A recombinant engineering bacterium for preparing recombinant albumin based on transcription factors and its application

Abstract

The present invention provides a recombinant engineered bacterium for preparing recombinant albumin based on transcription factors and its application, and relates to the field of genetic engineering technology. The present invention increases the expression of recombinant albumin by simultaneously overexpressing SUS1 gene and SUA7 gene into the strain while expressing recombinant albumin, and / or using CRISPR-Cas9 gene editing technology to target and knock out one or more genes in YHP1 and HDA1. The recombinant engineered bacterium of the present invention can significantly increase the yield of recombinant albumin and has great application value.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and in particular to a recombinant engineering bacterium for preparing recombinant albumin based on transcription factors and its application. Background Art

[0002] As the most commonly used eukaryotic protein expression system in academia and industry, Pichia pastoris has attracted widespread attention for its ability to efficiently produce a variety of recombinant proteins. It has many advantages: fast growth rate, easy to achieve high-density fermentation; simple culture medium components and culture conditions; easy to perform genetic manipulation, and there are commercial vector tools; good post-translational modification and protein secretion capabilities; low degree of glycosylation modification, far lower than that of Saccharomyces cerevisiae, and a variety of naturally occurring strong inducible and constitutive promoters.

[0003] Transcription is the first stage of gene expression and the main stage of gene regulation. Through the transcription process, cells can convert genetic information into RNA, and then synthesize proteins through the translation process. Such a process is essential for the normal function and development of cells. In the transcription process in the cell nucleus, transcription factors play a key regulatory role. They can enhance or reduce the transcription level of the target gene by binding to specific sequences, acting as activators or repressors.

[0004] Currently, there are methods to increase the yield of engineered bacteria by adjusting transcription factors. For example, CN111087459A increases the yield of artemisinin by overexpressing TCP transcription factors, and CN116836822A increases the yield of echinocandin compounds by introducing transcription factor McfJ into host cells. These examples provide new ideas for increasing the yield of recombinant engineered bacteria by adjusting transcription factors. Summary of the invention

[0005] The present invention provides a recombinant engineered bacterium for preparing recombinant albumin based on transcription factors and its application. The recombinant engineered bacterium achieves efficient expression of recombinant albumin by simultaneously overexpressing SUS1 gene and SUA7 gene and integrating them into the strain while expressing recombinant albumin, and / or utilizes CRISPR-Cas9 gene editing technology to knock out one or more genes in YHP1 and HDA1, thereby laying a foundation for the industrial application of recombinant albumin.

[0006] Transcription factors can be divided into positive regulatory factors and negative regulatory factors according to the results of their actions. SUS1 and SUA7 are positive regulatory factors and transcription activators, which can affect the assembly of the transcription initiation complex and the transcription efficiency, and determine whether a gene is expressed. They play a very important role in the transcriptional regulation of eukaryotic genes. Therefore, the transcription level of the target gene can be enhanced by overexpressing SUS1 and SUA7. YHP1 and HDA1 are negative regulatory factors and transcription repressors that can prevent gene transcription. Therefore, gene transcription can be promoted by knocking out YHP1 and HDA1, thereby increasing the expression of exogenous proteins.

[0007] The content of the present invention is as follows:

[0008] In a first aspect, the present invention provides a recombinant engineered bacterium for preparing recombinant albumin. The recombinant engineered bacterium, while expressing recombinant albumin, also includes the following operations: simultaneously overexpressing the SUS1 gene and the SUA7 gene and integrating them into the strain, and / or using CRISPR-Cas9 gene editing technology to target and knock out one or more genes in YHP1 and HDA1.

[0009] Furthermore, the amino acid sequence encoded by the SUS1 gene is shown as SEQ ID NO.1; the amino acid sequence encoded by the SUA7 gene is shown as SEQ ID NO.3; the amino acid sequence encoded by the YHP1 gene is shown as SEQ ID NO.5; and the amino acid sequence encoded by the HDA1 gene is shown as SEQ ID NO.7.

[0010] Furthermore, the SUS1 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 SUA7 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 YHP1 gene nucleotide sequence is a nucleotide sequence encoding the amino acid sequence described in SEQ ID NO.5; the HDA1 gene nucleotide sequence is a nucleotide sequence encoding the amino acid sequence described in SEQ ID NO.7.

[0011] The YHP1 gene nucleotide sequence is a nucleotide sequence encoding a protein of SEQ ID NO.5, the YHP1 gene nucleotide sequence is shown in SEQ ID NO.6, and the YHP1-gRNA sequence is shown in SEQ ID NO.14.

[0012] The HDA1 gene nucleotide sequence is a nucleotide sequence encoding a protein with a sequence of SEQ ID NO.7, the HDA1 gene nucleotide sequence is shown in SEQ ID NO.8, and the HDA1-gRNA sequence is shown in SEQ ID NO.15.

[0013] Preferably, the SUS1 gene nucleotide sequence is as shown in SEQ ID NO.2 or has 98% homology with the nucleotide sequence described in SEQ ID NO.2; the SUA7 gene nucleotide sequence is as shown in SEQ ID NO.4 or has 98% homology with the nucleotide sequence described in SEQ ID NO.4; the YHP1 gene nucleotide sequence is as shown in SEQ ID NO.6; and the HDA1 gene nucleotide sequence is as shown in SEQ ID NO.8.

[0014] Furthermore, in the engineered bacteria, the recombinant expression cassettes encoding SUS1 and SUA7 genes are on the same selection marker nucleic acid construct. In one embodiment of the present invention, the recombinant expression cassettes encoding SUS1 and SUA7 genes are on the same nucleic acid construct, and are screened with the geneticin G418 resistance tag.

[0015] Furthermore, the nucleic acid construct includes any one of pHIL-D2, pPIC3.5, pHIL-S1, pPIC9, pPink-LC, pPink-HC, pGAPZA, and pGAPZaA plasmids; the recombinant promoter is preferably a constitutive promoter, including any one or more of GAP promoter, TEF1 promoter, PGK1 promoter, and HGT1 promoter.

[0016] Furthermore, the recombinant engineered bacteria utilizes CRISPR-Cas9 gene editing technology to target and knock out one or more genes in YHP1 and HDA1, comprising the following steps:

[0017] S1: Construction of Cas9 and gRNA co-expression plasmid;

[0018] S2: Preparation of donor DNA;

[0019] S3: Preparation of competent cells;

[0020] S4: Construct a strain in which one or more genes in YHP1 and HDA1 are knocked out.

[0021] 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.

[0022] Furthermore, the recombinant albumin expressed by the recombinant engineered bacteria is selected from one of recombinant human albumin, recombinant feline albumin, recombinant canine albumin, recombinant bovine albumin, and recombinant horse albumin.

[0023] The NCBI accession number of the recombinant human albumin is NP_000468.1, and the nucleotide sequence encoding the amino acid sequence thereof is as shown in SEQ ID NO.9 or has at least 98% identity with the nucleotide sequence shown in SEQ ID NO.9;

[0024] The NCBI accession number of the recombinant feline albumin is NP_001009961.1, and the nucleotide sequence encoding the amino acid sequence thereof is shown in SEQ ID NO.10 or has at least 98% identity with the nucleotide sequence shown in SEQ ID NO.10;

[0025] The NCBI accession number of the recombinant canine albumin is NP_001003026.1, and the nucleotide sequence encoding the amino acid sequence thereof is shown in SEQ ID NO.11 or has at least 98% identity with the nucleotide sequence shown in SEQ ID NO.11;

[0026] The NCBI accession number of the recombinant bovine albumin is NP_851335.1, and the nucleotide sequence encoding the amino acid sequence thereof is shown in SEQ ID NO.12 or has at least 98% identity with the nucleotide sequence shown in SEQ ID NO.12;

[0027] The NCBI accession number of the recombinant horse albumin is NP_001075972.1, and the nucleotide sequence encoding its amino acid sequence is shown in SEQ ID NO.13 or has at least 98% identity with the nucleotide sequence shown in SEQ ID NO.13.

[0028] Furthermore, the Cas9 gene nucleotide sequence is shown in SEQ ID NO. 16, and the encoded amino acid sequence is shown in SEQ ID NO. 17. In one embodiment of the present invention, the recombinant expression cassette of Cas9 and gRNA is on a nucleic acid construct and screened with a blasticidin resistance tag.

[0029] The donor DNA uses Pichia pastoris genomic DNA as a template and uses overlap extension PCR technology to obtain left and right homologous arm fragments of one or more genes in YHP1 and HDA1.

[0030] Furthermore, it is characterized in that the engineered bacteria is selected from one or more of Pichia pastoris, Hansenula, Candida, and Saccharomyces cerevisiae. Preferably, the engineered bacteria is Pichia pastoris; more preferably, the Pichia pastoris is Pichia pastoris CBS7435.

[0031] On the other hand, the present invention also provides the use of the engineered bacteria in preparing recombinant albumin and / or increasing the expression level of recombinant albumin.

[0032] In another aspect, the present invention provides an aspect of preparing recombinant albumin, the method comprising: preparing recombinant albumin by fermentation using the recombinant engineered bacteria as described above. Specifically, the recombinant albumin is selected from at least one of recombinant human albumin, recombinant cat albumin, recombinant canine albumin, recombinant bovine albumin, and recombinant horse albumin.

[0033] In the last aspect, the present invention also provides recombinant albumin prepared by fermentation using the above-mentioned recombinant engineered bacteria.

[0034] The beneficial effects of the present invention include:

[0035] The present invention constructs a recombinant engineered bacterium for preparing recombinant albumin, which can significantly increase the expression of recombinant albumin, and the maximum yield of recombinant albumin can reach 192%. Different from the traditional method of directly increasing the copy number of the gene encoding the recombinant protein, the recombinant engineered bacterium of the present invention improves the transcription factors in the transcription process, knocks out one or more of the negative regulatory factors YHP1 and HDA1, and simultaneously overexpresses the positive regulatory factors SUS1 and SUA7 genes, and significantly enhances the heterologous protein secretion ability of the recombinant engineered strain by enhancing the transcription level of the target gene. The Pichia pastoris gene recombinant engineered bacterium shows strong potential in increasing the yield of proteins with high economic value and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] 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:

[0037] Figure 1 It is a schematic diagram of the structure of the recombinant expression cassette pART111 encoding the SUS1 recombinant nucleic acid molecule and the SUA7 recombinant nucleic acid molecule;

[0038] Figure 2 It is a schematic diagram of the structure of the recombinant expression cassette pART222 encoding the Cas9 recombinant nucleic acid molecule and the YHP1-gRNA recombinant nucleic acid molecule;

[0039] Figure 3 It is a schematic diagram of the structure of the recombinant expression cassette pART333 encoding the Cas9 recombinant nucleic acid molecule and the HDA1-gRNA recombinant nucleic acid molecule;

[0040] Figure 4Schematic diagrams of the structures of recombinant expression cassettes encoding recombinant albumin, wherein 4-1 is a schematic diagram of the structure of the recombinant expression cassette pPICZA-I encoding recombinant human albumin; 4-2 is a schematic diagram of the structure of the recombinant expression cassette pPICZA-II encoding recombinant cat albumin; 4-3 is a schematic diagram of the structure of the recombinant expression cassette pPICZA-III encoding recombinant canine albumin; 4-4 is a schematic diagram of the structure of the recombinant expression cassette pPICZA-IV encoding recombinant bovine albumin; 4-5 is a schematic diagram of the structure of the recombinant expression cassette pPICZA-V encoding recombinant horse albumin;

[0041] Figure 5 This is a schematic diagram of the results of expressing human recombinant albumin by each recombinant engineering bacteria in Example 5;

[0042] Figure 6 This is a schematic diagram of the results of expressing feline recombinant albumin by each recombinant engineering bacteria in Example 7;

[0043] Figure 7 This is a schematic diagram of the results of expressing canine recombinant albumin by each recombinant engineering bacteria in Example 9;

[0044] Figure 8 This is a schematic diagram of the results of expressing bovine recombinant albumin by each recombinant engineering bacteria in Example 11;

[0045] Fig. 9 This is a schematic diagram of the results of the expression of horse recombinant albumin by each recombinant engineered bacteria in Example 13. DETAILED DESCRIPTION

[0046] 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.

[0047] 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.

[0048] Host cell: refers to the cell that accepts foreign genes during transformation or transduction.

[0049] Engineered bacteria: Fungal cell strains that use genetic engineering methods to efficiently express exogenous genes.

[0050] In order to more clearly explain the overall concept of the present invention, 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 known in the art are not described.

[0051] If no specific conditions are specified in the examples, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer.

[0052] 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.

[0053] 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.

[0054] The sources of experimental materials and reagents involved in the present invention are as follows.

[0055] Strain: Pichia pastoris CBS7435 was purchased from the American Type Culture Collection (ATCC) on behalf of Ningbo Mingzhou Biotechnology Co., Ltd., also known as Komagataella phaffii NRRL Y-11430, ATCC Number: 76273™.

[0056] Expression vector: pPICZA was purchased from Hunan Fenghui Biotechnology Co., Ltd., item number JM006, specification 20μl; expression vector pGAPZA was purchased from Hunan Fenghui Biotechnology Co., Ltd., item number ZT226, specification 20μl; the following expression cassettes were constructed by the laboratory: 1) Expression cassette, which encodes SUS1 recombinant nucleic acid molecule and SUA7 recombinant nucleic acid molecule, its structural diagram is shown in Figure 1 2) An expression cassette encoding a Cas9 recombinant nucleic acid molecule and a YHP1-gRNA recombinant nucleic acid molecule, the structure of which is shown in FIG. Figure 2 3) An expression cassette encoding a Cas9 recombinant nucleic acid molecule and an HDA1-gRNA recombinant nucleic acid molecule, the structure of which is shown in FIG. Figure 3 4) an expression cassette encoding a recombinant nucleic acid molecule of recombinant human albumin, the structure of which is shown in FIG. Figure 4-1 5) An expression cassette encoding a recombinant nucleic acid molecule of recombinant feline albumin, the structure of which is shown in FIG. Figure 4-2 6) an expression cassette encoding a recombinant nucleic acid molecule of recombinant canine albumin, the structure of which is shown in FIG. Figure 4-3 7) An expression cassette encoding a recombinant nucleic acid molecule of recombinant bovine albumin, the structure of which is shown in FIG. Figure 4-4 8) An expression cassette encoding a recombinant nucleic acid molecule of recombinant horse albumin, the structure of which is shown in FIG. Figure 4-5 shown.

[0057] Enzymes and kits

[0058] 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 (Cat. No. 10131035, specification 20 ml), and Blasticidin (Cat. No. A1113902, specification 20 ml) were purchased from Thermo Fisher Scientific.

[0059] Culture medium

[0060] Escherichia coli culture medium LB (1% (w / v) tryptone, 0.5% (w / v) yeast extract, 1% (w / v) NaCl, pH 7.0).

[0061] 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).

[0062] 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.

[0063] Example 1: Construction of SUS1 and SUA7 co-expression strain

[0064] The recombinant expression cassettes encoding the SUS1 gene (nucleotide sequence such as SEQ ID NO.2, amino acid sequence such as SEQ ID NO.1) and the SUA7 gene (nucleotide sequence such as SEQ ID NO.4, amino acid sequence such as SEQ ID NO.3) were directly synthesized by a biological company and then connected into the plasmid pGAPZA to form an expression vector named pART110. The biological company was commissioned to replace the zeocin resistance gene in pART110 with the geneticin G418 resistance gene (nucleotide sequence as shown in SEQ ID NO.18) to form an expression vector named pART111.

[0065] The expression vector pART111 was linearized using DNA restriction endonuclease BglII, and CBS7435 was transformed by electroporation. The cells were spread on a selection plate YPDG containing geneticin G418 antibiotics and cultured at 30°C until colonies appeared. The positive transformants were identified as recombinant engineered bacteria 1 by colony PCR.

[0066] Example 2: Construction of Cas9 and gRNA co-expression vector

[0067] In this example, in order to establish a suitable genome editing CRISPR system, an attempt was made to use the dual promoter pHTX1 (nucleotide sequence as shown in SEQ ID NO.19) to construct a vector for co-expression of Cas9 and gRNA (Weninger A, HatzlAM, Schmid C, Vogl T, Glieder A. Combinatorial optimization of CRISPR / Cas9expression enables precision genome engineering in the methylotrophic yeast Pichia pastoris. J Biotechnol. 2016 Oct 10;235:139-49.).

[0068] The recombinant expression cassette encoding the Cas9 gene and the YHP1-gRNA gene (sequence as SEQ ID NO.14) was commissioned to be directly synthesized by a biological company, and then connected to the plasmid pPICZA digested with BglII and BamHI to form an expression vector named pART220. Using gene homologous recombination technology, the biological company was commissioned to replace the zeocin resistance gene in pART220 with the blasticidin resistance gene (nucleotide sequence as shown in SEQ ID NO.20) to form an expression vector named pART222.

[0069] The recombinant expression cassette encoding the Cas9 gene and the HDA1-gRNA gene (sequence as shown in SEQ ID NO.15) was commissioned to be directly synthesized by a biological company, and then connected to the plasmid pPICZA digested with BglII and BamHI to form an expression vector named pART330. Using gene homologous recombination technology, the biological company was commissioned to replace the zeocin resistance gene in pART330 with the blasticidin resistance gene (nucleotide sequence as shown in SEQ ID NO.20) to form an expression vector named pART333.

[0070] Example 3: Construction of YHP1 and HDA1 gene targeted knockout strains

[0071] In order to prepare the donor DNA for YHP1 knockout, the following operations were performed: using Pichia pastoris genomic DNA as a template, YHP1-UP-F / YHP1-UP-R and YHP1-DN-F / YHP1-DN-R as primers, PCR was performed to obtain the left and right homologous arm fragments of the YHP1 gene; then, using YHP1-UP-F and YHP1-DN-R as primers and a mixture of the left and right homologous arm fragments as a DNA template, overlap extension PCR technology was used to obtain the donor DNA for YHP1 knockout.

[0072] In order to prepare the donor DNA for HDA1 knockout, the following operations were performed: using Pichia pastoris genomic DNA as a template, HDA1-UP-F / HDA1-UP-R and HDA1-DN-F / HDA1-DN-R as primers, PCR was performed to obtain the left and right homologous arm fragments of the HDA1 gene; then, using HDA1-UP-F and HDA1-DN-R as primers and a mixture of the left and right homologous arm fragments as a DNA template, overlap extension PCR technology was used to obtain the donor DNA for HDA1 knockout.

[0073] The 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.

[0074] Table 1

[0075]

[0076] Table 2

[0077]

[0078] 2 μg of Cas9 and YHP1-gRNA co-expression vector and 2 μg of YHP1 knockout donor DNA were co-transformed into the recombinant engineered bacteria 1 in Example 1, coated on the selection plate YPDB containing blasticidin antibiotic, and cultured at 30°C until colonies appeared. The positive transformants were identified as recombinant engineered bacteria 2 by colony PCR.

[0079] 2 μg of Cas9 and HDA1-gRNA co-expression vector and 2 μg of HDA1 knockout donor DNA were co-transformed into the recombinant engineered bacteria 1 in Example 1, coated on a selection plate YPDB containing blasticidin antibiotic, and cultured at 30° C. until colonies appeared. The positive transformants were identified as recombinant engineered bacteria 3 by colony PCR.

[0080] 2 μg of Cas9 and HDA1-gRNA co-expression vector and 2 μg of HDA1 knockout donor DNA were co-transformed into the recombinant engineered bacteria 2 in Example 3, coated on the selection plate YPDB containing blasticidin antibiotic, and cultured at 30° C. until colonies appeared. The positive transformants were identified by colony PCR as the recombinant engineered bacteria 4 with YHP1 and HDA1 knockout.

[0081] Example 4: Construction of recombinant human albumin Pichia pastoris strain

[0082] The nucleic acid molecule encoding the signal peptide of the mating factor of Saccharomyces cerevisiae a (SEQ ID NO.21) and the nucleic acid molecule encoding the recombinant human albumin (nucleotide sequence as shown in SEQ ID NO.9) were commissioned to a biological company for direct synthesis, and then ligated into the plasmid pPICZA digested with EcoRI and NotI to form an expression vector named pPICZA-I.

[0083] The expression vector was linearized using DNA restriction endonuclease BglII and transformed with electroporation into Pichia pastoris CBS7435 and recombinant engineered bacteria 1, 2, 3, and 4. 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 to obtain recombinant engineered bacteria 5, 6, 7, 8, and 9.

[0084] Example 5: Expression level of recombinant human albumin in recombinant engineered bacteria

[0085] Recombinant engineered bacteria 5-9 were inoculated into 40 mL of BMGY culture medium and cultured overnight at 220 rpm and 30°C. The culture medium was centrifuged and the precipitate was resuspended in 40 mL of BMMY culture medium. Induction was started at 220 rpm and 25°C. 0.4% methanol was added every 24 h. After 72 h, the supernatant was collected by centrifugation at 10,000 rpm for 5 min. The supernatant was subjected to SDS-PAGE electrophoresis. The specific results are shown in Table 3 and Figure 5 shown.

[0086] The results showed that when SUS1 and SUA7 genes were overexpressed and YHP1 and HDA1 genes were knocked out at the same time, the expression level of recombinant human albumin in the recombinant engineered bacteria 5 was used as a control, and the expression level reached 176%.

[0087] Table 3

[0088]

[0089] Example 6: Construction of recombinant feline albumin Pichia pastoris strain

[0090] The nucleic acid molecule encoding the signal peptide of the mating factor of Saccharomyces cerevisiae a (SEQ ID NO.21) and the nucleic acid molecule encoding the recombinant feline albumin (nucleotide sequence as shown in SEQ ID NO.10) were commissioned to a biological company for direct synthesis, and then ligated into the plasmid pPICZA digested with EcoRI and NotI to form an expression vector named pPICZA-II.

[0091] The expression vector was linearized using DNA restriction endonuclease BglII and transformed with electroporation into Pichia pastoris CBS7435 and recombinant engineered bacteria 1, 2, 3, and 4. 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 to obtain recombinant engineered bacteria 10, 11, 12, 13, and 14.

[0092] Example 7: Expression level of recombinant feline albumin in recombinant engineered bacteria

[0093] 10-14 recombinant engineered bacteria were inoculated into 40 mL BMGY culture medium at 220 rpm and 30°C overnight. The culture medium was centrifuged and the precipitate was resuspended in 40 mL BMMY culture medium. Induction was started at 220 rpm and 25°C. 0.4% methanol was added every 24 h. After 72 h, the supernatant was collected by centrifugation at 10,000 rpm for 5 min. The supernatant was subjected to SDS-PAGE electrophoresis. The specific results are shown in Table 4 and Figure 6 shown.

[0094] The results showed that when SUS1 and SUA7 genes were overexpressed and YHP1 and HDA1 genes were knocked out at the same time, the expression level of recombinant feline albumin in the recombinant engineered bacteria 10 was used as a control, and the expression level reached 169%.

[0095] Table 4

[0096]

[0097] Example 8: Construction of recombinant Pichia canis albumin strain

[0098] The nucleic acid molecule encoding the signal peptide of the mating factor of Saccharomyces cerevisiae a (SEQ ID NO.21) and the nucleic acid molecule encoding the recombinant canine albumin (nucleotide sequence as shown in SEQ ID NO.11) were commissioned to a biological company for direct synthesis, and then ligated into the plasmid pPICZA digested with EcoRI and NotI to form an expression vector named pPICZA-III.

[0099] The expression vector was linearized using DNA restriction endonuclease BglII and transformed with electroporation into Pichia pastoris CBS7435 and recombinant engineered bacteria 1, 2, 3, and 4. 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 to obtain recombinant engineered bacteria 15, 16, 17, 18, and 19.

[0100] Example 9: Expression level of recombinant canine albumin in recombinant engineered bacteria

[0101] Recombinant engineered bacteria 15-19 were inoculated into 40 mL of BMGY culture medium and cultured overnight at 220 rpm and 30°C. The culture medium was centrifuged and the precipitate was resuspended in 40 mL of BMMY culture medium. Induction was started at 220 rpm and 25°C. 0.4% methanol was added every 24 h. After 72 h, the supernatant was collected by centrifugation at 10,000 rpm for 5 min. The supernatant was subjected to SDS-PAGE electrophoresis. The specific results are shown in Table 5 and Figure 7 shown.

[0102] The results showed that when SUS1 and SUA7 genes were overexpressed and YHP1 and HDA1 genes were knocked out at the same time, the expression level of recombinant canine albumin in the recombinant engineered bacteria 15 was used as a control, and the expression level reached 178%.

[0103] Table 5

[0104]

[0105] Example 10: Construction of recombinant bovine albumin Pichia pastoris strain

[0106] The nucleic acid molecule encoding the signal peptide of the mating factor of Saccharomyces cerevisiae a (SEQ ID NO.21) and the nucleic acid molecule encoding the recombinant bovine albumin (nucleotide sequence as shown in SEQ ID NO.12) were commissioned to a biological company for direct synthesis, and then ligated into the plasmid pPICZA digested with EcoRI and NotI to form an expression vector named pPICZA-IV.

[0107] The expression vector was linearized using DNA restriction endonuclease BglII and transformed with electroporation into Pichia pastoris CBS7435 and recombinant engineered bacteria 1, 2, 3, and 4. 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 to obtain recombinant engineered bacteria 20, 21, 22, 23, and 24.

[0108] Example 11: Expression level of recombinant bovine albumin in recombinant engineered bacteria

[0109] Recombinant engineered bacteria 20-24 were inoculated into 40 mL of BMGY culture medium and cultured overnight at 220 rpm and 30°C. The culture medium was centrifuged and the precipitate was resuspended in 40 mL of BMMY culture medium. Induction was started at 220 rpm and 25°C. 0.4% methanol was added every 24 h. After 72 h, the supernatant was collected by centrifugation at 10,000 rpm for 5 min. The supernatant was subjected to SDS-PAGE electrophoresis. The specific results are shown in Table 6 and Figure 8 shown.

[0110] The results showed that when SUS1 and SUA7 genes were overexpressed and YHP1 and HDA1 genes were knocked out at the same time, the expression level reached 192% using the recombinant bovine albumin expression level of the recombinant engineered bacteria 20 as a control.

[0111] Table 6

[0112]

[0113] Example 12: Construction of recombinant equine albumin Pichia pastoris strain

[0114] The nucleic acid molecule encoding the signal peptide of the mating factor of Saccharomyces cerevisiae a (SEQ ID NO.21) and the nucleic acid molecule encoding the recombinant horse albumin (nucleotide sequence as shown in SEQ ID NO.13) were commissioned to a biological company for direct synthesis, and then ligated into the plasmid pPICZA digested with EcoRI and NotI to form an expression vector named pPICZA-V.

[0115] The expression vector was linearized using DNA restriction endonuclease BglII and transformed with electroporation into Pichia pastoris CBS7435 and recombinant engineered bacteria 1, 2, 3, and 4. 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 to obtain recombinant engineered bacteria 25, 26, 27, 28, and 29.

[0116] Example 13: Expression level of recombinant horse albumin in recombinant engineered bacteria

[0117] Recombinant engineered bacteria 25-29 were inoculated into 40 mL of BMGY culture medium and cultured overnight at 220 rpm and 30°C. The bacterial solution was centrifuged and the precipitate was resuspended in 40 mL of BMMY culture medium. Induction was started at 220 rpm and 25°C. 0.4% methanol was added every 24 h. After 72 h, the supernatant was collected by centrifugation at 10,000 rpm for 5 min. The supernatant was subjected to SDS-PAGE electrophoresis. The specific results are shown in Table 7 and Fig. 9 shown.

[0118] The results showed that when SUS1 and SUA7 genes were overexpressed and YHP1 and HDA1 genes were knocked out at the same time, the expression level of recombinant horse albumin in the recombinant engineered bacteria 25 was used as a control, and the expression level reached 189%.

[0119] Table 7

[0120]

[0121] The above description is only an embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A recombinant engineered bacterium for preparing recombinant albumin based on transcription factors, characterized in that: While expressing recombinant albumin, the recombinant engineered bacteria also includes the following operations: simultaneously overexpressing the SUS1 gene and the SUA7 gene and integrating them into the strain, and using CRISPR-Cas9 gene editing technology to target and knock out one or more genes in YHP1 and HDA1, wherein the recombinant albumin is selected from at least one of recombinant human albumin, recombinant cat albumin, recombinant canine albumin, recombinant bovine albumin, and recombinant horse albumin, wherein the amino acid sequence encoded by the SUS1 gene is shown in SEQ ID NO.1; the amino acid sequence encoded by the SUA7 gene is shown in SEQ ID NO.3; the amino acid sequence encoded by the YHP1 gene is shown in SEQ ID NO.5; the amino acid sequence encoded by the HDA1 gene is shown in SEQ ID NO.7, and the recombinant engineered bacteria is Pichia pastoris.

2. The recombinant engineered bacterium according to claim 1, characterized in that The nucleotide sequence of the SUS1 gene is shown in SEQ ID NO.2; the nucleotide sequence of the SUA7 gene is shown in SEQ ID NO.4; the nucleotide sequence of the YHP1 gene is shown in SEQ ID NO.6; and the nucleotide sequence of the HDA1 gene is shown in SEQ ID NO.

8.

3. The recombinant engineered bacterium according to claim 1, characterized in that The recombinant expression cassette encoding one or more genes of SUS1, SUA7 is on the same nucleic acid construct of the selection marker or on the nucleic acid construct of different selection markers.

4. The recombinant engineered bacterium according to claim 3, characterized in that The nucleic acid construct comprises any one of pHIL-D2, pPIC3.5, pHIL-S1, pPIC9, pPink-LC, pPink-HC, pGAPZA and pGAPZaA plasmids.

5. The recombinant engineered bacterium according to claim 1, characterized in that The recombinant engineering bacteria utilizes CRISPR-Cas9 gene editing technology to target and knock out one or more genes in YHP1 and HDA1, including the following steps: S1: Construction of Cas9 and gRNA co-expression plasmid; S2: Preparation of donor DNA; S3: Preparation of competent cells; S4: Construct a strain in which one or more genes in YHP1 and HDA1 are knocked out.

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

7. A method for preparing recombinant albumin, characterized in that: The method comprises: preparing recombinant albumin by fermentation using the recombinant engineered bacteria as described in any one of claims 1 to 5.

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