with respect to SEQ ID NO: 28, and may include steps. In some embodiments of any of the methods described herein, the method may be performed in the absence of the addition of methanol. In some embodiments, the major carbon source for methanol-assimilating yeast cells may be dextrose, sucrose, xylose, lactose, maltose, isomaltose, arabinose, sugar alcohol, ethanol, acetate, or glycerol. In some embodiments, the major carbon source may be selected from the group consisting of glucose, sucrose, sorbitol, methanol, and glycerol. In some embodiments, ​ In embodiments, the main carbon source can be selected from the group consisting of glucose, sucrose, sorbitol, and glycerol. In some embodiments, the main carbon source can be oligosaccharides or polysaccharides (e.g., starch, pectin, cellulose, or hemicellulose). In some embodiments, the main carbon source for methanol-assimilating yeast cells can be a mixture of sugars (e.g., derived from cellulosic biomass or starch). In some embodiments, the methods provided herein enable an increase in the titer of a product (e.g., a protein). In some embodiments, the titer of the product (e.g., a protein) can be increased by at least 5% (e.g., at least 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50% %, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%

[0083] %, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% %, or more) compared to the corresponding method lacking the nucleic acid construct described herein. In some embodiments, the titer of the product (e.g., a protein) comprises the step of expressing a nucleic acid encoding a first product (e.g., a protein) operably linked to a first promoter element, wherein the first promoter element lacks any mutations at nucleotide positions corresponding to nucleotide positions 668 - 734 (e.g., nucleotide positions 673 - 729, nucleotide positions 678 - 724, nucleotide positions 683 - 719, or nucleotide positions 688 - 714) relative to SEQ ID NO: 28, and is at least 5% (e.g., at least 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50% %, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300% %, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% %, or more) increased compared to the corresponding method. The first promoter element is at nucleotide positions corresponding to nucleotide positions 668 - 734 (e.g., nucleotide positions 673 - 729, nucleotide positions 678 - 724, nucleotide positions 683 - 719, or nucleotide positions 688 - 714) relative to SEQ ID NO: 28, and lacks any mutations at these positions. In some embodiments, the titer of the product (e.g., a protein) is increased by at least 5% (e.g., at least 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50% at least 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50% , 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300 %, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 100 0%, or more) may increase. In some embodiments, the titer of the product (e.g., protein) comprises the step of expressing a nucleic acid construct comprising a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter element (e.g., encoding a first protein), and the first promoter element lacks any mutation selected from the group consisting of mutations corresponding to T146C, C154T, T303C, T426A, A433T, A435G, T530A, C572T, T59 6C, T617C, T688C, A696T, T702C, A709G, A712G, T 714G, A790G, A841T, and T862A with respect to SEQ ID NO: 28, and is at least 5% (e.g., at least 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more) increased compared to the corresponding method lacking any of the mutations. In some embodiments, the titer of the product (e.g., protein) comprises the step of expressing a nucleic acid construct comprising a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter element (e.g., encoding a first protein), and the first promoter element has the indicated nucleobases corresponding to the naturally occurring ones, and is at least 5% (e.g., at least 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more) increased compared to the corresponding method lacking any of the mutations. In some embodiments, the titer of the product (e.g., protein) comprises the step of expressing a nucleic acid construct comprising a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter element and the first promoter element has the indicated nucleobases corresponding to the naturally occurring Unless it is the same as the nucleobase of SEQ ID NO: 28, for SEQ ID NO: 28, 146C, 154 T, 303C, 426A, 433T, 435G, 530A, 572T, 596C, 617 C, 688C, 696T, 702C, 709G, 712G, 714G, 790G, 841 T, and any mutation selected from the group consisting of mutations corresponding to 862A is lacking, compared to the corresponding method, at least 5% (e.g., at least 6%, 7%, 8% , 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 5 00%, 600%, 700%, 800%, 900%, 1000%, or more than this) can be increased. In some embodiments, the titer of the product (e.g., protein) is the first promo ter element is operably linked to a nucleotide sequence (e.g., encoding a first protein ) comprising the step of expressing a nucleic acid construct, the first promoter ele ment lacks any mutation at the nucleotide positions corresponding to T146, C154, T303, T 426, A433, A435, T530, C572, T596, T617, T688, A 696, T702, A709, A712, T714, A790, A841, and T86 2, compared to the corresponding method, at least 5% (e.g., at least 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150 %, 200%, 250%, 300%, 350%, 400%, 500%, 600%, 700 %, 800%, 900%, 1000%, or more than this) can be increased. In some embodiments ​ In this state, the titer of the product (e.g., protein) involves expressing a nucleic acid construct containing a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter element, and the first promoter element lacks any mutation at nucleotide positions corresponding to nucleotide positions 146, 154, 303, 426, 433, 435, 530, 572, 596, 617, 688, 696, 702, 709, 712, 714, 790, 841, and 862 of SEQ ID NO: 28, and is increased by at least 5% (e.g., at least 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more) compared to the corresponding method. In some embodiments, the titer of the product (e.g., protein) involves expressing a nucleic acid construct containing a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter element, and the first promoter element lacks any mutation selected from the group consisting of mutations corresponding to T688C, A696T, T702C, A712G, and T7 14G of SEQ ID NO: 28, and is increased by at least 5% (e.g., at least 6%, 7%, 8%, 9%, 10 %, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 10 0%, 150%, 200%, 250%, 300%, 350%, 400%, 500%, 60 %.

[0084] In some embodiments, the titer of the product (e.g., protein) involves expressing a nucleic acid construct containing a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter element, and the first promoter element lacks any mutation selected from the group consisting of mutations corresponding to T688C, A696T, T702C, A712G, and T7 14G of SEQ ID NO: 28, and is increased by at least 5% (e.g., at least 6%, 7%, 8%, 9%, 10 %, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 10 0%, 150%, 200%, 250%, 300%, 350%, 400%, 500%, 60 %. In some embodiments, the titer of the product (e.g., protein) involves expressing a nucleic acid construct containing a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter element, and the first promoter element lacks any mutation selected from the group consisting of mutations corresponding to T688C, A696T, T702C, A712G, and T7 14G of SEQ ID NO: 28, and is increased by at least 5% (e.g., at least 6%, 7%, 8%, 9%, 10 %, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 10 (0%, 700%, 800%, 900%, 1000%, or more) may increase. In some embodiments, the titer of the product (e.g., protein) is comprised of a step of expressing a nucleic acid construct comprising a nucleotide sequence operably linked to a first promoter element (e.g., encoding a first protein), wherein the first promoter element lacks any mutation selected from the group consisting of mutations corresponding to 688C, 696T, 702C, 712G, and 714G relative to SEQ ID NO: 28, and compared to the corresponding method, is at least 5% (e.g., at least 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150 %, 200%, 250%, 300%, 350%, 400%, 500%, 600%, 700 %, 800%, 900%, 1000%, or more) may increase. In some embodiments, the titer of the product (e.g., protein) is comprised of a step of expressing a nucleic acid construct comprising a nucleotide sequence operably linked to a first promoter element, wherein the first promoter element lacks any mutation at the nucleotide positions corresponding to nucleotide positions T688, A696, T702, A712, and T714 relative to SEQ ID NO: 28, and compared to the corresponding method, is at can be increased by 800%, 900%, 1000%, or more. In some embodiments the titer of the product (e.g., protein) is determined by expressing a nucleic acid construct comprising a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter element, wherein the first promoter element lacks any mutations at nucleotide positions corresponding to nucleotide positions 688, 696, 702, 712, and 714 of SEQ ID NO: 28, and is at least 5% (e.g., at least 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 4 0%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 25 0%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 90 0%, 1000%, or more) greater than that of a corresponding method wherein the corresponding method is essentially identical to the reference method in all respects except for the identified differences. For example, the titer of the product (e.g., protein) can be determined by expressing a nucleic acid construct

[0085] Generally, "titer" is a measure of the amount of a substance in a solution. As used herein, the "titer" of a heme-binding protein refers to the total polypeptide amount, whether or not bound to heme, unless otherwise specified. The titer of a product (e.g., protein) can be measured by any suitable method, such as high performance liquid chromatography (HPLC), high performance liquid chromatography - mass spectrometry (HPLC-MS), enzyme-linked immunosorbent assay (ELISA), or ultraviolet and / or visible light spectroscopy. As used herein, a "corresponding method" is a method that is essentially identical to a reference method in all respects except for the identified differences. For example, expressing a nucleic acid construct comprising a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter element

[0086] As used herein, a "corresponding method" is a method that is essentially identical to a reference method in all respects except for the identified differences. For example, expressing a nucleic acid construct comprising a nucleotide sequence (e.g., A nucleus containing a potentially linked nucleotide sequence (e.g., encoding a first protein) A corresponding method for expressing a nucleic acid construct, wherein the first promoter element Lacks one or more mutations corresponding to mutations at nucleotide positions 668-734 relative to SEQ ID NO: 28 (e.g., nucleotide positions 67 3-729, nucleotide positions 678-724, nucleotide positions 683-719, and Also nucleotide positions 688-714). If it is a method, the corresponding method is a nucleic acid construct containing a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter element, and the nucleotide positions corresponding to nucleotide positions 66 8-734 relative to SEQ ID NO: 28 (e.g., nucleotide positions 673-729, nucleotide positions 678-72 4, nucleotide positions 683-719, or nucleotide positions 688-714) lacks any mutation corresponding to the mutation at the corresponding nucleotide position. Except for expressing the nucleic acid construct lacking any mutation corresponding to the mutation at the nucleotide position corresponding to SEQ ID NO: 28, in all aspects (e.g., the genetic composition of the cell, the temperature and number of cultures, etc.), it will be essentially the same as the reference method. For example, a corresponding method for expressing a nucleic acid construct containing a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter element, wherein the first promoter Element has T146C, C154T, T303C, T42 6A, A433T, A435G, T530A, C572T, T596C, T617C, T 688C, A696T, T702C, A709G, A712G, T714G, A790G relative to SEQ ID NO: 28. In all other aspects (e.g., the genetic composition of the cell, the temperature and number of cultures, etc.), it will be essentially the same as the reference method. For example, a corresponding method for expressing a nucleic acid construct containing a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter element, wherein the first promoter Element is T146C, C154T, T303C, T42 6A, A433T, A435G, T530A, C572T, T596C, T617C, T 688C, A696T, T702C, A709G, A712G, T714G, A790G relative to SEQ ID NO: 28. relative to SEQ ID NO: 28. , A841T, and T862A. If the method lacks multiple mutations, the corresponding method comprises: a nucleotide sequence operably linked to the first protein (e.g., encoding a first protein) A nucleic acid construct comprising the following repeat sequences: T146C, C154T, T303 relative to SEQ ID NO: 28. C, T426A, A433T, A435G, T530A, C572T, T596C, T6 17C, T688C, A696T, T702C, A709G, A712G, T714G, The group of mutations corresponding to A790G, A841T, and T862A In one embodiment, the nucleic acid construct is expressed in a manner that is consistent with the present invention, except that the nucleic acid construct lacks any of the mutations in the nucleic acid. The method is essentially the same as the reference method in terms of the genetic composition of the cells, the temperature and number of incubations, etc. For example, a nucleotide sequence operably linked to the first promoter element may be To express a nucleic acid construct comprising a sequence (e.g., encoding a first protein), A corresponding method, wherein the first promoter element is a nucleotide sequence corresponding to SEQ ID NO:28. Reotide positions T146, C154, T303, T426, A433, A435, T530 , C572, T596, T617, T688, A696, T702, A709, A712 , at nucleotide positions corresponding to T714, A790, A841, and T862 If the method lacks one or more mutations, the corresponding method comprises: A nucleotide sequence operably linked to the element (e.g., encoding a first protein) 28, comprising a nucleic acid construct comprising a nucleotide sequence at nucleotide position T146 relative to SEQ ID NO: 28. , C154, T303, T426, A433, A435, T530, C572, T596 , T617, T688, A696, T702, A709, A712, T714, A790 , any of the mutations at the nucleotide positions corresponding to A841 and T862, except for expressing a nucleic acid construct lacking any of them, in all aspects (e.g., the genetic composition of the cells, the temperature and number of cultures, etc.), would be essentially the same as the reference method. For example, a corresponding method for expressing a nucleic acid construct containing a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter element, wherein the first promoter element lacks one or more mutations selected from the group consisting of the mutations corresponding to 146C, 154T, 303C, 426A, 433T, 435G, 530A, 572T, 596C, 617C, 688C, 696T, 702C, 709G, 712G, 714G, 790G, 841T, and 862A with respect to SEQ ID NO: 28, in all aspects (e.g., the genetic composition of the cells, the temperature and number of cultures, etc.), would be essentially the same as the reference method. For example, a corresponding method for expressing a nucleic acid construct containing a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter element, wherein the first promoter element lacks one or more mutations selected from the group consisting of the mutations corresponding to 146C, 154T, 303C, 426A, 433T, 435G, 530A, 572T, 596C, 617C, 688C, 696T, 702C, 709G, 712G, 714G, 790G, 841T, and 862A with respect to SEQ ID NO: 28, except for expressing a nucleic acid construct lacking any of the mutations, in all aspects (e.g., the genetic composition of the cells, the temperature and number of cultures, etc.), would be essentially the same as the reference method. For example, a corresponding method for expressing a nucleic acid construct containing a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter element, wherein the first promoter element lacks one or more mutations selected from the group consisting of the mutations corresponding to 146C, 154T, 303C, 426A, 433T, 435G, 530A, 572T, 596C, 617C, 688C, 696T, 702C, 709G, 712G, 714G, 790G, 841T, and 862A with respect to SEQ ID NO: 28, except for expressing a nucleic acid construct lacking any of the mutations, in all aspects (e.g., the genetic composition of the cells, the temperature and number of cultures, etc.), would be essentially the same as the reference method. For example, a corresponding method for expressing a nucleic acid construct containing a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter element, wherein the first promoter element lacks one or more mutations selected from the group consisting of the mutations corresponding to 146C, 154T, 303C, 426A, 433T, 435G, 530A, 572T, 596C, 617C, 688C, 696T, 702C, 709G, 712G, 714G, 790G, 841T, and 862A with respect to SEQ ID NO: 28, except for expressing a nucleic acid construct lacking any of the mutations, in all aspects (e.g., the genetic composition of the cells, the temperature and number of cultures, etc.), would be essentially the same as the reference method. For example, a corresponding method for expressing a nucleic acid construct containing a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter element, wherein the first promoter element lacks one or more mutations selected from the group consisting of the mutations corresponding to 146C, 154T, 303C, 426A, 433T, 435G, 530A, 572T, 596C, 617C, 688C, 696T, 702C, 709G, 712G, 714G, 790G, 841T, and 862A with respect to SEQ ID NO: 28, except for expressing a nucleic acid construct lacking any of the mutations, in all aspects (e.g., the genetic composition of the cells, the temperature and number of cultures, etc.), would be essentially the same as the reference method. For example, a corresponding method for expressing a nucleic acid construct containing a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter element, wherein the first promoter element lacks one or more mutations selected from the group consisting of the mutations corresponding to 146C, 154T, 303C, 426A, 433T, 435G, 530A, 572T, 596C, 617C, 688C, 696T, 702C, 709G, 712G, 714G, 790G, 841T, and 862A with respect to SEQ ID NO: 28, A nucleic acid construct comprising a ligated nucleotide sequence (e.g., encoding a first protein) A corresponding method for expression, wherein the first promoter element lacks one or more mutations at nucleotide positions corresponding to nucleotide positions 146, 154, 303, 426, 433, 435, 5 30, 572, 596, 617, 688, 696, 702, 709, 712, 714, 7 90, 841, and 862 with respect to SEQ ID NO: 28, the corresponding method is a nucleic acid construct comprising a nucleotide sequence (e.g., encoding a first protein) operably linked to the first promoter element which lacks any of the mutations at nucleotide positions corresponding to nucleotide positions 146, 154, 303, 42 6, 433, 435, 530, 572, 596, 617, 688, 696, 702, 70 9, 712, 714, 790, 841, and 862 with respect to SEQ ID NO: 28, except for expressing a nucleic acid construct lacking any of the mutations at these nucleotide positions, in all aspects (e.g., the genetic composition of the cells, the temperature and number of cultures, etc.), will be essentially the same as the reference method. For example, a corresponding method for expressing a nucleic acid construct comprising a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter element, wherein the first promoter element lacks one or more mutations selected from the group consisting of mutations corresponding to T688C, A 696T, T702C, A712G, and T714G with respect to SEQ ID NO: 28, the corresponding method is a nucleotide sequence (e.g.,

[0087] encoding a first protein) operably linked to a first promoter element For example, a corresponding method for expressing a nucleic acid construct comprising a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter element, wherein the first promoter element lacks one or more mutations selected from the group consisting of mutations corresponding to T688C, A 696T, T702C, A712G, and T714G with respect to SEQ ID NO: 28, the corresponding method is a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter element If it is a method lacking one or more mutations selected from the group consisting of mutations corresponding to T688C, A A nucleic acid construct comprising a (encoding a protein), which is T688C, A Any of the mutations corresponding to 696T, T702C, A712G, and T714G Except for expressing a nucleic acid construct lacking any of the mutations included in the group, all aspects (For example, the genetic composition of cells, the temperature and number of cultures, etc.) will be essentially The same as the reference method. For example, for expressing a nucleic acid construct comprising a nucleotide sequence (for example, encoding a first protein) operably linked to a first promoter element The corresponding method, provided that the first promoter element lacks one or more mutations selected from the group consisting of the mutations corresponding to 688C, 696T, 702C, 712G, and 714G with respect to SEQ ID NO: 28 The corresponding method will be essentially the same as the reference method in all aspects (for example, the genetic composition of cells, the temperature and number of cultures, etc.) except for expressing a nucleic acid construct lacking any of the mutations included in the group consisting of the mutations corresponding to 688C, 696T, 702C, 712G, and 714G with respect to SEQ ID NO: 28. For example, for expressing a nucleic acid construct comprising a nucleotide sequence (for example, encoding a first protein) operably linked to a first promoter element The corresponding method, provided that the first promoter element lacks one or more mutations at the nucleotide positions corresponding to 688C, 696T, 702C, 712G, and 714G with respect to SEQ ID NO: 28 If the method is one that lacks one or more mutations selected from the group consisting of the mutations corresponding to 688C, 696T, 702C, 712G, and 714G with respect to SEQ ID NO: 28, the corresponding method Is a nucleic acid construct comprising a nucleotide sequence (for example, encoding a first protein) operably linked to a first promoter element That lacks any of the mutations included in the group consisting of the mutations corresponding to 688C, 696T, 702C, 712G, and 714G with respect to SEQ ID NO: 28. The corresponding method will be essentially the same as the reference method in all aspects (for example, the genetic composition of cells, the temperature and number of cultures, etc.) except for expressing a nucleic acid construct lacking any of the mutations included in the group Consisting of the mutations corresponding to 688C, 696T, 702C, 712G, and 714G with respect to SEQ ID NO: 28. For example, for expressing a nucleic acid construct comprising a nucleotide sequence (for example, encoding a first protein) operably linked to a first promoter element The corresponding method, provided that the first promoter element lacks one or more mutations at the nucleotide positions corresponding to nucleotide positions T688, A696, T702, A712, and T714 with respect to SEQ ID NO: 28 (For example, the genetic composition of cells, the temperature and number of cultures, etc.) will be essentially The same as the reference method. For example, for expressing a nucleic acid construct comprising a nucleotide sequence (for example, encoding a first protein) operably linked to a first promoter element The corresponding method for expressing a nucleic acid construct comprising a nucleotide sequence (for example, encoding a first protein) operably linked to a first promoter element Wherein the first promoter element lacks one or more mutations at the nucleotide positions corresponding to nucleotide positions T688, A696, T702, A712, and T714 with respect to SEQ ID NO: 28. If the method is one that lacks one or more mutations at the nucleotide positions corresponding to nucleotide positions T688, A696, T702, A712, and T714 with respect to SEQ ID NO: 28, the corresponding method The corresponding method, provided that the first promoter element lacks one or more mutations at the nucleotide positions corresponding to nucleotide positions T688, A696, T702, A712, and T714 with respect to SEQ ID NO: 28 If the method is one that lacks one or more mutations at the nucleotide positions corresponding to nucleotide positions T688, A696, T702, A712, and T714 with respect to SEQ ID NO: 28, the corresponding method A method that, except for expressing a nucleic acid construct comprising a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter element and lacking any of the mutations consisting of the mutations corresponding to T688, A696, T702, A712, and T714 with respect to SEQ ID NO: 28, would be essentially the same as the reference method in all aspects (e.g., the genetic composition of the cell, the temperature and number of times of culturing, etc.). For example, a corresponding method for expressing a nucleic acid construct comprising a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter element, provided that the first promoter element lacks one or more mutations at the nucleotide positions corresponding to nucleotide positions 688, 696, 702, 712, and 714 with respect to SEQ ID NO: 28, would be essentially the same as the reference method in all aspects (e.g., the genetic composition of the cell, the temperature and number of times of culturing, etc.), except for expressing a nucleic acid construct comprising a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter element and lacking any of the mutations consisting of the mutations corresponding to 688, 696, 702, 712, and 714 with respect to SEQ ID NO: 28. encoding a first protein), and lacking any of the mutations consisting of the mutations corresponding to T688, A696, T702, A712, and T714 with respect to SEQ ID NO: 28

[0088] Genetic manipulation of cells (e.g., yeast cells (e.g., methanol-utilizing yeast cells)) typically involves introducing a recombinant nucleic acid molecule (also referred to as a nucleic acid construct) into the cell. As described in this specification, recombinant nucleic acid molecules typically comprise at least one promoter element ​​​​​​​​​​​​​​​​ A product (e.g., a protein (e.g., a protein involved in heme biosynthesis, a heme-binding protein, or a transcription factor)) operably linked to an element (e.g., an inducible promoter element or a constitutive promoter element) and contains an exogenous nucleic acid encoding the product. In some embodiments, the recombinant nucleic acid molecule is a linear arrangement of two or more protein-coding sequences operably linked to the same or separate promoter elements (e.g., a first nucleic acid construct containing a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter, and a second nucleic acid construct containing a nucleotide sequence (e.g., encoding a second protein) operably linked to a second promoter, or a single promoter operably linked to a first nucleic acid construct containing a nucleotide sequence (e.g., encoding a first protein) and a second nucleic acid construct containing a nucleotide sequence (e.g., encoding a second protein)). Optionally, a recombinant nucleic acid molecule containing at least one promoter operably linked to a nucleotide sequence (e.g., encoding a protein) can be referred to as a cassette. The recombinant nucleic acid can include an expression element. The expression element includes nucleic acid sequences that direct and regulate the expression of the nucleic acid coding sequence. One example of an expression element is a promoter sequence . The expression element can also include introns, enhancer sequences, response elements, or inducer elements that regulate the expression of the nucleic acid. The expression element can be of bacterial, yeast, insect, mammalian, or viral origin and can be a vector (e.g., a plasmid, cosmid, phage, virus, or artificial chromosome). In some embodiments, the recombinant nucleic acid molecule is a linear arrangement of two or more protein-coding sequences operably linked to the same or separate promoter elements (e.g., a first nucleic acid construct containing a nucleotide sequence (e.g., encoding a first protein) operably linked to a first promoter, and a second nucleic acid construct containing a nucleotide sequence (e.g., encoding a second protein) operably linked to a second promoter, or a single promoter operably linked to a first nucleic acid construct containing a nucleotide sequence (e.g., encoding a first protein) and a second nucleic acid construct containing a nucleotide sequence (e.g., encoding a second protein)). Optionally, a recombinant nucleic acid molecule containing at least one promoter operably linked to a nucleotide sequence (e.g., encoding a protein) can be referred to as a cassette.

[0089] The recombinant nucleic acid can include an expression element. The expression element includes nucleic acid sequences that direct and regulate the expression of the nucleic acid coding sequence. One example of an expression element is a promoter sequence . The expression element can also include introns, enhancer sequences, response elements, or inducer elements that regulate the expression of the nucleic acid. The expression element can be of bacterial, yeast, insect, mammalian, or viral origin and can be a vector (e.g., a plasmid, cosmid, phage, virus, or artificial chromosome). The expression element can also include introns, enhancer sequences, response elements, or inducer elements that regulate the expression of the nucleic acid. The expression element can be of bacterial, yeast, insect, mammalian, or viral origin and can be a vector (e.g., a plasmid, cosmid, phage, virus, or artificial chromosome). - may contain combinations of elements from different origins.

[0090] Nucleic acids can be detected using any number of amplification techniques (e.g., PCR Primer: A Laboratory Manual, 1995, Die ffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, Cold Spring Harb or, NY; and U.S. Patent Nos. 4,683,195; 4,683,202 ; 4,800,159; and 4,965,188, see ), together with appropriate oligonucleotide (e.g., primer) pairs. Several modifications to the original PCR method have been developed and can be used to detect the selected nucleic acid.

[0091] Appropriate transcription factors, and nucleic acids encoding transcription factors (e.g., exogenous nucleic acids encoding transcription factors), include, for example, Mxr1 derived from Pichia pastoris. Representative Mxr1 nucleic acid sequences of P. pastoris can be found, for example, in GenBank accession number: DQ395124, while representative Mxr1 polypeptide sequences of P. pastoris can be found, for example, in GenBank accession number: ABD57365). In some embodiments, the transcription factor can be the Mit1 sequence derived from P. pastoris (see, for example, GenBank accession number: CAY70887). Appropriate transcription factors can also be from Hansenula polymorpha (e.g., Adr1; for example, for nucleic acid sequences see ). GenBank accession number: AEOI02000005, bases: 858873 - 8623 See also GenBank accession number 52 and for the amino acid sequence: ESX01253 (to be desired); and Candida boidinii (e.g., Trm1; e.g., GenBank accession number for the nucleic acid sequence: AB365355, and for the amino acid sequence: BAF99700; and Trm2; see also. For example, GenBank accession number for the nucleic acid sequence: AB548760, and for the amino acid sequence: BAJ07608 (to be desired). It may also be found in Transcription factors such as Mxr1 can usually be expressed at low levels. In some embodiments, it is desired to place an exogenous nucleic acid (e.g., a transcription factor) under the control of an inducible promoter.

[0092] In some embodiments, a transcription factor can bind to the promoter elements described herein and activate transcription from the promoter element. In some embodiments, when a nucleic acid sequence encoding a transcription factor is operably linked to the promoter element to which it binds, a positive feedback loop can be created that helps drive the expression of other nucleic acid sequences (e.g., a nucleic acid sequence encoding a protein) operably linked to the promoter. Non - limiting examples of transcription factors that can be used with the AOX1 promoter (e.g., a mutant AOX

[0093] 1 promoter) include Mxr1, Mit1, Adr1, Trm1, Trm2, and combinations thereof. In some embodiments, AOX 1 promoter) include Mxr1, Mit1, Adr1, Trm1, Trm2, and combinations thereof. In some embodiments, AOX 1 promoter) include Mxr1, Mit1, Adr1, Trm1, Trm2, and combinations thereof. In some embodiments, AOX 1 promoter) include Mxr1, Mit1, Adr1, Trm1, Trm2, and combinations thereof. In some embodiments, AOX 1 promoter) include Mxr1, Mit1, Adr1, Trm1, Trm2, and combinations thereof. In some embodiments, AOX 1 promoter) include Mxr1, Mit1, Adr1, Trm1, Trm2, and combinations thereof. In some embodiments, AOX 1 promoter) include Mxr1, Mit1, Adr1, Trm1, Trm2, and combinations thereof. Non - limiting examples of transcription factors that can be used with the AOX1 promoter (e.g., a mutant AOX 1 promoter) include Mxr1, Mit1, Adr1, Trm1, Trm2, and combinations thereof. In some embodiments, AOX Transcription factors that can be used with a promoter can include Mxr1. The MOX promoter - Non-limiting examples of transcription factors that can be used with a MOX promoter (e.g., a mutant MOX promoter) are Adr1. Non-limiting examples of transcription factors that can be used with an AOD1 promoter (e.g., a mutant AOD1 promoter) include Trm1, Trm2, or combinations thereof. In some embodiments, two methanol-regulated transcription factors (e.g., Mxr1 and M it1) can be operably linked to a methanol-inducible promoter element (e.g., pAOX1).

[0094] The recombinant nucleic acid molecules described herein may be stably integrated into the genome of a cell (e.g., a yeast cell (e.g., a methanol-assimilating yeast cell)), or may be expressed extrachromosomally from a replicating competent plasmid. Methods for achieving either are well known and routinely used in the art.

[0095] In addition, a first nucleic acid construct (e.g., a first protein (e.g., encoding a heme-binding protein)) comprising a nucleotide sequence operably linked to a promoter element (e.g., the promoter elements described herein) can be physically separated from a second nucleic acid construct comprising a nucleotide sequence (e.g., encoding a second protein (e.g., a transcription factor)) operably linked to a promoter element (e.g., the promoter elements described herein) (i.e., the first nucleic acid construct and the second nucleic acid construct can be completely separate molecules). Alternatively, the second nucleic acid construct can be physically separated from the first nucleic acid construct (i.e., the first nucleic acid construct and the second nucleic acid construct can be completely separate molecules). Alternatively, the p Operate on a promoter element (e.g., the promoter element described herein) Comprising a nucleotide sequence (e.g., encoding a first protein) operably linked thereto A first nucleic acid construct, and a nucleotide sequence (e.g., encoding a second protein) operably linked to a promoter element (e.g., the promoter element described herein) Can be incorporated within the same nucleic acid construct . In some embodiments, a first nucleic acid construct comprising a nucleotide sequence (e.g., encoding a first protein) operably linked to a promoter element Can be contiguous with a second nucleic acid construct comprising a nucleotide sequence (e.g., encoding a second protein) operably linked to a promoter element . Those skilled in the art will recognize that when a second nucleic acid construct (e.g., encoding a second protein) comprising a nucleotide sequence Is contiguous with a first nucleic acid construct (e.g., encoding a protein of interest) comprising a nucleotide sequence A single promoter, or a promoter element derived therefrom, can be used to drive transcription of both the nucleotide sequence (e.g., encoding a first protein, as well as a nucleic acid encoding a second protein) It will be appreciated that all of them can be used to drive all transcription . In the art, methods for introducing nucleic acids into cells (e.g., yeast cells (e.g., methanol-assimilating yeast cells )) are known, and include, without limitation, transduction, electroporation, biolistic particle delivery, and chemical transformation . In the art, methods for culturing cells (e.g., yeast cells (e.g., methanol-assimilating yeast cells)) are also known

[0096] In the art, methods for introducing nucleic acids into cells (e.g., yeast cells (e.g., methanol-assimilating yeast cells )) are known, and include, without limitation, transduction, electroporation, biolistic particle delivery, and chemical transformation . In the art, methods for culturing cells (e.g., yeast cells (e.g., methanol-assimilating yeast cells)) are also known . For example Pichia Protocols, Methods In Molecular Biology, 389, Cregg, Ed., 2007, 2nd E d., Humana Press, Inc. In some circumstances, the communications supported herein may be Methanol promotes the production of high levels of one or more products of interest (e.g., proteins). Although not required for efficient expression in Escherichia coli, methanol may be introduced into the culture medium. In some circumstances (e.g., those involved in the biosynthesis of heme), it may be desirable to add In the case where one or more nucleic acids encoding the enzyme(s) are expressed, Iron, or a pharma- ceutically or metabolically acceptable salt thereof (or GR It may be desirable to supplement with glycerol (AS salts).

[0097] The methods provided herein also include a step of purifying the expressed protein. As used herein, "enriched" protein refers to a protein that is enriched with the quality of the producing cells, on a dry weight basis. At least 5% (e.g., at least 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or or by dry weight, the production cell lysate (e.g., cell wall material or At least 10% (e.g., at least 15%, 20% , 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% , 75%, 80%, 90%, 95%, or 99% of the protein. As used herein, a "purified" protein is a protein that is free from the cellular components that naturally accompany it. Typically, a protein is a protein that is separated from a natural product by dry weight. Then, it is considered "purified" if it lacks at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, or 99%) of the other proteins and naturally occurring molecules with which it is associated.

[0098] As used herein, nucleic acids may include DNA and RNA, and may include nucleic acids containing one or more nucleotide analogs or backbone modifications. Nucleic acids may be single-stranded or double-stranded, which typically depends on their intended use. Additionally, nucleic acids and polypeptides that differ from a given sequence are also provided. Nucleic acids and polypeptides may have at least 50% sequence identity (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to a given nucleic acid sequence or polypeptide sequence. In some embodiments, a nucleic acid or polypeptide may have 100% sequence identity to a given nucleic acid sequence or polypeptide sequence.

[0099] In calculating the percent sequence identity, two sequences are aligned and the number of identical matches of nucleotides or amino acid residues between the two sequences is determined. The number of identical matches is divided by the length of the aligned region (i.e., the number of nucleotides or amino acid residues in the aligned region) and multiplied by 100 to arrive at the percent sequence identity value. The length of the aligned region is the length of a portion of one or both sequences - the full length of the shortest sequence. ​​​​​​​​​​​​​​​It will be appreciated that a single sequence may be of any size. Also, a single sequence may be expressed as more than one other sequence. Thus, across each aligned region, there may be different It will be appreciated that the percent sequence identity of each of the sequences may be different.

[0100] Alignment of two or more sequences to determine percent sequence identity In the present study, alignments of nucleic acid or polypeptide sequences are performed over their entire length. C, a computer program that allows for global alignment This can be performed using lustalW and default parameters (Chenna et al. al., 2003, Nucleic Acids Res., 31(13):3497-500). ClustalW is a method to measure identity, similarity, and The query sequence and one or more subject sequences are compared so that similarities and differences can be determined. Calculate the best matches of and align them. Maximize sequence alignment Thus, gaps of one or more residues may be For rapid, matched alignment of nucleic acid sequences, The default parameters (i.e., word size: 2; window size: 4; scoring method : percentage; number of top diagonals: 4; and gap penalty: 5) For alignment of multiple nucleic acid sequences, the following parameters may be used: :Gap opening penalty: 10.0;Gap extension penalty: 5.0; and weight transitions: yes can be used. Rapid, pairwise alignment of polypeptide sequences. For the evaluation, the following parameters are used: word size: 1; window size: 5; evaluation Determination method: percentage; number of top diagonals: 5; and gap penalty: 3 can be used. For multiple alignment of polypeptide sequences, the following parameters -: Weight matrix: blosum; gap opening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gap: on; hydrophilic residues: Gly, Pro, S er, Asn, Asp, Gln, Glu, Arg, and Lys; and residue-specific gap penalty: on can be used. ClustalW can be run, for example, on the Internet at the Baylor College of Medicine Search Lau ncher website, or at the European Bioinformatics I nstitute website.

[0101] Changes may be introduced into the nucleic acid molecule, thereby resulting in changes in the amino acid sequence of the encoded polypeptide . For example, the changes may be introduced into the nucleic acid coding sequence using mutagenesis (e.g., site-directed mutagenesis, PCR-mediated mutagenesis, transposon mutagenesis, chemical mutagenesis, UV mutagenesis, or radiation-induced mutagenesis), and nucleic acid molecules having such changes may be introduced into the nucleic acid coding sequence by chemically synthesizing such nucleic acids. Such nucleic acid changes may result in conservative amino acid substitutions and / or non-conservative amino acid substitutions at one or more amino acid residues. A "conservative amino acid substitution" is a substitution in which one amino acid residue is replaced with a different amino acid residue having a similar side chain (e.g., a frequency table for amino acid substitutions is provided ), or may be introduced into the nucleic acid coding sequence by chemically synthesizing nucleic acid molecules having such changes. Such nucleic acid changes may result in conservative amino acid substitutions and / or non-conservative amino acid substitutions at one or more amino acid residues. A "conservative amino acid substitution" is a substitution in which one amino acid residue is replaced with a different amino acid residue having a similar side chain (e.g., a frequency table for amino acid substitutions is provided ), resulting in conservative amino acid substitutions and / or non-conservative amino acid substitutions at one or more amino acid residues. A "conservative amino acid substitution" is a substitution in which one amino acid residue is replaced with a different amino acid residue having a similar side chain (e.g., a frequency table for amino acid substitutions is provided See, Dayhoff et al., 1978, Atlas of Protein Sequence and Structure, 5(Suppl. 3):345-352), and a non-conservative substitution is a substitution in which an amino acid residue is replaced with an amino acid residue having a non-similar side chain. Nucleic acid sequences and / or polypeptide sequences can, without limitation, be modified as described herein to increase expression (e.g., transcription and / or translation), tighten regulation release, deregulate, lose catabolite repression, specificity, secretion, heat stability, solvent stability, oxidative stability, protease resistance, catalytic activity, and / or modify color, etc., to improve one or more properties. As used herein, an "isolated" nucleic acid molecule is a nucleic acid molecule that, in nature, does not contain sequences flanking one or both ends of the nucleic acid from which the isolated nucleic acid molecule is derived

[0102] in the genome of the organism (e.g., cDNA, or genomic DNA fragments generated by PCR or restriction endonuclease digestion). Such isolated nucleic acid molecules are generally introduced into vectors (e.g., cloning vectors or expression vectors) for ease of manipulation or to create fusion nucleic acid molecules, as discussed in more detail below. In addition, isolated nucleic acid molecules can include engineered nucleic acid molecules, such as recombinant nucleic acid molecules or synthetic nucleic acid molecules. vectors or expression vectors) for ease of manipulation or to create fusion nucleic acid molecules, as discussed in more detail below. In addition, isolated nucleic acid molecules can include engineered nucleic acid molecules, such as recombinant nucleic acid molecules or synthetic nucleic acid molecules. The vectors described herein can be introduced into host cells. As used herein, a "host cell" refers to a particular cell into which a nucleic acid is introduced, and also includes progeny cells of such cells that carry the vector. Host cells can be any prokaryotic or eukaryotic cell

[0103] The vectors described herein can be introduced into host cells. As used herein, a "host cell" refers to a particular cell into which a nucleic acid is introduced, and also includes progeny cells of such cells that carry the vector. Host cells can be any prokaryotic or eukaryotic cell and It can be expressed. For example, the nucleic acid may be expressed in bacterial cells such as Escherichia coli (E. coli), in insect cells, yeast cells, or mammalian cells (Chinese hamster ovary cells (CHO) or COS cells, etc.). Those skilled in the art know other suitable host cells. Those skilled in the art know many methods, both in vivo and in vitro, for introducing the nucleic acid into the host cell. Without limitation, they include electroporation, calcium phosphate precipitation, polyethylene glycol (PEG) transformation, heat shock , lipofection, microinjection, and virus-mediated nucleic acid introduction.

[0104] The nucleic acid can be isolated using techniques defined in the art. For example, the nucleic acid can be isolated using, without limitation, recombinant nucleic acid techniques and / or polymerase chain reaction (PCR). For general PCR techniques, see, for example, PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor La boratory Press, 1995. Recombinant nucleic acid techniques include, for example, restriction enzyme digestion and ligation that can be used to isolate the nucleic acid. The isolated nucleic acid can also be chemically synthesized as a single nucleic acid molecule or a series of oligonucleotides.

[0105] The polypeptide can be purified from natural sources (e.g., biological samples) by known methods such as DEAE ion exchange, gel filtration, and hydroxyapatite chromatography. The polypeptide can also be, for example, expressed in an expression vector by expressing the nucleic acid. Additionally, purified polypeptides can be obtained by chemical synthesis. The purity of the peptide can be determined by any suitable method, for example, column chromatography, polyacrylamide gel electrophoresis, etc. This can be measured using mid-gel electrophoresis, or HPLC analysis.

[0106] The nucleic acid constructs described herein (e.g., the promoter elements described herein) A construct comprising a nucleotide sequence encoding a polypeptide operably linked to a Constructs or vectors comprising the expression construct or expression vector are also provided. Vectors are commercially available or can be prepared using recombinant DNA techniques as defined in the art. A construct or vector containing a nucleic acid can be made by operably linking such a nucleic acid. and may have an expression element linked to it, and a selectable marker (e.g., an antibiotic The nucleic acid-containing construct may further include a sequence encoding a gene encoding a nucleotide sequence (e.g., a gene encoding a nucleotide sequence ... The vector may be used to express a chimeric or fusion polypeptide (i.e., a heterologous polypeptide). A polypeptide operably linked to a tide, which is located at the N-terminus or C-terminus of the polypeptide. Representative heterologous polypeptides may include the encoded polypeptide. Heterologous polypeptides (e.g., 6xHis tags, Glutathione S-transferase (GST).

[0107] Nucleic acids can also be detected using hybridization. For the synthesis, see Sambrook et al. (1989, Molecular Cloning: A Laboratory Method anual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sec tions 7.37-7.57, 9.47-9.57, 11.7-11.8, and 11.45-11.57) are discussed in detail by Sambrook et al. Sambrook et al. disclose Southern blotting conditions suitable for oligonucleotide probes less than about 100 nucleotides (Sections 11.45-11.46). The Tm between a sequence less than 100 nucleotides in length and a second sequence can be calculated using the formula presented in Section 11.46. In addition, Sambrook et al. disclose Southern blotting conditions suitable for oligonucleotide probes greater than about 100 nucleotides (see Sections 9.47-9.54). The Tm between a sequence greater than 100 nucleotides in length and a second sequence can be calculated using the formula presented in Sections 9.50-9.51 of Sambrook et al.

[0108] The conditions under which a membrane containing nucleic acid is prehybridized, hybridized, and washed to remove excess probe and nonspecifically bound probe can play a significant role in the stringency of hybridization. Such hybridization and washing can be carried out under moderately stringent or highly stringent conditions, as appropriate. For example, washing conditions can be made more stringent by decreasing the salt concentration in the wash solution and / or by increasing the temperature at which washing is carried out. By way of example only, highly stringent conditions Typically, it involves washing the membrane in 0.2× SSC at 65 °C.

[0109] In addition, the interpretation of the amount of hybridization is affected by, for example, the specific activity of the labeled oligonucleotide probe, the number of probe-binding sites on the template nucleic acid to which the probe hybridizes, and the amount of exposure of the autoradiograph or other detection medium. One of ordinary skill in the art can contemplate any number of hybridization and wash conditions for use in examining the hybridization of probe nucleic acid molecules to immobilized target nucleic acids, but it will be readily appreciated that it is important to examine the hybridization of a probe to a target nucleic acid under identical hybridization conditions, wash conditions, and exposure conditions. Preferably, the target nucleic acids are on the same membrane.

[0110] If hybridization to one nucleic acid is at least 5-fold (e.g., at least 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, or 100-fold) less than hybridization to another nucleic acid, the nucleic acid molecule is considered to hybridize to one nucleic acid but not to another nucleic acid. The amount of hybridization may be quantified directly on the membrane, for example, using a PhosphorImager or Densitometer (Molecular Dynamics, Sunnyvale, CA), or may be quantified by autoradiography.

[0111] Polypeptides can be detected using antibodies. Techniques for detecting polypeptides using antibodies include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation including reduction and immunofluorescence. The antibody may be a polyclonal antibody or a monoclonal antibody. Antibodies having specific binding affinity for a polypeptide can be produced using methods well known in the art. The antibody can be conjugated to a solid support such as a microtiter plate using methods known in the art. In the presence of the polypeptide, an antibody-polypeptide complex is formed.

[0112] Detection (e.g., detection of an amplification product, a hybridization complex, or a polypeptide ) is usually achieved using a detectable label. The term "label" is intended to encompass the use of both direct labels and indirect labels. Detectable labels include enzymes, cofactors, fluorescent substances, luminescent substances, bioluminescent substances, and radioactive substances.

[0113] Methods are described herein that can be used to generate strains lacking sequences for selection (i.e., lacking a selectable marker). These methods involve the use of circular plasmid DNA vectors and linear DNA sequences; the circular plasmid DNA vectors contain a selectable marker and an origin of DNA replication (also known as an autonomously replicating sequence (ARS)), and the linear DNA sequences contain sequences for integration into the Pichia genome by homologous recombination. In addition, linear DNA molecules include, without limitation, heme-binding L egH, dehydrin, phytase, protease catalase, lipase, peroxidase, amylase, transglutaminase, oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, ethanol, lactic acid, butanol, One or more enzymes involved in the pathway for the production of low molecular weight adipic acid or succinic acid, or an antibody against any such protein, one or more may include a nucleic acid sequence encoding the protein of interest.

[0114] Cells (e.g., yeast cells (e.g., methanol-assimilating yeast cells (e.g., Pichia))) are transformed with both DNA molecules, and the transformants can be selected by the presence of a selectable marker on the circular plasmid. The transformants can then be screened, for example, using PCR, for integration of the linear DNA molecule into the genome. Once a transformant containing a linear DNA molecule without the marker has been identified, the cells can be grown in the absence of selection for the circular plasmid. In the absence of selection, the plasmid carrying the marker is not stably maintained, and the plasmid is often lost very rapidly after selection is relaxed. The resulting strain carries the integrated linear DNA in the absence of the heterologous sequence for selection. Thus, this method can be used to construct strains (e.g., Pichia strains) lacking a selectable marker (e.g., a heterologous selectable marker) with little to no effect on the yield of the recombinant product (e.g., protein). )

[0115] According to the present disclosure, conventional molecular biology methods, microbiology methods, biochemistry methods, and recombinant DNA techniques within the scope of the art can be employed. Such techniques are well described in the literature. In the following examples, the materials and methods of the present disclosure are further illustrated However, this does not limit the scope of the methods and compositions recited in the claims. It is not so.

[0116] Exemplary embodiments Embodiment 1. A nucleic acid construct comprising a first alcohol oxidase promoter element wherein the first alcohol oxidase promoter element corresponds to SEQ ID NO: 28 and contains a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 668 to 734. A nucleic acid construct. Embodiment 2. The nucleic acid construct according to Embodiment 1, wherein the first alcohol oxidase promoter element contains a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 673 to 729 with respect to SEQ ID NO: 28. wherein the first alcohol oxidase promoter element contains a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 673 to 729 with respect to SEQ ID NO: 28. A nucleic acid construct according to Embodiment 1. Embodiment 3. The nucleic acid construct according to Embodiment 1, wherein the first alcohol oxidase promoter element contains a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 678 to 724 with respect to SEQ ID NO: 28. wherein the first alcohol oxidase promoter element contains a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 678 to 724 with respect to SEQ ID NO: 28. A nucleic acid construct according to Embodiment 1. Embodiment 4. The nucleic acid construct according to Embodiment 1, wherein the first alcohol oxidase promoter element contains a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 683 to 719 with respect to SEQ ID NO: 28. wherein the first alcohol oxidase promoter element contains a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 683 to 719 with respect to SEQ ID NO: 28. A nucleic acid construct according to Embodiment 1. Embodiment 5. The nucleic acid construct according to Embodiment 1, wherein the first alcohol oxidase promoter element contains a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 688 to 714 with respect to SEQ ID NO: 28. wherein the first alcohol oxidase promoter element contains a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 688 to 714 with respect to SEQ ID NO: 28. A nucleic acid construct according to Embodiment 1. Embodiment 6. The first alcohol oxidase promoter element corresponds to SEQ ID NO: 28 For the nucleotide positions corresponding to any of nucleotide positions 668 to 734 The nucleic acid construct according to Embodiment 1, which contains two or more mutations at the nucleotide positions corresponding to any of nucleotide positions 668 to 734. Embodiment 7. The first alcohol oxidase promoter element is SEQ ID NO: 28 For the nucleotide positions corresponding to any of nucleotide positions 668 to 734 The nucleic acid construct according to Embodiment 1, which contains three or more mutations at the nucleotide positions corresponding to any of nucleotide positions 668 to 734. Embodiment 8. The first alcohol oxidase promoter element is SEQ ID NO: 28 For the nucleotide positions corresponding to any of nucleotide positions 668 to 734 The nucleic acid construct according to Embodiment 1, which contains four or more mutations at the nucleotide positions corresponding to any of nucleotide positions 668 to 734. Embodiment 9. The first alcohol oxidase promoter element is SEQ ID NO: 28 For the nucleotide positions corresponding to any of nucleotide positions 668 to 734 The nucleic acid construct according to Embodiment 1, which contains five or more mutations at the nucleotide positions corresponding to any of nucleotide positions 668 to 734. Embodiment 10. The first alcohol oxidase promoter has the sequence of SEQ ID NO: 29 The nucleic acid construct according to Embodiment 1. Embodiment 11. A nucleic acid construct containing the first alcohol oxidase promoter element wherein the first alcohol oxidase promoter element contains one or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to T146, C154, T303, T426, A433, A435, T530, C 572, T596, T617, T688, A696, T702, A709, A712, T 714, A790, A841, and T862 with respect to SEQ ID NO: 28. A nucleic acid construct. Embodiment 12. The first alcohol oxidase promoter element is SEQ ID NO: 2 ​ For 8, T146, C154, T303, T426, A433, A435, T530 , C572, T596, T617, T688, A696, T702, A709, A712 , consisting of nucleotide positions corresponding to T714, A790, A841, and T862 An embodiment comprising two or more mutations at nucleotide positions selected from the group The nucleic acid construct according to embodiment 11. Embodiment 13. The first alcohol oxidase promoter element has SEQ ID NO: 2 For 8, T146, C154, T303, T426, A433, A435, T530 , C572, T596, T617, T688, A696, T702, A709, A712 , consisting of nucleotide positions corresponding to T714, A790, A841, and T862 An embodiment comprising three or more mutations at nucleotide positions selected from the group The nucleic acid construct according to embodiment 11. Embodiment 14. The first alcohol oxidase promoter element has SEQ ID NO: 2 For 8, T146, C154, T303, T426, A433, A435, T530 , C572, T596, T617, T688, A696, T702, A709, A712 , consisting of nucleotide positions corresponding to T714, A790, A841, and T862 An embodiment comprising four or more mutations at nucleotide positions selected from the group The nucleic acid construct according to embodiment 11. Embodiment 15. The first alcohol oxidase promoter element has SEQ ID NO: 2 For 8, T146, C154, T303, T426, A433, A435, T530 , C572, T596, T617, T688, A696, T702, A709, A712 , nucleotides positions corresponding to T714, A790, A841, and T862, An embodiment comprising 5 or more mutations at nucleotide positions selected from the group The nucleic acid construct according to claim 11. Embodiment 16. The first alcohol oxidase promoter element has SEQ ID NO: 2 8, one or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to T688, A696, T702, A712, and T714 The nucleic acid construct according to any one of embodiments 11 to 15, comprising The nucleic acid construct according to any one of embodiments 11 to 15, comprising Embodiment 17. The first alcohol oxidase promoter element has SEQ ID NO: 2 8, one or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to T688, A696, T702, A712, and T714 The nucleic acid construct according to any one of embodiments 11 to 15, comprising 2 or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to T688, A696, T702, A712, and T714 The nucleic acid construct according to any one of embodiments 11 to 15, comprising Embodiment 18. The first alcohol oxidase promoter element has SEQ ID NO: 2 8, one or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to T688, A696, T702, A712, and T714 The nucleic acid construct according to any one of embodiments 11 to 15, comprising 3 or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to T688, A696, T702, A712, and T714 The nucleic acid construct according to any one of embodiments 11 to 15, comprising Embodiment 19. The first alcohol oxidase promoter element has SEQ ID NO: 2 8, one or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to T688, A696, T702, A712, and T714 The nucleic acid construct according to any one of embodiments 11 to 15, comprising 4 or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to T688, A696, T702, A712, and T714 The nucleic acid construct according to any one of embodiments 11 to 15, comprising Embodiment 20. The first alcohol oxidase promoter element has SEQ ID NO: 2 For 8, the nucleic acid constructs according to any one of Embodiments 11 to 15, which contain mutations at nucleotide positions corresponding to T688, A696, T702, A712, and T714. Embodiment 21. A nucleic acid construct containing a first alcohol oxidase promoter element, wherein the first alcohol oxidase promoter element has one or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to 146, 154, 303, 426, 433, 435, 530, 572, 596, 617, 688, 696, 702, 709, 712, 714, 790, 841, and 8 62 with respect to SEQ ID NO: 28. Embodiment 22. The nucleic acid construct according to Embodiment 21, wherein the first alcohol oxidase promoter element has two or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to 146, 154, 303, 426, 433, 435, 530, 572, 596, 617, 688, 696, 702, 709, 712, 714, 790, 841, and 8 62 with respect to SEQ ID NO: 28. Embodiment 23. The nucleic acid construct according to Embodiment 21, wherein the first alcohol oxidase promoter element has three or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to 146, 154, 303, 426, 433, 435, 530, 572, 596, ​​​​​​​​​​​​Four or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to 146, 154, 303, 426, 433, 435, 530, 572, 596, 617, 688, 696, 702, 709, 712, 714, 790, 841, and 862 with respect to 8, of the nucleic acid construct according to embodiment 21. Embodiment 25. The nucleic acid construct according to embodiment 21, wherein the first alcohol oxidase promoter element has five or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to 146, 154, 303, 426, 433, 435, 530, 572, 596, 617, 688, 696, 702, 709, 712, 714, 790, 841, and 862 with respect to 8 of SEQ ID NO: 2. Embodiment 26. The nucleic acid construct according to any one of embodiments 21 to 25, wherein the first alcohol oxidase promoter element contains one or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to 688, 696, 702, 712, and 714 with respect to 8 of SEQ ID NO: 2. Embodiment 27. The nucleic acid construct according to any one of embodiments 21 to 25, wherein the first alcohol oxidase promoter element contains two or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to 688, 696, 702, 712, and 714 with respect to 8 of SEQ ID NO: 2. Embodiment 28. The nucleic acid construct according to any one of embodiments 21 to 25, wherein the first alcohol oxidase promoter element has nucleotide positions corresponding to 688, 696, 702, 712, and 714 with respect to 8 of SEQ ID NO: 2 comprising three or more mutations at nucleotide positions selected from the group consisting of a nucleic acid construct according to any one of embodiments 21 to 25. Embodiment 29. The first alcohol oxidase promoter element has SEQ ID NO: 2 8, and contains four or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to 688, 696, 702, 712, and 714 a nucleic acid construct according to any one of embodiments 21 to 25. Embodiment 30. The first alcohol oxidase promoter element has SEQ ID NO: 2 8, and contains a mutation at the nucleotide position corresponding to 688, 696, 702, 712, and 714, a nucleic acid construct according to any one of embodiments 21 to 25. 8, and contains a mutation at the nucleotide position corresponding to 688, 696, 702, 712, and 714, a nucleic acid construct according to any one of embodiments 21 to 25. a nucleic acid construct according to any one of embodiments 21 to 25. Embodiment 31. A nucleic acid construct comprising a first alcohol oxidase promoter element wherein the first alcohol oxidase promoter element has SEQ ID NO: 28 and contains one or more mutations selected from the group consisting of mutations corresponding to T146C, C154T, T303C, T426A, A433T, A435G, T530A, C572T, T596C, T617C, T688C, A696T, T702 C, A709G, A712G, T714G, A790G, A841T, and T862A a nucleic acid construct. Embodiment 32. The first alcohol oxidase promoter element has SEQ ID NO: 2 8, and contains T146C, C154T, T303C, T426A, A433T, A435 G, T530A, C572T, T596C, T617C, T688C, A696T, T7 02C, A709G, A712G, T714G, A790G, A841T, and T86 2A comprising two or more mutations selected from the group consisting of mutations corresponding to 2A and the nucleic acid construct according to embodiment 31. Embodiment 33. The first alcohol oxidase promoter element is SEQ ID NO: 2 8, with respect to T146C, C154T, T303C, T426A, A433T, A435 G, T530A, C572T, T596C, T617C, T688C, A696T, T7 02C, A709G, A712G, T714G, A790G, A841T, and T86 comprising three or more mutations selected from the group consisting of mutations corresponding to 2A and the nucleic acid construct according to embodiment 31. Embodiment 34. The first alcohol oxidase promoter element is SEQ ID NO: 2 8, with respect to T146C, C154T, T303C, T426A, A433T, A435 G, T530A, C572T, T596C, T617C, T688C, A696T, T7 02C, A709G, A712G, T714G, A790G, A841T, and T86 comprising four or more mutations selected from the group consisting of mutations corresponding to 2A and the nucleic acid construct according to embodiment 31. Embodiment 35. The first alcohol oxidase promoter element is SEQ ID NO: 2 8, with respect to T146C, C154T, T303C, T426A, A433T, A435 G, T530A, C572T, T596C, T617C, T688C, A696T, T7 02C, A709G, A712G, T714G, A790G, A841T, and T86 comprising five or more mutations selected from the group consisting of mutations corresponding to 2A and the nucleic acid construct according to embodiment 31. Embodiment 36. The first alcohol oxidase promoter element is SEQ ID NO: 2 8, and contains one or more mutations selected from the group consisting of T688C, A696T, T702C, A712G, and T714G, any one of Embodiments 1 to 35 The nucleic acid construct described in one. Embodiment 37. The first alcohol oxidase promoter element is SEQ ID NO: 2 8, and contains two or more mutations selected from the group consisting of T688C, A696T, T702C, A712G, and T714G, any one of Embodiments 1 to 35 The nucleic acid construct described in one. Embodiment 38. The first alcohol oxidase promoter element is SEQ ID NO: 2 8, and contains three or more mutations selected from the group consisting of T688C, A696T, T702C, A712G, and T714G, any one of Embodiments 1 to 35 The nucleic acid construct described in one. Embodiment 39. The first alcohol oxidase promoter element is SEQ ID NO: 2 8, and contains four or more mutations selected from the group consisting of T688C, A696T, T702C, A712G, and T714G, any one of Embodiments 1 to 35 The nucleic acid construct described in one. Embodiment 40. The first alcohol oxidase promoter element is SEQ ID NO: 2 8, and contains the mutations T688C, A696T, T702C, A712G, and T71 4G, the nucleic acid construct described in any one of Embodiments 1 to 35. Embodiment 41. A nucleic acid construct containing the first alcohol oxidase promoter element wherein the first alcohol oxidase promoter element is SEQ ID NO: 28 8, and contains one or more mutations selected from the group consisting of T688C, A696T, T702C, A712G, and T714G, any one of Embodiments 1 to 35 The nucleic acid construct described in one. Embodiment 42. A nucleic acid construct containing the first alcohol oxidase promoter element wherein the first alcohol oxidase promoter element is SEQ ID NO: 28 In contrast, a nucleic acid construct comprising one or more mutations selected from the group consisting of mutations corresponding to 146C, 154T, 303C, 426A, 433T, 435G, 530A, 5 72T, 596C, 617C, 688C, 696T, 702C, 709G, 712G, 7 14G, 790G, 841T, and 862A. Nucleic acid construct. Embodiment 42. The first alcohol oxidase promoter element is SEQ ID NO: 2 8, and comprises two or more mutations selected from the group consisting of mutations corresponding to 146C, 154T, 303C, 426A, 433T, 435G, 530A , 572T, 596C, 617C, 688C, 696T, 702C, 709G, 712G , 714G, 790G, 841T, and 862A, the nucleic acid construct according to Embodiment 41. Embodiment 43. The first alcohol oxidase promoter element is SEQ ID NO: 2 8, and comprises two or more mutations selected from the group consisting of mutations corresponding to 146C, 154T, 303C, 426A, 433T, 435G, 530A , 572T, 596C, 617C, 688C, 696T, 702C, 709G, 712G , 714G, 790G, 841T, and 862A, the nucleic acid construct according to Embodiment 41. Embodiment 44. The first alcohol oxidase promoter element is SEQ ID NO: 2 8, and comprises two or more mutations selected from the group consisting of mutations corresponding to 146C, 154T, 303C, 426A, 433T, 435G, 530A , 572T, 596C, 617C, 688C, 696T, 702C, 709G, 712G , 714G, 790G, 841T, and 862A, the nucleic acid construct according to Embodiment 41. Embodiment 45. The first alcohol oxidase promoter element has SEQ ID NO: 2 8, with 146C, 154T, 303C, 426A, 433T, 435G, 530A , 572T, 596C, 617C, 688C, 696T, 702C, 709G, 712G , 714G, 790G, 841T, and 862A, and comprises five or more mutations selected from the group consisting of the nucleic acid construct according to Embodiment 41. Embodiment 46. The first alcohol oxidase promoter element has SEQ ID NO: 2 8, and comprises one or more mutations selected from the group consisting of 688C, 696T, 702C, 712G, and 714G the nucleic acid construct according to any one of Embodiments 1 to 45. Embodiment 47. The first alcohol oxidase promoter element has SEQ ID NO: 2 8, and comprises two or more mutations selected from the group consisting of 688C, 696T, 702C, 712G, and 714G the nucleic acid construct according to any one of Embodiments 1 to 45. Embodiment 48. The first alcohol oxidase promoter element has SEQ ID NO: 2 8, and comprises three or more mutations selected from the group consisting of 688C, 696T, 702C, 712G, and 714G the nucleic acid construct according to any one of Embodiments 1 to 45. Embodiment 49. The first alcohol oxidase promoter element has SEQ ID NO: 2 8, and comprises four or more mutations selected from the group consisting of 688C, 696T, 702C, 712G, and 714G the nucleic acid construct according to any one of Embodiments 1 to 45. Embodiment 50. The first alcohol oxidase promoter element has mutations 688C, 696T, 702C, 712G, and 714G relative to SEQ ID NO: 2 and is the nucleic acid construct according to any one of Embodiments 1 to 45. Embodiment 51. The first alcohol oxidase promoter element is an alcohol oxidase promoter element derived from a promoter selected from the group consisting of AOX1, AOX2, AOD1, MOX, MOD1, and MOD2, and is the nucleic acid construct according to any one of Embodiments 1 to 50. Embodiment 52. The first alcohol oxidase promoter element is an alcohol oxidase 1 (AOX1) promoter element, and is the nucleic acid construct according to any one of Embodiments 1 to 51. Embodiment 53. The first alcohol oxidase promoter element has at least 90% sequence identity to SEQ ID NO: 2 and is the nucleic acid construct according to any one of Embodiments 1 to 52. Embodiment 54. The first alcohol oxidase promoter element has at least 95% sequence identity to SEQ ID NO: 2 and is the nucleic acid construct according to any one of Embodiments 1 to 52. Embodiment 55. Further comprising a nucleotide sequence, wherein the nucleotide sequence is operably linked to the first alcohol oxidase promoter element, and is the nucleic acid construct according to any one of Embodiments 1 to 5 4. Embodiment 56. The nucleotide sequence encodes a first protein, and is the nucleic acid construct according to Embodiment 55. Embodiment 57. The first protein is exogenous to the methanol-assimilating yeast cell, and is the nucleic acid construct according to Embodiment 56. ​ Embodiment 58. The nucleic acid construct according to Embodiment 56 or Embodiment 57, wherein the first protein is heterologous to the methanol-assimilating yeast cell. Embodiment 59. The nucleic acid construct according to any one of Embodiments 56 to 58, wherein the first protein is selected from the group consisting of an antibody or a fragment thereof, an enzyme, a regulatory protein, a peptide hormone, a blood coagulation protein, a cytokine, a cytokine inhibitor, and a heme-binding protein. Embodiment 60. The nucleic acid construct according to any one of Embodiments 56 to 59, wherein the first protein is a heme-binding protein. Embodiment 61. The nucleic acid construct according to Embodiment 60, wherein the heme-binding protein is selected from the group consisting of globin, cytochrome, cytochrome c oxidase, ligninase, catalase, and peroxidase. Embodiment 62. The nucleic acid construct according to Embodiment 60, wherein the heme-binding protein is selected from the group consisting of androglobin, chlorocruorin, cytoglobin, erythrocruorin, flavohemoglobin, globin E, globin X, globin Y, hemoglobin, histoglobin, leghemoglobin, myoglobin, neuroglobin, non-symbiotic hemoglobin, protoglobin, and truncated hemoglobin. Embodiment 63. The nucleic acid construct according to Embodiment 60, wherein the heme-binding protein is non-symbiotic hemoglobin. Embodiment 64. The nucleic acid construct according to Embodiment 60, wherein the heme-binding protein is leghemoglobin. Embodiment 65. The nucleic acid construct according to Embodiment 60, wherein the heme-binding protein comprises an amino acid sequence having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 1 to 27. ​​​​​​​​​​​​​​The nucleic acid construct according to item 60. Embodiment 66. The nucleic acid construct according to any one of Embodiments 1 to 65, wherein the first alcohol oxidase promoter element contains a recognition sequence for a transcription factor. Embodiment 67. A cell comprising a first nucleic acid construct, wherein the first nucleic acid construct is the nucleic acid construct according to any one of Embodiments 1 to 66. Embodiment 68. The cell according to Embodiment 67, which is a yeast cell. Embodiment 69. The cell according to Embodiment 68, wherein the yeast cell is a methanol-assimilating yeast cell. Embodiment 70. The cell according to Embodiment 69, wherein the methanol-assimilating yeast cell is a cell of the genus Pichia, Candida, Hansenula, or Torulopsis. Embodiment 71. The cell according to Embodiment 69 or 70, wherein the methanol-assimilating yeast cell is a Pich...

Claims

1. A nucleic acid construct comprising a first alcohol oxidase promoter element, wherein the first alcohol oxidase promoter element comprises four or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to T146, C154, T303, T426, A433, A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790, A841, and T862 relative to SEQ ID NO:

28.

2. The nucleic acid construct described in claim 1, wherein the first alcohol oxidase promoter element comprises four or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to T688, A696, T702, A712, and T714 relative to SEQ ID NO:

28.

3. A nucleic acid construct as described in claim 1 or 2, wherein the first alcohol oxidase promoter element contains mutations at nucleotide positions corresponding to T688, A696, T702, A712, and T714 relative to sequence number 28.

4. A nucleic acid construct comprising a first alcohol oxidase promoter element, wherein the first alcohol oxidase promoter element comprises two or more mutations selected from the group consisting of mutations corresponding to T146C, C154T, T303C, T426A, A433T, A435G, T530A, C572T, T596C, T617C, T688C, A696T, T702C, A709G, A712G, T714G, A790G, A841T, and T862A relative to SEQ ID NO:

28.

5. The nucleic acid construct described in claim 4, wherein the first alcohol oxidase promoter element comprises two or more mutations selected from the group consisting of mutations corresponding to T688C, A696T, T702C, A712G, and T714G relative to SEQ ID NO:

28.

6. A nucleic acid construct as described in claim 4 or 5, wherein the first alcohol oxidase promoter element includes mutations corresponding to T688C, A696T, T702C, A712G, and T714G relative to sequence number 28.

7. A nucleic acid construct described in any one of claims 1 to 6, wherein the first alcohol oxidase promoter element has at least 90% sequence identity to SEQ ID NO:

28.

8. A nucleic acid construct described in any one of claims 1 to 7, further comprising a nucleotide sequence, wherein the nucleotide sequence is operably linked to the first alcohol oxidase promoter element.

9. The nucleic acid construct described in claim 8, wherein the nucleotide sequence encodes a first protein.

10. The nucleic acid construct described in claim 9, wherein the first protein is selected from the group consisting of an antibody or fragment thereof, an enzyme, a regulatory protein, a peptide hormone, a blood coagulation protein, a cytokine, a cytokine inhibitor, and a heme-binding protein.

11. A nucleic acid construct described in claim 9 or 10, wherein the first protein is a heme-binding protein.

12. The nucleic acid construct described in claim 11, wherein the heme-binding protein is leghemoglobin.

13. The nucleic acid construct described in claim 11, wherein the heme-binding protein comprises an amino acid sequence having at least 90% sequence identity to any of the amino acid sequences of SEQ ID NOs: 1 to 27.

14. The nucleic acid construct described in claim 11, wherein the heme-binding protein comprises an amino acid sequence identical to any of the amino acid sequences of SEQ ID NOs: 1 to 27.

15. A nucleic acid construct described in any one of claims 1 to 14, wherein the nucleic acid construct is capable of expressing a coding sequence operably linked to the first alcohol oxidase promoter element.

16. A nucleic acid construct comprising a first alcohol oxidase promoter element and a second alcohol oxidase promoter element, wherein the first alcohol oxidase promoter element and the second alcohol oxidase promoter element each comprise one or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to T146, C154, T303, T426, A433, A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790, A841, and T862 relative to SEQ ID NO:28, and the nucleic acid construct further comprises a first nucleotide sequence operably linked to the first alcohol oxidase promoter element and a second nucleotide sequence operably linked to the second alcohol oxidase promoter element.

17. The nucleic acid construct of claim 16, wherein the first alcohol oxidase promoter element and the second alcohol oxidase promoter element each comprise one or more mutations at nucleotide positions selected from the group consisting of nucleotide positions corresponding to T688, A696, T702, A712, and T714 relative to SEQ ID NO:

28.

18. A nucleic acid construct described in claim 16 or 17, wherein the first alcohol oxidase promoter element and the second alcohol oxidase promoter element each contain mutations at nucleotide positions corresponding to T688, A696, T702, A712, and T714 relative to SEQ ID NO:

28.

19. A nucleic acid construct described in any one of claims 16 to 18, wherein the first nucleotide sequence encodes a transcriptional activator and the second nucleotide sequence encodes an enzyme, a regulatory protein, or a heme-binding protein.

20. A cell comprising a first nucleic acid construct, wherein the first nucleic acid construct is a nucleic acid construct described in any one of claims 1 to 19.

21. The cell described in claim 20, further comprising a second nucleic acid construct comprising a second nucleotide sequence, wherein the second nucleic acid construct is operably linked to the first alcohol oxidase promoter element or a second promoter element.

22. A method for producing a protein in a cell, comprising: Expressing the nucleic acid construct of any one of claims 1 to 19, wherein the first alcohol oxidase promoter element is operably linked to a nucleotide sequence encoding the protein. A method comprising:

23. The method of claim 22, carried out in the absence of added methanol.

24. The method of claim 22, carried out in the presence of added methanol.

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