A recombinant engineered bacterium for preparing recombinant equine albumin and its application

Abstract

The present invention provides a recombinant engineered bacterium for preparing recombinant equine albumin and its application, relating to the field of genetic engineering technology. The present invention increases the expression level of recombinant equine albumin by, while expressing recombinant equine albumin, targetedly knocking out one or more of the genes SED1 and TIP1 associated with cell wall formation, and / or integrating overexpression of one or more of the genes PAC10 and YDJ1 associated with promoting protein folding into the strain. The recombinant engineered bacterium of the present invention can significantly increase the yield of recombinant equine albumin, reaching up to 51.27 g / L. This is a 5.38-fold increase in yield compared to unmodified recombinant engineered bacteria, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, in particular to a recombinant engineering bacterium for preparing recombinant equine albumin and an application thereof. Background Art

[0002] As the most abundant protein in serum, equine serum albumin (ESA) possesses diverse biological functions. It maintains plasma colloidal osmotic pressure, ensuring balanced distribution of fluid inside and outside blood vessels. It binds to and transports a variety of endogenous and exogenous substances, such as fatty acids, hormones, and drugs, regulating their metabolism and physiological effects within the body. It also possesses antioxidant properties, scavenging free radicals and protecting cells from oxidative damage.

[0003] In the pharmaceutical field, horse serum albumin has a wide range of applications. In drug development, it can be used as a drug carrier to improve drug pharmacokinetic properties, enhance drug stability and targeting, and enhance drug efficacy. In cell culture, horse serum albumin is a key component of cell culture media, providing essential nutrients and growth factors for cell growth, promoting cell proliferation and survival. In biotechnology, it is used in experiments such as protein purification and immunoassays, serving as a valuable experimental reagent.

[0004] Traditional methods for extracting equine serum albumin primarily rely on isolation and purification from horse serum. However, this method has numerous limitations. The limited availability of horse serum, limited by factors such as the number of horses and the frequency of blood collection, makes it difficult to meet the demands of large-scale production. Furthermore, extracting albumin from animal serum carries the potential risk of contamination with pathogens, such as viruses and bacteria, which could pose a health threat to users. Furthermore, the complex and costly nature of traditional extraction methods hinders widespread application.

[0005] To address the limitations of traditional extraction methods, recombinant expression technology has emerged. By constructing recombinant engineered equine serum albumin (ESA), large-scale, low-cost production of ESA can be achieved. Recombinant expression technology enables production under controlled conditions, reducing the risk of pathogen contamination and improving product safety and quality stability. Currently, with the rapid development of the pharmaceutical and biotechnology industries, the demand for ESA is growing. The development of recombinant engineered ESA bacteria has significant practical significance and broad market prospects.

[0006] Heterologous protein expression is crucial for a variety of biopharmaceutical and industrial applications. As one of the most successful eukaryotic protein expression systems, Pichia pastoris has experienced rapid development and garnered widespread attention over the past 20 years, generating significant economic and social value. Current methods for efficiently expressing exogenous proteins primarily include exogenous gene modification, bacterial strain modification, and fermentation optimization. From the perspective of exogenous gene modification, it mainly includes the optimization of exogenous gene codons, the increase of exogenous gene integration copy number and the selection of guide peptides; from the perspective of strain modification, it mainly includes the knockout of protein degradation enzyme genes, the introduction of folding factors, the introduction of Vitreoscilla hemoglobin (Vgb) genes, and the knockout of glycerol transporter genes; fermentation level optimization includes adjusting the temperature and dissolved oxygen level in environmental factors, adding auxiliary expression substances such as non-inhibitory carbon sources, vitamin C, certain amino acids and oleic acid, and fermentation process optimization includes the study of the initial induction bacteria amount and methanol feeding method (Zhu Wen, Hu Youjia, Xie Liping. Relevant strategies and research progress of efficient expression of exogenous proteins in Pichia pastoris [J], Chinese Journal of Pharmaceuticals 2018, 49 (4): 417-425). CN118580980A discloses a cell wall defective human albumin recombinant engineering bacteria, whose human albumin production is significantly improved. These optimizations of Pichia pastoris provide a basis for the efficient expression of horse albumin. Summary of the Invention

[0007] The present invention improves the yield of equine albumin by increasing the number of exogenous gene copies, introducing an α-mating factor signal peptide, and modifying the bacterial strain. Furthermore, the present invention selectively introduces protein folding genes for the endoplasmic reticulum while simultaneously knocking out cell wall genes in the target bacterial strain to achieve the purpose of improving protein expression. The specific scheme of the present invention is as follows:

[0008] In a first aspect, the present invention provides a recombinant engineered bacterium for preparing recombinant equine albumin. The recombinant engineered bacterium, while expressing recombinant equine albumin, also comprises the following operations: targeted knockout of one or more of the genes SED1 and TIP1 related to cell wall composition, and / or overexpression and integration of one or more of the genes PAC10 and YDJ1 related to promoting protein folding into the strain, thereby achieving efficient expression of recombinant equine albumin, laying the foundation for the industrial application of recombinant equine albumin.

[0009] Preferably, the operation includes at least any one of the following:

[0010] (1) Knockout of SED1 and TIP1 genes;

[0011] (2) Knockout of SED1 and TIP1 genes, and overexpression of PAC10 gene;

[0012] (3) Knockout of SED1 and TIP1 genes, and overexpression of YDJ1 gene;

[0013] (4) Knockout of SED1 and TIP1 genes, and overexpression of PAC10 and YDJ1 genes.

[0014] Furthermore, the amino acid sequence encoded by the SED1 gene is shown as SEQ ID NO.1, and the nucleotide sequence of the SED1 gene is shown as SEQ ID NO.2; the amino acid sequence encoded by the TIP1 gene is shown as SEQ ID NO.3, and the nucleotide sequence of the TIP1 gene is shown as SEQ ID NO.4.

[0015] The amino acid sequence encoded by the PAC10 gene is shown as SEQ ID NO.5. The nucleotide sequence of the PAC10 gene is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO.5, including sequences currently disclosed in databases and nucleotide sequences optimized according to actual needs. Preferably, the nucleotide sequence is shown as SEQ ID NO.6 or has at least 95% homology with the nucleotide sequence shown in SEQ ID NO.6.

[0016] The amino acid sequence encoded by the YDJ1 gene is shown as SEQ ID NO.7, and the nucleotide sequence of the YDJ1 gene is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO.7, including sequences currently disclosed in databases and nucleotide sequences optimized according to actual needs. Preferably, the nucleotide sequence is shown as SEQ ID NO.8 or has at least 95% homology with the nucleotide sequence shown in SEQ ID NO.8.

[0017] Furthermore, the amino acid sequence of the horse albumin is shown in SEQ ID NO. 9. The horse albumin is synthesized after codon optimization and cloned into an expression vector to construct a recombinant plasmid.

[0018] Furthermore, the recombinant horse albumin nucleotide sequence is such as SEQ ID NO.10 or a nucleotide sequence having at least 95% homology with SEQ ID NO.10.

[0019] Furthermore, the exogenous gene expressed by the recombinant engineered bacteria can also be selected from one of other recombinant albumins. Preferably, the recombinant albumin includes one of recombinant human albumin, recombinant feline albumin, recombinant canine albumin, recombinant bovine albumin, recombinant sheep albumin, and recombinant porcine albumin.

[0020] 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. Furthermore, the Pichia pastoris strain includes at least one of X-33, GS115, GS190, GS200, JC220, JC254, KM71, M-C100-3, SMD1163, SMD1165, and SMD1168. In a specific embodiment of the present invention, the donor strain is Pichia pastoris X-33.

[0021] Furthermore, the expression vector of the recombinant engineered bacteria includes any one or more of pHIL-D2, pAO815, pPIC3K, pPICZ, pHWO10, pGAPZ, pPIC9K, pPICZα, and pGAPα. Furthermore, the expression vector is a multi-copy insertion expression vector, preferably, the multi-copy insertion expression vector is selected from at least one of pPIC3.5K, pPIC9K, and pAO815.

[0022] Furthermore, the promoter of the recombinant engineered bacteria includes any one or more of the AOX1 promoter, GAP promoter, TEF1 promoter, GCW14 promoter and AOX2 promoter. In a specific embodiment of the present invention, the promoter is the AOX1 promoter.

[0023] Furthermore, the recombinant engineered bacteria utilizes CRISPR-Cas9 gene editing technology and homologous recombination technology to target and knock out one or more genes in SED1 and TIP1.

[0024] Furthermore, the recombinant engineered bacteria utilizes CRISPR-Cas9 gene editing technology to target and knock out one or more genes in SED1 and TIP1, comprising the following steps:

[0025] 1-1: Construction of Cas9 and gRNA co-expression plasmid;

[0026] 1-2: Prepare donor DNA;

[0027] 1-3: Prepare competent cells;

[0028] 1-4: Construct a strain in which one or more genes in SED1 and TIP1 are knocked out.

[0029] Furthermore, the donor DNA uses Pichia pastoris genomic DNA as a template and utilizes overlap extension PCR technology to obtain left and right homologous arm fragments of one or more genes in SED1 and TIP1.

[0030] Furthermore, the homologous recombination technology is EasyGeno seamless cloning technology, and the recombinant engineering bacteria utilizes EasyGeno seamless cloning technology to target and knock out one or more genes in SED1 and TIP1, including the following steps:

[0031] 2-1: Linearize the vector;

[0032] 2-2: Prepare insert fragments, which include upstream and downstream fragments of SED1 and TIP1 genes respectively;

[0033] 2-3: Perform a recombination reaction, where the vector: fragment molar ratio is ≈1:2-3;

[0034] 2-4: Directly transform the recombination reaction product into competent cells.

[0035] Furthermore, the number of copies of the nucleotide sequence encoding recombinant horse albumin in the recombinant engineered bacteria is 4 to 7. In an embodiment of the present invention, the number of copies of the nucleotide sequence encoding recombinant horse albumin in the recombinant engineered bacteria is 6.

[0036] Furthermore, the recombinant engineered bacteria of the present invention also include a drug-resistant gene segment. The drug-resistant gene segment is used for screening the recombinant engineered bacteria. In an embodiment of the present invention, the drug-resistant segment is one or more of the following: a geneticin G418 resistance gene, a kanamycin resistance gene, an ampicillin resistance gene, a His4 gene, and a blasticidin resistance gene.

[0037] Furthermore, the recombinant engineered bacteria also includes a signal peptide sequence, which 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 the Saccharomyces cerevisiae α mating factor signal peptide; preferably, the nucleotide sequence encoding the Saccharomyces cerevisiae α mating factor (α-factor) signal peptide is shown in SEQ ID NO.11.

[0038] The second aspect of the present invention provides the use of the recombinant engineered bacteria in preparing recombinant equine albumin and / or increasing the expression level of recombinant equine albumin.

[0039] The third aspect of the present invention provides recombinant horse albumin prepared by fermentation using the above-mentioned recombinant engineered bacteria.

[0040] In a fourth aspect, the present invention provides a method for culturing the recombinant engineered bacteria, wherein the culture temperature is 26-30°C, the methanol concentration is 0.4-0.6%, and the induction time is 70-74 hours.

[0041] In a specific embodiment of the present invention, the culture temperature in the method is 28° C., the methanol concentration is 0.5%, and the induction time is 72 h.

[0042] The beneficial effects of the present invention include:

[0043] The present invention constructs a recombinant engineered bacterium for preparing recombinant horse albumin. While expressing recombinant horse albumin, one or more of the genes SED1 and TIP1 related to cell wall composition are targeted for knockout, and / or one or more of the genes PAC10 and YDJ1 related to promoting protein folding are overexpressed and integrated into the strain. The present invention transforms the recombinant engineered bacterium by promoting protein folding and cell wall defects, thereby achieving the purpose of increasing the production of recombinant horse albumin. The recombinant engineered bacterium of the present invention significantly increases the expression level of recombinant horse albumin, and can achieve a maximum production of recombinant horse albumin of 51.27 g / L, which is 5.38 times higher than that of the unmodified recombinant engineered bacterium. The recombinant engineered bacterium shows strong potential in increasing the production of proteins with high economic value and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] 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:

[0045] Figure 1 is a schematic diagram of the structure of the recombinant expression cassette encoding recombinant horse albumin;

[0046] Figure 2 This is an SDS-PAGE gel electrophoresis diagram of the recombinant engineered bacteria 5-10 expressing recombinant horse albumin in the example. DETAILED DESCRIPTION

[0047] Recombinant engineered bacteria: a fungal cell line that uses genetic engineering methods to efficiently express exogenous genes.

[0048] In order to more clearly illustrate the overall concept of the present invention, the following is a detailed description of the embodiments in conjunction with the accompanying drawings. 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.

[0049] If no specific conditions are specified in the examples, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer.

[0050] Unless otherwise specified, in the following embodiments, the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased from the market.

[0051] Molecular biology experimental methods not specifically described in the following examples were performed according to the kits and product instructions, or with reference to the methods described in the book Molecular Cloning Laboratory Manual (3rd edition) by J. Sambrook.

[0052] The sources of experimental materials and reagents involved in the present invention are as follows.

[0053] Strain: Pichia pastoris X-33, purchased from Thermo Fisher Scientific. This strain inherently possesses resistance to prokaryotic antibiotics, such as resistance to kanamycin and ampicillin. X-33 is a His+ strain, grows faster, and can tolerate high copy numbers.

[0054] Expression vector and expression cassette: customized by Yunzhou Biotechnology Co., Ltd.

[0055] Enzymes and kits

[0056] Restriction endonucleases BgIII, NotI, and BamHI were purchased from Thermo Fisher Scientific; plasmid extraction kit, agarose gel recovery kit, and DNA product purification kit were purchased from Tiangen Biotechnology Co., Ltd.; kanamycin, geneticin, and blasticidin were purchased from Thermo Fisher Scientific; SYBR Green Master Mixture was purchased from Bio-Rad; EasyGeno seamless cloning kit was purchased from Tiangen Biotechnology Co., Ltd.; alkaline phosphatase (CIP) was purchased from New England Biolabs; and DH5α competent cells were purchased from Tiangen Biotechnology Co., Ltd.

[0057] culture medium

[0058] Escherichia coli solid culture medium LB: 1% (w / v) tryptone, 0.5% (w / v) yeast extract, 1.5% (w / v) agar, 1% (w / v) NaCl, pH 7.0.

[0059] Escherichia coli liquid culture medium LB: 1% (w / v) tryptone, 0.5% (w / v) yeast extract, 1% (w / v) NaCl, pH 7.0.

[0060] Yeast solid medium YPD: 1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose, and 2% (w / v) agar.

[0061] Yeast liquid medium YPD: 1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose, PBS buffer, pH = 7.0.

[0062] Yeast medium BMGY: 1% (w / v) yeast extract, 2% (w / v) peptone, 1.34% (w / v) YNB, 0.00004% (w / v) biotin, 0.23% (w / v) K2HPO4, 1.18% (w / v) KH2PO4 (0.23% (w / v) K2HPO4 and 1.18% (w / v) KH2PO4 can be directly replaced by PBS solution).

[0063] Yeast induction medium BMMY: 1% (w / v) yeast extract, 2% (w / v) peptone, 1.34% (w / v) YNB, 0.00004% (w / v) Biotin, 0.5% methanol (V / V), 1.34% (w / v) YNB, 0.00004% (w / v) biotin, 0.23% (w / v) K2HPO4, 1.18% (w / v) KH2PO4 (0.23% (w / v) K2HPO4 and 1.18% (w / v) KH2PO4 can be directly replaced by PBS solution).

[0064] Buffer: 0.283% (w / v) Na2HPO4, 0.4% (w / v) NaH2PO4, pH=7.4.

[0065] Example 1: Construction of recombinant equine albumin Pichia pastoris strain

[0066] The nucleic acid molecule encoding the signal peptide of the mating factor of Saccharomyces cerevisiae a (SEQ ID NO. 11) and the nucleic acid molecule encoding the recombinant horse albumin (nucleotide sequence shown in SEQ ID NO. 10) were commissioned to a biological company for direct synthesis, and the kanamycin resistance gene was changed to the geneticin G418 resistance gene (nucleotide sequence shown in SEQ ID NO. 12). The kanamycin resistance gene was then ligated into the plasmid pPIC9K digested with the restriction sites EcoRI and NotI to form an expression vector named pPIC9K-rESA (see Figure 1 shown).

[0067] The expression vector was linearized with the DNA restriction endonuclease NotI (enzyme digestion system is provided in Table 1) and transformed into Pichia pastoris X-33 by electroporation. After incubation at 30°C for 2 hours, the strain was plated onto YPDK plates containing 50 μg / mL kanamycin and cultured at 28°C for 48–72 hours. Sterile water was added to the plate and the strain was plated onto YPD plates containing 250 μg / mL G418. After culture at 28°C for 48–72 hours, positive single colonies were selected for subsequent expression. X-33 strains selected from the YPD plates were picked with a sterile 10 μL pipette tip for rESA expression. After shake flask culture, the expression level of recombinant equine albumin was determined by SDS-PAGE.

[0068] Shake flask culture steps: take the recombinant engineered bacteria and inoculate them into 100 mL of BMGY medium, and culture them at 28 °C and 210 rpm until the OD 600 = 1.0, aspirate 1 mL of the bacterial suspension for subsequent SDS-PAGE analysis, collect the entire suspension, centrifuge at 10,000 rpm for 5 min at room temperature, discard the supernatant aseptically, add 100 mL of BMMY medium to fully resuspend the cells, and incubate at 28°C, 220 rpm, adding a certain amount of methanol every 24 h to maintain a constant methanol concentration of 0.5%. After 72 h, centrifuge at 10,000 rpm for 5 min, and collect the supernatant.

[0069] Table 1

[0070]

[0071] Example 2: Determination of recombinant horse albumin copy number

[0072] The qPCR method (real-time quantitative PCR) was used to calculate the copy number of recombinant equine albumin: 5 recombinant engineered bacteria expressing high amounts of recombinant equine albumin prepared in Example 1 were selected, and the yeast genomic DNA extraction kit (TIANGEN DP325) was used to prepare the DNA of the strain to be tested to ensure the purity (A 260 / A 280 ≈1.8-2.0) and concentration (≥50 ng / μL). Primers were designed for the target gene recombinant equine albumin (rESA) gene sequence (SEQ ID NO. 10) in the pPIC9K vector and the single-copy gene GAPDH in Pichia pastoris. The primer sequences are shown in Table 2. rESA and GAPDH were cloned into the same vector to construct a plasmid containing the target gene and the internal reference gene. Serial dilutions (10 9 -10 6 100 copies / μL) was used as a standard. The qPCR amplification system is shown in Table 3.

[0073] Table 2

[0074]

[0075] Table 3

[0076]

[0077] Amplification procedure: 95°C pre-denaturation for 5 min → 40 cycles (95°C for 30 s → 60°C for 30 s → 72°C for 30 s) → melting curve analysis. Calculate the copy number ratio of the target gene to the reference gene based on the standard curve to obtain the rESA copy number.

[0078] The corresponding results of the yield and copy number of the recombinant engineering bacteria with higher yield are shown in Table 4.

[0079] Table 4

[0080]

[0081] As can be seen from Table 4, when the copy number is 4-7, the expression levels of the strains are not much different. When the copy number exceeds 5, the yield no longer increases significantly, and even decreases. It is speculated that this may be due to excessive protein retention in the endoplasmic reticulum or Golgi apparatus, triggering an unfolded protein reaction and resulting in blockage of the secretory pathway. To solve this problem, the present invention further modified the recombinant engineered bacteria 5, overexpressed genes related to protein folding, and knocked out genes related to cell wall formation to promote the secretion of recombinant horse albumin.

[0082] Example 3: Construction of cell wall gene SED1 and TIP1 gene deletion vector

[0083] The EasyGeno seamless cloning kit was used to amplify Pichia pastoris genomic DNA to construct the Pichia pastoris SED1 gene deletion vector pPICZαA-ΔSED1. The Zeocin resistance gene was replaced with a blasticidin resistance gene (nucleotide sequence shown in SEQ ID NO. 13). The construction of pPICZαA-ΔSED1 was commissioned to a biotechnology company. The primer sequences are shown in Table 5. The amplified pPICZαA-ΔSED1 vector was digested with BamHI monocleavage enzyme and purified with alkaline phosphatase to obtain the purified pPICZαA-ΔSED1 linear vector. The linearized vector pPICZαA-ΔSED1 was seamlessly cloned and ligated according to the EasyGeno kit instructions. After heat shock transformation, the vector was transformed into DH5α cells on LB plates coated with blasticidin (25 μg / mL) to select for positive transformants. The positive transformants were transformed with pPICZαA-ΔSED1 into the recombinant engineered bacteria 6 using the electroporation method to screen for blasticidin (500 μg / mL)-resistant clones to obtain the recombinant engineered bacteria 6.

[0084] The PCR amplification system is the same as shown in Table 3, the specific nucleotide sequences of the upstream primers and the downstream primers are shown in Table 5, and the EasyGeno seamless cloning system is shown in Table 6.

[0085] Table 5

[0086]

[0087] Table 6

[0088]

[0089] The EasyGeno seamless cloning kit was used to amplify Pichia pastoris genomic DNA to construct the Pichia pastoris TIP1 gene deletion vector pPICZαA-ΔTIP1. The Zeocin resistance gene was replaced with a blasticidin resistance gene (nucleotide sequence shown in SEQ ID NO. 13). The pPICZαA-ΔTIP1 construction process was commissioned to a biotechnology company. The amplified pPICZαA-ΔTIP1 vector was digested with BamHI monocleavage enzyme and purified with alkaline phosphatase to obtain the linearized pPICZαA-ΔTIP1 vector. The linearized pPICZαA-ΔTIP1 vector was seamlessly cloned and ligated according to the EasyGeno kit instructions. After heat shock transformation, the vector was transformed into DH5α cells onto LB plates coated with blasticidin (25 μg / mL) to select positive transformants. Positive transformants were then electroporated into recombinant engineered strain 6 to select for blasticidin (500 μg / mL)-resistant colonies, resulting in recombinant engineered strain 7.

[0090] Example 4: Construction of expression strains for protein folding genes PAC10 and YDJ1

[0091] A biotechnology company was commissioned to synthesize recombinant expression cassettes encoding the PAC10 gene (nucleotide sequence such as SEQ ID NO.6, amino acid sequence such as SEQ ID NO.5) and the YDJ1 gene (nucleotide sequence such as SEQ ID NO.8, amino acid sequence such as SEQ ID NO.7). The recombinant expression cassettes were then ligated into the plasmid pGAPZA (containing the Zeocin resistance gene) to construct expression vectors, named pART1-1 and pART1-2, respectively. The recombinant expression cassettes encoding the PAC10 and YDJ1 genes were ligated into the same plasmid pGAPZA (containing the Zeocin resistance gene) to construct an expression vector, named pART1-3.

[0092] The expression vectors pART1-1, pART1-2, and pART1-3 were linearized using the DNA restriction endonuclease BgIII and transformed into the recombinant engineered bacteria 7 by electroporation. The cells were spread on a YPDG selective plate containing Zeocin and cultured at 28°C until colonies appeared. The positive transformants were identified by colony PCR as recombinant engineered bacteria 8-10. The specific information is shown in Table 7.

[0093] Table 7

[0094]

[0095] Example 5: Expression level of recombinant horse albumin in recombinant engineered bacteria

[0096] 5L fermenter culture: 5-10 recombinant engineered bacteria were inoculated into 100 mL of BMGY medium, cultured at 28°C, 210 rpm for 16-24 h, and then transferred to 500 mL of BMGY medium (inoculation ratio of 1:10) and cultured until OD 600 =3.0. Add 2-3 L of BMGY (no more than 60% by volume) to a 5 L fermenter and adjust the pH to 6.0. Autoclave at 121°C for 20 minutes. After cooling, connect pH and DO electrodes and calibrate (pH using 4.0 and 6.86 standard buffers; the DO electrode is 100% calibrated to saturated air). Temperature: 28-30°C, initial agitation: 300-500 rpm, aeration: 0.5-1.0 vvm (volume ratio / minute), and pH: automatically controlled (using 28% ammonia or 25% phosphoric acid, target pH 5.0). Inoculate the fermenter with 10% seed solution and incubate for 18-24 hours. Then, supplement with 50% glycerol at a rate of 90 mL / h to maintain DO ≥ 20% until the cell wet weight reaches 200 g / L. Drain all residual glycerol and add BMMY medium at 28°C. Add methanol to a final concentration of 0.5% and incubate at 220 rpm. Add a certain amount of methanol every 24 hours to keep the methanol concentration constant at 0.5%. After 72 hours, centrifuge at 10,000 rpm for 5 minutes and collect the supernatant. The supernatant was subjected to SDS-PAGE electrophoresis. The electrophoretic pattern is as follows: Figure 2 The specific results are shown in Table 8.

[0097] The results showed that when one or more genes among SED1 and TIP1 were knocked out, and / or one or more genes among PAC10 and YDJ1 were overexpressed, the expression level of recombinant equine albumin was significantly increased. Among them, when the SED1 and TIP1 genes were knocked out and the PAC10 and YDJ1 genes were overexpressed, the expression level of recombinant equine albumin reached a maximum of 51.27 g / L, an increase of 5.38 times.

[0098] Table 8

[0099]

[0100] The foregoing is merely an embodiment of the present invention and is not intended to limit the present invention. It will be apparent to those skilled in the art that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention are intended to be included within the scope of the claims of the present invention.

Claims

1. A recombinant engineered bacterium for producing recombinant equine albumin, wherein the recombinant engineered bacterium, while expressing recombinant equine albumin, further comprises at least one of the following operations: (1) Knockout of SED1 and TIP1 genes; (2) Knockout of SED1 and TIP1 genes, and overexpression of PAC10 gene; (3) Knockout of SED1 and TIP1 genes, and overexpression of YDJ1 gene; (4) Knockout of SED1 and TIP1 genes, and overexpression of PAC10 and YDJ1 genes, in, The amino acid sequence encoded by the SED1 gene is shown in SEQ ID NO.1; the amino acid sequence encoded by the TIP1 gene is shown in SEQ ID NO.3; the amino acid sequence encoded by the PAC10 gene is shown in SEQ ID NO.5; and the amino acid sequence encoded by the YDJ1 gene is shown in SEQ ID NO.

7. The recombinant engineering bacteria is Pichia pastoris, and the copy number of the recombinant horse albumin in the recombinant engineering bacteria is 6.

2. The recombinant engineered bacterium according to claim 1, characterized in that The nucleotide sequence of the SED1 gene is shown in SEQ ID NO.2; the nucleotide sequence of the TIP1 gene is shown in SEQ ID NO.4; the nucleotide sequence of the PAC10 gene is shown in SEQ ID NO.6; and the nucleotide sequence of the YDJ1 gene is shown in SEQ ID NO.

8.

3. The recombinant engineered bacterium according to claim 1, characterized in that The expression vector of the recombinant engineered bacteria includes any one or more of pHIL-D2, pAO815, pPIC3K, pPICZ, pHWO10, pGAPZ, pPIC9K, pPICZα, and pGAPα; the promoter of the recombinant engineered bacteria includes any one or more of AOX1 promoter, GAP promoter, TEF1 promoter, GCW14 promoter, and AOX2 promoter.

4. The recombinant engineered bacterium according to claim 1, characterized in that The recombinant engineered bacteria utilizes CRISPR-Cas9 gene editing technology and homologous recombination technology to target and knock out one or more genes in SED1 and TIP1.

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

6. A method for preparing recombinant equine albumin, characterized in that: The method comprises: preparing recombinant horse albumin by fermentation using the recombinant engineered bacteria according to any one of claims 1 to 4.

7. A method for culturing the recombinant engineered bacteria according to any one of claims 1 to 4, characterized in that: The culture temperature in the method is 26-30° C., and the methanol concentration is 0.4-0.6%.

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