Expression constructs and methods for genetically engineering methylotrophic yeasts

Abstract

The present application relates to expression constructs and methods for genetically engineering methylotrophic yeasts. The present invention provides methods and materials for genetically engineering methylotrophic yeasts.

Description

[0001] This application is a divisional application of the invention patent application filed on May 11, 2016, with application number "201680029580.4" and titled "Expression construct and method for genetically engineered methyltrophic yeast". Technical Field

[0002] This invention generally relates to DNA constructs and methods for genetically engineering methyltrophic yeast using such DNA constructs. Background Technology

[0003] Methyltrophic yeasts such as Pichia pastoris ( Pichia pastoris This is typically used to express recombinant proteins. This invention provides constructs that can be used to efficiently express one or more polypeptides in methyltrophic yeast. Summary of the Invention

[0004] This invention describes Pichia pastoris ( P. pastoris The strain is used to overexpress the transcription activator Mxr1 starting from the AOX1 promoter to increase the expression levels of transgenes also expressed starting from the AOX1 promoter, thereby significantly increasing the recombinant yield of one or more proteins. Furthermore, the expression of Mxr1 starting from the AOX1 promoter creates a positive feedback loop that allows other transgenes to be expressed starting from the AOX1 promoter in the absence of the normally specific inducer methanol when the repressor carbon source is depleted. Mxr1 expression leads to a significant increase in protein yield.

[0005] On one hand, the present invention provides a methyltrophic yeast cell comprising a recombinant nucleic acid molecule. The recombinant nucleic acid molecule typically comprises a foreign nucleic acid encoding a transcriptional activator operably linked to at least one methanol-inducible promoter element. A representative methyltrophic yeast may be *Candida* (…). Candida ), Hansenula genus ( Hansenula ), Pichia pastoris ( Pichia ) or genus *Gnaphalium* ( Toruplosis A representative methyltrophic yeast is Pichia pastoris.

[0006] In some embodiments, the recombinant nucleic acid molecule is stably integrated into the genome of a methyltrophic yeast cell. In some embodiments, the recombinant nucleic acid molecule is expressed extrachromosomally by a replicative plasmid.

[0007] In some embodiments, the exogenous nucleic acid encoding the transcription activator comprises the Mxr1 sequence from *Pichia pastoris* and the sequence from *Hansenula polymorpha*. Hansenula polymorpha The Adr1 sequence from Candida botrytis cinerea ( Candida boidiniiThe Trm1 sequence of *Candida botrytis* and the Trm2 sequence of *Candida botrytis* are shown. Representative nucleic acids encoding transcription activators are shown in DQ395124. Representative transcription activators have amino acid sequences as shown in ABD57365.

[0008] In some embodiments, at least one methanol-inducible promoter element is an alcohol oxidase 1 (AOX1) promoter element from *Pichia pastoris*, an AOD1 promoter element from *Candida botrytis*, a MOX promoter element from *Hansenula polymorpha*, or a promoter element from *Pichia pastoris*. Pichia methanolica The MOD1 promoter element, the DHAS promoter element from Pichia pastoris, the FLD1 promoter element from Pichia pastoris, or the PEX8 promoter element from Pichia pastoris are all from Pichia pastoris.

[0009] In some embodiments, the methyltrophic yeast cell further includes a nucleic acid molecule comprising at least one heterologous nucleic acid encoding a polypeptide operatively linked to at least one methanol-inducible promoter element. In some embodiments, the at least one heterologous nucleic acid encodes one or more polypeptides involved in the biosynthesis of iron cofactors, such as heme (e.g., ALA synthase, ALA dehydratase, bile pigmentogen deaminase, UPG III synthase, UPG III decarboxylase, CPG oxidase, PPG oxidase, and / or ferrous chelate). In some embodiments, the one or more polypeptides involved in iron cofactor biosynthesis are linked to at least one methanol-inducible promoter element.

[0010] On the other hand, the present invention provides a method for expressing heterologous peptides in cells. This method generally includes: providing methyltrophic yeast cells as described herein; introducing a recombinant nucleic acid molecule into the methyltrophic yeast cells, said recombinant nucleic acid molecule comprising at least one heterologous nucleic acid encoding a peptide operatively linked to at least one promoter element of Pichia pastoris alcohol oxidase 1 (AOX1); and culturing said cells under conditions suitable for expressing the recombinant nucleic acid molecule, thereby expressing the heterologous peptide.

[0011] In some embodiments, cell culture conditions include the addition of iron or a pharmaceutically or metabolically acceptable salt thereof. In some embodiments, the introduction step includes techniques such as transduction, electroporation, gene gun, or chemical transformation. In some embodiments, the culture step includes culturing cells in the presence of methanol.

[0012] On the other hand, the present invention provides recombinant organisms comprising transcription activators operably linked to their activated promoters. In some embodiments, the present invention provides recombinant organisms expressing polypeptides operably linked to said promoters. Furthermore, the present invention provides methods for expressing polypeptides from inducible promoters without the addition of inducers, as described herein.

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the relevant methods and compositions pertain. While similar or equivalent methods and materials may be used in practice or testing of the relevant methods and compositions, suitable methods and materials are described below. Furthermore, materials, methods, and examples are illustrative only and not limiting. The entire contents of all publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference. Attached Figure Description

[0014] Figure 1 This is a schematic diagram illustrating the steps involved in the heme biosynthesis pathway.

[0015] Figure 2 This is a schematic diagram of the plasmid used to construct the production strain MXY0183.

[0016] Figure 3 This is a schematic diagram showing the production of production strains MXY0183 and MXY0207 from the parent strain Bg11.

[0017] Figure 4 This is a schematic diagram showing plasmids pGAB and pMx354.

[0018] Figure 5 This is a schematic diagram showing the production of antibiotics from parent strain Bg11 by antibiotics selected from production strains MXY0291 and MXY0338.

[0019] Figure 6 This is a schematic diagram illustrating the introduction of a linear DNA fragment containing Mxr1 and LegH variant 3 through co-transformation to generate strain MXY0291.

[0020] Figure 7 This is a schematic diagram showing a linear construct expressing LegH under the control of the non-pAOX1 constitutive promoter in native Pichia pastoris.

[0021] Figure 8 These are photographs showing phenotypic changes associated with strain MXY0183. The images show shake flasks at the start of induction (0 hours) and 72 hours after induction. 1, MXY0051; 2, MXY0118; 3, MXY0183.

[0022] Figure 9 The figures show LegH production from modified Pichia pastoris strains. Figure A is an SDS gel showing lysates of Pichia pastoris strains grown in shake flasks: 51, MXY0051; 118, MXY0118; 183, MXY0183. Figure B is a table comparing LegH production from strains MXY0118, MXY0183, and MXY0207.

[0023] Figure 10 Experimental data for strain MXY0206 are shown. Figure A is a photograph of shake-flask cultures of strains MXY0183 (left) and MXY0206 (right) after 48 hours of growth with inhibited carbon source expression. Figure B is a photograph of cell clumps of shake-flask cultures of strains MXY0183 (left) and MXY0206 (right) after 48 hours of growth in BMY medium. Figure C is a graph showing the relative yield of heme-loaded LegH (in the absence of any inducer).

[0024] Figure 11 This is a summary table showing the relative yields of the strains described herein grown in a 2L fermenter in the presence of methanol and glycerol or methanol and glucose. Detailed Implementation

[0025] This invention provides nucleic acid constructs that allow genetically engineered cells to enhance recombinant expression of peptides. In some embodiments, nucleic acid constructs are provided that allow genetically engineered cells to enhance recombinant expression of peptides from an inducible promoter in the absence of an inducible molecule. Not bound by any particular mechanism, the methods described in this aspect generate a positive feedback loop in which low-level natural expression of a transcription activator induces a promoter operably linked to the transcription activator. This results in increased expression of the transcription activator as well as one or more target peptides, wherein the target peptides are operably linked to the same inducible promoter.

[0026] This article provides nucleic acid constructs that allow for the genetic engineering of methyltrophic yeast cells. Although this article uses Pichia pastoris (i.e., Pichia pastoris). (P. pastoris) The method is illustrated by example, but other species of the genus Pichia pastoris or any species from the genera Candida, Hansenula, Pichia pastoris and Globosa can be used.

[0027] Genetically engineered methyltrophic yeast cells typically involve introducing recombinant nucleic acid molecules into the cells. As described herein, recombinant nucleic acid molecules typically include exogenous nucleic acids encoding transcription activators operatively linked to at least one inducible promoter element.

[0028] The recombinant nucleic acid molecules used in the methods described herein are typically DNA, but RNA molecules may be used in appropriate contexts. As used herein, “exogenous” means any nucleic acid sequence introduced into the cell genome from an external source, which may be the same or different organisms or synthetically produced nucleic acids. For example, an exogenous nucleic acid may be a nucleic acid from a microorganism (e.g., a genus or species of methyltrophic yeast) that is introduced into a different genus or species of methyltrophic yeast. However, an exogenous nucleic acid may also be a nucleic acid from methyltrophic yeast that, despite the existence of the corresponding native nucleic acid sequence, is recombinantly introduced into methyltrophic yeast as an additional copy. For example, *Pichia pastoris* contains an endogenous nucleic acid encoding the Mxr1 transcription activator; additional *Pichia pastoris* Mxr1 nucleic acid (e.g., recombinantly introduced into *Pichia pastoris* or modified endogenous *Pichia pastoris* Mxr1 nucleic acid) is considered exogenous.

[0029] Transcription activators and the nucleic acids encoding them (e.g., exogenous nucleic acids encoding transcription activators) are known in the art. For example, the transcription activator from Pichia pastoris is the Mxr1 sequence, but suitable transcription activators can also be found in Hansenula polymorpha (Adr1 sequence; see, for example, the nucleic acid sequences of GenBank accession numbers AEOI02000005, bases 858873 to 862352 and the amino acid sequence of GenBank accession number ESX01253) and Candida botrytis cinerea (Trm1 sequence; see, for example, the nucleic acid sequence of GenBank accession number AB365355 and the amino acid sequence of GenBank accession number BAF99700; Trm2 sequence; see, for example, the nucleic acid sequence of GenBank accession number AB548760 and the amino acid sequence of GenBank accession number BAJ07608). Representative Pichia pastoris Mxr1 nucleic acid sequences can be found, for example, in GenBank accession number DQ395124, while representative Pichia pastoris Mxr1 polypeptide sequences can be found, for example, in GenBank accession number ABD57365.

[0030] Transcriptional activators such as Mxr1 can be expressed normally at low levels. Therefore, it is desirable to place exogenous nucleic acids (i.e., transcriptional activators) under the control of inducible promoters. As used herein, "operably linked" means that a promoter or other expression element is positioned relative to the nucleic acid coding sequence in such a way that it directs or regulates nucleic acid expression (e.g., within a frame).

[0031] Many inducible promoters can be used when genetically engineering methyltrophic yeasts. For example, methanol-inducible promoters or promoter elements thereof can be used. Methanol-inducible promoters are known in the art. For example, a commonly used methanol-inducible promoter from *Pichia pastoris* is the promoter of the alcohol oxidase 1 (AOX1) gene or a portion thereof, which strongly transcribes in response to methanol. However, other methanol-inducible promoters or promoter elements thereof can be used, including but not limited to the alcohol oxidase (AOD1) promoter from *Candida botrytis* (see, for example, GenBank accession number YSAAOD1A), the alcohol oxidase (MOX) promoter from *Hansenula polymorpha* (see, for example, GenBank accession number X02425), and the MOD1 or MOD2 promoter from *Pichia methanata* (see, for example, Raymond et al., 1998, *Yeast*, 14:11-23; and Nakagawa et al., 1999, *Yeast*). 15:1223-30), the DHAS promoter from *Pichia pastoris* (see, for example, GenBank accession number FJ752551) or its promoter elements, the formaldehyde dehydrogenase (FLD1) promoter from *Pichia pastoris* (see, for example, GenBank accession number AF066054), or the PEX8 promoter from *Pichia pastoris* (see, for example, Kranthi et al., 2010, *Yeast*, 27:705-11). In some embodiments, the transcription activator is the Mit1 sequence from *Pichia pastoris* (see, for example, GenBank accession number CAY70887). All of these promoters are known to be induced by methanol.

[0032] Those skilled in the art will understand that the recombinant nucleic acid molecules described herein can be stably integrated into the genome of methyltrophic yeast cells, or can be expressed extrachromosomally by replicative plasmids. Methods for achieving either are well known and routinely used in the art.

[0033] As demonstrated herein, the methanol-regulated transcription activators in Pichia pastoris can bind to the AOX1 promoter and synergize with Mxr1 to activate transcription of the AOX1 promoter. In some embodiments, the two methanol-regulated transcription activators (e.g., Mxr1 and Mit1) can be operatively linked to methanol-inducible promoter elements.

[0034] As described herein, strains comprising recombinant nucleic acid molecules can be used to regulate (e.g., overexpress) a second recombinant nucleic acid molecule in methyltrophic yeast cells. The second recombinant nucleic acid molecule may include, for example, one or more heterologous nucleic acids encoding one or more target polypeptides. Similar to exogenous nucleic acids encoding transcription activators, heterologous nucleic acids refer to any nucleic acid sequence that is not natural relative to the genome or the genome of an organism (e.g., a heterologous nucleic acid may be a nucleic acid from a microorganism (e.g., a genus or species of methyltrophic yeast) that has been introduced into different genera or species of methyltrophic yeast).

[0035] To illustrate simply, a heterologous nucleic acid encoding one or more target polypeptides can be a nucleic acid involved in the biosynthesis of heme cofactors. This article exemplifies nucleic acids encoding eight different enzymes involved in heme biosynthesis, sequenced and annotated from the *Pichia pastoris* genome. For example, heterologous nucleic acids encoding ALA synthase, ALA dehydratase, bile pigmentogen deaminase, UPG III synthase, UPG III decarboxylase, CPG oxidase, PPG oxidase, and ferrochelate can be expressed in the methyltrophic yeast strains described herein. To genetically engineer methyltrophic yeast to contain more than one heterologous nucleic acid (e.g., transgenic), combinations of methanol-inducible and constitutive promoters or their elements can be used to further enhance the expression of this nucleic acid.

[0036] Previous studies in *Saccharomyces cerevisiae* have identified ALA dehydratase and bile pigmentogen deaminase as rate-limiting enzymes in heme biosynthesis (see, for example, Hoffman et al., 2003, *Biochem. Biophys. Res. Commun.*, 310(4):1247-53). However, heterologous expression of heme enzymes from *Pichia pastoris* individual promoters failed to overcome the limitations associated with the expression of recombinant proteins containing heme cofactors (see Kraner et al., 2015, *Microb. Cell Fact.*, 13;14:4). As described in this paper, efficient expression of protein-containing recombinant heme in *Pichia pastoris* was achieved by co-expressing the entire heme biosynthesis pathway from a methanol-inducible promoter, although it is understandable that one or more genes involved in the heme biosynthesis pathway can be expressed from one or more constitutive promoters.

[0037] In addition to enzymes involved in the biosynthesis of iron cofactors, it should be understood that nucleic acids encoding members of the globulin family, including phytohemoglobin (PF00042 in the Pfam database), may exist. In the embodiments described herein, a nucleic acid encoding soybean leghemoglobin (LegH) is present. LegH is a protein that binds to an iron cofactor (heme), giving it a characteristic absorption at 415 nm and a distinct red color. This LegH protein (also known as LGB2) is naturally present in soybean root nodules (see, for example, UniprotKB accession number P02236), and the nucleic acid sequence used herein has been codon-optimized for expression in Pichia pastoris. See, for example, WO 2014 / 110539 and WO 2014 / 110532.

[0038] Optionally, the heteronucleotide encoding the target polypeptide can be, for example, but not limited to, dehydrated proteins, phytases, proteases, catalases, lipases, peroxidases, amylases, transglutaminases, oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases, or antibodies against such polypeptides. In other embodiments, the heteronucleotide can encode one or more enzymes involved in the synthesis pathways of small molecules such as ethanol, lactate, butanol, adipic acid, or succinate.

[0039] Similar to exogenous nucleic acids encoding transcription activators, heterologous nucleic acids encoding target polypeptides can be operatively linked to inducible promoter elements (e.g., methanol-inducible promoter elements), or they can be operatively linked to constitutive promoters or constitutive promoter elements. Inducible promoters and their elements have been discussed above. Constitutive promoters and constitutive promoter elements are known in the art. For example, a commonly used constitutive promoter from Pichia pastoris is the promoter or a portion thereof from the transcription elongation factor EF-1α gene (TEF1), which is strongly transcribed constitutively. However, other constitutive promoters or their promoter elements may be used, including but not limited to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter from Pichia pastoris (see, for example, GenBank accession number U62648.1), the promoter GCW14p (PAS_chr1-4_0586) from the potential glycosylphosphatidylinositol (GPI)-anchored protein (see, for example, GenBank accession number XM_002490678), or the promoter from the 3-phosphate glycerate kinase gene (PGK1) from Pichia pastoris (see, for example, GenBank accession number AY288296).

[0040] Similar to the recombinant nucleic acid molecules described in this article, the second recombinant nucleic acid molecule can be stably integrated into the genome of methyltrophic yeast cells, or it can be expressed extrachromosomally by a replicative plasmid.

[0041] Those skilled in the art should understand that combinations of inducible (e.g., methanol-inducible) and constitutive promoters (or their promoter elements) can be used to further increase the expression of any nucleic acid operatively linked thereto.

[0042] Those skilled in the art will understand that the heteronucleotide encoding the target polypeptide (operably linked to a promoter element) can be isolated from the recombinant nucleic acid molecule described herein, or can be adjacent to a foreign nucleic acid encoding a transcription activator operably linked to a promoter element contained within the recombinant nucleic acid molecule described herein. Those skilled in the art should also understand that if the second nucleic acid molecule is adjacent to the recombinant nucleic acid molecule described herein, a single promoter or its promoter element can be used to drive the transcription of two or more genes (e.g., a foreign nucleic acid encoding a transcription activator and one or more heteronucleotides encoding the target polypeptide).

[0043] Methods for introducing nucleic acids into methyltrophic yeast cells are known in the art, including but not limited to transduction, electroporation, biolistic particle delivery, and chemical transformation.

[0044] Furthermore, methods for culturing methyltrophic yeast cells are known in the art. See, for example, the Pichia pastoris protocol: Molecular biology methods (Pichia Protocols, Methods in Molecular Biology ), 389, ed. Cregg, 2007, 2nd edition, Humana Press, Inc. In some cases, it may be necessary to introduce or add methanol to the culture medium, although, as demonstrated herein, methanol is not required to obtain high levels of expression of one or more target peptides. In some cases (e.g., when expressing one or more nucleic acids encoding enzymes involved in iron cofactor biosynthesis), iron or pharmaceutically or metabolically acceptable (or GRAS) salts may be required.

[0045] Pichia pastoris strains are capable of growing on methanol as the sole carbon source. Methanol utilization is initiated by the conversion of methanol to formaldehyde through the action of alcohol oxidases. The methyltrophic yeast, Pichia pastoris, contains two alcohol oxidase genes, AOX1 and AOX2. Strains with reduced alcohol oxidase activity (“slow methanol utilization” or MutS strains) typically produce more recombinant proteins expressed by the AOX1 promoter than strains without reduced alcohol oxidase activity. Strains with mutations in both AOX genes and a complete lack of alcohol oxidase activity cannot metabolize methanol but can still express proteins from the AOX1 promoter upon methanol induction. These strains retain the ability to grow using other carbon sources but can still express heterologous proteins from the AOX1 promoter upon the addition of methanol. Because these strains do not metabolize methanol (“subtractive methanol utilization” or Mut- strains), less methanol is required to induce protein expression, and strains carrying these mutations avoid problems associated with methanol feeding in large-scale fermentation. See, for example, Chiruvolu et al., 1997, Enzyme Microb. Technol., 21:277-83. This paper demonstrates that LegH expression starting from the AOX1 promoter in Mut- strains significantly increases LegH production. Therefore, methyltrophic yeasts with mutations in both the AOX1 and AOX2 genes can be used in the methods described herein.

[0046] The target protein, or a complex containing one or more target proteins (e.g., heme-bound LegH, dehydrated proteins, phytases, proteases, catalases, lipases, peroxidases, amylases, transglutaminases, oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases, or antibodies), can be purified from yeast cells. Methods for purifying polypeptides are known in the art. As used herein, a “purified” polypeptide is a polypeptide that has been isolated or purified from the cellular component naturally associated with it. Typically, a polypeptide is considered “purified” when it is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, or 99%) dry weight, separated from the polypeptide and the naturally occurring molecules naturally associated with it. Synthetic polypeptides are “purified” because they are naturally separated from the polypeptide naturally associated with them.

[0047] As used herein, nucleic acids may include DNA and RNA, and include nucleic acids containing one or more nucleotide analogs or backbone modifications. Nucleic acids may be single-stranded or double-stranded, typically depending on their intended use. The present invention also provides nucleic acids and polypeptides that differ from a given sequence. Nucleic acids and polypeptides may have at least 50% sequence identity relative to a given nucleic acid or polypeptide sequence (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).

[0048] When calculating the percentage of sequence identity, two sequences are aligned, and the number of identical nucleotide or amino acid residues between the two sequences is determined. This number of identical matches is divided by the length of the aligned region (i.e., the number of aligned nucleotide or amino acid residues) and multiplied by 100 to obtain the percentage of sequence identity. It should be understood that the length of the aligned region can be a portion of one or two sequences, with a maximum length equal to the full length of the shortest sequence. It can also be understood that a single sequence can be aligned with more than one other sequence; therefore, each aligned region can have a different percentage of sequence identity.

[0049] The computer program ClustalW with default parameters can be used to align two or more sequences to determine the percentage of sequence identity, thus allowing nucleic acid or peptide sequences to be aligned across their entire length (global alignment). Chenna et al., 2003, Nucleic Acids Res., 31(13):3497-500. ClustalW calculates the best match between the query and one or more target sequences and aligns them to determine identity, similarity, and differences. Vacancies can be inserted into the query sequence, target sequence, or both to maximize sequence alignment. For rapid pairing and alignment of nucleic acid sequences, the default parameters can be used (i.e., word size: 2; window size: 4; scoring method: percentage; top diagonal number: 4; vacancy penalty: 5); for aligning multiple nucleic acid sequences, the following parameters can be used: vacancy open penalty: 10.0; vacancy expansion penalty: 5.0; and weight conversion: yes. To quickly align peptide sequences, the following parameters can be used: word size: 1; window size: 5; scoring method: percentage; top diagonal: 5; and vacancy penalty: 3. For multiple alignments of peptide sequences, the following parameters can be used: weight matrix: blosum; vacancy opening penalty: 10.0; vacancy expansion penalty: 0.05; hydrophilic gap: on; hydrophilic residues: glycine (Gly), proline (Pro), serine (Ser), asparagine (Asn), aspartic acid (Asp), glutamine (Gln), glutamic acid (Glu), arginine (Arg), and lysine (Lys); and specific residue vacancy penalty: on. For example, ClustalW can run on the Baylor College of Medicine Search Launcher website or on the European Bioinformatics Institute website on the World Wide Web.

[0050] Changes can be introduced into nucleic acid molecules, resulting in alterations to the amino acid sequence of the encoded polypeptide. These changes can be introduced into the nucleic acid coding sequence, for example, through mutagenesis (e.g., site-directed mutagenesis, PCR-mediated mutagenesis) or by chemically synthesizing nucleic acid molecules with such changes. These nucleic acid changes can lead to conserved and / or non-conserved amino acid substitutions of one or more amino acid residues. A “conserved amino acid substitution” is a substitution in which an amino acid residue is replaced by a different amino acid residue with a similar side chain (see, for example, Dayhoff et al. (1978, Atlas of Protein Sequence and Structure), 5(Supplement 3): 345-352), which provides a frequency table for amino acid substitutions), and a non-conserved substitution is a substitution in which an amino acid residue is replaced by an amino acid residue without a similar side chain. As described herein, nucleic acid and / or polypeptide sequences can be modified to improve one or more properties, including but not limited to enhanced expression (e.g., transcription and / or translation), more stringent regulation, dysregulation, reduced catabolite repression, modification specificity, secretion, thermal stability, solvent stability, oxidative stability, protease resistance, catalytic activity, and / or color.

[0051] As used herein, an "isolated" nucleic acid molecule is a nucleic acid molecule whose genome does not contain naturally occurring side-terminals of one or both ends of the nucleic acid (e.g., cDNA or genomic DNA fragments produced by PCR or restriction endonuclease digestion). Such isolated nucleic acid molecules are typically introduced into vectors (e.g., cloning or expression vectors) to facilitate manipulation or the production of fusion nucleic acid molecules, as discussed in more detail below. Additionally, isolated nucleic acid molecules may include engineered nucleic acid molecules such as recombinant or synthetic nucleic acid molecules.

[0052] Nucleic acids can be isolated using conventional techniques in the art. For example, any method can be used to isolate nucleic acids, including but not limited to recombinant nucleic acid techniques and / or polymerase chain reaction (PCR). Conventional PCR techniques are described, such as PCR Primer: A Laboratory Manual, edited by Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleic acid techniques include, for example, restriction enzyme digestion and ligation that can be used to isolate nucleic acids. Isolated nucleic acids can also be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides.

[0053] Peptides can be purified from natural sources (e.g., biological samples) using known methods such as DEAE ion exchange, gel filtration, and hydroxyapatite chromatography. Peptides can also be purified, for example, by expressing nucleic acids in an expression vector. Alternatively, purified peptides can be obtained through chemical synthesis. The purity of a peptide can be measured using any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC.

[0054] The present invention also provides constructs or vectors containing nucleic acids (e.g., nucleic acids encoding polypeptides). Constructs or vectors, including expression constructs or vectors, are commercially available or can be manufactured using recombinant DNA techniques conventional in the art. Nucleic acid-containing constructs or vectors may have expression elements operatively linked to such nucleic acids and may also include sequences encoding selectability markers (e.g., antibiotic resistance genes). Nucleic acid-containing constructs or vectors may encode chimeric or fusion polypeptides (i.e., polypeptides operatively linked to heterologous polypeptides, which may be located at the N-terminus or C-terminus of the polypeptide). Representative heterologous polypeptides are those that can be used to purify the encoded polypeptide (e.g., 6xHis tags, glutathione S-transferase (GST)).

[0055] Expression elements comprise nucleic acid sequences that direct and regulate the expression of nucleic acid coding sequences. One embodiment of an expression element is a promoter sequence. Expression elements may also include introns, enhancer sequences, response elements, or inducible elements that regulate nucleic acid expression. Expression elements may be of bacterial, yeast, insect, mammalian, or viral origin, and vectors may contain combinations of elements from different sources.

[0056] As described herein, the vector can be introduced into a host cell. As used herein, "host cell" refers to the specific cell into which the nucleic acid is introduced, and also includes the progeny of such cells carrying the vector. The host cell can be any prokaryotic or eukaryotic cell. For example, nucleic acids can be introduced into bacterial cells (e.g., *Escherichia coli*). E. coli Nucleic acid can be expressed in insect cells, yeast cells, or mammalian cells (e.g., Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art. Many methods for introducing nucleic acids into host cells, both in vivo and in vitro, are well known to those skilled in the art, including but not limited to electroporation, calcium phosphate precipitation, polyethylene glycol (PEG) conversion, heat shock, lipid transfection, microinjection, and virus-mediated nucleic acid transfer.

[0057] Nucleic acids can be detected using any number of amplification techniques (see, for example, PCR Primer: A Laboratory Manual, 1995, Dieffenbach & Dveksler, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York); and U.S. Patents 4,683,195, 4,683,202, 4,800,159, and 4,965,188) and a suitable pair of oligonucleotides (e.g., primers). Numerous modifications to the original PCR have been developed and can be used for nucleic acid detection.

[0058] Nucleic acids can also be detected using hybridization. Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Chapters 7.37–7.57, 9.47–9.57, 11.7–11.8, and 11.45–11.57) discuss hybridization between nucleic acids in detail. Sambrook et al. disclosed Southern blotting conditions suitable for oligonucleotide probes shorter than approximately 100 nucleotides (Sections 11.45–11.46). The Tm between a sequence shorter than 100 nucleotides and a second sequence can be calculated using the formula provided in Section 11.46. Sambrook et al. additionally disclosed Southern blotting conditions for oligonucleotide probes longer than approximately 100 nucleotides (see Sections 9.47–9.54). The Tm between a sequence longer than 100 nucleotides and a second sequence can be calculated using the formula provided by Sambrook et al. in Sections 9.50-9.51.

[0059] The conditions under which nucleic acid-containing membranes are pre-hybridized and hybridized, as well as the conditions for washing the membranes to remove excess and non-specifically bound probes, play a crucial role in the tightness of hybridization. This hybridization and washing can be performed under appropriate moderate or highly stringent conditions. For example, more stringent washing conditions can be achieved by decreasing the salt concentration in the washing buffer and / or increasing the washing temperature. To illustrate simply, highly stringent conditions typically involve washing the membrane at 65°C in 0.2X SSC.

[0060] Furthermore, the interpretation of hybridization levels may be affected by factors such as the specific activity of the labeled oligonucleotide probe, the number of probe-binding sites on the template nucleic acid to which the probe has hybridized, and the autoradiographic exposure or other detection media. Those skilled in the art will readily understand that while any number of hybridization and washing conditions can be used to examine the hybridization of the probe nucleic acid molecule with the immobilized target nucleic acid, it is more important to examine the hybridization of the probe with the target nucleic acid under the same hybridization, washing, and exposure conditions. Preferably, the target nucleic acids are on the same membrane.

[0061] A nucleic acid molecule is considered to have hybridized with one nucleic acid but not with another if it hybridizes with one nucleic acid at least 5 times more than with another (e.g., at least 6, 7, 8, 9, 10, 20, 50, or 100 times more). The amount of hybridization can be quantified on a membrane or directly from the autoradiography (e.g., Phosphor Imager) or densitometer (Molecular Dynamics, Sunnyvale, CA) used.

[0062] Antibodies can be used to detect peptides. Techniques for detecting peptides using antibodies include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. Antibodies can be polyclonal or monoclonal. Antibodies with specific binding affinity for peptides can be prepared using methods known in the art. The antibodies can be attached to a solid support, such as a microtiter plate, using methods known in the art. In the presence of the peptide, an antibody-peptide complex is formed.

[0063] Detection is typically accomplished using detectable labels (such as amplification products, hybridization complexes, or peptides). The term "label" is intended to include both direct and indirect labeling. Detectable labels include enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.

[0064] Methods for preparing strains lacking selectable sequences (i.e., lacking selectable markers) are described herein. These methods involve using a circular plasmid DNA vector and a linear DNA sequence; the circular plasmid DNA vector contains a selectable marker and an origin of DNA replication (also known as an autonomous replication sequence (ARS)), and the linear DNA sequence contains a sequence integrated into the Pichia pastoris genome via homologous recombination. The linear DNA molecule may additionally include nucleic acid sequences encoding one or more target proteins, such as, but not limited to, heme-binding LegH, dehydratin, phytase, protease, catalase, lipase, peroxidase, amylase, transglutaminase, oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, one or more enzymes involved in the pathways of small molecule (such as ethanol, lactic acid, butanol, adipic acid, or succinic acid) production, or antibodies against any such proteins.

[0065] Pichia pastoris cells can be transformed with both a DNA molecule selected by the presence of a selection marker on a circular plasmid and a transformant. Transformants can then be screened using, for example, PCR targeting the integration of the linear DNA molecule into the genome. Once a transformant with correctly integrated, unlabeled linear DNA molecule is identified, the cells can be grown in the absence of a selection circular plasmid. Because the labeled plasmid cannot be stably maintained in the absence of selection, it is typically lost very quickly after selection relaxation. The resulting strain carries the integrated linear DNA in the absence of a heterologous selection sequence. Therefore, this method can be used to construct Pichia pastoris strains lacking selection markers (e.g., heterologous selection markers) with minimal to no impact on recombinant protein yield.

[0066] According to the present invention, conventional molecular biology, microbiology, biochemistry, and recombinant DNA techniques within the scope of the art can be used. These techniques are well explained in the literature. The invention will be further described in the following examples, which do not limit the scope of the related methods and compositions described in the claims.

[0067] Example

[0068] Part A. Materials and Methods

[0069] Example 1 - Polymerase Chain Reaction

[0070] The target gene was amplified from genomic DNA or plasmid DNA templates using Phusion Hi-fidelity DNA polymerase (New England Biolabs). In short, 0.6 μM of forward and reverse primers were incubated with 10–50 ng of template DNA and 400 μM of nucleotide mixture, respectively, in the presence of 1–2 U of Phusion DNA polymerase. The reaction conditions were as follows:

[0071]

[0072] Example 2 - Plasmid Construction via Ligation

[0073] In the presence of T4 DNA ligase (New England Biolabs), incubate approximately 50-100 ng of restriction endonuclease-digested plasmid and more than 3 molar amounts of PCR-amplified insert fragment. Ligation is performed at 16°C for more than 2 hours. Transform 2 μl of the ligation reaction mixture into DH10B electrocompetent E. coli cells (…). E. coli )middle.

[0074] Example 3 - Transformation into E. coli ElectroMax DH10B T1 phage-resistant competent cells

[0075] Using a 1.7 kV micropulser (BioRad) and 1 mm-pitch electroporation cuvettes (BioRad, catalog number 165-2089), 1.5–2 μl of the ligation mixture was transformed into 20 μl of ElectroMax DH10B T1 phage-resistant competent cells (Invitrogen, catalog number 12033-015). After the pulse, 1 ml of SOC was added to the cells, and the cells were incubated at 37°C with shaking at 200 rpm for 1 hour. 10 μl of the recovered mixture was plated onto LB agar plates containing 100 μg / ml ampicillin. The plates were incubated overnight at 37°C.

[0076] Example 4 - Linearized plasmid DNA for transformation into Pichia pastoris P. pastoris )

[0077] Digest plasmid DNA in 1x CutSmart buffer with SfiI restriction endonuclease (New England Laboratory, catalog number R0123L) at 50°C for 1–4 hours, or digest with PmeI restriction endonuclease (New England Laboratory, catalog number R0560L) in 1x CutSmart buffer at 37°C for 1–4 hours. Purify the linearized plasmid from a 0.8% agarose gel using the Zymoclean Gel DNA Recovery Kit (Zymo Research, catalog number D4002). Elute the DNA in 20 μl of water.

[0078] Example 5 - Pichia pastoris (P. pastoris) P. pastoris ) Transformation - Preparation of competent cells

[0079] The selected *Pichia pastoris* strain was grown to mid-exponential growth (~2 OD) in 25 ml YPD medium. Cells were collected by centrifugation at 930 × g for 15 min. The cell pellet was resuspended in 2 ml of 80% YPD and 200 mM HEPES (pH 6.8). 75 μl of 1M DTT was added. The resuspended cell pellet was mixed at 100 rpm for 25 min at 30 °C. 40 ml of ice-cold sterile water was added to the suspension, and cells were collected by centrifugation at 1125 x g for 15 min and placed on ice. The cell pellet was resuspended in 40 ml of ice-cold water and collected as previously described for two additional washing steps. The cell pellet was then resuspended in 20 ml of ice-cold 1M sorbitol and collected by centrifugation as previously described. The final cell pellet was resuspended in 0.3 ml of ice-cold sterile sorbitol, aliquoted, and frozen at -80 °C.

[0080] Example 6 - Transformation into Pichia pastoris

[0081] Approximately 30–100 ng of linearized plasmid DNA was transformed into 30 μl of electrocompetent Pichia pastoris cells using a GenePulser (BioRad) set to 1.15 kV and a GenePulser electroporator (BioRad) with a 1 mm spacing. P. pastorisAdd 1 ml of YPD / 1M sorbitol and mix with the cells at a 1:1 ratio. Incubate the cells at 30°C with shaking at 100 rpm for 3 hours to recover. Spread 100 μl of the recovered mixture onto a YPD plate containing an appropriate antibiotic, and spread the remaining cells onto another YPD plate containing an appropriate antibiotic. Incubate the plates at 30°C for 48 hours. Streak the primary transformation plate onto a YPD plate with an appropriate antibiotic and incubate the plate at 30°C for 48 hours. Cover the single colonies onto antibiotic-containing YPD plates and perform colony PCR or gDNA preparation using a patch to confirm integration into the chromosome. Culture the strain in a shake flask for further analysis.

[0082] Example 7 - Growth culture of LegH in shake flask for production

[0083] The strain from fresh patches was inoculated onto BMGY growth medium (BMY supplemented with 0.75% glycerol) and grown overnight at 30°C with shaking at 200 rpm. The next day, LegH expression was induced with methanol by diluting the ON culture with BMMY medium (BMY + 1% methanol) supplemented with 0.1 mM ferric citrate (III). The culture grew to an OD600 of 0.5–0.7. Antifoaming agent was added to a final concentration of 0.01%. The culture was grown for a total of 72 hours; methanol was added every 24 hours by adding 1 / 10 shake flask volume of 10x BMMY medium (BMY + 10% methanol). After 72 hours of induction, cells were harvested by centrifugation.

[0084] Example 8 - Shake Flask Culture Medium

[0085] BMY medium was prepared by dissolving 10 g of yeast extract and 20 g of soybean peptone in 790 ml of water. The mixture was sterilized by autoclaving and cooled to room temperature. 100 ml of 1M potassium phosphate buffer (pH 6.0) and 100 ml of 10X amino acid-free yeast nitrogen source (13.4 g YNB powder per 100 ml; Sigma-Aldrich) were filtered and sterilized (PES with a pore size of 0.2 μm) and added to the medium. pH adjustment was not required.

[0086] BMY culture medium components

[0087]

[0088] Dissolve the following components in water and autoclave.

[0089] Low-permeability culture medium in shake flasks

[0090]

[0091] Example 9 - Fermentation medium and feed

[0092] Dissolve the components described below and adjust the volume with water. These components are FCC food grade or equivalent. Sterilize the culture medium by autoclaving, steaming, or an equivalent method.

[0093] Low-permeability culture medium containing 95 g / L glycerol for fermentation

[0094]

[0095] After sterilization, allow the culture medium to cool to room temperature and add the following substances:

[0096]

[0097] The trace metal PTM1 solution can be obtained from the powder mixture from Sunrise Science (catalog number 4052-AB-1L). Mix bag A and bag B in 950 mL of water and add 5 mL of sulfuric acid. Some precipitation is expected during mixing; filter the mixture sterile (using 0.2 μm pore size PES) and store at 4°C protected from light.

[0098] Vitamin solution formula

[0099]

[0100] Alternatively, trace metal PTM1 can be prepared as follows:

[0101]

[0102] The components were mixed together, filtered, sterilized, and stored at room temperature. A glycerol feed mixture was prepared by dissolving 17.5 g of AmberFerm 4000 in 320 mL of water with stirring. The water-Amberferm mixture was added to 850 g of glycerol and vigorously stirred until homogeneous. This feed mixture was then autoclaved.

[0103] Glycerin feed solution

[0104]

[0105] The methanol feed is prepared using 99-100% methanol supplemented with 12 mL / L PTM1 solution.

[0106] Example 10 - Laboratory-scale hyperaerobic transfer fermentation protocol

[0107] Seed Shake Bottle Program

[0108] In a sterile biosafety cabinet, mix the hypoosmolar medium and BMY at a hypoosmolar:BMY ratio of 9:1. Add 12.5 g / L glycerol to the medium. Use USP food-grade glycerol / glycerol (99.7% purity in 50% v / v (63% w / w) glycerol / water solution) and autoclave. Add Sigma 204 or equivalent antifoaming agent to the medium at a concentration of 0.25 mL / L. Recover the glycerol seed bottle, spray externally with 70% IPA or ethanol for about 5 minutes, and thaw at room temperature in the biosafety cabinet. Inoculate the glycerol seed bottle into isolated shake flasks; use 1 mL inoculation bottle for every 1 L of shake flask medium. Incubate the culture at 30°C with shaking for 24 hours (200 RPM, 1 second). A ratio of actual medium volume to nominal shake flask volume of 1:10 to 1:5 is generally used. Adding 250–500 mL of medium to a 2.8 L nominal volume flask is generally successful. After 24 hours of growth, measure the OD at 600 nm. If the OD is 15 or higher, use the culture to inoculate the fermenter. If the OD value is less than 15, culture the culture for another 1-2 hours and measure the OD again. If the OD does not reach 15 after 15 to 30 hours, the seed flask is considered a failure.

[0109] Fermentation scheme

[0110] The fermentation medium and seed culture are prepared as described herein. The initial volume should be approximately 40% of the maximum fermenter volume; for example, if the maximum working volume of the fermenter is 10 L, the initial volume should be 4 L. This is because the process will approach the maximum working volume at the end of fermentation. The fermenter is inoculated with shake flask medium at a 10% inoculum-to-fermenter ratio; for example, if there is 4 L of initial medium in the fermenter, the fermenter is inoculated with approximately 0.4 L of shake flask seed culture. The total volume in the fermenter at this point is called the T0 volume, for example, 4.4 L in this representative example. Process control includes the following: a temperature of 30°C; dissolved oxygen control via a stirring-aeration cascade to maintain a 20% saturation setpoint; and pH control via the addition of 28% NH4OH, with the setpoint depending on the stage of the process.

[0111] Phased (From vaccination to glycerol consumption, signaled by a DO spike):

[0112] Based on the responsiveness of PID control to dissolved oxygen, a strong dissolved oxygen spike or a rapid decrease in agitation and aeration rates, or a combination of both, can be observed when cells consume glycerol present in the culture medium. When this occurs, a feed-batch phase is initiated. The feed-batch phase lasts approximately 20 hours, but a maximum of 24 hours is considered acceptable. The pH setpoint is 5.0. The wet cell weight at the end of the feed-batch phase is approximately 220 g / L.

[0113] Replenishment in batches Start the glycerol feed at T0 volume to achieve a pure glycerol concentration of 12-14 g / L / hr. Maintain this combined regime until the wet cell weight reaches approximately 35 g / L, which will take approximately 7-10 hours. The pH setpoint is 5.0.

[0114] Productization stage Sampling should be taken before the start of the transition phase. The methanol feed should begin based on the T0 volume to achieve a pure methanol concentration of 1 g / L / hr, continuing until a methanol concentration of 1-2 g / L is reached in the fermentation broth. During the remainder of fermentation, the methanol feed rate should be adjusted to maintain a methanol concentration of 0.25-1 g / L in the broth. Based on the T0 volume, the glycerol complex should be reduced linearly over 2 hours from 12-14 g / L / hr pure glycerol to 8-9 g / L / hr pure glycerol. A gradual reduction in the feed rate every 20 minutes is also acceptable. The pH set point should be changed to 3.5, and fermentation should be allowed to adjust naturally to the new set point (i.e., without acid addition).

[0115] Production stage(Gradual reduction from the end of glycerol feed to the end of fermentation): The pH set point is 3.5. Maintain a methanol concentration of 0.25–1 g / L in the fermentation broth. Based on the T0 volume, maintain the glycerol feed rate at 8–9 g / L / hr of pure glycerol. Sampling is performed approximately every 12 hours. Samples are centrifuged at 4°C with 4000–7000 RCF, and the supernatant is gently poured off. This supernatant is stored in a separate test tube. Three samples of the pelleted feed and 5 mL of supernatant from each time point are frozen at -80°C. If 15–20% dissolved oxygen cannot be maintained during production, the glycerol feed rate can be reduced to 5 g / L / hr of pure glycerol, even at maximum aeration and stirring speeds, based on the T0 volume. Fermentation ends 60 hours after inoculation. On a 1000L scale, the harvesting process includes shutting off the feed and aeration, cooling the fermentation broth to 8°C, and concentrating the paste using a sharp object or disc centrifuge. Harvesting typically takes about 5-10 hours and does not result in a noticeable loss of product quality. For laboratory-scale samples, an additional 50 mL sample is collected at the end, in addition to the initial 3 × 5 mL sample. Wet cell weight >450 g / L, the rotating particles appear pink, unlike the whiter rotating particles from the pre-induced sample. After approximately 6-12 hours of induction, the broth begins to change color from white to a more pronounced pink.

[0116] Construction of production strains in Part B

[0117] Production strain MXY0183

[0118] Example 11 - Cloning each enzyme in the heme biosynthesis pathway into a pGAN or pGAZ integration vector

[0119] pGAN (with a nat select marker) and pGAZ (with a zeocin select marker) were purchased from Biogrammatics, Inc. (Carlsbad, CA). Each gene was placed under the control of the AOX1 promoter, and the FDH terminator was placed directly after the stop codon of each gene. Genes in the heme biosynthesis pathway were amplified by PCR from wild-type Pichia pastoris strains or by subcloning from previous constructs.

[0120] The heme biosynthesis pathway, including enzymes involved in heme biosynthesis, is described in [link to relevant documentation]. Figure 1 Intermediates produced during the biosynthesis of heme are shown in the box, and the enzymes catalyzing each step are shown on the right. For example, *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). S. cerevisiae The rate-limiting enzyme step shown is indicated by an underline.

[0121] The genes for ALA synthase, ALA dehydratase, UPGIII synthase, UPGIII decarboxylase, CPG oxidase, and PPG oxidase were amplified by PCR using primers containing the BsaI restriction endonuclease recognition site. The oligonucleotides were synthesized by ElimBiopharm.

[0122]

[0123] Genes were amplified from genomic DNA using a fusion high-fidelity DNA polymerase (Phusion High-Fidelity DNA Polymerase) (New England Laboratory, catalog number M0530L). PCR products were obtained and purified using DNA Clean & Concentrator-5 (catalog number D4004), and the DNA was eluted in 25 μl of water. Vector DNA, pGAZ and pGAN, and PCR products were digested in 50 μl reaction volumes with BsaI (New England Laboratory, catalog number R0535S) at 37°C.

[0124] Linearized vectors and digested PCR products were purified from 0.8% agarose gels using the Zymoclean Gel DNA Recovery Kit (ZymoResearch catalog number D4002). DNA was eluted in 20 μl of H2O. 10 μl of ligation reaction mixture was prepared using T4 DNA ligase (New England Laboratories, catalog number M0202S) at 16°C overnight.

[0125] Subcloning of the PBD and ferrochelate genes from previously constructed plasmids: pJAZ PBD was digested for 5 minutes at 37°C with BstBI (Bsp119I) (ThermoScientific, FD0124) and NotI (ThermoScientific, FD0596) in 1x rapid digestion buffer. pJAZ_Ferroch was digested for 5 minutes at 37°C with MfeI (MunI, ThermoScientific, FD0753) and NotI (ThermoScientific, FD0596) in 1x rapid digestion buffer.

[0126] The digestion product was purified from 0.8% agarose gel using the Zymoclean Gel DNA Recovery Kit (Zymo Research catalog number D4002). DNA was eluted in 20 μl of H2O.

[0127] The ligation reaction was established using T4 DNA ligase (New England Laboratory, catalog number M0202S) in 10 μl of reactants after overnight incubation at 16°C.

[0128] By electroporation using a MicroPulser (BioRad) set to 1.7 kV, 1.5 μl of the ligation mixture was transformed into 20 μl of ElectroMax DH10B T1 anti-phage competent cells (Yingjie, catalog number 12033-015); the cells were incubated at 37°C for 1 hour at 1 ml SOC with shaking at 200 rpm. 10 μl of the recovered mixture was plated onto LB agar plates containing 100 μg / ml ampicillin. The plates were incubated overnight at 37°C. The presence of colony insertion was screened by colony PCR. The gene sequence was confirmed.

[0129]

[0130] Example 12 - Assembling the heme biosynthesis gene on a plasmid to integrate it into the gene of Pichia pastoris mutS Because of the group

[0131] Primers containing restriction endonuclease sites were used to PCR amplify the entire cassette “promoter-gene-terminator” to assemble a plasmid that integrates into the Pichia pastoris genome.

[0132] Paoxl was assembled on the pGAN plasmid (pMx327). _ ups _ FDHterm-Paoxl _ UPD _ FDHterm-Paoxl _ CPO _ FDH item

[0133] The UPS and UPD cassettes were cloned using pGAN-CPGoxidase (pMx312) as a vector. The UPG III synthase cassette was amplified from pMx310 by primer PCR to the AOX1 promoter / FDH1 terminator containing NheI and AphI restriction endonuclease recognition sites, accordingly:

[0134]

[0135] Accordingly, the UPG III decarboxylase cassette was amplified from pMx311 by primer PCR to an AOX1 promoter / FDH1 terminator containing SphI and AgeI for restriction endonuclease recognition sites and:

[0136]

[0137] Plasmid DNA was amplified using Phusion High-Fidelity DNA polymerase (New England Laboratories, catalog number M0530L).

[0138] The obtained PCR products were amplified using DNA Clean & Concentrator-5 (Zymo Research, catalog number D4004), and the DNA was eluted in 25 µl H2O.

[0139] pGAN-CPGoxidase (pMx312), named the vector, was digested overnight at 37°C with NheI-HF (New England Laboratory, catalog number R3131S) and AgeI-HF (New England Laboratory, catalog number R3552S) in 1x CutSmart buffer.

[0140] UPG III synthase kit PCR products were digested overnight at 37°C in 1x CutSmart buffer with NheI-HF (New England Laboratory, catalog number R3131S) and Sph1-HF (New England Laboratory, catalog number R3182S).

[0141] PCR products from the UPG III decarboxylase kit were digested overnight at 37°C in 1x CutSmart buffer using Spl-HF (New England Laboratory, catalog number R3182S) and AgeI-HF (New England Laboratory, catalog number R3552S).

[0142] The digested vector and PCR products were purified from 0.8% agarose gel using the Zymoclean Gel DNA Recovery Kit (Zymo Research, catalog number D4002). DNA was eluted in 20 μl H2O.

[0143] Three ligations were established in 10 μl of UPG III synthase kits digested with NheI-SphI, UPG III decarboxylase kits digested with SphI-AgeI, and vectors digested with NheI-AgeI, using T4 DNA ligase (New England Laboratory, catalog number M0202S) at 16°C.

[0144] 1.5 μl of the ligation mixture was transformed into 20 μl of ElectroMax DH10B T1 anti-phage competent cells (Yingjie, catalog number 12033-015) using a MicroPulser (BioRad) set to 1.7 kV via electroporation. Cells were incubated at 37°C for 1 hour at 1 ml SOC with shaking at 200 rpm. 10 μl of the recovered mixture was plated onto LB agar plates containing 100 μg / ml ampicillin. The plates were incubated overnight at 37°C. Colony presence was screened by colony PCR. The ligation sequence between the vector and insert was confirmed.

[0145] Assembly Paox1 _ ALAsynthase _ FDH1term-Paox1 _ PPGoxidase _ FDH1term- Paox1 _ Fc _ FDH1term box - Paox _ PBD _ FDH1term box (pMx330)

[0146] a. PCR amplification of the gene cassette:

[0147] The ALA synthase kit was used with primers to amplify PCR from pMx310 to the corresponding recognition sites of the NheI and XhoI restriction endonucleases, AOX1 promoter / FDH1 terminator:

[0148]

[0149] The PPG oxidase kit was amplified from pMx313 by primer PCR to the AOX1 promoter / FDH1 terminator containing XhoI and AflII restriction endonuclease recognition sites:

[0150]

[0151] Ferrous chelate PCR was used to amplify pMx323 to the AOX1 promoter / FDH1 terminator containing recognition sites for AflII and AgeI restriction endonucleases using primers.

[0152]

[0153] The G418 marker was amplified by PCR from the pJAG plasmid purchased from Biogrammatics using the following primers:

[0154]

[0155] Plasmid DNA was amplified using Phusion High-Fidelity DNA polymerase (New England Laboratory, catalog number M0530L). PCR products were obtained and purified using DNA Clean & Concentrator-5 (Zymo Research, catalog number D4004), and the DNA was eluted in 25 μl of H2O.

[0156] b. Preparation of carriers

[0157] pGAZ-PBD (pMx321), named the vector, was digested overnight at 37°C in 1x CutSmart buffer with NheI-HF (New England Laboratory, catalog number R3131S) and XhoI (New England Laboratory, catalog number R0146S).

[0158] pGAZ-ALAsyn-PBD (pMx328) was digested overnight at 37°C in 1×NEBuffer 3.1 with MluI (New England Laboratory, catalog number R0198S) and BbvCI (New England Laboratory, catalog number R0601S).

[0159] pGAG-ALAsyn-PBD (pMx332) was digested in 1x CutSmart buffer at 37°C using XhoI (New England Laboratory, catalog number R0146S) and AgeI-HF (New England Laboratory, catalog number R3552S).

[0160] c. Prepare intermediate structural builders and assemble the final box.

[0161] The digested vector and PCR products were purified from 0.8% agarose gel using the Zymoclean Gel DNA Recovery Kit (Zymo Research, catalog number D4002). DNA was eluted in 20 μl H2O.

[0162] The pGAZ-PBD (pMx321) vector, digested with NheI-XhoI restriction endonuclease, was ligated overnight at 16°C with T4 DNA ligase (New England Laboratory, catalog number M0202S) in 10 μl of the ALA synthase PCR product digested with the same enzyme to produce the pGAZ-ALAsyn.-PBD plasmid (pMx328).

[0163] pGAG-ALAsyn-PBD (pMx332), digested with XhoI and AgeI-HF restriction endonucleases, was ligated in three different ways with PCR products from PPG oxidase and ferrous chelate ligase kits digested with XhoI, AflII and AflII, AgeI-HF restriction endonucleases, respectively, to produce pGAG-ALAs synthase-PBD oxidase (pMx330).

[0164] Electroporation was performed using a MicroPulser (BioRad) set to 1.7 kV. 1.5 μl of the ligation mixture was transformed into 20 μl of ElectroMax DH10B T1 anti-phage competent cells (Yingjie, catalog number 12033-015). Cells were incubated at 37°C for 1 hour at 1 ml SOC with shaking at 200 rpm. 10 μl of the recovered mixture was plated onto LB agar plates containing 100 μg / ml ampicillin. The plates were incubated overnight at 37°C. Colony presence was screened by colony PCR. The ligation sequence between the vector and the insert was confirmed.

[0165]

[0166] Example 13 - Integration of a linearized plasmid with a gene cassette into the genome of Pichia pastoris Bg11

[0167] The plasmid used to generate the production strain MXY0183 was... Figure 2 The steps taken to modify the production strain MXY0183 are shown in the diagram. Figure 3 middle.

[0168] The first enzyme to be introduced into the genome of Pichia pastoris Bg11 was ALAD. Using PmeI restriction enzymes (New England Biolabs), ALAD (pMX229), driven by pAOX1, was introduced. Figure 2 i and Figure 3 The plasmid was linearized. The linearized plasmid was purified from 0.8% agarose gel as described above and transformed into Pichia pastoris using homologous recombination at the native AOX1 locus, thereby producing strain MXY099. Figure 3 ).

[0169] Using SfiI restriction endonuclease, the pAOX1 promoter containing the gene named pMX282 was removed. Figure 2 ii and Figure 3 The plasmid containing two copies of the soybean LegH gene (optimized for Pichia pastoris; SEQ ID NO:3) under the control of ALAD was linearized. The linearized plasmid was purified from 0.8% agarose gel as described above and transformed into Pichia pastoris containing ALAD, thereby producing strain MXY0118. Figure 3 Using qPCR, it was determined that strain MXY0118 contained several copies of the LegH gene, likely due to the multiplexing of plasmid pMX282 during recombination.

[0170] The plasmid pMX327, containing genes encoding uroporphyrinogen III synthase (UPS), uroporphyrinogen III decarboxylase (UPD), and porphyrinogen III oxidase (CPO) under the control of the AOX1 promoter (the enzymes catalyze steps 4, 5, and 6, respectively), was linearized using the SfiI restriction endonuclease. Figure 2 iii and Figure 3 ) and introduced into MXY0118, thereby producing strain MXY0170 ( Figure 3 ).

[0171] Genes encoding ALA synthase (ALAS), protoporphyrin III oxidase (PPO), ferrochelate (FC), and bile pigmentogen deaminase (PBD) from the Pichia pastoris genome (which catalyze steps 1, 7, 8, and 3, respectively) were assembled into plasmid pMX330. Figure 2 iv and Figure 3 pMX330 was linearized with the SfiI restriction endonuclease and transformed into MXY0170, resulting in the production of strain MXY0183. Figure 3 The genotype of MXY0183 was confirmed using PCR and qPCR.

[0172] Production strain MXY0207

[0173] Example 14 - Construction of pGAB expression vector

[0174] pGAB expression vector ( Figure 4 ) by using Aspergillus terreus ( Aspergillus terreus The open reading frame of the blast fungicide deaminase (BSD) gene was replaced with the open reading frame of the Zeocin resistance gene in the pGAZ vector (BioGrammatics, Inc., Carlsbad, CA) to construct a transformant that allows selection of a plasmid carrying the antibiotic blast fungicide S.

[0175] As described in this article, BSD open reading frames were amplified from commercially synthesized DNA molecules using oligonucleotide primers MxO0476 and MxO0477 via high-fidelity polymerase chain reaction.

[0176]

[0177] BSD PCR products were separated by electrophoresis on a 1% agarose gel in 1xTBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.3) and visualized using SYBR safe DNA gel staining (Life Sciences, Carlsbad, CA). The desired DNA fragments were separated from the agarose gel and the DNA was recovered using the Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA).

[0178] The purified BSD PCR product and pGAZ vector were digested with 10 units of MluI and 10 units of BbvCI restriction endonuclease (New England Biolabs) at 37°C for 1 hour in 1xNE Buffer 3.1 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 100 μg / ml BSA, pH 7.9 at 25°C). The digested DNA product was recovered by gel electrophoresis as described above.

[0179] Purified MluI and BbvCI-digested BSD PCR products and pGAZ vector were reacted with 400 units of T4 DNA ligase (New England Biolabs) in 20 µl of 1x T4 DNA ligase reaction buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, pH 7.5 at 25°C) and cultured at 20 µl for 2 h at 16°C. Electroporation of competent E. coli DH10B cells was performed using 2 µl of ligation reaction, and antibiotic-resistant transformants were selected on LSB agar plates supplemented with 100 µg / µl ampicillin.

[0180] Example 15 - Construction of Mxr1 expression vector

[0181] The Mxr1 expression vector pMx354 was constructed by introducing the Mxr1 open reading frame into the pGAB vector. Figure 4 The Mxr1 open reading frame inserts into the pGAB, and translation begins immediately downstream of methanol-inducible alcohol oxidase 1 (AOX1) from Pichia pastoris, with the transcription terminator from the Pichia pastoris FDH1 gene following the translation stop signal.

[0182] The open reading frame encoding the Mxr1 protein was amplified from genomic DNA isolated from *Pichia pastoris* strain Bg11 MutS (purchased from BioGrammatics, Inc., Carlsbad, CA). The Mxr1 open reading frame was amplified from *Pichia pastoris* genomic DNA using primers MxO0495 (TTT TGC GGC CGC ATG AGC AAT CTA CCC CCA ACT TTT G (SEQ ID NO:45)) and MxO0496 (AAA AGC GGC CGC CTA GAC ACC ACC ATC TAG TCG GTT (SEQ ID NO:46)), which were attached to the NotI restriction endonuclease recognition site. Amplification was performed using polymerase chain reaction (PCR), as described herein.

[0183] The amplified Mxr1 PCR product and pGAB vector were digested with 10 units of NotI restriction endonuclease (New England Biolabs) at 37°C in 1x NE Buffer 3.1 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 100 μg / ml BSA, pH 7.9 at 25°C) for 1 hour. Following digestion, the NotI-digested pMx352 vector was treated with 5 units of phosphatase (New England Biolabs) at 37°C in 1x phosphatase buffer (50 mM Bis-Tris-propane-HCl, 1 mM MgCl2, 0.1 mM ZnCl2, pH 6 at 25°C) for 15 minutes.

[0184] NotI-digested amplified pMx352 vector Mxr1 fragment and pMx352 vector were separated by electrophoresis on a 1% agarose gel in 1xTBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.3) and visualized using SYBR safe DNA gel staining (Life Sciences, Carlsbad, CAN). The desired DNA fragments were separated from the agarose gel and the DNA was recovered using the Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CAN).

[0185] NotI digested fragments containing the Mxr1 open reading frame were introduced into pGABs at the NotI site downstream of the AOX1 promoter via ligation. A mixture of 137 ng NotI digested DNA encoding the Mxr1 open reading frame and 60 ng NotI digested, phosphatase-treated pMx352 was reacted in 20 µl of 1x T4 DNA ligase reaction buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, pH 7.5 at 25°C) using 400 units of T4 DNA ligase (New England Biolabs), and cultured at 16°C for 2 h in 20 µl of the reaction. Electroporation of competent E. coli DH10B cells was performed using 2 µl of the ligation reaction, and antibiotic-resistant transformants were selected on LSB agar plates supplemented with 100 µg / µl ampicillin. The plates were incubated overnight at 37°C. Colonies containing the insert were screened by PCR using primers MxO0495 and MxO0496. The sequence of the final vector was confirmed by DNA sequencing.

[0186] During cloning, six additional amino acid sequences were introduced into the N-terminus of Mxr1. The open reading frame of Mxr1 is shown below the "Nucleic Acid Sequence" section, and the amino acid residues from the clone are underlined. Pichia pastoris producing strains containing the six additional amino acids at the N-terminus and Pichia pastoris strains containing wild-type Mxr1 (i.e., without the six additional amino acids at the N-terminus) are indistinguishable in the fermenter.

[0187] Example 16 - Construction of natural Mxr1 expression vector

[0188] A plasmid containing the Mxr1 transcriptional regulatory gene controlled by the pAOX1 promoter (named pMX354) was used as a template for PCR amplification. The 3' end of the AOX1 promoter, the LegH open reading frame, and the AOX1 terminator were amplified from pMX354 using primers MxO0617 and MxO0647 as shown below. The AOX1 terminator, linker, and 5' end of the AOX1 promoter were amplified from pMX382 using primers MxO0618 and MxO0646.

[0189]

[0190] PCR products were obtained and purified using DNA Clean & Concentrator-5, and the DNA was eluted in 12 µl H2O. The purified PCR products were then bound and used as templates for subsequent rounds of PCR amplification using primers MxO0617 and MxO0618. The resulting PCR product consisted of the 3' end of the AOX1 promoter, the Mxr1 open reading frame, the AOX1 terminator, the short adapter sequence, and the 5' end of the AOX1 promoter. The PCR products were obtained and purified as described herein. The purified PCR products were cloned into the pCR™-Blunt II-TOPO® vector using the ZeroBlunt® TOPO® PCR Cloning Kit (Ingenie, catalog number K2800-20) to create the pMX402 vector.

[0191] Example 17 - Construction of Pichia pastoris strains MXY0206 and MXY0207

[0192] pMx354 Mxr1 expression vector ( Figure 4 It was introduced into strain MXY0183 through DNA transformation. Figure 3 ).

[0193] The pMx354 vector (1.5 µg) was linearized by digestion of the unique PmeI site in the AOX1 promoter sequence with 20 units of PmeI restriction endonuclease (New England Biolabs) at 37°C in 1x NE Buffer 4 (50 mM potassium acetate, 20 mM trihydroxyaminomethane acetate, 10 mM magnesium acetate, 1 mM MTT, pH 7.9 at 25°C).

[0194] The PmeI-digested pMX354 vector was purified by gel electrophoresis and recovered using the Zymoclean gel DNA recovery kit described above. The linearized pMX354 vector was introduced into strain MXY0183 by transformation and selection on medium containing blastomycin. Two independent clones were obtained by transformation and were named MXY0206 and MXY0207, respectively. PCR confirmed the presence of additional copies of Mxr1 under the control of the AOX1 promoter in these strains.

[0195] Production strain MXY0291

[0196] Example 18 - Construction of strains MXY0213 and MXY0260

[0197] Figure 5The steps taken to construct an antibiotic-free, marker-free strain MXY0213 containing seven heme biosynthetic pathway enzymes are shown. A linear DNA fragment containing the variant Mxr1 (with six additional amino acids at the N-terminus) was introduced using co-transformation, ensuring homology with the pAOX1 promoter at each end. Figure 5 and Figure 6 i) The linear Mxr1 expression cassette and the pIL75 plasmid were simultaneously introduced into the Pichia pastoris strain MXY213 via co-transformation. The pIL75 vector carried the panARS autonomous replication sequence (Liachko & Dunham, 2014, FEMSYeast Res., 14:364-7), thus preserving the plasmid vector (without integration into the genome of the transformed cells) and the kanMX marker for selecting transformants with the antibiotic G418. Transformed cells were selected on G418-added medium to ensure the presence of the kanMX marker on the pIL75 plasmid. Pichia pastoris transformants were screened by colony PCR to select transformants with the pIL75 plasmid and correctly integrated Mxr1 expression cassette.

[0198] Example 19 - Co-transformation to introduce the LegH expression cassette into Pichia pastoris

[0199] Plasmids containing different Pichia pastoris codon-optimized variants of the soybean protein LegH gene (variant 3; SEQ ID NO:5) under the control of the pAOX1 promoter named pMX399 were used as the source of templates for gene PCR amplification. The backbone from the TOPO clone plasmid pMX401 was amplified by PCR. Inserts and vectors were assembled using the Gibson kit (NEB Gibson Assembly Kit) to produce plasmid pMX422. This plasmid was used as a template for subsequent rounds of PCR amplification using primers MxO0617 and MxO0618.

[0200]

[0201] The resulting PCR product consists of the 3' end of the AOX1 promoter (in the direction from 5' to 3'), the LegH variant 3 open reading frame, the AOX1 terminator, the short adapter sequence, and the 5' end of the AOX1 promoter. Figure 6 (ii) The PCR products were obtained and purified by agarose gel electrophoresis as described herein.

[0202] Transformants with LegH expression cassettes integrated into the genome were screened by PCR and the LegH gene copy number was characterized by qPCR.

[0203] Example 20 - Conversion of plasmid vector with selectable markers

[0204] In the LegH expression cassette, clones with high copy numbers obtained via colony PCR and qPCR were shown. By relaxing antibiotic selection, the pIL75 plasmid was no longer needed for selection on G418. Transformants were streaked into single colonies on G418-free medium. Because the panARS plasmid is not stably maintained under selection-deficient conditions, pIL75 was rapidly lost from the transformed cells in this scenario. The resulting Pichia pastoris strain MXY0291 contained a sequence for LegH expression with a copy number similar to MXY0207, but lacked a heterologous sequence for selection.

[0205] Production strains MXY0330, MXY0333 and MXY0338

[0206] Example 21 - Construction of strain MXY0306

[0207] Genotypic PCR of strain MXY0291 revealed that a portion of the CPG oxidase coding sequence was deleted during the strain's construction. The full-length CPG oxidase coding region was restored by replacing the truncated copy. In short, a linear DNA fragment containing the pAOX1 promoter and the full-length CPG oxidase coding region was generated by PCR amplification from plasmid pMX312 using primers MxO0866 and MxO0867 as shown below.

[0208]

[0209] A linear pAOX1-CPG oxidase DNA fragment was introduced into strain MXY0291 via co-transformation with the pIL75 plasmid. Transformants were selected on G418 medium and then screened by PCR for the appearance of the full-length CPG oxidase coding region. Isolates containing the full-length CPG oxidase were identified, and subsequently, plasmid vectors requiring selection on G418 were immobilized as described above. This strain was named MXY0306 (see [link to strain name]). Figure 5 ).

[0210] Example 22 - Linear construct of hybrid promoter strain

[0211] LegH variant 3 is expressed under the guidance of each of the three native Pichia pastoris constitutive promoters shown in this paper. Linear constructs are as follows: Figure 7As shown, the construct contains the 3' half of the promoter, LegH variant 3, and the FDH1 transcription terminator. This is followed by an antibiotic resistance cassette containing the pTEF promoter from *Ashbya gossypii*, the acetamase gene from *Aspergillus nidulans*, and the TEF terminator from *Ashbya gossypii*. Finally, the construct contains the 5' half of the promoter. This linear cassette was amplified using the oligonucleotide primers listed in the table below to produce constructs containing hundreds of base pairs at the 5' and 3' ends (homological to their respective promoters in the native *Pichia pastoris* genome).

[0212] Primers for amplifying linear constructs

[0213]

[0214] Competent MXY0306 cells were transformed using each linear cassette, while transformants containing the amdS selection cassette were selected based on their growth ability on agar plates containing acetamide (as the sole nitrogen source). These strains were purified, isolated, and confirmed by PCR to contain LegH (a constitutively controlled promoter). Figure 5 ).

[0215] Example 23 - Nucleic Acid Sequence

[0216] The Mxr1 nucleic acid sequence (the underlined nucleotides encode the 6 N-terminal amino acids introduced during cloning) (SEQ ID NO:1)

[0217]

[0218]

[0219] Mxr1 protein sequence (the underlined 6 amino acids introduced during cloning) (SEQ ID NO:2)

[0220]

[0221] LegH nucleic acid sequence of Pichia pastoris with optimized codons (SEQ ID NO:3)

[0222]

[0223] The LegH amino acid sequence of Pichia pastoris codon-optimized (SEQ ID NO:4)

[0224]

[0225] Nucleic acid sequence of LegH variant 3 of Pichia pastoris with optimized codons (SEQ ID NO:5)

[0226]

[0227] The 3-amino acid sequence of the LegH variant of Pichia pastoris with optimized codons (SEQ ID NO:6)

[0228]

[0229] Pichia pastoris pAOX1 promoter (SEQ ID NO:7)

[0230]

[0231] pGAP promoter of Pichia pastoris (SEQ ID NO:8)

[0232]

[0233] Pichia pastoris pGCW14 promoter (SEQ ID NO:9)

[0234]

[0235] pTEF1 promoter of Pichia pastoris (SEQ ID NO:10)

[0236]

[0237] Heme biosynthetic enzyme 1-ALA synthase (SEQ ID NO:11)

[0238]

[0239]

[0240] Heme biosynthetic enzyme 2-ALA synthase (SEQ ID NO:12)

[0241]

[0242] Heme biosynthetic enzyme 3-bilirubinogen deaminase (SEQ ID NO:13)

[0243]

[0244]

[0245] Heme biosynthetic enzyme 4-uroporphyrinogen III synthase (SEQ ID NO:14)

[0246]

[0247] The heme biosynthetic enzyme 5-uroporphyrinogen III decarboxylase (SEQ ID NO:15)

[0248]

[0249] The biosynthetic enzyme of heme, 6-uroporphyrinogen III oxidase (SEQ ID NO:16)

[0250]

[0251]

[0252] The heme biosynthetic enzyme 7-protoporphyrinogen oxidase (SEQ ID NO:17)

[0253]

[0254] Heme biosynthetic enzyme 8-ferrochelate (SEQ ID NO:18)

[0255]

[0256]

[0257] Part C. Results and Discussion

[0258] Example 24 - Characterization of strain MXY0183

[0259] The optimal growth conditions for strain MXY0183 include a target pH of 3.0 to 6.0 and a temperature of 28°C to 35°C. For LegH protein production, strain MXY0183 must be viable and grow aerobically for 6 consecutive days.

[0260] The expression of genes associated with strain MXY0183 caused phenotypic changes in the strain. Figure 8 Photographs of shake flasks are shown at the start of induction (0 hours) and 72 hours after induction. Designated shake flask #1 contains the host strain MXY0051. Designated shake flasks #2 and #3 contain one of an intermediate strain (e.g., MXY0118, containing more than 10 copies of the LegH gene and ALA dehydrogenase from the heme biosynthesis pathway) and a producing strain (e.g., MXY0183, containing more than 10 copies of the LegH gene and 8 enzymes from the heme biosynthesis pathway), respectively. The distinctive red color in shake flask #3 after 72 hours indicates the production of LegH through heme binding.

[0261] After growth in shake flasks, as shown above, Pichia pastoris strains MXY0051, MXY0118, and MXY0183 were lysed and the proteins ran on SDS sol. Figure 9 A). The arrows indicate the location of the LegH protein. A comparison of LegH produced in strains MXY0183 and MXY0118 is shown. Figure 9 In B, this confirms the heme loading efficiency of strain MXY0183 for LegH protein.

[0262] Example 25 - Characterization of strain MXY0207

[0263] Next, experiments were conducted to determine the benefits of overexpressing transcription activators in the presence of genes encoding eight enzymes involved in heme biosynthesis. Strains MXY0183, containing more than 10 copies of LegH sequences and genes encoding the eight enzymes involved in heme biosynthesis, and sister strains MXY0206 and MXY0207, containing more than 10 copies of LegH sequences, genes encoding the eight enzymes involved in heme biosynthesis, and the Mxr1 transcription activator, were grown in shake flask cultures in the presence of glycerol (the repressive carbon source for these strains). A photograph of the shake flask cultures after 48 hours is shown below. Figure 10 As shown in Figure A, a photograph of cell clumps after 48 hours of growth on BMY medium without an additional carbon source is shown in Figure A. Figure 10 As shown in B; these experiments demonstrate that when the inhibiting carbon source is consumed in the growth medium of the strain (in which Mxr1 is also expressed from the AOX1 promoter), transgenes (e.g., hemease) under the control of the AOX1 promoter are significantly expressed in the absence of an inducing carbon source. The relative yield of heme-loaded LegH in shake-flask cultures grown in the absence of an inducer is shown in Figure B. Figure 10 As shown in C. These experiments demonstrate that, in the absence of methanol induction, significant production of recombinant heme-loaded proteins is achieved in Pichia pastoris strains (where Mxr1 expression is also driven by the AOX1 promoter) via transgenes driven by the AOX1 promoter, in the absence of methanol induction.

[0264] Selected strains were grown in a 2L fermenter, and the relative yields of LegH and heme-loaded LegH were determined. Figure 11 Compared to strain MXY0183, strain MXY0207 produces more LegH and is able to produce enough heme to very efficiently load the LegH protein.

[0265] Example 26 - Characterization of strain MXY0291

[0266] As described in Examples 18 to 20, strain MXY0291 was constructed to reproduce the LegH production capacity of MXY0207 and did not contain antibiotic resistance genes. It was determined that strain MXY0291 contained 16 copies of LegH variant 3, Mxr1, and 7 of the 8 heme biosynthesizers. When grown in a 2L fermenter, this strain exhibited increased LegH production compared to MXY0207. This increase was observed in both induction media containing methanol / glycerol and methanol / glucose (d-glucose). Figure 11 ).

[0267] Example 27 - Characterization of hybrid promoter strains

[0268] In strains already containing several copies of LegH, all heme biosynthesizers, and the transcription factor Mxr1 under the control of the promoter pAOX1 (hereinafter referred to as MXY0291), additional copies of soybean hyperhemoglobin (LegH) were expressed under three different constitutive promoters: pGAP, pGCW14, and pTEF1. When induced with methanol in the presence of glucose (i.e., D-glucose), both the constitutive promoter and pAOX1 drove LegH expression, while only the pAOX1 promoter drove heme enzyme expression. This resulted in a further increase in LegH production compared to the previous strain MXY0291. Figure 11 ).

[0269] It should be understood that although the relevant methods and compositions have been described herein in conjunction with many different aspects, the foregoing description of each aspect is intended to illustrate, and not limit, the scope of the relevant methods and compositions. Other aspects, advantages, and modifications are within the scope of the following claims.

[0270] The methods and compositions disclosed in this invention can be used in conjunction with the products of the disclosed methods and compositions, can be used to prepare the products of the disclosed methods and compositions, or can be used as products of the disclosed methods and compositions. These and other materials are disclosed herein, and it should be understood that combinations, subsets, interactions, groups, etc., of these methods and compositions are disclosed. That is, although specific references to various individual and common combinations and arrangements of these compositions and methods may not be explicitly disclosed, each has been given particular consideration and description herein. For example, if a particular related composition or method is disclosed and discussed, and many compositions or methods are discussed, then each combination and arrangement of compositions and methods has been given particular consideration unless specifically indicated otherwise. Similarly, any subset or combination of these has also been given particular consideration and disclosed.

Claims

1. A methyltrophic yeast cell comprising a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule comprises: a) A first exogenous nucleic acid encoding a methanol expression regulator Mxr1 transcriptional activator from *Pichia pastoris*, said Mxr1 transcriptional activator being operatively linked to at least one methanol-inducible promoter element, said at least one methanol-inducible promoter element comprising a sequence binding to said Mxr1 transcriptional activator, wherein said at least one methanol-inducible promoter element comprises a first alcohol oxidase 1 (AOX1) methanol-inducible promoter element from *Pichia pastoris*; and b) A second exogenous nucleic acid encoding a heterologous polypeptide, said exogenous polypeptide being operatively linked to a second alcohol oxidase 1 AOX1 methanol-inducible promoter element from Pichia pastoris, said second alcohol oxidase 1 AOX1 methanol-inducible promoter element comprising a sequence that binds to said Mxr1 transcription activator.

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