Recombinant engineering bacteria for preparing recombinant human albumin based on transport and secretion pathway and its application

Abstract

The present invention provides a recombinant engineering bacterium for preparing recombinant human albumin based on the transport secretion pathway and its application, and relates to the field of genetic engineering technology. The present invention integrates one or more of the endoplasmic reticulum-related genes SRP54 and ERJ5, the vesicle transport-related genes YOS1 and SLY1, and the Golgi apparatus-related genes GOS1 and YIP1 into the genome of the recombinant engineering bacterium through gene recombination technology, thereby realizing efficient secretory expression of recombinant human albumin. The method provided by the present invention significantly improves the expression amount of recombinant human albumin and has broad application prospects.

Description

Technical Field

[0001] The present application relates to the field of genetic engineering technology, and in particular to a recombinant engineering bacterium for preparing recombinant human albumin based on a transport-secretion pathway and its application. Background Art

[0002] Pichia pastoris is one of the most widely used model microorganisms in the fermentation industry. The exogenous gene expression system using it as a host has developed rapidly in recent years and is widely used in various fields such as biomedicine, cosmetics, feed, industrial enzyme preparations and basic research. It has advantages among many secretory expression systems. The secretory pathway of the Pichia pastoris expression system is mainly as follows: after the target gene is transcribed into mRNA, the nascent peptide chain is synthesized on the ribosome, transported to the endoplasmic reticulum through the protein channel on the endoplasmic reticulum membrane for folding and modification, and after the quality inspection element in the secretion system detects the correct folding, it is then transported to the Golgi apparatus for glycosylation modification to form a protein with a specific structure and function, and finally secreted outside the cell through vesicle transport.

[0003] With the development of science and technology, there are more and more methods to improve the secretion of heterologous proteins, among which the most traditional method is to increase the copy number of the recombinant protein encoding gene. In theory, as the copy number of the target gene increases, the protein expression should increase accordingly; however, in practice, when the copy number increases to a certain number, due to factors such as the obstruction of the transport and secretion pathways, the production efficiency of the protein is limited, and its expression decreases instead, which is not conducive to achieving the purpose of increasing production.

[0004] As studies on the secretory expression of heterologous proteins in yeast have shown, the expression levels of different proteins vary greatly, and some heterologous proteins have limited secretion capacity, which limits the production efficiency of proteins. Therefore, it is crucial to improve the secretion efficiency and yield of proteins by modifying genes related to the protein transport and secretion pathway of Pichia pastoris and maintaining the smoothness of the protein secretion pathway. This provides a new idea for increasing the yield of target proteins. Summary of the invention

[0005] In view of the problems and shortcomings in the prior art, the present invention solves the problem of reduced heterologous protein production due to excessive increase in copy number by modifying the transport and secretion pathway of the host bacteria. This provides a new direction for increasing the content of heterologous protein. The content of the present invention is as follows:

[0006] In a first aspect, the present invention provides a recombinant engineered bacterium for preparing recombinant human albumin based on the transport-secretion pathway. The recombinant engineered bacterium, while expressing the recombinant albumin, also comprises the following operations: integrating one or more recombinant expression cassettes of endoplasmic reticulum-related genes, vesicle transport-related genes, and Golgi apparatus-related genes into the genome of the recombinant engineered bacterium through gene recombination technology, wherein: the endoplasmic reticulum-related genes are selected from one or more of SRP54 and ERJ5; the vesicle transport-related genes are selected from one or more of YOS1 and SLY1; the Golgi apparatus-related genes are selected from one or more of GOS1 and YIP1.

[0007] Furthermore, the amino acid sequence encoded by the SRP54 gene is shown in SEQ ID NO.1; the amino acid sequence encoded by the ERJ5 gene is shown in SEQ ID NO.3; the amino acid sequence encoded by the YOS1 gene is shown in SEQ ID NO.5; the amino acid sequence encoded by the SLY1 gene is shown in SEQ ID NO.7; the amino acid sequence encoded by the GOS1 gene is shown in SEQ ID NO.9; and the amino acid sequence encoded by the YIP1 gene is shown in SEQ ID NO.11.

[0008] Furthermore, the SRP54 gene nucleotide sequence is a nucleotide sequence encoding the amino acid sequence described in SEQ ID NO.1, including a nucleotide sequence that has been disclosed or a nucleotide sequence optimized according to actual needs; the ERJ5 gene nucleotide sequence is a nucleotide sequence encoding the amino acid sequence described in SEQ ID NO.3, including a nucleotide sequence that has been disclosed or a nucleotide sequence optimized according to actual needs; the YOS1 gene nucleotide sequence is a nucleotide sequence encoding the amino acid sequence described in SEQID NO.5; the SLY1 gene nucleotide sequence is a nucleotide sequence encoding the amino acid sequence described in SEQ ID NO.7; the GOS1 gene nucleotide sequence is a nucleotide sequence encoding the amino acid sequence described in SEQ ID NO.9; the YIP1 gene nucleotide sequence is a nucleotide sequence encoding the amino acid sequence described in SEQ ID NO.11.

[0009] Preferably, the SRP54 gene nucleotide sequence is as shown in SEQ ID NO.2 or has 98% homology with the nucleotide sequence shown in SEQ ID NO.2; the ERJ5 gene nucleotide sequence is as shown in SEQ ID NO.4 or has 98% homology with the nucleotide sequence shown in SEQ ID NO.4; the YOS1 gene nucleotide sequence is as shown in SEQ ID NO.6 or has 98% homology with the nucleotide sequence shown in SEQ ID NO.6; the SLY1 gene nucleotide sequence is as shown in SEQ ID NO.8 or has 98% homology with the nucleotide sequence shown in SEQ ID NO.8; the GOS1 gene nucleotide sequence is as shown in SEQ ID NO.10 or has 98% homology with the nucleotide sequence shown in SEQ ID NO.10; the YIP1 gene nucleotide sequence is as shown in SEQ ID NO.12 or has 98% homology with the nucleotide sequence shown in SEQ ID NO.12.

[0010] Furthermore, the recombinant engineered bacteria contains at least one copy of an expression cassette encoding recombinant human albumin. Preferably, the number of copies of the nucleotide sequence encoding recombinant human albumin in the recombinant engineered bacteria is 2 to 6. In an embodiment of the present invention, the number of copies of the nucleotide sequence encoding recombinant human albumin in the recombinant engineered bacteria is 4.

[0011] Furthermore, the recombinant engineered bacteria also include at least one of a promoter sequence and a screening signal peptide sequence. Preferably, the expression cassette includes: an AOX1 promoter, an a-factor signal peptide and a human albumin nucleotide sequence.

[0012] Furthermore, the recombinant engineered bacteria of the present invention further include drug resistance gene fragments. The drug resistance gene fragments are used for screening recombinant engineered bacteria. In an embodiment of the present invention, the drug resistance fragments are one or more of bleomycin, blasticidin resistance, hygromycin B, and geneticin G418 resistance genes.

[0013] Furthermore, the recombinant engineered bacteria also include a signal peptide sequence, and the signal peptide sequence is used for the secretory expression of exogenous proteins in the recombinant engineered bacteria. In an embodiment of the present invention, the signal peptide sequence in the recombinant engineered bacteria is a mating factor signal peptide of Saccharomyces cerevisiae a; preferably, the nucleotide sequence encoding the mating factor signal peptide of Saccharomyces cerevisiae a is shown in SEQ ID NO.13.

[0014] Furthermore, the recombinant expression cassette encoding at least one gene among SRP54, ERJ5, YOS1, SLY1, GOS1, and YIP1 is on a nucleic acid construct of the same selection marker or on a nucleic acid construct of a different selection marker.

[0015] Furthermore, the recombinant engineered bacteria is selected from one or more of Pichia pastoris, Hansenula, Candida, and Saccharomyces cerevisiae. Preferably, the recombinant engineered bacteria is Pichia pastoris; more preferably, the Pichia pastoris is CBS7435.

[0016] The second aspect of the present invention provides the use of the recombinant engineered bacteria in preparing recombinant albumin and / or increasing the expression level of recombinant albumin.

[0017] The third invention of the present invention provides a method for preparing the recombinant engineered bacteria, characterized in that the method includes the step of integrating one or more recombinant expression cassettes of endoplasmic reticulum-related genes, vesicle transport-related genes, and Golgi apparatus-related genes into the genome of the recombinant engineered bacteria through gene recombination technology, wherein: the endoplasmic reticulum-related genes are selected from one or more of SRP54 and ERJ5; the vesicle transport-related genes are selected from one or more of YOS1 and SLY1; the Golgi apparatus-related genes are selected from one or more of GOS1 and YIP1.

[0018] In a fourth aspect, the present invention provides a method for preparing recombinant albumin, the method comprising: a step of preparing the recombinant albumin by fermentation using the recombinant engineered bacteria.

[0019] The beneficial effects of the present invention include but are not limited to:

[0020] The present invention provides a recombinant engineering bacterium for preparing recombinant human albumin based on the transport and secretion pathway, which can significantly increase the expression of human recombinant albumin. The present invention strengthens the protein transport pathway and maintains smooth secretion by overexpressing one or more of the endoplasmic reticulum-related genes SRP54 and ERJ5, the vesicle transport-related genes YOS1 and SLY1, and the Golgi apparatus-related genes GOS1 and YIP1, thereby significantly enhancing the secretion capacity of heterologous proteins and solving the problem of decreased yield due to excessive increase in copy number. The method shows strong potential in increasing the yield of proteins with high economic value and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0022] Figure 1 It is a schematic diagram of the structure of the recombinant expression cassette pART301 encoding the recombinant nucleic acid molecule of human albumin;

[0023] Figure 2 It is a schematic diagram of the structure of the recombinant expression cassette pART302 encoding the SRP54 recombinant nucleic acid molecule;

[0024] Figure 3 It is a schematic diagram of the structure of the recombinant expression cassette pART303 encoding the ERJ5 recombinant nucleic acid molecule;

[0025] Figure 4 It is a schematic diagram of the structure of the recombinant expression cassette pART304 encoding the SRP54 recombinant nucleic acid molecule and the ERJ5 recombinant nucleic acid molecule;

[0026] Figure 5 It is a schematic diagram of the structure of the recombinant expression cassette pART305 encoding the YOS1 recombinant nucleic acid molecule;

[0027] Figure 6 It is a schematic diagram of the structure of the recombinant expression cassette pART306 encoding the SLY1 recombinant nucleic acid molecule;

[0028] Figure 7 It is a schematic diagram of the structure of the recombinant expression cassette pART307 encoding the YOS1 recombinant nucleic acid molecule and the SLY1 recombinant nucleic acid molecule;

[0029] Figure 8 It is a schematic diagram of the structure of the recombinant expression cassette pART308 encoding the GOS1 recombinant nucleic acid molecule;

[0030] Fig. 9 It is a schematic diagram of the structure of the recombinant expression cassette pART309 encoding the YIP1 recombinant nucleic acid molecule;

[0031] Fig.10 It is a schematic diagram of the structure of the recombinant expression cassette pART310 encoding the GOS1 recombinant nucleic acid molecule and the YIP1 recombinant nucleic acid molecule;

[0032] Fig.11 This is the result diagram of the qPCR standard curve of the HIS4 gene copy number detection in Example 1;

[0033] Fig.12 This is the result diagram of the qPCR standard curve of the rHA gene copy number detection in Example 1;

[0034] Fig.13 This is a graph showing the results of the recombinant human albumin expressed by the recombinant engineering bacteria in Example 4. DETAILED DESCRIPTION

[0035] The present invention is described in detail below with reference to examples, but the present invention is not limited to these examples. Unless otherwise specified, the raw materials and catalysts in the examples of the present invention are purchased through commercial channels.

[0036] Recombination: Broadly speaking, any gene exchange process that causes genotype changes is called recombination.

[0037] Recombinant engineered bacteria: a fungal cell strain that uses genetic engineering methods to efficiently express exogenous genes.

[0038] Expression cassette: refers to a gene expression system that contains all the necessary elements required for the expression of foreign proteins, including promoter, foreign gene cloning site, signal peptide sequence, mature peptide coding sequence of the target protein, terminator, selection marker, etc.

[0039] Vector: refers to autonomous DNA that can bring foreign DNA into host cells for replication or ultimately expression of foreign gene DNA. It is mainly divided into cloning vectors and expression vectors. The former is mainly used for gene replication and amplification, while the latter is mainly used for the expression of target genes.

[0040] In order to more clearly explain the overall concept of the present application, the following is described in detail in conjunction with the accompanying drawings of the specification by way of embodiment. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0041] If no specific conditions are specified in the examples, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer.

[0042] Unless otherwise specified, in the following embodiments, the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0043] The molecular biology experimental methods not specifically described in the following examples were all carried out according to the kits and product instructions, or with reference to the methods described in the book Molecular Cloning Experiment Guide (3rd edition) by J. Sambrook.

[0044] The sources of experimental materials and reagents involved in the present invention are as follows.

[0045] Strain: Pichia pastoris CBS7435 was purchased from the American Type Culture Collection (ATCC) on behalf of Ningbo Mingzhou Biotechnology Co., Ltd. It is also known as Komagataella phaffii NRRL Y-11430, ATCC Number: 76273™.

[0046] Expression vector: The expression vector pPICZA was purchased from Hunan Fenghui Biotechnology Co., Ltd., catalog number JM006; the following expression cassettes were constructed in the laboratory: 1) Expression cassette, which encodes at least one recombinant nucleic acid molecule of human albumin, the structural diagram of which is shown in Figure 1 2) an expression cassette encoding a recombinant nucleic acid molecule of SRP54, the structure of which is shown in FIG. Figure 2 3) An expression cassette encoding an ERJ5 recombinant nucleic acid molecule, the structure of which is shown in FIG. Figure 3 4) an expression cassette encoding a SRP54 recombinant nucleic acid molecule and an ERJ5 recombinant nucleic acid molecule, the structure of which is shown in FIG. Figure 4 5) An expression cassette encoding a YOS1 recombinant nucleic acid molecule, the structure of which is shown in FIG. Figure 5 6) An expression cassette encoding a SLY1 recombinant nucleic acid molecule, the structure of which is shown in FIG. Figure 6 7) An expression cassette encoding a YOS1 recombinant nucleic acid molecule and a SLY1 recombinant nucleic acid molecule, the structure of which is shown in FIG. Figure 7 8) An expression cassette encoding a GOS1 recombinant nucleic acid molecule, the structure of which is shown in FIG. Figure 8 9) An expression cassette encoding a YIP1 recombinant nucleic acid molecule, the structure of which is shown in FIG. Fig. 9 10) An expression cassette encoding a GOS1 recombinant nucleic acid molecule and a YIP1 recombinant nucleic acid molecule, the structure of which is shown in FIG. Fig.10 shown.

[0047] Enzymes and kits

[0048] Restriction endonuclease BglII (Cat. No. R0144S, specification 2000 units) was purchased from NEB; plasmid extraction kit (Cat. No. DP103-02, specification 50 preps), agarose gel recovery kit (Cat. No. DP209-02, specification 50 preps), DNA product purification kit (Cat. No. DP204-02, specification 50 preps), yeast genomic DNA extraction kit (Cat. No. DP307-02, specification 50 preps) were purchased from Tiangen Biotechnology Co., Ltd.; PrimeSTAR high-fidelity DNA polymerase (Cat. No. R047A, specification 100 Rxns) was purchased from Takara; Zeocin (Cat. No. R250-01, specification 10 ml), Geneticin G418 (Cat. No. 10131035, specification 20 ml), Blasticidin (Cat. No. A1113902, specification 20 ml), Hygromycin B B (Cat. No. 10687010, specification 20 ml) was purchased from Thermo Fisher Scientific.

[0049] Culture medium

[0050] Escherichia coli culture medium LB (1% (w / v) tryptone, 0.5% (w / v) yeast extract, 1% (w / v) NaCl, pH 7.0).

[0051] Yeast medium YPD (1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose); yeast screening medium YPDZ (YPD + different concentrations of zeocin); YPDG (YPD + different concentrations of Geneticin); YPDB (YPD + different concentrations of Blasticidin); YPDH (YPD + different concentrations of Hygromycin B).

[0052] Yeast medium BMGY (1% (w / v) yeast extract, 2% (w / v) peptone, 1.34% (w / v) YNB, 0.00004% (w / v) Biotin, 1% glycerol (V / V)), yeast induction medium BMMY (1% (w / v) yeast extract, 2% (w / v) peptone, 1.34% (w / v) YNB, 0.00004% (w / v) Biotin, 0.4% methanol (V / V)), Note: YNB is yeast nitrogen base (Yeast Nitrogen Base); Biotin is biotin.

[0053] Example 1: Construction of recombinant human albumin Pichia pastoris strain

[0054] The nucleic acid molecule encoding the signal peptide of the mating factor of Saccharomyces cerevisiae a (SEQ ID NO.13) and the nucleic acid molecule encoding the recombinant human albumin (amino acid sequence such as SEQ ID NO.14, nucleotide sequence such as SEQ ID NO.15) were commissioned to be directly synthesized by a biological company, and then ligated into the plasmid pPICZA digested with EcoRI and NotI to form an expression vector named pART301 (such as Figure 1 as shown).

[0055] The expression vector was linearized with DNA restriction endonuclease BglII and transformed into Pichia pastoris CBS7435 by electroporation. The cells were spread on selection plates YPDZ containing different high concentrations of zeocin antibiotics and cultured at 30°C until colonies appeared. Positive transformants were identified by colony PCR and stored in glycerol tubes.

[0056] The genomic DNA of randomly selected transformants was extracted using a yeast genome extraction kit, and real-time fluorescence quantitative PCR was performed using a double standard curve method using vectors containing the endogenous gene HIS4 and the exogenous gene recombinant human albumin in single copy form, respectively, to establish a standard curve and detect the copy number of recombinant human albumin in each transformant.

[0057] The pPIC9K vector (purchased from Invitrogen) containing a single copy of the HIS4 gene and the pART301 vector containing a single copy of the recombinant human albumin gene were used as standards to establish a double standard curve. The starting plasmid copy number was 109, and a 10-fold dilution was performed, with a total of 6 dilution gradients, and 2 parallel samples in each gradient were used as the standard curve for amplification. The extracted genome of each copy of the transformant was diluted to 10 ng / μL as a PCR template.

[0058] The fluorescence quantitative PCR reaction system is shown in Table 1, and the specific nucleotide sequences of the upstream primer and the downstream primer are shown in Table 2.

[0059] Table 1

[0060]

[0061] Table 2

[0062]

[0063] Fluorescence quantitative PCR reaction parameters:

[0064] Stage 1: 95°C for 3 min; Stage 2: 95°C for 5 sec, 60°C for 20 sec, repeated for 40 cycles; Stage 3: 95°C for 10 sec, 72°C for 5 min, 97°C for 1 sec.

[0065] Substitute the cycle number Ct of each copy transformant test solution into the standard curve (HIS4 gene standard curve is as follows Fig.11 As shown, the rHA gene standard curve is as follows Fig.12 The copy number concentrations of the starting templates of the endogenous gene HIS4 and the exogenous gene rHA in the genome were obtained, and the copy number results of the transformants obtained by the screening after transformation were calculated by the ratio of the copy number of the exogenous gene starting template to the copy number of the endogenous gene starting template. The specific information is shown in Table 3.

[0066] Table 3

[0067]

[0068] Example 2: Construction of gene expression vectors related to protein transport and secretion pathways

[0069] The nucleic acid molecule encoding geneticin G418 (SEQ ID NO.16) is connected to pPICZA to replace the Zeocin resistance gene to form a plasmid with geneticin G418 resistance, and the nucleic acid molecule encoding the constitutive promoter GAP and at least one of the endoplasmic reticulum-related genes SRP54 and ERJ5 is connected to the plasmid to form an expression vector pART302 containing the SRP54 gene, an expression vector pART303 containing the ERJ5 gene, and an expression vector pART304 containing the SRP54 gene and the ERJ5 gene, respectively.

[0070] The nucleic acid molecule encoding blasticidin (SEQ ID NO.17) was connected to pPICZA to replace the Zeocin resistance gene to form a blasticidin resistance gene, and the nucleic acid molecule encoding the constitutive promoter GAP and at least one of the vesicle transport related genes YOS1 and SLY1 were connected to the plasmid to form an expression vector pART305 containing the YOS1 gene, an expression vector pART306 containing the SLY1 gene, and an expression vector pART307 containing the YOS1 gene and the SLY1 gene, respectively.

[0071] A nucleic acid molecule encoding Hygromycin B (SEQ ID NO.18) is connected to pPICZA to replace the Zeocin resistance gene to form a plasmid having Hygromycin B resistance, and a nucleic acid molecule encoding a constitutive promoter GAP and at least one of the Golgi apparatus-related genes GOS1 and YIP1 are connected to the plasmid to form an expression vector pART308 containing the GOS1 gene, an expression vector pART309 containing the YIP1 gene, and an expression vector pART310 containing the GOS1 gene and the YIP1 gene, respectively.

[0072] Among them, the amino acid sequence of SRP54 is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2; the amino acid sequence of ERJ5 is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4; the amino acid sequence of YOS1 is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.6; the amino acid sequence of SLY1 is shown in SEQ ID NO.7, and the nucleotide sequence is shown in SEQ ID NO.8; the amino acid sequence of GOS1 is shown in SEQ ID NO.9, and the nucleotide sequence is shown in SEQ ID NO.10; the amino acid sequence of YIP1 is shown in SEQ ID NO.11, and the nucleotide sequence is shown in SEQ ID NO.12.

[0073] Example 3: Construction of a recombinant human albumin strain co-expressing genes related to the transport and secretion pathway

[0074] The different endoplasmic reticulum, vesicle transport and Golgi apparatus related gene expression vectors were combined and optimized, and the recombinant engineered bacteria 4 in Example 1 were transformed respectively, spread on selection plates containing different antibiotics, and cultured at 30° C. until colonies appeared. The positive transformants were identified by colony PCR. The specific transformation conditions are shown in Table 4.

[0075] Table 4

[0076]

[0077] Example 4: Expression level of human albumin in each recombinant engineered bacteria

[0078] Recombinant engineered bacteria 1 to 9 (specific information is shown in Tables 3 and 4) were taken respectively, inoculated into 40 mL of BMGY culture medium, cultured at 220 rpm and 30°C overnight, the bacterial solution was centrifuged, the precipitate was resuspended in 40 mL of BMMY culture medium, induced at 220 rpm and 25°C, 0.4% methanol was added every 24 h, centrifuged at 10,000 rpm for 5 min after 72 h, and the supernatant was collected. The supernatant was subjected to SDS-PAGE electrophoresis, and the specific results are shown in Tables 5 and Fig.13 shown.

[0079] The results showed that: taking the recombinant human albumin expression level of recombinant engineered bacteria 1 as a control, when the copy number of recombinant human albumin was 4, the expression level of recombinant engineered bacteria 4 that did not overexpress genes related to protein transport and secretion pathways was only 57%, and the protein secretion capacity was limited, which restricted the protein production efficiency; while on the basis of recombinant engineered bacteria 4, when the endoplasmic reticulum-related genes SRP54 and ERJ5, vesicle transport-related genes YOS1 and SLY1, and Golgi apparatus-related genes GOS1 and YIP1 were overexpressed at the same time, the expression level of recombinant engineered bacteria 9 was significantly increased, reaching 356%.

[0080] Table 5

[0081]

[0082] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. A recombinant engineering bacterium for preparing recombinant human albumin based on the transport and secretion pathway, characterized in that: The recombinant engineering bacteria, while expressing recombinant human albumin, also includes the following operations: integrating one or more recombinant expression cassettes of endoplasmic reticulum-related genes, vesicle transport-related genes, and Golgi apparatus-related genes into the genome of the recombinant engineering bacteria by gene recombination technology for overexpression, wherein the overexpressed gene is selected from any one of the following: A. The endoplasmic reticulum-related gene is the SRP54 gene, the vesicle transport-related gene is the YOS1 gene, and the Golgi apparatus-related gene is the GOS1 gene; B. The endoplasmic reticulum-related gene is the ERJ5 gene, the vesicle transport-related gene is the SLY1 gene, and the Golgi apparatus-related gene is the YIP1 gene; C. The endoplasmic reticulum-related genes are SRP54 gene and ERJ5 gene, the vesicle transport-related gene is SLY1 gene, and the Golgi apparatus-related gene is YIP1 gene; D. The endoplasmic reticulum-related genes are SRP54 gene and ERJ5 gene, the vesicle transport-related genes are YOS1 gene and SLY1 gene, and the Golgi apparatus-related gene is YIP1 gene; E. The endoplasmic reticulum-related genes are SRP54 gene and ERJ5 gene, the vesicle transport-related genes are YOS1 gene and SLY1 gene, and the Golgi apparatus-related genes are GOS1 gene and YIP1 gene; The amino acid sequence encoded by the SRP54 gene is shown in SEQ ID NO.1; the amino acid sequence encoded by the ERJ5 gene is shown in SEQ ID NO.3; the amino acid sequence encoded by the YOS1 gene is shown in SEQ ID NO.5; the amino acid sequence encoded by the SLY1 gene is shown in SEQ ID NO.7; the amino acid sequence encoded by the GOS1 gene is shown in SEQ ID NO.9; the amino acid sequence encoded by the YIP1 gene is shown in SEQ ID NO.11; The recombinant engineering bacteria is Pichia pastoris; The recombinant engineering bacteria contain 4 copies of expression cassettes encoding recombinant human albumin.

2. The recombinant engineered bacterium according to claim 1, characterized in that The nucleotide sequence of the SRP54 gene is shown in SEQ ID NO.2; the nucleotide sequence of the ERJ5 gene is shown in SEQ ID NO.4; the nucleotide sequence of the YOS1 gene is shown in SEQ ID NO.6; the nucleotide sequence of the SLY1 gene is shown in SEQ ID NO.8; the nucleotide sequence of the GOS1 gene is shown in SEQ ID NO.10; and the nucleotide sequence of the YIP1 gene is shown in SEQ ID NO.

12.

3. The recombinant engineered bacterium according to claim 1, characterized in that The recombinant engineering bacteria further comprises at least one of a promoter sequence and a screening signal peptide sequence.

4. The recombinant engineered bacterium according to claim 1, characterized in that The recombinant expression cassette encoding at least one gene among SRP54, ERJ5, YOS1, SLY1, GOS1, and YIP1 is on the nucleic acid construct of the same selection marker or on the nucleic acid construct of different selection markers.

5. Use of the recombinant engineered bacteria according to any one of claims 1 to 4 in preparing recombinant human albumin and / or increasing the expression level of recombinant human albumin.

6. A method for preparing the recombinant engineered bacteria according to any one of claims 1 to 4, characterized in that: The method comprises the steps of integrating one or more recombinant expression cassettes of endoplasmic reticulum-related genes, vesicle transport-related genes, and Golgi apparatus-related genes into the genome of a recombinant engineering bacterium expressing human albumin by gene recombination technology for overexpression, wherein the overexpressed gene is selected from any one of the following: A. The endoplasmic reticulum-related gene is the SRP54 gene, the vesicle transport-related gene is the YOS1 gene, and the Golgi apparatus-related gene is the GOS1 gene; B. The endoplasmic reticulum-related gene is the ERJ5 gene, the vesicle transport-related gene is the SLY1 gene, and the Golgi apparatus-related gene is the YIP1 gene; C. The endoplasmic reticulum-related genes are SRP54 gene and ERJ5 gene, the vesicle transport-related gene is SLY1 gene, and the Golgi apparatus-related gene is YIP1 gene; D. The endoplasmic reticulum-related genes are SRP54 gene and ERJ5 gene, the vesicle transport-related genes are YOS1 gene and SLY1 gene, and the Golgi apparatus-related gene is YIP1 gene; E. The endoplasmic reticulum-related genes are SRP54 gene and ERJ5 gene, the vesicle transport-related genes are YOS1 gene and SLY1 gene, and the Golgi apparatus-related genes are GOS1 gene and YIP1 gene; The amino acid sequence encoded by the SRP54 gene is shown in SEQ ID NO.1; the amino acid sequence encoded by the ERJ5 gene is shown in SEQ ID NO.3; the amino acid sequence encoded by the YOS1 gene is shown in SEQ ID NO.5; the amino acid sequence encoded by the SLY1 gene is shown in SEQ ID NO.7; the amino acid sequence encoded by the GOS1 gene is shown in SEQ ID NO.9; the amino acid sequence encoded by the YIP1 gene is shown in SEQ ID NO.11; The recombinant engineering bacteria is Pichia pastoris; The recombinant engineering bacteria contain 4 copies of expression cassettes encoding recombinant human albumin.

7. A method for preparing recombinant human albumin, characterized in that: The method comprises the step of preparing recombinant human albumin by fermentation using the recombinant engineered bacteria as described in any one of claims 1 to 4.

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