Recombinant engineering bacterium for preparing recombinant mararia albumin and application of recombinant engineering bacterium

Abstract

The invention provides a recombinant engineering bacterium for preparing recombinant equine albumin and application of the recombinant engineering bacterium, and relates to the technical field of genetic engineering. According to the invention, one or more of genes SED1 and TIP1 related to cell wall composition is / are knocked out in a targeted manner while the recombinant equine albumin is expressed, and / or one or more of genes PAC10 and YDJ1 related to protein folding promotion is / are overexpressed and integrated into a strain, so that the expression quantity of the recombinant equine albumin is improved. According to the recombinant engineering bacterium disclosed by the invention, the yield of the recombinant equine albumin can be obviously improved, the highest yield can reach 51.27 g / L, and compared with an unmodified recombinant engineering bacterium, the yield is improved by 5.38 times, and the recombinant engineering bacterium has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to a recombinant engineering bacterium for preparing recombinant horse albumin and its application. Background Art

[0002] Equine Serum Albumin (ESA), as the most abundant protein in serum, has diverse biological functions. It can maintain the plasma colloid osmotic pressure and ensure the balanced distribution of fluids inside and outside blood vessels; it can bind to and transport various endogenous and exogenous substances, such as fatty acids, hormones, drugs, etc., and regulate the metabolism and physiological effects of these substances in the body; it also has antioxidant properties, capable of scavenging free radicals and protecting cells from oxidative damage.

[0003] In the pharmaceutical field, Equine Serum Albumin has extensive application value. In drug research and development, it can be used as a drug carrier to improve the pharmacokinetic properties of drugs, enhance the stability and targeting of drugs, and enhance drug efficacy. In cell culture, Equine Serum Albumin is an important component of cell culture media, which can provide necessary nutrients and growth factors for cell growth, and promote cell proliferation and survival. In the field of biotechnology, it can be used in experiments such as protein purification and immunoassay, and is an important experimental reagent.

[0004] Traditional methods for extracting Equine Serum Albumin mainly involve separation and purification from horse serum. However, this method has many limitations. The source of horse serum is limited, restricted by factors such as the number of horses and blood collection frequency, and it is difficult to meet the demand for large-scale production. Moreover, extracting albumin from animal serum poses a potential risk of pathogen contamination, such as viruses, bacteria, etc., which may pose a threat to the health of users. In addition, the process of traditional extraction methods is complex and the cost is high, which is not conducive to the widespread application of products.

[0005] To address the limitations of traditional extraction methods, recombinant expression technology has emerged. By constructing a recombinant engineering bacterium for Equine Serum Albumin, large-scale and low-cost production of Equine Serum Albumin can be achieved. Recombinant expression technology enables production under controllable conditions, reduces the risk of pathogen contamination, and improves the safety and quality stability of products. Currently, with the rapid development of the pharmaceutical and biotechnology industries, the demand for Equine Serum Albumin is increasing day by day. Constructing a recombinant engineering bacterium for Equine Serum Albumin has important practical significance and broad market prospects.

[0006] Heterologous expression of proteins is of great significance in various biopharmaceutical industries and industrial applications. As one of the most successful eukaryotic protein expression systems, Pichia pastoris has experienced rapid development and received extensive attention in the past 20 years, generating huge economic and social value. At present, the methods for highly expressing foreign proteins mainly include foreign gene modification, strain modification, and fermentation level optimization. From the perspective of foreign gene modification, it mainly includes optimization of foreign gene codons, increase in the integration copy number of foreign genes, and selection of leader peptides; from the perspective of strain modification, it mainly includes knocking out protein degrading enzyme genes, introducing protein folding promoting factors, introducing Vitreoscilla hemoglobin (Vgb) genes, and knocking out glycerol transporter genes; fermentation level optimization includes adjusting temperature and dissolved oxygen levels 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 research on the initial induced cell mass and methanol feeding method (Zhu Wen, Hu Youjia, Xie Liping. Related strategies and research progress of highly expressing foreign proteins in Pichia pastoris [J], Chinese Journal of Pharmaceuticals 2018, 49(4): 417-425). CN118580980A discloses a recombinant engineering bacterium with defective cell walls, and its human albumin production is significantly increased. These optimizations of Pichia pastoris provide a basis for highly expressing equine albumin. Summary of the Invention

[0007] The present invention improves the production of equine albumin through three aspects: increasing the copy number of foreign genes, introducing the α mating factor signal peptide, and strain modification. Further, the present invention selects to introduce protein folding genes into the endoplasmic reticulum and simultaneously knock out cell wall genes in the target strain to achieve the purpose of improving protein expression. The specific scheme of the present invention is as follows: In the first aspect of the present invention, there is provided a recombinant engineering bacterium for preparing recombinant equine albumin. While expressing recombinant equine albumin, the recombinant engineering bacterium further 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 realizing the high-efficiency expression of recombinant equine albumin and laying a foundation for the industrial application of recombinant equine albumin.

[0008] Preferably, the operation at least includes any one of the following: (1) Knock out the SED1 and TIP1 genes; (2) Knock out the SED1 and TIP1 genes and overexpress the PAC10 gene; (3) Knock out the SED1 and TIP1 genes and overexpress the YDJ1 gene; (4) Knock out the SED1 and TIP1 genes, and overexpress the PAC10 and YDJ1 genes.

[0009] Furthermore, the amino acid sequence encoded by the SED1 gene is shown in SEQ ID NO.1, and the nucleotide sequence of the SED1 gene is shown in SEQ ID NO.2; the amino acid sequence encoded by the TIP1 gene is shown in SEQ ID NO.3, and the nucleotide sequence of the TIP1 gene is shown in SEQ ID NO.4.

[0010] The amino acid sequence encoded by the PAC10 gene is shown in SEQ ID NO.5, and the nucleotide sequence of the PAC10 gene is the nucleotide sequence encoding the amino acid sequence of SEQ ID NO.5, including the sequences publicly available in the current database and the nucleotide sequences optimized according to actual needs. Preferably, the nucleotide sequence is shown in SEQ ID NO.6 or has at least 95% homology with the nucleotide sequence shown in SEQ ID NO.6.

[0011] The amino acid sequence encoded by the YDJ1 gene is shown in SEQ ID NO.7, and the nucleotide sequence of the YDJ1 gene is the nucleotide sequence encoding the amino acid sequence of SEQ ID NO.7, including the sequences publicly available in the current database and the nucleotide sequences optimized according to actual needs. Preferably, the nucleotide sequence is shown in SEQ ID NO.8 or has at least 95% homology with the nucleotide sequence shown in SEQ ID NO.8.

[0012] Furthermore, the amino acid sequence of the horse albumin is shown in SEQ ID NO.9. After codon optimization, the horse albumin is synthesized and cloned onto an expression vector to construct a recombinant plasmid.

[0013] Furthermore, the nucleotide sequence of the recombinant horse albumin is SEQ ID NO.10 or a nucleotide sequence having at least 95% homology with SEQ ID NO.10.

[0014] Furthermore, the foreign gene expressed by the recombinant engineering bacteria can also be selected from one of other recombinant albumins. Preferably, the recombinant albumin includes one of recombinant human albumin, recombinant cat albumin, recombinant dog albumin, recombinant bovine albumin, recombinant ovine albumin, and recombinant porcine albumin.

[0015] Furthermore, the recombinant engineering bacteria are selected from one or more of Pichia pastoris, Hansenula anomala, Candida sp., and Saccharomyces cerevisiae. Preferably, the recombinant engineering bacteria are Pichia pastoris. Further, the Pichia pastoris strains include 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.

[0016] Furthermore, the expression vectors of the recombinant engineering bacteria include any one or more of pHIL-D2, pAO815, pPIC3K, pPICZ, pHWO10, pGAPZ, pPIC9K, pPICZα, and pGAPα. Further, the expression vector is a multi-copy insertion type expression vector. Preferably, the multi-copy insertion type expression vector is selected from at least one of pPIC3.5K, pPIC9K, and pAO815.

[0017] Furthermore, the promoters of the recombinant engineering bacteria include any one or more of AOX1 promoter, GAP promoter, TEF1 promoter, GCW14 promoter, and AOX2 promoter. In a specific embodiment of the present invention, the promoter is AOX1 promoter.

[0018] Furthermore, the recombinant engineering bacteria use CRISPR-Cas9 gene editing technology and homologous recombination technology to targetedly knockout one or more genes of SED1 and TIP1.

[0019] Furthermore, the recombinant engineering bacteria use CRISPR-Cas9 gene editing technology to targetedly knockout one or more genes of SED1 and TIP1, including the following steps: 1-1: Construct a co-expression plasmid of Cas9 and gRNA; 1-2: Prepare donor DNA; 1-3: Prepare competent cells; 1-4: Construct a strain that knocks out one or more genes of SED1 and TIP1.

[0020] Furthermore, for the donor DNA, using the Pichia pastoris genomic DNA as a template, one or more gene left and right homologous arm fragments of SED1 and TIP1 are obtained by overlapping extension PCR technology.

[0021] Furthermore, the homologous recombination technology is EasyGeno seamless cloning technology. The recombinant engineering bacteria use EasyGeno seamless cloning technology to targetedly knockout one or more genes of SED1 and TIP1, including the following steps: 2-1: Linearize the vector; 2-2: Prepare the insertion fragments, which respectively include the upstream and downstream fragments of the SED1 and TIP1 genes; 2-3: Conduct a recombination reaction, where the molar ratio of vector:fragment ≈ 1:2-3; 2-4: Directly transform the recombination reaction product into competent cells.

[0022] Furthermore, the copy number of the nucleotide sequence encoding recombinant equine albumin in the recombinant engineering bacteria is 4-7. In the examples of the present invention, the copy number of the nucleotide sequence encoding recombinant equine albumin in the recombinant engineering bacteria is 6.

[0023] Furthermore, the recombinant engineering bacteria of the present invention further include a drug resistance gene fragment. The drug resistance gene fragment is used for the screening of recombinant engineering bacteria. In the examples of the present invention, the drug resistance fragment is one or more of the geneticin G418 resistance gene, kanamycin resistance gene, ampicillin resistance gene, His4 gene, and blasticidin resistance gene. Furthermore, the recombinant engineering bacteria also include a signal peptide sequence, and the signal peptide sequence is used for the secretory expression of foreign proteins in the recombinant engineering bacteria. In the examples of the present invention, the signal peptide sequence in the recombinant engineering 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 as shown in SEQ ID NO.11.

[0024] In the second aspect of the present invention, there is provided the use of the recombinant engineering bacteria in the preparation of recombinant equine albumin and / or the increase of the expression level of recombinant equine albumin.

[0025] In the third aspect of the present invention, there is provided recombinant equine albumin prepared by fermenting the above-mentioned recombinant engineering bacteria.

[0026] In the fourth aspect of the present invention, there is provided a method for culturing the recombinant engineering bacteria, wherein the culture temperature in the method is 26-30 °C, the methanol concentration is 0.4-0.6%, and the induction time is 70-74 h.

[0027] In the specific examples 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.

[0028] The beneficial effects of the present invention include: The present invention constructs a recombinant engineering bacterium for preparing recombinant horse albumin. While expressing recombinant horse albumin, one or more genes related to cell wall composition, such as SED1 and TIP1, are targeted for knockout, and / or one or more genes related to promoting protein folding, such as PAC10 and YDJ1, are overexpressed and integrated into the strain. The present invention modifies the recombinant engineering bacterium from two aspects: promoting protein folding and cell wall defect, so as to achieve the purpose of increasing the yield of recombinant horse albumin. The recombinant engineering bacterium of the present invention significantly increases the expression level of recombinant horse albumin, and the yield of recombinant horse albumin can reach up to 51.27 g / L at most. Compared with the unmodified recombinant engineering bacterium, the yield is increased by 5.38 times. This recombinant engineering bacterium shows great potential in improving the yield of proteins with high economic value and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The drawings described herein are used to provide a further understanding of the present invention and form a part of the present invention. The schematic 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: Figure 1 It is a schematic structural diagram of a recombinant expression cassette encoding recombinant horse albumin; Figure 2 It is an SDS-PAGE gel electrophoresis diagram of the recombinant engineering bacterium 5-10 expressing recombinant horse albumin in the example. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] Recombinant engineering bacterium: A bacterial cell line in which foreign genes are highly expressed by genetic engineering methods.

[0031] In order to more clearly illustrate the overall concept of the present invention, the following will be described in detail by way of examples in conjunction with the drawings of the specification. In the following description, a large number of specific details are given 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.

[0032] For those not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer.

[0033] Unless otherwise specified, in the following embodiments, for reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through commercial purchase.

[0034] The molecular biology experimental methods not specifically described in the following examples are all carried out according to the kits and product instructions, or are carried out with reference to the methods described in "Molecular Cloning: A Laboratory Manual" (Third Edition) by J. Sambrook.

[0035] The sources of experimental materials and reagents involved in the present invention are as follows.

[0036] Strain: Pichia pastoris X-33, purchased from Thermo Fisher Scientific. This strain itself has resistance to antibiotics of prokaryotes, such as resistance to kanamycin and ampicillin, and X-33 is a His+ strain, which grows faster and can tolerate high copy numbers.

[0037] Expression vector and expression cassette: Customized from GenScript Biotech Corporation.

[0038] Enzymes and kits Restriction endonucleases BgI II, Not I, and BamH I were purchased from Thermo Fisher Scientific; plasmid extraction kit, agarose gel recovery kit, and DNA product purification kit were purchased from Tiangen Biotech 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 Biotech Co., Ltd.; alkaline phosphatase (CIP) was purchased from NEB; DH5α competent cells were purchased from Tiangen Biotech Co., Ltd.

[0039] Media E. coli solid medium LB: 1% (w / v) tryptone, 0.5% (w / v) yeast extract, 1.5% (w / v) agar, 1% (w / v) NaCl, pH 7.0.

[0040] E. coli liquid medium LB: 1% (w / v) tryptone, 0.5% (w / v) yeast extract, 1% (w / v) NaCl, pH 7.0.

[0041] Yeast solid medium YPD: 1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose, 2% (w / v) agar.

[0042] Yeast liquid medium YPD: 1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose, PBS buffer, pH = 7.0.

[0043] 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 (where 0.23% (w / v) K2HPO4 and 1.18% (w / v) KH2PO4 can be directly replaced by PBS solution).

[0044] 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 (where 0.23% (w / v) K2HPO4 and 1.18% (w / v) KH2PO4 can be directly replaced by PBS solution).

[0045] Buffer: 0.283% (w / v) Na2HPO4, 0.4% (w / v) NaH2PO4, pH = 7.4. Example 1: Construction of recombinant Pichia pastoris strain expressing equine albumin The nucleic acid molecule encoding the mating factor signal peptide of Saccharomyces cerevisiae (SEQ ID NO.11) and the nucleic acid molecule encoding recombinant equine albumin (nucleotide sequence as shown in SEQ ID NO.10) were directly synthesized by a biological company, and the kanamycin resistance gene was changed to the geneticin G418 resistance gene (nucleotide sequence as shown in SEQ ID NO.12), and then ligated into the plasmid pPIC9K digested with the restriction enzyme sites EcoRI and NotI to form an expression vector, named pPIC9K-rESA (as Figure 1 shown).

[0046] The expression vector was linearized using the DNA restriction enzyme NotI (the digestion system is shown in Table 1), electrotransformed into Pichia pastoris X-33, incubated at 30 °C for 2 h and then spread on a YPDK plate containing 50 μg / mL kanamycin, cultured at 28 °C for 48 - 72 h, added with a sterile water scraper, and spread on a YPD plate containing 250 μg / mL G418, and cultured at 28 °C for 48 - 72 h. Positive monoclonal colonies were screened for subsequent expression. The X-33 strain screened on the YPD plate was picked with a sterilized 10 μL pipette tip for the expression of rESA, and the expression level of recombinant equine albumin was determined by SDS-PAGE after shake flask culture.

[0047] Shaking flask culture steps: Recombinant engineering bacteria were separately inoculated into 100 mL of BMGY medium and cultured at 28 °C and 210 rpm until OD 600 = 1.0. 1 mL of the bacterial solution was taken for subsequent SDS-PAGE analysis. All the bacterial solution was collected, centrifuged at 10000 rpm for 5 min at room temperature, the supernatant was discarded aseptically, 100 mL of BMMY medium was added to fully suspend the bacterial cells, and the cells were cultured at 28 °C and 220 rpm. A certain amount of methanol was added every 24 h to keep the methanol concentration constant at 0.5%. After 72 h, the cells were centrifuged at 10000 rpm for 5 min, and the supernatant was collected.

[0048] Table 1

[0049] Example 2: Determination of the copy number of recombinant horse albumin The copy number of recombinant horse albumin was calculated by qPCR (real-time quantitative PCR): Five recombinant engineering bacteria with high expression levels of recombinant horse albumin prepared in Example 1 were selected, and the DNA of the strains to be tested was prepared using a yeast genomic DNA extraction kit (TIANGEN DP325) to ensure the purity (A 260 / A 280 ≈ 1.8 - 2.0) and concentration (≥ 50 ng / μL). Primers were designed for the gene sequence of the target gene recombinant horse albumin (rESA) (SEQ ID NO.10) in the pPIC9K vector and the single-copy gene GAPDH gene of 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, and were serially diluted (10 9 -10 6 copies / μL) as the standard. The qPCR amplification system is shown in Table 3.

[0050] Table 2

[0051] Table 3

[0052] Amplification program: Pre-denaturation at 95 °C for 5 min → 40 cycles (95 °C for 30 s → 60 °C for 30 s → 72 °C for 30 s) → Melting curve analysis. The copy number of rESA was obtained by calculating the ratio of the copy numbers of the target gene and the internal reference gene according to the standard curve.

[0053] The corresponding results of the yields and copy numbers of the recombinant engineering bacteria with higher yields are shown in Table 4.

[0054] Table 4

[0055] As can be seen from Table 4, when the copy number is 4 - 7, the expression levels of the strains are not significantly 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 the retention of excessive proteins in the endoplasmic reticulum or Golgi apparatus, triggering the unfolded protein response and resulting in blockage of the secretion pathway. To solve this problem, the present invention further modifies recombinant engineering bacterium 5, overexpresses genes related to protein folding, and simultaneously knocks out genes related to cell wall formation to promote the secretion of recombinant equine albumin.

[0056] Example 3: Construction of gene deletion vectors for cell wall components SED1 and TIP1 Using the EasyGeno seamless cloning kit, the Pichia pastoris SED1 gene deletion vector pPICZαA - ΔSED1 was constructed by amplifying the Pichia pastoris genomic DNA. In this process, the Zeocin resistance gene was replaced with the blasticidin resistance gene (nucleotide sequence shown in SEQ ID NO.13). The steps of constructing pPICZαA - ΔSED1 were entrusted to a biological company. The primer sequences are shown in Table 5. The amplified pPICZαA - ΔSED1 vector was digested with BamH I single - cutter enzyme and then treated with alkaline phosphatase to purify the linear vector to obtain the purified pPICZαA - ΔSED1 linear vector. The linear vector pPICZαA - ΔSED1 was ligated by seamless cloning according to the EasyGeno kit instructions and then heat - shocked and transformed into DH5α, and the transformed cells were spread on LB plates containing blasticidin (25 μg / mL) to screen for positive transformants. The obtained positive transformants were used to transfer pPICZαA - ΔSED1 into recombinant engineering bacterium 6 by electroporation, and clones resistant to blasticidin (500 μg / mL) were screened to obtain recombinant engineering bacterium 6.

[0057] The PCR amplification system is the same as shown in Table 3. The specific nucleotide sequences of the upstream and downstream primers are shown in Table 5, and the EasyGeno seamless cloning system is shown in Table 6.

[0058] Table 5

[0059] Table 6

[0060] The EasyGeno seamless cloning kit was used to amplify the genomic DNA of Pichia pastoris to construct the Pichia pastoris TIP1 gene deletion vector pPICZαA-ΔTIP1, in which the Zeocin resistance gene was replaced with the Blasticidin resistance gene (nucleotide sequence as shown in SEQ ID NO.13). The steps of constructing pPICZαA-ΔTIP1 were entrusted to a biological company. The amplified pPICZαA-ΔTIP1 vector was digested with the single restriction enzyme BamH I and then alkaline phosphatase was added to purify the linear vector to obtain the pPICZαA-ΔTIP1 linear vector. The linear vector pPICZαA-ΔTIP1 was ligated by seamless cloning according to the EasyGeno kit instructions and then heat shock transformed into DH5α, and the transformed cells were spread on an LB plate containing Blasticidin (25 μg / mL) to screen for positive transformants. The obtained positive transformants were used to transform pPICZαA-ΔTIP1 into the recombinant engineering strain 6 by electroporation, and Blasticidin (500 μg / mL) resistant clones were screened to obtain the recombinant engineering strain 7.

[0061] Example 4: Construction of protein folding gene PAC10 and YDJ1 expression strains A biological company was entrusted to synthesize the recombinant expression cassettes encoding the PAC10 gene (nucleotide sequence as shown in SEQ ID NO.6, amino acid sequence as shown in SEQ ID NO.5) and the YDJ1 gene (nucleotide sequence as shown in SEQ ID NO.8, amino acid sequence as shown in SEQ ID NO.7) respectively, and the recombinant expression cassettes were 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 two genes PAC10 and YDJ1 were ligated into the same plasmid pGAPZA (containing the Zeocin resistance gene) to construct an expression vector, named pART1-3.

[0062] The expression vectors pART1-1, pART1-2, and pART1-3 were linearized using the DNA restriction enzyme BgI II, and then electrotransformed into the recombinant engineering strain 7 respectively. The cells were spread on the selective plate YPDG containing Zeocin and cultured at 28 °C until colonies appeared. The positive transformants were identified by colony PCR as the recombinant engineering strains 8-10, and the specific information is shown in Table 7.

[0063] Table 7

[0064] Example 5: Expression level of recombinant equine albumin in recombinant engineering bacteria 5L Fermenter Culture: 5 - 10 of the recombinant engineering bacteria were respectively inoculated into 100 mL of BMGY medium and cultured at 28 °C and 210 rpm for 16 - 24 h, then transferred to 500 mL of BMGY medium (inoculation ratio 1:10) and cultured until OD 600 = 3.0. 2 - 3 L of BMGY (volume not exceeding 60%) was added to the 5L tank, and the pH was adjusted to 6.0. Autoclaved at 121 °C for 20 minutes. After cooling, connect the pH and DO electrodes and calibrate (pH with 4.0 and 6.86 standard buffer solutions; DO electrode calibrated to 100% saturated air). Temperature: 28 - 30 °C, initial stirring: 300 - 500 rpm, aeration rate: 0.5 - 1.0 vvm (volume ratio / minute), pH: automatically controlled (adjusted with 28% ammonia water or 25% phosphoric acid, target pH 5.0). The seed solution was inoculated into the fermenter at an inoculation amount of 10% and cultured for 18 - 24 h, then 50% glycerol was supplemented at a rate of 90 mL / h to maintain DO ≥ 20% until the wet cell weight reached 200 g / L. Drain the residual glycerol, add BMMY medium and culture at 28 °C, add methanol to a final concentration of 0.5%, and culture at 220 rpm. A certain amount of methanol was supplemented every 24 h to keep the methanol concentration constant at 0.5%. After 72 h, centrifuge at 10000 rpm for 5 min and collect the supernatant. The supernatant was detected by SDS-PAGE electrophoresis, and the electrophoresis pattern was as Figure 2 shown, and the specific results are shown in Table 8.

[0065] The results showed that when one or more genes of SED1 and TIP1 were knocked out, and / or one or more genes of PAC10 and YDJ1 were overexpressed, the expression level of recombinant equine albumin increased significantly. Among them, when knocking out the genes of SED1 and TIP1 and overexpressing the genes of PAC10 and YDJ1 simultaneously, the highest expression level of recombinant equine albumin reached 51.27 g / L, which was increased by 5.38 times.

[0066] Table 8

[0067] The above are only the embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the scope of the claims of the present invention.

Claims

1. A recombinant engineering bacterium for preparing recombinant equine albumin, which, while expressing recombinant equine albumin, further comprises at least any one of the following operations: (1) Knocking out the SED1 and TIP1 genes; (2) Knocking out the SED1 and TIP1 genes and overexpressing the PAC10 gene; (3) Knocking out the SED1 and TIP1 genes and overexpressing the YDJ1 gene; (4) Knocking out the SED1 and TIP1 genes and overexpressing the PAC10 and YDJ1 genes.

2. The recombinant engineering bacterium according to claim 1, wherein The amino acid sequence encoded by the SED1 gene is as shown in SEQ ID NO.1; the amino acid sequence encoded by the TIP1 gene is as shown in SEQ ID NO.3; the amino acid sequence encoded by the PAC10 gene is as shown in SEQ ID NO.5; the amino acid sequence encoded by the YDJ1 gene is as shown in SEQ ID NO.

7.

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

8.

4. The recombinant engineering bacterium according to claim 1, wherein The recombinant engineering bacterium is selected from one or more of Pichia pastoris, Hansenula anomala, Candida spp., and Saccharomyces cerevisiae.

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

6. The recombinant engineering bacteria according to claim 1, characterized in that, The recombinant engineering bacterium uses the CRISPR-Cas9 gene editing technology and homologous recombination technology to target and knock out one or more of the SED1 and TIP1 genes.

7. The recombinant engineering bacterium according to claim 1, characterized in that, The copy number of recombinant equine albumin in the recombinant engineering bacterium is 4 to 7.

8. Use of the recombinant engineering bacterium according to any one of claims 1-7 in the preparation of recombinant equine albumin and / or increasing the expression level of recombinant equine albumin.

9. A method for preparing recombinant equine albumin, characterized in that, The method includes: fermenting with the recombinant engineering bacterium according to any one of claims 1-7 to prepare recombinant equine albumin.

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

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