CN115247135A - Enhancing the transport process from Golgi to extracellular protein and improving the yield of pichia pastoris extracellular glucose oxidase - Google Patents

Abstract

The invention provides a gene for improving the yield of Glucose Oxidase (GOD) in pichia pastoris and application thereof. Specifically, the invention provides a construction method of a Glucose Oxidase (GOD) production strain, wherein the activity of a vesicle secretion promoting factor is enhanced or the vesicle secretion promoting factor is introduced into an original strain. Experiments prove that the extracellular GOD yield of the pichia pastoris is obviously improved by coexpressing the VAMP4 gene participating in a vesicle transport pathway from a Golgi body to a plasma membrane.

Description

Enhancing the transport process from Golgi to extracellular protein and improving the yield of pichia pastoris extracellular glucose oxidase

Technical Field

The invention relates to the field of biotechnology. In particular to a method for improving the yield of pichia pastoris extracellular glucose oxidase by strengthening the transport process of Golgi to extracellular protein.

Background

Glucose oxidase (GOD, EC 1.1.3.4) is a flavin glycoprotein in nature, as a homodimeric molecule, capable of oxidizing β -D glucose to gluconic acid, with molecular oxygen as an electron acceptor to produce hydrogen peroxide. In recent years, GOD, as a mature commercial industrial enzyme, is increasingly used in industries such as food, textile bleaching and dyeing, and in emerging industries such as glucose biosensors and environmental protection fuels, but the low yield of GOD is a major limiting factor for large-scale industrial application.

The pichia pastoris expression system is used as a foreign protein expression system, and has the advantages of post-translational processing of the expression protein, easy purification of the foreign protein, high-density fermentation, low culture cost and the like, so the pichia pastoris is selected as a common expression host for GOD in recent years. However, more and more researches prove that the obstruction of the yeast secretory pathway becomes one of the bottlenecks in realizing the high-efficiency expression of the foreign protein, wherein the transportation of vesicles is needed for the protein from the endoplasmic reticulum to the Golgi apparatus and then from the Golgi apparatus to the cytoplasmic membrane, and the low efficiency of vesicle transportation causes part of the foreign protein to be accumulated in the cells.

In conclusion, there is a need in the art to provide a method for improving vesicle transport efficiency, and increasing the yield of glucose oxidase.

Disclosure of Invention

The invention aims to provide a method for enhancing the transport process from a Golgi apparatus to extracellular protein and improving the yield of Pichia pastoris extracellular glucose oxidase.

In a first aspect of the invention, there is provided a method of increasing expression and/or activity of Glucose Oxidase (GOD) in a yeast cell, comprising the steps of:

enhancing expression and/or activity of a vesicle secretagogue or a gene thereof in a yeast cell in a starting strain to thereby obtain a Glucose Oxidase (GOD) producing strain having enhanced expression of GOD and/or activity of GOD, wherein,

the vesicular secretagogue comprises one or more proteins selected from the group consisting of:

(1) A protein having an amino acid sequence as set forth in any one of SEQ ID No. 1-6;

(2) 1-6 by substitution, deletion or addition of one or more amino acid residues, and the protein with the vesicle secretor promoting factor activity and derived from the (1); and/or

(3) The homology of the amino acid sequence and any sequence shown in SEQ ID NO. 1-6 is more than or equal to 85 percent (preferably more than or equal to 90 percent, 95 percent and more preferably more than or equal to 98 percent), and the protein has the vesicle secretion promoting factor activity.

In another preferred example, the vesicular secretagogue comprises a protein derived from (1) and formed by adding 1 to 5 amino acid residues to both ends of an amino acid sequence shown in any one of SEQ ID NO. 1 to 6, and having the activity of the vesicular secretagogue.

In another preferred embodiment, the vesicular secretagogue comprises at least a protein having an amino acid sequence shown in SEQ ID No. 1.

In another preferred embodiment, the vesicular secretagogue comprises at least 2 (preferably 3, 4, 5) proteins having an amino acid sequence as set forth in any one of SEQ ID No. 1-6.

In another preferred embodiment, the vesicular secretagogue is selected from one or more of the proteins of the amino acid sequences shown in SEQ ID No. 1, 2 and/or 5.

In another preferred embodiment, the vesicular secretagogue is selected from one or more of the following: VAMP4, SEC4, EXO84P, EXO70P, STX1-4, YPT32; preferably, it is selected from VAMP4, SEC4, EXO84P.

In another preferred embodiment, the enhancing activity of the vesicle secretagogue comprises:

(1) Improving the expression quantity of the vesicle secretagogue and/or the activity of the protein; and/or

(2) Reducing degradation and/or inactivation of vesicular secretagogues.

In another preferred example, the increasing the expression level of the vesicular secretagogue comprises: up-regulating the expression of the gene encoding the vesicular secretagogue.

In another preferred embodiment, the vesicular secretagogue is exogenous or endogenous.

In another preferred embodiment, the vesicular secretagogue is endogenous.

In another preferred embodiment, the gene encoding the vesicular secretagogue is selected from the group consisting of: a cDNA sequence, a genomic sequence, or a combination thereof.

In another preferred embodiment, said up-regulating the expression of a gene encoding a vesicular secretagogue comprises up-regulating the transcription level and/or translation level of said encoding gene.

In another preferred embodiment, the enhancement of the activity of the vesicular secretagogue can be achieved by one or a combination of the following methods: expressing a gene encoding said protein, either homologous or heterologous, and/or increasing the copy number of said encoding gene in said strain, and/or engineering regulatory sequences (e.g. promoters) of said encoding gene to enhance the rate of transcription (e.g. the rate of transcription initiation), and/or modifying the translational regulatory region of the messenger RNA carrying said encoding gene to enhance the translational strength, and/or modifying the encoding gene itself to enhance mRNA stability, protein stability, release of feedback inhibition of the protein.

In another preferred embodiment, said enhancing the activity of the vesicular secretagogue comprises overexpressing said vesicular secretagogue in a strain.

In another preferred embodiment, the content and/or activity of vesicular secretagogues in said GOD-producing strain is increased by at least 20%, preferably by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or 200% compared to the starting strain.

In another preferred embodiment, the GOD in said GOD producing strain has an increase in total intracellular and extracellular enzyme content (or yield) and/or activity of at least 2%, preferably at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or 200% compared to the starting strain.

In another preferred embodiment, the extracellular enzyme content (or yield) and/or activity of GOD in said GOD-producing strain is increased by at least 3%, preferably by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or 200% as compared to the starting strain.

In another preferred embodiment, the secretion rate of GOD in said GOD producing strain is increased by at least 5%, preferably by at least 10%, 20%, 30%, 40%, 50%, 60%, 80%, 90% or 100% compared to the starting strain.

In another preferred example, the construction method includes the steps of:

(a1) Providing an expression vector carrying a gene encoding a vesicular secretagogue;

(b1) Transferring the expression vector into an original strain to obtain a recombinant strain; and

(c1) And (3) culturing the recombinant strain.

In another preferred embodiment, the method further comprises determining the GOD yield of the resulting strain.

In another preferred embodiment, the starting strain is a yeast cell.

In another preferred embodiment, the yeast cell is selected from the group consisting of pichia pastoris and saccharomyces cerevisiae.

In another preferred embodiment, the yeast cell is Pichia pastoris (Pichia pastoris).

In another preferred embodiment, the starting strain is Pichia pastoris (Pichia pastoris) GS115.

In another preferred embodiment, said starting strain expresses a GOD wild type or a mutant thereof.

In another preferred embodiment, the genome of said starting strain has integrated into it an expression cassette for a GOD downstream of the AOX promoter.

In another preferred embodiment, the expression cassette of the GOD further comprises a signal peptide and/or a promoter.

In another preferred embodiment, the GOD mutant has a gene sequence as shown in SEQ ID No. 7 at positions 33-615.

In another preferred embodiment, each of the corresponding GOD protein-encoding genes has at least 92%, preferably at least 95%, more preferably at least 98%, 99% homology between the genomes of the starting strains.

In another preferred example, said GOD producing strain is a GOD highly producing strain.

In another preferred embodiment, the "high yield" refers to that the expression level or production capacity of GOD protein of the strain is increased by at least 20%, preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or 200% compared with the original strain.

In a second aspect of the present invention, there is provided a GOD producing strain in which the activity of vesicular secretagogues is enhanced, wherein the vesicular secretagogues comprise one or more proteins selected from the group consisting of:

(1) A protein having an amino acid sequence as set forth in any one of SEQ ID No. 1-6;

(2) 1-6 by substitution, deletion or addition of one or more amino acid residues, and the protein with the vesicle secretor promoting factor activity and derived from the (1); and/or

(3) The homology of the amino acid sequence and any sequence shown in SEQ ID NO. 1-6 is more than or equal to 85 percent (preferably more than or equal to 90 percent, 95 percent, more preferably more than or equal to 98 percent), and the protein has the vesicle secretion promoting factor activity.

In another preferred embodiment, the GOD producing strain is prepared by the method of the first aspect of the present invention.

In another preferred embodiment, said GOD-producing strain is used for the production of GOD and/or downstream products that are precursors to GOD.

In another preferred embodiment, said GOD producing strain expresses a GOD wild type or GOD mutant.

In another preferred example, the GOD producing strain has integrated into its genome an expression cassette comprising a GOD downstream of the AOX promoter.

In another preferred embodiment, the expression cassette of the GOD further comprises a signal peptide and/or a promoter.

In another preferred embodiment, the GOD mutant has a gene sequence as shown in SEQ ID No. 7 at positions 33-615.

In another preferred embodiment, each of the corresponding GOD protein-encoding genes has at least 92%, preferably at least 95%, more preferably at least 98%, 99% homology between the genomes of the starting strains.

In another preferred example, said GOD producing strain is a GOD highly producing strain.

In another preferred embodiment, the "high yield" refers to that the expression level or production capacity of GOD protein of the strain is increased by at least 20%, preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or 200% compared with the original strain.

In a third aspect of the present invention, there is provided a use of the GOD-producing strain according to the second aspect of the present invention for producing GOD and/or downstream products that are precursors to GOD.

In a fourth aspect of the present invention, there is provided a method for preparing a GOD protein, comprising the steps of:

1) Fermentatively culturing the GOD producing strain of claim 3 to thereby obtain a GOD protein; and

2) Optionally obtaining the GOD protein from the fermentation culture system of 1).

In a fifth aspect of the present invention, there is provided a method for enhancing the GOD protein production capacity of a strain, comprising the steps of:

enhancing the activity of vesicular secretagogues in the starting strain, wherein the vesicular secretagogues comprise one or more proteins selected from the group consisting of:

(1) A protein having an amino acid sequence as set forth in any one of SEQ ID No. 1-6;

(2) 1-6 by substitution, deletion or addition of one or more amino acid residues, and the protein with the vesicle secretor promoting factor activity and derived from the (1); and/or

(3) The homology of the amino acid sequence and any sequence shown in SEQ ID NO. 1-6 is more than or equal to 85 percent (preferably more than or equal to 90 percent, 95 percent, more preferably more than or equal to 98 percent), and the protein has the vesicle secretion promoting factor activity.

In another preferred example, the method comprises the steps of: the expression of the gene coding for the vesicular secretagogue is up-regulated in the starting strain.

In another preferred embodiment, the vesicle secretagogue is selected from one or more of the following group: VAMP4, SEC4, EXO84P, EXO70P, STX1-4, YPT32; preferably, it is selected from VAMP4, SEC4, EXO84P.

In a sixth aspect of the present invention, there is provided a use of a vesicular secretagogue or a gene promoter thereof for enhancing the GOD protein production capacity of a strain, the vesicular secretagogue comprising one or more proteins selected from the group consisting of:

(1) A protein having an amino acid sequence as set forth in any one of SEQ ID No. 1-6;

(2) 1-6 by substitution, deletion or addition of one or more amino acid residues, and the protein with the vesicle secretor promoting factor activity and derived from the (1); and/or

(3) The homology of the amino acid sequence and any sequence shown in SEQ ID NO. 1-6 is more than or equal to 85 percent (preferably more than or equal to 90 percent, 95 percent, more preferably more than or equal to 98 percent), and the protein has the vesicle secretion promoting factor activity.

In another preferred embodiment, the accelerator is selected from the group consisting of: a small molecule compound, a nucleic acid molecule, a polypeptide, a small molecule ligand, or a combination thereof.

In another preferred embodiment, the nucleic acid molecule is selected from the group consisting of: miRNA, shRNA, siRNA, or a combination thereof.

In another preferred embodiment, the strain is pichia pastoris.

In a seventh aspect of the present invention, there is provided a vesicular secretagogue, wherein the vesicular secretagogue is a protein selected from one or more of the following group:

(1) A protein having an amino acid sequence as set forth in any one of SEQ ID No. 1-6;

(2) 1-6 by substitution, deletion or addition of one or more amino acid residues, and the protein with the vesicle secretor promoting factor activity and derived from the (1); and/or

(3) The homology of the amino acid sequence and any sequence shown in SEQ ID NO. 1-6 is more than or equal to 85 percent (preferably more than or equal to 90 percent, 95 percent, more preferably more than or equal to 98 percent), and the protein has the vesicle secretion promoting factor activity.

In another preferred embodiment, the vesicle secretion promoting factor at least comprises a protein having an amino acid sequence as shown in SEQ ID No. 1.

In another preferred embodiment, the vesicular secretagogue comprises at least 2 (preferably 3, 4, 5) proteins having an amino acid sequence as set forth in any one of SEQ ID No. 1-6.

In another preferred embodiment, the vesicular secretagogue is selected from the proteins of the amino acid sequences shown in SEQ ID No. 1, 2 and/or 5.

In an eighth aspect of the invention there is provided a polynucleotide encoding a vesicle secretagogue, the polynucleotide encoding a vesicle secretagogue according to the seventh aspect of the invention.

In a ninth aspect of the invention there is provided a vector comprising a polynucleotide according to the eighth aspect of the invention.

In a tenth aspect of the invention, there is provided a use of the vesicular secretagogue according to the seventh aspect of the invention, the polynucleotide encoding the vesicular secretagogue according to the eighth aspect of the invention, the vector according to the ninth aspect of the invention for preparing the GOD producing strain according to the second aspect of the invention.

In another preferred embodiment, the strain is pichia pastoris.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows a schematic diagram of the construction of pAOX-UH-SEC4/EXO84P/EXO70P/STX1-4/YPT32 plasmid. Wherein, the "inserted genes" represent SEC4, EXO84P, EXO70P, STX1-4, YPT32 genes, respectively.

FIG. 2 shows a schematic diagram of the pAOX-UH-VAMP4 plasmid construction.

FIG. 3 shows the construction process of recombinant bacteria E5-VAMP4/SEC4/EXO84P/EXO70P/STX1-4/YPT 32. "inserted genes" represent VAMP4, SEC4, EXO84P, EXO70P, STX1-4, YPT32 sequences, respectively.

FIG. 4 shows the electrophoresis of PCR products of 6 genes of interest. M is DL15000 DNA Marker; lane 1; lane 2; lane 3; lane 4: EXO70P; lane 5; lane 6.

FIG. 5 shows (A) growth curves of each recombinant bacterium in shake flasks and (B) extracellular GOD yield of each recombinant bacterium at 144h of induction.

FIG. 6 shows the total intracellular (A) GOD production and (B) secretion rate of each recombinant bacterium at 144h induction.

Detailed Description

The inventor finds that the expression quantity of the specific vesicle secretion promoting factor in the pichia pastoris is improved through a large number of experiments, and the enzyme yield of the pichia pastoris GOD production strain can be greatly improved. Specifically, vesicle secretion promoting factors involved in the transport pathway from golgi to plasma membrane vesicles, such as VAMP4, SEC4, EXO84P and the like, are overexpressed in the GOD production strain, and the total intracellular and extracellular enzyme yield and secretion rate of the GOD can be remarkably improved. Experiments show that the extracellular enzyme yield of the GOD production strain over expressing the secretagogue gene VAMP4 is improved by 24.1 percent compared with that of the original strain E5. The present invention has been completed based on this finding.

Term(s) for

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of 8230or" consisting of 8230.

As used herein, the term "exogenous" refers to a system that includes a substance that was not originally present. For example, an exogenous gene or sequence broadly refers to a gene or fragment thereof that is not contained in the chromosome or plasmid of a strain itself, and is "exogenous" to a strain when a coding gene that is not originally present in the strain is introduced into the strain by transformation or the like. Where a "non-endogenous" gene or sequence generally refers to a particular gene or sequence that is contained within the chromosome of the same strain or a different strain, but not within the chromosome of the particular wild or mutant strain serving as the host cell.

As used herein, "nucleic acid sequence" refers to an oligonucleotide, nucleotide, or polynucleotide and fragments or portions thereof, and may also refer to genomic or synthetic DNA or RNA, which may be single-stranded or double-stranded, representing either the sense strand or the antisense strand.

As used herein, a protein or polynucleotide "derivative" refers to an amino acid sequence having one or more amino acid or nucleotide changes or a polynucleotide sequence encoding it. The alteration may comprise a deletion, insertion or substitution of an amino acid or a nucleotide in the amino acid sequence or the nucleotide sequence. Derivatives may have "conservative" changes, where the substituted amino acid has similar structural or chemical properties as the original amino acid, such as the substitution of isoleucine with leucine. Derivatives may also have non-conservative changes, such as replacement of glycine with tryptophan. As used herein, "insertion" or "addition" refers to a change in an amino acid sequence or nucleotide sequence resulting in an increase of one or more amino acids or nucleotides compared to the naturally occurring molecule. "substitution" refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides.

As used herein, "complementary" or "complementation" refers to the natural binding of polynucleotides by base pairing under the conditions of allowable salt concentration and temperature. Ext>ext> forext>ext> exampleext>ext>,ext>ext> theext>ext> sequenceext>ext> "ext>ext> Cext>ext> -ext>ext> Text>ext> -ext>ext> Gext>ext> -ext>ext> Aext>ext>"ext>ext> mayext>ext> bindext>ext> toext>ext> theext>ext> complementaryext>ext> sequenceext>ext> "ext>ext> Gext>ext> -ext>ext> Aext>ext> -ext>ext> Cext>ext> -ext>ext> Text>ext>"ext>ext>.ext>ext> The complementarity between the two single stranded molecules may be partial or complete. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.

As used herein, "homology" refers to the degree of complementarity, which may be partial homology or complete homology. "partial homology" refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. Inhibition of such hybridization can be detected by hybridization (Southern blot or Northern blot) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes compete for and inhibit the binding of fully homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that nonspecific binding is allowed under the conditions of reduced stringency, because specific or selective interaction is required for binding of two sequences to each other.

Sequence identity is determined by comparing two aligned sequences along a predetermined comparison window (which may be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of the reference nucleotide sequence or protein) and determining the number of positions at which identical residues occur. Typically, this is expressed as a percentage. The measurement of sequence identity of nucleotide sequences is a method well known to those skilled in the art.

As used herein, "isolated" refers to separating a substance from its original environment (which, if it is a natural substance, is the natural environment). If the polynucleotide or protein in the natural state in the living cell is not isolated or purified, but the same polynucleotide or protein is isolated or purified if it is separated from other substances coexisting in the natural state.

The term "polynucleotide encoding a protein" is meant to include polynucleotides encoding the protein and polynucleotides including additional coding and/or non-coding sequences.

Vesicle secretion promoting factor and encoding gene thereof

As used herein, "vesicle secretagogues", "vesicle secretagogues of the present invention" refer to proteins that facilitate transport of vesicles from the golgi to the plasma membrane, such as VAMP4, SEC4, EXO84P, EXO70P, STX1-4, YPT32 (see table 1 for details).

The vesicle associated membrane protein (VAMP 4) gene encodes a vesicle associated membrane protein, the v-SNARE (synaptic vesicle receptor) required for the fusion of secretory vesicles with the plasma membrane. It has been shown that after extracellular fusion they are internalized by vesicles from the plasma membrane and then recycled to new secretory vesicles through the endosome and golgi apparatus. Therefore, the v-SNARE protein encoded by VAMP4 plays an important role in membrane fusion trafficking between different vesicles and plasma membranes in promoting protein secretion to the extracellular space. The GTP-binding protein encoded by SEC4 also plays a crucial role in the final phase of secretion, and in a stable state, it exists in a soluble form in cells, can bind to late Golgi secretory vesicles and participate in fusion with the plasma membrane, and it has been reported in the related literature that the protein encoded by STX1-4 can also participate in fusion with the plasma membrane. EXO84P is an essential component of the final stages of the yeast secretory pathway as well as EXO70P, an essential protein that plays a dual role in spliceosome assembly and exocytosis, mediates secretory vesicle polarization and targets the active site of exocytosis, and EXO70P can be localized to an extracellular site independent of actin function by direct binding to the polar determinants of the cell cortex. The protein encoded by YPT32 is an essential component for the transport in the secretory pathway, which may be involved in the formation of secretory vesicles of the trans-golgi apparatus.

TABLE 1 information of the secretagogue genes and their amino acid sequences

Glucose oxidase GOD

Glucose oxidase can catalyze the oxidation of glucose to produce hydrogen peroxide and glucono-delta-lactone, or to produce gluconic acid. Thus, the glucose oxidase mutants of the present invention can be applied in a wide range of fields including, but not limited to: producing gluconic acid; as food preservatives and color stabilizers; for the production of hydrogen peroxide for textile bleaching; used for manufacturing a glucometer for detecting the blood sugar concentration of a diabetic patient and the like, and has more ideal enzyme activity, catalytic efficiency or substrate affinity compared with the wild type. The method for improving the expression and/or activity of Glucose Oxidase (GOD) in yeast cells can be used for improving the expression level of a GOD wild type protein or a mutant protein thereof, wherein the sequence of the mutant protein has at least 92%, preferably at least 95%, more preferably at least 98% and 99% homology with the sequence of the GOD wild type protein.

An amino acid sequence of a wild-type GOD from Aspergillus niger is shown in SEQ ID No. 22.

The GOD production strain has 8 copies of V20W + T30V mutant GOD genes (the number of the GOD genes is EC 1.1.3.4), and the amino acid sequence of the mutant GOD (GOD mutant) is shown in the 33 th to 615 th positions of SEQ ID NO. 7. Contains the V20W + T30V mutation: the amino acid at the 20 th position in the wild type GOD amino acid sequence is mutated from Val to Trp, and the amino acid at the 30 th position is mutated from Thr to Val.

Note: the GAS' signal peptide sequence (amino acids 1-32) is underlined.

The construction of GOD-producing bacteria containing glucose oxidase mutants can be found in patent application CN 110628738B.

The invention relates to a construction method

In the invention, by utilizing a genetic engineering method, relevant factors in the transport from a Golgi body to a plasma membrane vesicle in the pichia pastoris are selected to modify the secretion pathway of the pichia pastoris, so that the secretion yield of the GOD is improved.

The invention further provides a construction method of the GOD production strain, which comprises the following steps: enhancing the activity of vesicle secretagogue in the original strain.

In another preferred example, the construction method includes the steps of:

(a1) Providing an expression vector carrying a gene encoding a vesicular secretagogue;

(b1) Transferring the expression vector into an original strain to obtain a recombinant strain; and

(c1) And (3) culturing the recombinant strain.

The term "enhance" as used herein refers to increasing, enhancing, augmenting or elevating the expression and/or activity of a protein, for example, a protein. In the present invention, the enhancement of the activity of a protein associated with spinosyn synthesis may include increasing the expression level of a protein associated with spinosyn synthesis and/or the activity of the protein itself, and/or reducing the degradation of a protein associated with spinosyn synthesis. In view of the teachings of the present invention and the prior art, it will also be understood by those skilled in the art that "enhancing" as used herein may also include enhancing the activity of a protein by expressing an exogenously encoded gene.

In a specific embodiment, enhancing the activity of a protein may enhance the self-activity of the protein by the exogenous addition of a substance or mutation.

In particular embodiments, enhancing the activity of a protein, including increasing the amount of the protein in a strain, can be achieved by expressing an endogenous or heterologous coding gene for the protein, and/or increasing the copy number of the coding gene, and/or modifying a regulatory sequence of the coding gene (e.g., modifying a promoter of the coding gene to increase the rate of transcription initiation), and/or modifying a translational regulatory region or a rare codon of messenger RNA carrying the coding gene to increase translational strength, and/or modifying the coding gene itself to increase mRNA stability, protein stability, release of feedback inhibition of the protein, and the like.

As described above, the control sequences include a promoter capable of initiating transcription, any operator sequence for transcriptional control, sequences encoding suitable mRNA ribosome binding domains, sequences which control termination of transcription and translation. Modifications to regulatory sequences include, but are not limited to, such as: modifications introduced by deletions, insertions, conservative or non-conservative mutations, or combinations thereof in a polynucleotide sequence may also be made by replacing the original polynucleotide sequence with a polynucleotide sequence having enhanced activity.

A vector is a DNA construct that includes a polynucleotide sequence encoding a target protein operably linked to suitable control sequences so that the target protein can be expressed in a host cell. The vector may replicate or function independently of the host cell genome, or may be integrated into the genome of the host cell, after being transferred into a suitable host cell. These vectors may not be particularly limited as long as the vector is replicable in host cells, and it may be constructed using any vector known in the art. Examples of vectors include natural or recombinant plasmids, cosmids, viruses, and phages. For example, pWE15, pET, pUC vectors and the like.

In addition, by inserting the vector into the chromosome of the host cell, a polynucleotide encoding the endogenous target protein on the chromosome can be replaced with a modified polynucleotide. Insertion of the polynucleotide into the chromosome can be performed using any method known in the art, including, but not limited to, such as: by homologous recombination. Polynucleotides include DNA and RNA encoding target proteins, which may be inserted into the chromosome of a host cell in any form so long as they are capable of expression in the host cell. Including, but not limited to, such as: the polynucleotide may be introduced into the host cell in its native state, and/or in the form of an expression cassette.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. Representative examples are: escherichia coli, streptomyces, agrobacterium; fungal cells such as yeast; plant cells, and the like. In higher eukaryotic cells, transcription is enhanced if enhancer sequences are inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to increase transcription of a gene.

In a preferred form of the invention, the polynucleotide is expressed in a yeast cell, preferably a Pichia cell. Preferably, the 3' end of the polynucleotide further comprises a signal peptide and/or a promoter. Such signal peptides include, but are not limited to: saccharomyces cerevisiae alpha mating factor (alpha-MF), pichia acid phosphatase (PHO 1) signal peptide, etc. The promoter includes but is not limited to: AOX promoter, and the like.

Transformation of a host cell with recombinant DNA may be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, such as E.coli, competent cells capable of DNA uptake can be harvested after the exponential growth phase and treated by the CaCl2 method using procedures well known in the art. Another approach is to use MgCl2. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformant can be cultured by a conventional method to express the protein encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant protein in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art.

In a specific embodiment, the starting strain (recombinant strain E5 expressing GOD) in the present invention is constructed as follows:

the GOD gene containing the V20W + T30V mutation linked to the signal peptide GAS' was inserted downstream of the pAOX promoter of the pPIC9K vector to construct plasmid pGG30B.

The plasmid pGG30B is integrated into the His site of the genome of Pichia pastoris GS115 and screened by antibiotic concentration gradient,obtaining recombinant Pichia pastoris E5 (kan) containing 8 copies of the GOD gene ^R HIS4)。

In a specific embodiment, the genes of 6 vesicle secretion promoting factors, i.e., VAMP4, SEC4, EXO84P, EXO70P, STX1-4 and YPT32, in the cytoplasmic membrane vesicle trafficking pathway from the Golgi apparatus of the starting strain G/GS115 are inserted into the pAOX-UH vector at the downstream of the pAOX promoter to obtain a co-expression vector. Integrating the vector to the genome of the recombinant strain E5 in a homologous recombination mode to construct the vesicle factor co-expression strain.

It is understood that the vesicular secretagogue in the present invention may be exogenous or endogenous, as long as it can promote the extracellular enzyme production of GOD by the GOD-producing strain, and is not particularly limited. In addition, the vesicle secretion promoting factor can also be a protein which has the vesicle secretion promoting factor activity and the homology of the sequence of the vesicle secretion promoting factor with any one sequence shown in SEQ ID NO. 1-6 is more than or equal to 85 percent (preferably more than or equal to 90 percent, 95 percent and more preferably more than or equal to 98 percent) through gene mutation or other genetic engineering modification modes.

In a preferred embodiment of the invention, the vesicular secretion promoting factor is derived from pichia pastoris, which is a GOD (human immunodeficiency virus) production strain, and a co-expression production strain of the vesicular secretion promoting factor and the GOD is constructed by means of gene recombination. It is understood that one or more vesicle secretagogues, including but not limited to VAMP4, SEC4, EXO84P, can be expressed in the GOD-producing strain by genetic recombination or other genetic engineering means, thereby increasing the yield, activity and/or secretion rate of the GOD.

The main advantages of the invention include

(1) The invention unexpectedly discovers for the first time that the enhancement of the activity of a specific vesicular secretagogue in pichia pastoris (for example, the improvement of the expression level of the vesicular secretagogue) can greatly improve the total yield of proteins inside and outside a GOD cell of the pichia pastoris, particularly the yield of the extracellular GOD.

(2) The GOD yield of the GOD production strain prepared by the construction method is obviously improved compared with that of the original strain.

(3) The extracellular GOD yield of the GOD production strain prepared by the construction method is obviously improved compared with that of the original strain.

The invention is further illustrated by the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specifying the detailed conditions in the following examples, generally according to conventional conditions such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

Materials and methods

1.1 materials

1.1.1 strains and vectors

The recombinant bacterium E5 (GOD gene number is EC 1.1.3.4) secreting and expressing GOD and the expression vector pAOX-UH are both constructed and preserved in the laboratory, wherein the E5 is recombinant pichia pastoris (kan) which takes GS115 as a starting bacterium, has 8 copies of V20W + T30V mutant GOD genes and has signal peptide of GAS ^R HIS 4). Pichia pastoris GS115, E.coli DH 5. Alpha. Were deposited in the laboratory.

1.1.2 Primary reagents and instruments

All tool enzymes used in the experiments, including Exnase II, restriction enzymes (SalI, ndeI, etc.), and Standard nucleic acid molecular weight Marker were purchased from TaKaRa Biotech Ltd. All molecular cloning kits, including plasmid extraction kits, PCR purification kits, and yeast genomic DNA extraction kits were purchased from Axygen corporation. Visible spectrophotometer 721 was obtained from the third analytical instrument works of shanghai; constant temperature shaker TS-S200B was purchased from Shanghai Producer of laboratory instruments, inc.

1.1.3 culture Medium

YPD liquid medium, BMGY liquid medium, BMMY liquid medium, and the respective medium configurations can be found in the Pichia pastoris instruction manual of Invitrogen.

1.2 methods

1.2.1 construction of recombinant plasmids and corresponding recombinant bacteria

Construction of 32 recombinant plasmids 1.2.1.1pAOX-UH-VAMP4/SEC4/EXO84P/EXO70P/STX1-4/YPT

The NCBI database accession numbers of 6 secretagogue gene sequences VAMP4, SEC4, EXO84P, EXO70P, STX1-4 and YPT32 in the vesicle trafficking pathway from Golgi to cytoplasmic membrane are:

VAMP4(PAS_FragB_0011，333bp)；

SEC4(PAS_chr3_0143，615bp)；

EXO84P(PAS_chr4_0078，2181bp)；

EXO70P(PAS_chr4_0695，1866bp)；

STX1-4(PAS_chr1-4_0294，861bp)；

YPT32(PAS_chr1-4_0528，663bp)。

the G/GS115 genome DNA is used as a template to amplify 6 target genes respectively, and PCR amplification primers of each gene are shown in Table 2.

TABLE 2 primers

Due to the introduction of Not I and Xba I enzyme cutting sites at the upstream and downstream of SEC4, EXO84P, EXO70P, STX1-4 and YPT32 genes, amplified target gene fragments can be subjected to NotI and Xba I double enzyme cutting purification, then are subjected to ligation reaction with a linearized vector pAOX-UH which is also subjected to NotI/Xba I double enzyme cutting purification (see figure 1, wherein an 'insertion fragment' represents SEC4, EXO84P, EXO70P, STX1-4 and YPT32 5 different genes), the constructed recombinant plasmids are subjected to heat shock transformation into Escherichia coli DH5 alpha, single colonies are picked on an LB plate, and colony PCR verification is carried out on positive transformants by using primers X-AOF/AOXTT-R2, so that correct strains are verified and sent for sequencing.

Since the VAMP4 gene fragment contains Not I restriction site, the gene co-expression vector can Not be constructed by introducing Not I/Xba I restriction site into the gene fragment and connecting the gene fragment with a linearized vector, so that the construction method of the recombinant plasmid can be constructed by connecting VAMP4 with the Sal I linearized plasmid pAOX-UH by the method of Clon Express II One Step Cloning Kit, and the construction method is shown in FIG. 2. And thermally shocking and transforming the constructed recombinant plasmid into escherichia coli DH5 alpha, selecting a single colony on an LB (Langmuir-Blodgett) plate, carrying out colony PCR (polymerase chain reaction) verification on a positive transformant by using a primer AOX-F/AOXTT-R2, and sending a strain with correct verification to sequencing.

1.2.1.2 Construction of 6 gene co-expression bacteria

After the co-expression vector pAOX-UH-VAMP4/SEC4/EXO84P/EXO70P/STX1-4/YPT32 is linearized by BlnI/XhoI double enzyme digestion, the electric shock is transformed into competent cells of a recombinant bacterium E5, the competent cells are spread on a YPD plate containing hygromycin (the final concentration is 100 mu g/mL) to be cultured for 48h, and positive transformants are obtained by screening and are respectively named as E5-VAMP4, E5-SEC4, E5-EXO84P, E5-EXO70P, E5-STX1-4 and E5-YPT32. The linearized fragment can be integrated into the genome of the recombinant strain E5 by means of homologous recombination (FIG. 3).

1.2.2 Induced expression and correlation analysis of 6 secretion-promoting recombinant bacteria

1.2.2.1 Shake flask fermentation of 6 secretion-promoting recombinant bacteria

Inoculating 6 kinds of recombinant bacteria in YPD culture medium, culturing for 20 hr, taking out a small amount of bacteria, transferring to BMGY culture medium, culturing for about 18 hr to make OD ₆₀₀ = 4-6, collect thalli centrifugally, and use BMMY culture medium to resuspend to OD ₆₀₀ Induction culture was performed in an amount of about 1.2, 1mL was sampled every 24 hours, 1% methanol was supplemented, and the dry weight of the cells and the intracellular and extracellular enzyme production levels of the recombinant bacteria were measured, using E5 as a control.

1.2.2.2 determination of Dry Cell Weight (DCW)

Taking fermentation liquor, diluting to a certain multiple, and measuring OD of thallus ₆₀₀ According to OD ₆₀₀ Relationship to dry weight: DCW (g/L) =0.24 × OD ₆₀₀ +1.23(R ² = 0.994), DCW is calculated.

1.2.2.3 determination of the extracellular and intracellular enzyme production of recombinant bacteria

Treating fermentation liquor: centrifuging 1mL of fresh fermentation broth taken from the shake flask at 12000rpm for 3min, and transferring the supernatant fermentation broth to a 1.5mL EP tube for determination of the extracellular enzyme yield A2; 500. Mu.L of fresh fermentation broth taken from the flask was washed with Breaking Buffer, resuspended at 12000rpm, centrifuged for 5min, and the supernatant removed. mu.L of Breaking Buffer was added to resuspend the cells, followed by an equal volume (500. Mu.L) of 0.5mm pickled glass beads. After the cells were disrupted by a cryomill, they were centrifuged at 12000rpm for 10min, and the supernatant was transferred to a new 1.5mL centrifuge tube for determination of the intracellular enzyme yield A1.

The volume enzyme activity of GOD is measured by an end-point method: 2.5mL of o-dianisidine, 0.3mL of 18% glucose and 0.1mL of 90U/mL of horseradish peroxide were sequentially added to a 10mL centrifuge tube, and after incubation at 37 ℃ for 5min, the centrifuged supernatant enzyme solutions were added to 5 centrifuge tubes, and after reaction for 3min, 2mL of sulfuric acid was added to terminate the reaction, and OD was measured with the reaction without the enzyme solution as a control ₅₃₀ The light absorption value of (2).

Enzyme yield and OD according to volume ₅₃₀ To calculate the volumetric enzyme activity: GOD volumetric enzyme production (U/mL) = (0.1578 XOD) ₅₃₀ —0.0033)×V/V ₀ Wherein V is the total volume of the reaction, V ₀ The volume of enzyme solution added.

GOD yield per microbial cell (U/g.DCW) = volumetric enzyme yield/DCW × 1000, wherein DCW refers to the dry weight of the microbial cells.

( GOD enzyme activity definition: the enzyme amount for catalyzing 1 mu mol of beta-D-glucose to generate gluconic acid per minute is one enzyme activity unit U at 37 ℃. )

The total intracellular and extracellular enzyme yield A0 of the unit thalli is the sum of the intracellular enzyme yield A1 and the extracellular enzyme yield A2.

Example 1 construction of recombinant plasmids and corresponding recombinant bacteria

Using G/GS115 genome DNA as a template, carrying out PCR amplification to obtain 6 target genes, namely VAMP4 (333 bp), SEC4 (615 bp), EXO84P (2181 bp) and EXO70P (1866 bp); STX1-4 (861 bp), YPT32 (663 bp), amplification results are shown in FIG. 4, and the fragment lengths are as expected. 6 target genes are respectively connected with a vector pAOX-UH which is linearized by enzyme digestion to obtain 6 secretion-promoting recombinant plasmids, and sequencing results show that 6 recombinant plasmids are successfully constructed. Then, each recombinant plasmid is transformed into E5 by electric shock, and positive transformants are obtained by screening.

Example 2 induced expression and correlation analysis of secretion-promoting recombinant bacteria

2.1 Growth curve and enzyme yield curve of 6 secretion-promoting recombinant bacteria

As can be seen from FIG. 5A, the growth trends of the secretion-promoting recombinant bacteria E5-VAMP4/SEC4/EXO84P/EXO70P/STX1-4/YPT32 are substantially consistent with those of the control bacteria E5, which indicates that the overexpression of the secretion-promoting genes VAMP4, SEC4, EXO84P, EXO70P, STX1-4 and YPT32 has little obvious influence on the growth of the bacteria.

And (3) measuring the extracellular enzyme yield A2 of unit thalli of different recombinant bacteria, as shown in figure 5B, the extracellular enzyme yields of the unit thalli of the three secretion-promoting recombinant bacteria except E5-VAMP4/SEC4/EX084P are higher than that of the control bacterium E5, and the enzyme yields of the other recombinant bacteria cannot reach the extracellular enzyme yield level of the control bacterium E5. As can be seen from Table 3, the yield of extracellular enzyme per cell of E5-VAMP4 reaches the maximum of 28053.3U/g.DCW at 144h of methanol induction, which is 1.24 times higher than that of E5, and is 24.1% higher than that of E5. In addition, the extracellular enzyme yield of the recombinant bacteria E5-SEC4 and E5-EX084P is respectively 23705.4 and 23200.8U/g.DCW, which are respectively 4.9 percent and 2.6 percent higher than that of the original bacteria E5.

2.2 Comparison of intracellular and extracellular total enzyme yield and secretion rate of 6 secretion-promoting recombinant bacteria

Calculating the total enzyme yield A0 of each recombinant bacterium in the extracellular and intracellular unit, the intracellular enzyme yield A1 of each recombinant bacterium in the induction period of 144h, and the secretion rate (= the extracellular enzyme yield A2/the total enzyme yield A0 multiplied by 100 percent in the extracellular and intracellular unit).

As is clear from FIGS. 6A, 6B and Table 3, the total intracellular and extracellular enzyme yields A0 of E5-VAMP4 per unit cell was 36222.5U/g.DCW, which is 1.11 times higher than that of the control E5, i.e., the yield was increased by 11.3% as compared with A0 of the control E5, at 144 hours of induction. Compared with the control bacterium, the yield of the E5-VAMP4 unit intracellular enzyme is 0.82 times of that of the control bacterium E5, and is reduced by 17.8 percent relative to the A1 of the control bacterium E5. In addition, the secretion rate of E5-VAMP4 was 77.4%, which was improved compared to the secretion rate of E5 (69.50%) of the control bacterium (about 1.11 times the secretion rate of E5).

The results show that the co-expression of the VAMP4 gene is not only beneficial to improving the total enzyme yield inside and outside the cell of the thallus, but also beneficial to improving the secretion rate of the recombinant bacteria, so that the GOD enzyme produced inside the cell is effectively secreted outside the cell. Therefore, the experimental result proves that the VAMP4 gene in the transport pathway from the Golgi apparatus to the plasma membrane vesicle in the pichia pastoris is over-expressed, so that the improvement of the GOD enzyme yield is obviously promoted.

When the induction lasts for 144 hours, the intracellular and extracellular total enzyme yields of E5-EX084P and E5-SEC4 are 33073.6 and 34218.8U/g.DCW respectively, which are respectively improved by 1.6 percent and 5.1 percent compared with the control bacterium E5; compared with the control bacterium E5, the secretion rate of the recombinant bacterium is basically equal, so the extracellular enzyme production level of the two recombinant bacteria is not obviously improved.

Compared with the control bacterium E5, the total enzyme yield inside and outside the cell of the rest recombinant bacteria E5-EXO70P/STX1-4/YPT32 is not improved, and as can be seen from figure 5B, the secretion rate is slightly lower than that of the control bacterium E5, which indicates that the obstruction of the secretion pathway is still one of the limiting bottlenecks of the enzyme production levels of the recombinant bacteria.

TABLE 3 Unit bacterial enzyme yield of recombinant bacteria (144 h methanol induction)

Conclusion

On the basis of 8-copy recombinant bacteria E5 for expressing GOD in a secretory mode, VAMP4, SEC4, EXO84P, EXO70P, STX1-4 and YPT32 genes are respectively overexpressed, wherein the yield of extracellular GOD enzymes of fermentation supernatant of E5-VAMP4/SEC4/EXO84P is improved at the shake flask fermentation level, the recombinant bacteria integrated with the VAMP4 gene are improved most obviously and reach 24.1%, and the residual coexpression bacteria of the E5-EXO70P/STX1-4/YPT32 genes are not improved compared with a control bacteria E5.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

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Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp

85 90 95

Leu Asn Ala Tyr Gly Asp Ile Phe Gly Ser Ser Val Asp His Ala Tyr

100 105 110

Glu Thr Val Glu Leu Ala Thr Asn Asn Gln Thr Ala Leu Ile Arg Ser

115 120 125

Gly Asn Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr

130 135 140

Arg Pro His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn

145 150 155 160

Glu Gly Trp Asn Trp Asp Asn Val Ala Ala Tyr Ser Leu Gln Ala Glu

165 170 175

Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe

180 185 190

Asn Ala Ser Cys His Gly Val Asn Gly Thr Val His Ala Gly Pro Arg

195 200 205

Asp Thr Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala

210 215 220

Val Glu Asp Arg Gly Val Pro Thr Lys Lys Asp Phe Gly Cys Gly Asp

225 230 235 240

Pro His Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val

245 250 255

Arg Ser Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro

260 265 270

Asn Leu Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser

275 280 285

Gln Asn Gly Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His

290 295 300

Lys Gly Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ala

305 310 315 320

Ala Gly Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly

325 330 335

Met Lys Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu

340 345 350

Pro Val Gly Leu Asn Leu Gln Asp Gln Thr Thr Ala Thr Val Arg Ser

355 360 365

Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala

370 375 380

Thr Phe Asn Glu Thr Phe Gly Asp Tyr Ser Glu Lys Ala His Glu Leu

385 390 395 400

Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly

405 410 415

Gly Phe His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg

420 425 430

Asp Trp Ile Val Asn His Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp

435 440 445

Thr Ala Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr

450 455 460

Arg Gly Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu His His Phe

465 470 475 480

Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln

485 490 495

Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met

500 505 510

Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr

515 520 525

Asp Ala Asp Leu Ser Ala Trp Thr Glu Tyr Ile Pro Tyr His Phe Arg

530 535 540

Pro Asn Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met

545 550 555 560

Gly Gly Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu

565 570 575

Arg Val Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val

580 585 590

Met Thr Val Phe Tyr Ala Met Ala Leu Lys Ile Ser Asp Ala Ile Leu

595 600 605

Glu Asp Tyr Ala Ser Met Gln

610 615

<210> 8

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atctgaatag cgccgtcgac atgtcttctt cagtcccata tgaccc 46

<210> 9

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atgatgatga tgatggtcga ttatgtgaaa tgaacaacaa taggaacga 49

<210> 10

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ttgcggccgc aaatggcatc aagaggcaca tcaaggca 38

<210> 11

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gctctagagc tcaacaacaa gacgatttgg tgtcgttgg 39

<210> 12

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ttgcggccgc aaatgggagg ggattactct ttacgaaag 39

<210> 13

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gctctagagc ttagaaagcg tatgtatctt ccattaaaaa gtc 43

<210> 14

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ttgcggccgc aaatgcaacg aatcccaatt gatttgg 37

<210> 15

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gctctagagc ttaaatggaa tctagcattc tatcaaaacc gc 42

<210> 16

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ttgcggccgc aaatgagtaa ccagtataat ccgtatgagc ag 42

<210> 17

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gctctagagc ctatcttccc cagtttccga cacc 34

<210> 18

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ttgcggccgc aaatgtcaaa cagtgctgaa gattactctt ac 42

<210> 19

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gctctagagc ctaacaacat gaatccttct tgattttact atc 43

<210> 20

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

ggaagctgcc ctgtcttaaa cctt 24

<210> 21

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

aatgaagcct gcatctctca ggc 23

<210> 22

<211> 583

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

Ser Asn Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Asp Val Ser

1 5 10 15

Gly Arg Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu

20 25 30

Thr Thr Ala Ala Arg Leu Thr Glu Asn Pro Asn Ile Ser Val Leu Val

35 40 45

Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp

50 55 60

Leu Asn Ala Tyr Gly Asp Ile Phe Gly Ser Ser Val Asp His Ala Tyr

65 70 75 80

Glu Thr Val Glu Leu Ala Thr Asn Asn Gln Thr Ala Leu Ile Arg Ser

85 90 95

Gly Asn Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr

100 105 110

Arg Pro His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn

115 120 125

Glu Gly Trp Asn Trp Asp Asn Val Ala Ala Tyr Ser Leu Gln Ala Glu

130 135 140

Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe

145 150 155 160

Asn Ala Ser Cys His Gly Val Asn Gly Thr Val His Ala Gly Pro Arg

165 170 175

Asp Thr Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala

180 185 190

Val Glu Asp Arg Gly Val Pro Thr Lys Lys Asp Phe Gly Cys Gly Asp

195 200 205

Pro His Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val

210 215 220

Arg Ser Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro

225 230 235 240

Asn Leu Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser

245 250 255

Gln Asn Gly Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His

260 265 270

Lys Gly Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ala

275 280 285

Ala Gly Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly

290 295 300

Met Lys Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu

305 310 315 320

Pro Val Gly Leu Asn Leu Gln Asp Gln Thr Thr Ala Thr Val Arg Ser

325 330 335

Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala

340 345 350

Thr Phe Asn Glu Thr Phe Gly Asp Tyr Ser Glu Lys Ala His Glu Leu

355 360 365

Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly

370 375 380

Gly Phe His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg

385 390 395 400

Asp Trp Ile Val Asn His Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp

405 410 415

Thr Ala Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr

420 425 430

Arg Gly Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu His His Phe

435 440 445

Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln

450 455 460

Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met

465 470 475 480

Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr

485 490 495

Asp Ala Asp Leu Ser Ala Trp Thr Glu Tyr Ile Pro Tyr His Phe Arg

500 505 510

Pro Asn Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met

515 520 525

Gly Gly Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu

530 535 540

Arg Val Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val

545 550 555 560

Met Thr Val Phe Tyr Ala Met Ala Leu Lys Ile Ser Asp Ala Ile Leu

565 570 575

Glu Asp Tyr Ala Ser Met Gln

580

Claims (10)

1. A method of increasing expression and/or activity of Glucose Oxidase (GOD) in a yeast cell, comprising the steps of:

enhancing expression and/or activity of a vesicle secretagogue or a gene thereof in a yeast cell in a starting strain to thereby obtain a Glucose Oxidase (GOD) producing strain having enhanced expression of GOD and/or activity of GOD, wherein,

the vesicular secretagogue comprises one or more proteins selected from the group consisting of:

(1) A protein having an amino acid sequence as set forth in any one of SEQ ID No. 1-6;

(2) 1-6 by substitution, deletion or addition of one or more amino acid residues, and the protein with the vesicle secretor promoting factor activity and derived from the (1); and/or

(3) The homology of the amino acid sequence and any sequence shown in SEQ ID NO. 1-6 is more than or equal to 85 percent (preferably more than or equal to 90 percent, 95 percent, more preferably more than or equal to 98 percent), and the protein has the vesicle secretion promoting factor activity.

2. The method of claim 1, wherein the vesicular secretagogue is selected from one or more of the proteins having the amino acid sequences shown in SEQ ID No. 1, 2 and/or 5.

3. A GOD-producing strain having enhanced activity of a vesicular secretagogue, wherein the vesicular secretagogue comprises one or more proteins selected from the group consisting of:

(1) A protein having an amino acid sequence as set forth in any one of SEQ ID No. 1-6;

(2) 1-6 by substitution, deletion or addition of one or more amino acid residues, and the protein with the vesicle secretor promoting factor activity and derived from the (1); and/or

(3) The homology of the amino acid sequence and any sequence shown in SEQ ID NO. 1-6 is more than or equal to 85 percent (preferably more than or equal to 90 percent, 95 percent and more preferably more than or equal to 98 percent), and the protein has the vesicle secretion promoting factor activity.

4. A method of producing a GOD protein, comprising the steps of:

1) Fermentatively culturing the GOD producing strain of claim 3, thereby obtaining a GOD protein; and

2) Optionally obtaining the GOD protein from the fermentation culture system of 1).

5. A method for enhancing the GOD protein production capacity of a strain, comprising the steps of:

enhancing the activity of vesicular secretagogues in a starting strain, wherein the vesicular secretagogues comprise one or more proteins selected from the group consisting of:

(1) A protein having an amino acid sequence as set forth in any one of SEQ ID No. 1-6;

(2) 1-6 by substitution, deletion or addition of one or more amino acid residues, and the protein with the vesicle secretor promoting factor activity and derived from the (1); and/or

(3) The homology of the amino acid sequence and any sequence shown in SEQ ID NO. 1-6 is more than or equal to 85 percent (preferably more than or equal to 90 percent, 95 percent, more preferably more than or equal to 98 percent), and the protein has the vesicle secretion promoting factor activity.

6. Use of a vesicular secretagogue or a gene promoter therefor for enhancing the GOD protein production capacity of a strain, the vesicular secretagogue comprising one or more proteins selected from the group consisting of:

(1) A protein having an amino acid sequence as set forth in any one of SEQ ID No. 1-6;

(2) 1, protein which is formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence shown in any one of SEQ ID NO. 1-6, has the vesicle secretor promoting factor activity and is derived from the protein (1); and/or

(3) The homology of the amino acid sequence and any sequence shown in SEQ ID NO. 1-6 is more than or equal to 85 percent (preferably more than or equal to 90 percent, 95 percent, more preferably more than or equal to 98 percent), and the protein has the vesicle secretion promoting factor activity.

7. A vesicular secretagogue, which is a protein selected from one or more of the following proteins:

(1) A protein having an amino acid sequence as set forth in any one of SEQ ID No. 1-6;

(2) 1-6 by substitution, deletion or addition of one or more amino acid residues, and the protein with the vesicle secretor promoting factor activity and derived from the (1); and/or

(3) The homology of the amino acid sequence and any sequence shown in SEQ ID NO. 1-6 is more than or equal to 85 percent (preferably more than or equal to 90 percent, 95 percent and more preferably more than or equal to 98 percent), and the protein has the vesicle secretion promoting factor activity.

8. A polynucleotide encoding a vesicular secretagogue according to claim 7, wherein the polynucleotide encodes the vesicular secretagogue.

9. A vector comprising the polynucleotide of claim 8.

10. Use of the vesicular secretagogue according to claim 7, the polynucleotide encoding a vesicular secretagogue according to claim 8, the vector according to claim 9 for the preparation of the GOD-producing strain according to claim 3.

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