Pichia pastoris bioengineering bacteria for relieving collagen degradation and construction method and use thereof

Abstract

The application discloses a pichia pastoris bioengineering bacterium for relieving degradation of recombinant collagen, a preparation method and application thereof. Through bioinformatics analysis, gene knockout and function verification screening, three protease genes capable of relieving degradation of collagen in a pichia pastoris host are obtained, namely, chr1-4_0611, chr1-4_0362 and chr2-2_0380. Knocking out the protease chr1-4_0611 can make the total protein yield accumulation increase to 3.5 times of that of a control bacterium, and the proportion of a target band is greater than 50%, which can provide good theoretical and technical guidance for effective expression of other recombinant proteins in the pichia pastoris host.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a Pichia pastoris bioengineered strain that alleviates collagen degradation, its construction method, and its uses. Background Technology

[0002] Collagen, as a natural polymer material, not only possesses excellent biocompatibility, degradability, and low immunogenicity, but also boasts high tensile strength and hemostatic properties. These advantages have made collagen a rising star in the medical aesthetics industry. Recombinant humanized collagen is based on the original sequence of human collagen. Highly water-soluble and bioactive portions are selected and recombined or tandemly repeated, with codon optimization to obtain the recombinant humanized collagen sequence. Large-scale production can be achieved by selecting suitable hosts and fermentation technologies. Increasing research confirms that recombinant humanized collagen exhibits good water solubility and high bioactivity, outperforming natural human collagen, and has broad application prospects in biomedical materials, cosmetics, and health foods.

[0003] Pichia pastoris, as a biosafe host, possesses advantages such as non-pathogenicity, no endotoxin production, and the ability to obtain high yields of recombinant proteins through high-density fermentation. However, due to Pichia pastoris' powerful degradation system, most recombinant proteins undergo varying degrees of degradation during expression in the host, significantly reducing the yield of the target protein and posing substantial challenges to downstream separation and purification processes.

[0004] Currently, there are three main ways to alleviate the degradation of recombinant proteins in Pichia pastoris hosts: (1) constructing protease gene-deficient hosts; for example, in 2013, Wu Min et al. knocked out multiple protease genes in Pichia pastoris hosts, including the PEP4 gene encoding protease A, the PRB1 gene encoding protease B, and the YPS gene family (yapsin family members). They found that when YPS1 and PEP4 genes were knocked out simultaneously, the fusion protein [HSA / PTH(1-34)] of recombinant human serum albumin (HSA) and parathyroid hormone (1-34) [PTH(1-34)] could maintain about 80% integrity, while knocking out YPS1 alone had a weak effect on alleviating protein degradation; this conclusion is consistent with the conclusion of Marc WT et al. in 2004 that knocking out YPS1 alone had almost no effect on alleviating protein degradation; in 1999, Thomas Boehmd et al. expressed endostatin with C-terminal lysine retention after knocking out the kex1 gene in the Pichia pastoris host. Similarly, in 2016, Suma Sreenivas et al. alleviated 80% of the C-terminal arginine degradation of double-stranded glargine insulin by knocking out the kex1 gene in the Pichia pastoris host. These two studies show that KEX1 knockout strains can be used as tools to express proteins whose C-terminus are easily degraded by acidic carboxypeptidase.(2) By mutation or deletion of some protease cleavage sequences; for example: In 2008, Zhang Qingfeng et al. found that the NANP motif exposed at the N-terminus was the main cause of protein instability when expressing the circospore protein PfCSP of Plasmodium falciparum. By adding LRKPKHKKLKQPADGNPDPNANPNVDP to the N-terminus, they obtained a single full-length protein consistent with the theory. In 2014, Holger Spiegel et al. identified the protease cleavage sequences EKRK and PEVK at the N-terminus and C-terminus of the recombinant multi-subunit malaria vaccine candidate protein, respectively. After targeted modification, they finally achieved the production of a full-length recombinant vaccine candidate protein without significant degradation. In 2014, MA Tsygankov et al. found that truncated recombinant gamma interferon with higher stability and immunomodulatory activity was obtained by deleting 10 or 12 amino acids containing trypsin-like protease recognition sites at the C-terminus or by simultaneously introducing cysteine ​​or asparagine at the C-terminus. In 2016, Jia Hao et al. improved the stability and immunomodulatory activity of interferon IFN-α / IgG. In FC fusion proteins, the N-glycosylation site 297N in the Fc region is mutated to 297Q, which prolongs its half-life. In 2018, Zhang Yunfeng et al. mutated the Y in the N-terminal peptide YVEF of Streptomyces griseus trypsin SGT to hydrophobic amino acids A, L, M, F, V, P, I, and W. They found that when the mutation was FVEF, the expression level and enzyme activity of SGT were significantly increased. They speculated that the higher hydrophobicity of FVEF (F>Y) allows the N-terminal sequence to be translated and folded at a more suitable rate. This phenomenon of providing sufficient time for the nascent peptide chain to fold correctly into specific domains by keeping the N-terminal sequence at a lower translation rate exists in most proteins.(3) By adding protease inhibitors; for example, in 2000, Kaoru Kobayashi et al. studied the effects of adding different protease inhibitors to Pichia pastoris hosts on the degradation of recombinant human serum albumin and summarized the types of proteases inhibited by different inhibitors: Antipain is a serine protease and cysteine ​​protease inhibitor; Bestatin is an aminopeptidase inhibitor; Chymostatin is a cysteine ​​protease and serine protease inhibitor; DFP (diisopropylfluorophosphate) is a serine protease inhibitor; E-64 is a cysteine ​​protease inhibitor; Elastatinal is a serine protease inhibitor; Leupeptin is a serine protease and cysteine ​​protease inhibitor; Pepstatin A is an aspartic protease inhibitor; PMSF (phenylmethylsulfonyl fluoride) is a serine protease inhibitor; Phosphoramidon is a metalloproteinase inhibitor; and soybean trypsin inhibitor is a serine protease inhibitor.

[0005] In summary, no single approach can prevent degradation of all types of recombinant proteins expressed in Pichia pastoris. This is because the Pichia pastoris host primarily contains three classes of proteases: cytoplasmic proteases, vacuolar proteases, and secretion-related proteases. Vacuole proteases are responsible for most cellular protein hydrolysis, especially under nutrient deprivation conditions. Vacuole proteases are involved in cell differentiation, precursor maturation and vacuolar protease activation, general protein degradation (especially under nutrient stress), exogenous peptide metabolism, and other functions. Currently, there are eight known vacuolar proteases: two endopeptides: protease yscA (PrA, PEP4) and protease yscB (PrB); two carboxypeptidases: carboxypeptidases Y and S (CpY, CpS); two aminopeptidases: aminopeptidases I and aminopeptidases Co (ApI, ApCo); and dipeptidyl aminopeptidases B (DPAP-B). Each protease has a different degradation site, and there are also unknown proteases. This means that to mitigate the degradation of recombinant collagen, it is necessary to explore or develop specific methods tailored to each protease.

[0006] It should be noted that the information disclosed in this background section is only for understanding the background technology of the present application concept, and therefore, the above description is not considered to constitute prior art information. Summary of the Invention

[0007] To address the above problems, the present invention provides a Pichia pastoris bioengineered strain that alleviates the degradation of recombinant collagen. The engineered strain is obtained by knocking out the protease gene chr1-4_0611, chr1-4_0362 or chr2-2_0380 in the Pichia pastoris genome. Knocking out the chr1-4_0611, chr1-4_0362, or chr2-2_0380 genes can increase the accumulation of the target protein by 6% to 68%. This invention also publicly verifies for the first time the function of the three genes chr1-4_0611, chr1-4_0362, and chr2-2_0380 in alleviating protein degradation. Furthermore, chr1-4_0611, chr1-4_0362, and chr2-2_0380 all belong to the metalloproteinase family. This explains that the addition of EDTA to alleviate collagen degradation is directly related to the inhibition of metalloproteinase activity, and also proves that the metalloproteinases chr1-4_0611, chr1-4_0362, and chr2-2_0380 are involved in the degradation of collagen or other proteins in the Pichia pastoris host.

[0008] The present invention provides a bioengineered bacterium, wherein the bioengineered bacterium is Pichia pastoris with any one or more proteases of chr1-4_0611, chr1-4_0362 or chr2-2_0380 knocked out.

[0009] Among them, chr1-4_0611, chr1-4_0362 or chr2-2_0380 are aminopeptidase, metalloproteinase and metalloproteinase respectively, and their nucleotide sequences are shown as Seq ID NO.1, Seq ID NO.4 and Seq ID NO.6 respectively.

[0010] Furthermore, the Pichia pastoris is any one of GS115, KM71H, SMD1168H, and SMD1163.

[0011] Another aspect of the present invention provides the use of the above-mentioned bioengineered bacteria in the preparation of recombinant collagen.

[0012] Furthermore, the collagen is type I, type II, type III, or type XVII collagen.

[0013] The following embodiments use only these two examples for illustration, but this application is not limited to these.

[0014] Another aspect of the present invention provides a method for preparing recombinant collagen, wherein the method involves using the above-mentioned Pichia pastoris as a fermentation strain, culturing, and collecting the fermentation product to obtain recombinant collagen.

[0015] Furthermore, the collagen is any one of type I, type II, type III, or type XVII collagen.

[0016] Furthermore, the Pichia pastoris is any one of GS115, KM71H, SMD1168H, or SMD1163.

[0017] Furthermore, the method specifically involves: constructing gene-knockout Pichia pastoris, screening out successfully knocked-out strains, transforming plasmids containing collagen gene expression fragments into the knockout strains, inoculating them into a culture medium for culture, fermenting, and collecting the fermentation products to obtain collagen.

[0018] Furthermore, the gene knockout method is to use the CRISPR gene editing system, Cre / loxp, or Flp / FRT for knockout. Those skilled in the art can use these methods for gene knockout based on existing knowledge.

[0019] Furthermore, methods for culturing Pichia pastoris are well known in the art, and can also be found in the following examples.

[0020] Beneficial technical effects: We reduced collagen degradation by constructing gene knockout mechanisms, specifically the gene chr1-4_0611 (Seq ID NO.1), chr1-4_0362 (Seq ID NO.4), or chr2-2_0380 (Seq ID NO.6) from Pichia pastoris. Please refer to the following examples for details. Attached Figure Description

[0021] Figure 1 DAS1TT and Cas9 gene retrieval results Figure 2 pPICZH-ARS-DASTT1-Cas9 colony PCR verification Figure 3 P HTX and 2-gRNA gene retrieval results Figure 4 pPICZH-panARS-DASTT1-cas9-HTX-HH-gRNA1-2-HDV colony PCR verification Figure 5 1. Results of gRNA retrieval from genes 3-10 Figure 6 pPICZH-panARS-DASTT1-cas9-HTX-HH-gRNA1-1 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10-HDV colony PCR verification Figure 7 Donor DNA retrieval results Figure 8 Transformation status of gene knockout plasmid after electroporation into KM71H Figure 9 KM71H gene knockout 1-2 colony PCR verification Figure 10 KM71H gene knockout 3-4 colony PCR verification Figure 11 KM71H gene knockout 5-6 colony PCR verification Figure 12 KM71H gene knockout 7-8 colony PCR verification Figure 13 KM71H gene knockout 9-10 colony PCR verification Figure 14 KM71H knockout plasmid loss results Figure 15 Results of KM71HΔ1-10 knockout bacteria transformed with pPIC9KM-C3A1 plasmid Figure 16 KM71HΔ1-2 / pPIC9KM-C3A1 colony PCR results Figure 17 KM71HΔ3-4 / pPIC9KM-C3A1 colony PCR results Figure 18 KM71HΔ5-6 / pPIC9KM-C3A1 colony PCR results Figure 19 KM71HΔ7-8 / pPIC9KM-C3A1 colony PCR results Figure 20 KM71HΔ9-10 / pPIC9KM-C3A1 colony PCR results Figure 21 SDS-PAGE results of collagen expression in KM71HΔ1-4 / pPIC9KM-C3A1 knockout bacteria Figure 22 SDS-PAGE results of collagen expression in KM71HΔ5-8 / pPIC9KM-C3A1 knockout bacteria Figure 23 SDS-PAGE results of collagen expression in KM71HΔ9-10 / pPIC9KM-C3A1 knockout bacteria Figure 24 Comparison of KM71HΔ1-10 / pPIC9KM-C3A1 knockout bacteria collagen shake-flask fermentation results Figure 25 Genome extraction results of KM71HΔ1 / Δ4 / Δ6 / pPIC9KM-C3A1 knockout bacteria Figure 26 Relative C3A1 gene copy number in KM71HΔ1 / Δ4 / Δ6 / pPIC9KM-C3A1 knockout bacteria Figure 27 KM71HΔ1 / Δ4 / Δ6 / pPIC9KM-C3A1 Knockout Microbial Tank Fermentation SDS-PAGE Comparison Figure 28 Transformation status of gene knockout plasmid after electroporation into SMD1168H Figure 29 SMD1168H gene knockout 1 / 4 / 6 colony PCR verification Figure 30 SMD1168H knockout plasmid loss results Figure 31 Results of transformation of SMD1168HΔ1 / Δ4 / Δ6 knockout bacteria into pPIC9KM-C3A1 plasmid Figure 32 SMD1168HΔ1 / Δ4 / Δ6-C3A1 knockout bacterial colony PCR verification Figure 33 SDS-PAGE results of SMD1168HΔ1 / Δ4 / Δ6-C3A1 collagen expression Figure 34 Comparison of SMD1168HΔ1 / Δ4 / Δ6-C3A1 knockout bacteria collagen shake-flask fermentation results Figure 35 SMD1168HΔ1 / Δ4 / Δ6-C3A1 knockout bacteria genome extraction results Figure 36 Relative C3A1 gene copy number in SMD1168HΔ1 / Δ4 / Δ6-C3A1 knockout bacteria Figure 37 SMD1168HΔ1 / Δ4 / Δ6 / pPIC9KM-C3A1 Knockout Bacterial Tank Fermentation SDS-PAGE Comparison Figure 38 KM71HΔ1Δ4 / pPIC9KM-C3A1 colony PCR results Figure 39 SDS-PAGE results of collagen expression in KM71HΔ1Δ4 / pPIC9KM-C3A1 knockout bacteria Figure 40 Comparison of shake-flask fermentation results between KM71HΔ1Δ4 / pPIC9KM-C3A1 knockout strain and control strain Detailed Implementation

[0022] Our main research and development steps are as follows: (1) Identify the target gene sequence to be knocked out through bioinformatics and functional analysis; (2) Construct a gene knockout system, including but not limited to the CRISPR gene editing system, Cre / loxp, and Flp / FRT; (3) Constructing homologous arm sequences for target gene repair; (4) Electroporation was performed into Pichia pastoris competent cells to perform gene knockout and verify the knockout and plasmid loss; (5) After obtaining the knockout bacteria, the plasmid containing the collagen sequence was transformed into the knockout bacteria for screening and expression verification.

[0023] In the previous experiments, we tested the effects of the above-mentioned methods of alleviating protein degradation on collagen. (1) We tried to express the full length of type III collagen in SMD1163 and SMD1168H hosts, named C3A1. However, the growth rate of the knockout bacteria was too slow, and no obvious effect was observed during fermentation. Instead, fewer main bands were obtained. (2) We tried to add 1 mM trypsin inhibitor and 1 mM pepstatin inhibitor during fermentation, but no obvious effect was observed. Although no obvious effect was observed during fermentation, the metal ion chelating agent EDTA was unexpectedly found to have a significant effect on alleviating collagen degradation during the storage of the liquid. The purity of the purified liquid increased from 89.9% (control group) to 95.1% (EDTA group). Therefore, we speculated that EDTA may inhibit the activity of a certain metal ion-dependent protease. In order to further verify our hypothesis, we searched and analyzed all proteases in the Pichia pastoris genome and selected some protease genes for knockout verification.

[0024] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the process equipment or apparatus not specifically specified in the following embodiments are all conventional equipment or apparatus in the art. Furthermore, it should be understood that one or more method steps mentioned in the present invention do not exclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps, unless otherwise stated; it should also be understood that the combined connection relationship between one or more devices / apparatus mentioned in the present invention does not exclude the existence of other devices / apparatus before or after the combined devices / apparatus, or the insertion of other devices / apparatus between these explicitly mentioned two devices / apparatus, unless otherwise stated. Moreover, unless otherwise stated, the numbering of each method step is only a convenient tool for identifying each method step, and not for limiting the order of the method steps or limiting the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0025] Example 1: Selection of protease gene from Pichia pastoris host Using the online eukaryotic genome annotation platform http: / / bioinformatics.psb.ugent.be / webtools / bogas, we retrieved the genome information of Pichia pastoris and obtained 105 genes annotated as proteases. Functional and location screening of these 105 protease genes revealed that only 27 genes belong to vacuolar proteases, which may be involved in the degradation of exogenous proteins. These 27 genes are roughly divided into four categories: metalloproteinases, aminopeptidases, aspartic proteases, and protease B (Table 1). In the SMD1168 host, protease A (PrA) is missing, while in the SMD1163 host, both protease A and protease B are missing. However, previous results showed that knocking out these two genes did not reveal a significant mitigation effect. Therefore, we mainly selected 5 metalloproteinases, 3 aminopeptidases, and 2 aspartic protease genes (Table 2) for knockout and functional verification.

[0026] Table 1. Vacuole proteases annotated in the Pichia pastoris genome Table 2. Ten selected proteases Seq ID NO.1 chr1-4_0611 Vacuolar aminopeptidase Y, 1530bp Seq ID NO.2 chr3_0299 Aspartic protease, 1584 bp Seq ID NO.3 chr1-4_0347 Putative metalloprotease, 2076 bp Seq ID NO.4 chr1-4_0362 Putative metalloprotease, 1944 bp Seq ID NO.5 chr1-1_0379 GPI-anchored aspartyl protease (yapsin)involved in protein processing, 1782 bp Seq ID NO.6 chr2-2_0380 Zinc-dependent metallopeptidase yscII, 2766bp Seq ID NO.7 chr2-1_0487 Leucyl aminopeptidase (leukotriene A4hydrolase) with epoxide hydrolase activity, 1986 bp Seq ID NO.8 chr4_0546 Putative metalloprotease, 3156 bp Seq ID NO.9 chr3_0896 Dipeptidyl aminopeptidase, 2451bp Seq ID NO.10, chr3_0953 Putative metalloprotease, 2973 bp Example 2: Construction of gene knockout plasmids using the CRISPR / Cas9 system First, the gene synthesis-related plasmid pPICZαH-ARS was used, which contains an autonomously replicated sequence (ARS) derived from CR382126.1Kluyveromyces lactis NRRL Y-1140 chromosome F.

[0027] pPICZαH-HH-gRNA1-2-HDV: 5' cleaving hammerhead (HH) ribozyme is added before the designed spRNA and sructure RNA, and 3' cleaving hepatitis delta virus (HDV) ribozyme is added after it to process the transcribed sgRNA and release the mature sgRNA.

[0028] The Ezup Column Yeast Genomic DNA Purification Kit (Shanghai Sangon Biotech) was used to extract Pichia pastoris genome. Primers DAS1TT-F and DAS1TT-R were designed using CE Design software to retrieve the DAS1TT terminator sequence from the Pichia pastoris genome. The PCR system is shown in Table 3. The PCR results are as follows: Figure 1 (1) As shown; using pET28a-cas9 plasmid as template, and Cas9-F and Cas9-R as primers, the Cas9 sequence from Streptococcus pyogenes, optimized for human codon preference, was retrieved. The system is shown in Table 3. The PCR results are as follows. Figure 1 (2) As shown; then, using DAS1TT-F and Cas9-R as primers, the Cas9 fragment and the DAS1TT fragment were ligated by overlap extension PCR. The system is shown in Table 4, and the PCR results are as follows. Figure 1 As shown in (3), the Cas9-DAS1TT combined fragment was successfully amplified. Simultaneously, the pPICZαH-ARS plasmid was digested with Pme I and Not I, as shown in Table 5. Finally, a one-step cloning method was used to ligate the pPICZαH-ARS (Pme I + Not I) fragment, the Cas9 fragment, and the DAS1TT fragment, as shown in Table 6. After colony growth, colony PCR verification was performed using primers DAS1TT-F and Cas9-R. The results of the colony PCR verification are shown in Table 6. Figure 2 As shown in the figure, a band of approximately 4354 bp was successfully amplified, indicating that the plasmid pPICZH-ARS-DASTT1-Cas9 was successfully constructed.

[0029] Table 3 Primer-based fragment PCR system Table 4. Overlap Amplification System Table 5. Pme I and Not I double digestion system of pPICZαH-ARS plasmid Table 6 One-step cloning ligation system Table 7. Primer sequences for constructing pPICZαH-ARS-Cas9 The following describes plasmid construction using the protease gene 2 as an example: Primers HH-gRNA1-2-F and HDV-R were designed to extract the HH-gRNA1-2-HDV sequence from the synthesized plasmid pPICZαH-HH-gRNA1-2-HDV. The PCR system is shown in Table 9. The PCR results are as follows: Figure 3 (1) As shown; using pHTX1-F and pHTX1-R as primers, the pHTX1 bidirectional promoter sequence on the Pichia pastoris genome was retrieved. The system is shown in Table 9, and the PCR results are as follows. Figure 3 (2) As shown; the two fragments above were ligated using overlap PCR, and the system is shown in Table 10. The PCR results are as follows. Figure 3 As shown in (3), the pHTX1-HH-gRNA1-2-HDV sequence was successfully amplified. Simultaneously, the plasmid pPICZH-ARS-DASTT1-Cas9 was double-digested with Age I and Not I, as shown in Table 11. After digestion, the fragments were recovered from the gel and ligated using a one-step cloning method, as shown in Table 12. After transformants grew, colony PCR was performed using primers pHTX1-F and HDV-R for verification. The results of the colony PCR are shown below. Figure 4 As shown in the figure, the target band of about 750 bp was successfully amplified, and the plasmid pPICZH-panARS-DASTT1-cas9-HTX-HH-gRNA1-2-HDV was obtained.

[0030] During the construction of the above plasmids, appropriate restriction sites were introduced after each element. After successful plasmid construction, only the gRNA sequences of different protease genes needed to be replaced by the Kpn I and Age I restriction sites, while other universal elements remained unchanged, making the construction process simple and convenient. Therefore, following a similar gene primer design (Table 13) and amplification, the sgRNA sequences of protease genes numbered 1, 3, 4, 5, 6, 7, 8, 9, and 10 were successfully amplified, as shown in the results. Figure 5 As shown, it was then ligated into the particle pPICZH-panARS-DASTT1-cas9-HTX-HH-gRNA1-2-HDV, which had been digested with KpnI and AgeI. After transformants grew, colony PCR was performed for verification, and the results are shown below. Figure 6 As shown, the target band of the corresponding size was successfully amplified, and the following bands were obtained sequentially: pPICZH-panARS-DASTT1-cas9-HTX-HH-gRNA1-1-HDV, pPICZH-panARS-DASTT1-cas9-HTX-HH-gRNA1-3-HDV, pPICZH-panARS-DASTT1-cas9-HTX-HH-gRNA1-4-HDV, pPICZH-panARS-DASTT1-cas9-HTX-HH-gRNA1-5-HDV, and pPICZH-panARS-DASTT1-cas9-HTX-HH-gRNA1-4-HDV. S-DASTT1-cas9-HTX-HH-gRNA1-6-HDV, pPICZH-panARS-DASTT1-cas9-HTX-HH-gRNA1-7-HDV, pPICZH-panARS-DASTT1-cas9-HTX -HH-gRNA1-8-HDV, pPICZH-panARS-DASTT1-cas9-HTX-HH-gRNA1-9-HDV, pPICZH-panARS-DASTT1-cas9-HTX-HH-gRNA1-10-HDV.

[0031] Table 8 Primer sequences for constructing pPICZaH-ARS-Cas9-HH-gRNA1-2-HDV Table 9 Primer-based fragment PCR system Table 10 Overlap Amplification System Table 11. Age I and Not I double digestion system of pPICZH-DASTT1-Cas9 plasmid Table 12 One-step cloning ligation system Table 13 Different sgRNA sequences The upper and lower homologous arms of 10 different proteases on the genome were retrieved using primers shown in Table 14 and recovered by column chromatography. The upper and lower homologous arms were then ligated using up-F and down-R primers via overlap. The results are as follows: Figure 7 As shown in the figure, a homologous arm fragment of approximately 2160 bp was successfully amplified. The product was purified and recovered using a gel extraction purification kit for later use.

[0032] Table 14 Donor DNA Amplification Primers Example 3: Knockout of the protease gene in the Pichia pastoris KM71H host After all knockout plasmids and donor DNA have been constructed, Pichia pastoris competent cells are prepared and genes are knocked out. The method for preparing knockout competent cells is as follows: (1) Pichia pastoris KM71H was streaked onto YPD plates and cultured at 30°C for 2-3 days. A single colony was then picked and inoculated into 3 mL of YPD medium and cultured overnight at 30°C and 250 rpm. The next day, 1 mL of the bacterial culture was transferred to 50 mL of YPD medium and cultured in a shaker at 30°C and 250 rpm until OD500 was reached. 600 Reaching 0.8~1; (2) In a clean bench, dispense the above culture medium into sterile 50 mL centrifuge tubes, centrifuge at 25°C and 500 g for 5 min, discard the supernatant in the clean bench, add 9 mL of BEDS solution (3% (v) ethylene glycol, 5% (v) dimethyl sulfoxide, 1 M D-sorbitol, 10 mM bicine-NaOH to bring the volume to 100 mL) and 1 mL of 1 M dithiothreitol solution to resuspend the cells, place them in a shaker and incubate at 30°C and 150 rpm for 5 min; (3) After incubation, centrifuge at 25°C and 500 g for 5 min, and discard the supernatant in a clean bench; (4) Add 400 μL of BEDS to resuspend the cells, and dispense 100 μL / vial into 1.5 mL centrifuge tubes for later use.

[0033] After successful preparation of competent cells, one cell line was taken from each cell line, and 500 ng of pPICZH-panARS-DASTT1-cas9-HTX-HH-gRNAs-HDV plasmid and 1 μg of donor DNA fragment were added. The mixture was gently pipetted and transferred to a 0.2 cm electroporation cuvette, and incubated on ice for 2 min. After incubation, electroporation was performed with the following parameters: 2 kV, 4 ms. Immediately after electroporation, the cells were resuspended in resuscitation medium (0.5 mL 1 M sorbitol, 0.5 mL YPD), and then incubated at 30°C for 3 h in a shaker. After incubation, the bacterial culture was plated on 0.2 g / L HYG-YPD plates and incubated upside down at 30°C for 3–4 days. Transformants were then verified by colony PCR. The transformation status of each knockout gene is shown below. Figure 8 As shown in Table 15, a certain number of transformants were obtained from each strain. Then, 24 transformants were selected for yeast colony PCR verification. The primer sequences are shown in Table 15, and the PCR results are as follows: Figures 9-13As shown in Table 16, the theoretical band lengths after gene knockout are as follows. PCR results show that knockout genes 1-10 successfully amplified bands of approximately 2413 bp, 2309 bp, 2192 bp, 2258 bp, 2268 bp, 2218 bp, 2131 bp, 2216 bp, 2197 bp, and 2211 bp, respectively. A single band in the bp indicates successful knockout of genes 1-10. The corresponding knockout bacteria are named KM71HΔ1 / pPICZH-panARS-DASTT1-cas9-HTX-HH-gRNA1-1-HDV, KM71HΔ2 / pPICZH-panARS-DASTT1-cas9-HTX-HH-gRNA1-2-HDV, KM71HΔ3 / pPICZH-panARS-DASTT1-cas9-HTX-HH-gRNA1-3-HDV, KM71HΔ4 / pPICZH-panARS-DASTT1-cas9-HTX-HH-gRNA1-4-HDV, and KM71HΔ5 / pPICZH-panARS-DASTT1-cas9-HTX-HH-gRNA1-4-HDV, respectively. HH-gRNA1-5-HDV, KM71HΔ6 / pPICZH-panARS-DASTT1-cas9-HTX-HH-gRNA1-6-HDV, KM71HΔ7 / pPICZH-panARS-DASTT1-cas9-HTX-HH-gRNA1-7-HDV, KM71HΔ8 / pPICZH-panAR S-DASTT1-cas9-HTX-HH-gRNA1-8-HDV, KM71HΔ9 / pPICZH-panARS-DASTT1-cas9-HTX-HH-gRNA1-9-HDV, and KM71HΔ10 / pPICZH-panARS-DASTT1-cas9-HTX-HH-gRNA1-10-HDV.

[0034] Table 15 Primers for colony PCR knockout verification of each knockout gene Table 16 Length of each knockout gene and length of the gene after knockout Example 4 Obtaining knockout bacteria by losing the knockout plasmid After confirming successful gene knockout, the plasmid KM71HΔ10 / pPICZH-panARS-DASTT1-cas9-HTX-HH-gRNA1s-HDV containing the Cas9 editing gene is passaged multiple times. This reduces the metabolic stress on the host and avoids the toxicity of continuous Cas9 protein expression to cells. The specific experimental method is as follows: Verified colonies are inoculated into 3 mL of YPD medium and cultured overnight. The next day, 10 μL of the bacterial culture is inoculated into a new 15 mL test tube containing 3 mL of YPD and cultured overnight. After culture, 1 μL of the bacterial culture is streaked onto a YPD plate and incubated upside down at 30°C for approximately 3 days. The resulting transformants are then spotted onto YPD plates containing 0.2 g / L hygromycin and those without hygromycin. Colonies that grow on YPD-free plates but not on 0.2 g / L hygromycin plates are considered to have successfully lost the plasmid and can be preserved as a strain, or validated by streaking and then preserved again. The plasmid loss results of the above 10 protease gene knockout bacteria are as follows: Figure 14 As shown in the figure, all 10 knockout bacteria produced colonies that could grow on YPD-free plates but not on 0.1 g / L hygromycin plates, indicating that plasmid loss was successful in these 10 bacterial strains. The recombinant bacteria obtained were named KM71HΔ1, KM71HΔ2, KM71HΔ3, KM71HΔ4, KM71HΔ5, KM71HΔ6, KM71HΔ7, KM71HΔ8, KM71HΔ9 and KM71HΔ10, respectively.

[0035] Example 5: Construction and screening of KM71H knockout bacteria containing recombinant collagen gene The collagen expression effect was verified using the recombinant plasmid pPIC9KM-C3A1 constructed in patent application number CN2024117088441. The recombinant plasmid pPIC9KM-C3A1 was linearized by single enzyme digestion with Sal I. The enzyme digestion system (50 μL) was: 10× Q-cut buffer 5 μL, Sac I or Sal 1 μL, plasmid 1~2.0 μg, and ddH2O to make up to 50 μL. The enzyme digestion reaction was carried out at 37℃ for 2~4 h. After the enzyme digestion, the digested product was purified and recovered using the Tiangen large-scale PCR product recovery kit for later use.

[0036] Single colonies of *Pichia pastoris* KM71H and *Pichia pastoris* KM71HΔ1~Δ10 were picked from streak plates and inoculated into sterile shake tubes containing 3 mL of YPD medium. The colonies were incubated overnight at 30°C and 250 rpm. The following day, 1 mL of the seed culture was transferred to a 500 mL Erlenmeyer flask containing 50 mL of YPD medium and incubated at 30°C and 250 rpm until OD500 was reached.600 The concentration was set to 1.0. The bacterial culture was then collected in 50 mL round-bottom spiral centrifuge tubes and centrifuged at 1500 g for 5 min at 4°C, discarding the supernatant. The cells were resuspended in 40 mL of pre-chilled sterile water and centrifuged at 1500 g for 5 min at 4°C, discarding the supernatant. This sterile water washing step was repeated once. The cells were then resuspended in 10 mL of pre-chilled 1 M sorbitol and centrifuged at 1500 g for 5 min at 4°C, discarding the supernatant. 300 μL of pre-chilled 1 M sorbitol was then added, and the mixture was gently pipetted to mix. Competent cells of each bacterial species were aliquoted into 1.5 mL centrifuge tubes at 100 μL each for later use.

[0037] Add 10 μL of the purified and recovered product to the prepared competent cells, incubate on ice for 5 min, then transfer the mixture to a 0.2 cm electroporation cuvette. Set the electroporator to SC2 program for electroporation. After electroporation, add 1 mL of pre-chilled 1 M sorbitol, then incubate on a shaker at 30°C and 150 rpm for 1 h. Spread the mixture to a concentration of 0.3 mg / mL. −1 On the G418 YPD plate, after the transformants have grown ( Figure 15 Colony PCR verification was performed, and the results of the colony PCR were as follows: Figures 16-20 As shown in the figure, the PCR successfully amplified a C3A1 band of approximately 3087 bp, indicating that all linearized products were successfully integrated into the yeast genome.

[0038] Positive colonies grown on the plates were selected for expression strain verification and screening. The specific steps are as follows: (1) Pick the colonies that can grow on high concentration G418 plates and put them into a 96-well deep plate containing 300 μL YPD medium. Incubate at 30℃ and 250 rpm for 12~24 h.

[0039] (2) Transfer 10-100 μL of bacterial culture to two 48-well deep-well plates containing 500 μL of BMGY medium and incubate at 30°C and 250 rpm for 12-18 h.

[0040] (3) Centrifuge at 4000 rpm for 10 min, discard the supernatant, add 500 μL of BMMY medium, induce at 28℃ and 250 rpm for 24~48 h, and add 1% methanol at 24 h.

[0041] (4) After induction, centrifuge at 4000 rpm for 10 min, and use a multi-channel pipette to aspirate 20 μL of supernatant into each 96-well PCR tube. Add 5 μL of 5×Sample Loading Buffer and denature at room temperature for 5 min, then centrifuge at 4000 rpm for 10 min. Aspirate 10 μL of supernatant for SDS-PAGE analysis. After Coomassie Brilliant Blue staining and destaining, the tubes are ready for photographing.

[0042] SDS-PAGE screening results are as follows Figures 21-23 As shown in the figure, the target band was observed in all knockout bacteria between 95 and 140 kDa. However, some knockout genes may have affected the growth of the strains, resulting in thinner target bands. The strains were then selected for shake-flask fermentation to compare the effects. These strains were named KM71HΔ1 / pPIC9KM-C3A1, KM71HΔ2 / pPIC9KM-C3A1, and KM71HΔ3 / pPIC9KM-C3A1, respectively. 3A1, KM71HΔ4 / pPIC9KM-C3A1, KM71HΔ5 / pPIC9KM-C3A1, KM71HΔ6 / pPIC9KM-C3A1, KM71HΔ7 / pPIC9KM-C3A1, KM71HΔ8 / pPIC9KM-C3A1, KM71HΔ9 / pPIC9KM-C3A1 and KM71HΔ10 / pPIC9KM-C3A1.

[0043] Example 6: Verification of the degradation mitigation effect of knockout bacteria (KM71H) The above-mentioned strains were sequentially inoculated into 3 mL of YPD medium and cultured overnight, then transferred to 50 mL of BMGY medium and cultured until OD. 600 The OD values ​​reached 4.0–6.0, and the culture was transferred to 100 mL of BMMY medium with an initial OD of 2.0 for induction of protein expression. The results are as follows: Figure 24 As shown in the figure, C3A1 was well expressed in all knockout bacteria. Among them, the target bands of the knockout bacteria KM71HΔ1 / pPIC9KM-C3A1, KM71HΔ4 / pPIC9KM-C3A1 and KM71HΔ6 / pPIC9KM-C3A1 were significantly improved compared with the control, which may be because the knockout of these genes did alleviate the degradation of C3A1.

[0044] To rule out whether the differences in expression levels were caused by different gene integration copy numbers, we extracted the genomes of the three knockout bacteria and the control bacteria. Figure 25 The relative copy number of the C3A1 gene was determined by qPCR, and the results are as follows: Figure 26As shown in the figure, the relative copy number of C3A1 in these knockout bacteria is consistent with that of the control strain, both around 1.0. This indicates that the thickening of the target band in KM71HΔ1 / pPIC9KM-C3A1, KM71HΔ4 / pPIC9KM-C3A1, and KM71HΔ6 / pPIC9KM-C3A1 is indeed due to reduced degradation.

[0045] To further verify the effectiveness of these knockout bacteria, we conducted on-tank fermentation expression validation again. Specifically, we streaked Pichia pastoris KM71H / pPIC9KM-C3A1, Pichia pastoris KM71HΔ1 / pPIC9KM-C3A1, Pichia pastoris KM71HΔ4 / pPIC9KM-C3A1, and Pichia pastoris KM71HΔ6 / pPIC9KM-C3A1 into a container containing 0.3 mg / mL of Pichia pastoris KM71HΔ6 / pPIC9KM-C3A1. −1 G418 was cultured on YPD plates at 30°C for 72 h. Single colonies were picked and inoculated into 50 mL of BMG medium and cultured until the OD600 reached 4.0–6.0. ​​Then, a 10% inoculum was transferred to 100 mL of BMG medium and cultured until the OD reached 8.0–12.0. The secondary seed culture was then inoculated into a 5 L fermenter, and 4.35 mL·L⁻¹ of BMG medium was added simultaneously. −1 PTM 1 trace elements and 4.35 mL·L −1 Initiate fermentation with 0.02% biotin. When DO rapidly recovers to 50-70%, begin glycerol feeding at a rate of 10-20 mL / h. −1 ·L −1 The DO content was maintained at 25-30% by fine-tuning the glycerol feed rate. Feeding was stopped after 5-6 hours, and the mixture was "starved" for 30-60 minutes before methanol induction began. The methanol flow rate was 0.5-7.2 mL / h. −1 ·L −1 The induction temperature was 28℃, and samples were taken during the process to detect the wet weight of the bacterial cells and the protein concentration.

[0046] Seed culture medium BMG (1 L): 10 g glycerol, 2.3 g K2HPO4, 11.81 g KH2PO4, bring to a final volume of 900 mL, sterilize at 121℃ for 15 min, cool, and then add 100 mL 13.4% YNB and 2 mL 0.02% biotin. Fermentation medium BSM (1 L): 80 g glycerol, 26.7 mL H3PO4, 1.175 g CaSO4, 18.2 g K2SO4, 7.28 g MgSO4, 4.13 g KOH, dissolved in water and brought to a final volume of 2000 mL. After sterilization, add 4.35 mL PTM1. Fed growth medium: 50% (w / v) glycerol (containing 12 mL·L⁻¹) −1 PTM1) Fermentation induction medium: 100% methanol (containing 12 mL·L⁻¹) −1 PTM1).

[0047] PTM1 ingredients (1 L): CuSO4·5H2O 6.0 g, NaI 0.08 g, MnSO4·H2O 3.0 g, Na2MoO4·2H2O 0.2 g, H3BO3 0.02 g, CoCl2 0.5 g, ZnCl2 20.0 g, FeSO4·7H2O 65.0 g, H2SO4 5 mL·L −1 Filter 0.2 g of biotin to sterilize it and store it at 4°C for later use.

[0048] Protein expression during the fermentation process of the four strains is as follows: Figure 27 As shown in the figure, the expression level of C3A1 protein increased significantly with the extension of fermentation time. Among them, the expression level of the knockout strain Pichia pastoris KM71HΔ1 / pPIC9KM-C3A1 exceeded that of the control strain (10.36 g·L⁻¹). −1 It reached 13.0 g·L⁻¹ in 72 h. −1 The protein expression levels of the knockout bacteria Pichia pastoris KM71HΔ4 / pPIC9KM-C3A1 and Pichia pastoris KM71HΔ6 / pPIC9KM-C3A1 were 9.0 g·L⁻¹. −1 and 8.6 g·L −1 We analyzed the reasons and hypothesized that the knockout of genes 4 and 6 affected the growth of the strain, resulting in a poorer growth state during fermentation compared to the control, with a cell wet weight of only 125 g·L⁻¹ at 72 h. −1 and 124 g·L −1The wet cell weights of Pichia pastoris KM71HΔ1 / pPIC9KM-C3A1 and the control strain reached 153 g / L and 165 g / L, respectively. Therefore, the low protein expression levels of Pichia pastoris KM71HΔ4 / pPIC9KM-C3A1 and Pichia pastoris KM71HΔ6 / pPIC9KM-C3A1 were due to the small cell size. The protein production of 1, 4, and 6 units of knockout bacteria was calculated to be 0.084 g·g⁻¹. −1 0.072 g·g −1 and 0.068 g·g −1 The amounts were wild mushrooms (0.064 g·g). −1 The knockout values ​​were 1.31, 1.13, and 1.06 times higher than those of genes 1, 4, and 6, further validating the effect of knocking out genes 1, 4, and 6 in alleviating C3A1 protein degradation.

[0049] Table 17 Comparison of expression levels of knockout bacteria in a 5L fermenter Example 7: Knockout of the protease gene in the Pichia pastoris SMD1168H host To verify the effect of the aforementioned protease gene on alleviating collagen degradation in other hosts, we electroporated the knockout plasmid into the SMD1168H host. SMD1168H competent cells were prepared according to the method in Example 3. One competent cell line was taken from each host, and 500 ng of pPICZH-panARS-DASTT1-cas9-HTX-HH-gRNAs-HDV plasmid and 1 μg of donor DNA fragment were added. The cells were gently mixed by pipetting and transferred to a 0.2 cm electroporation cuvette and incubated on ice for 2 min. After incubation, electroporation was performed with the following parameters: 2 kV, 4 ms. Immediately after electroporation, the cells were resuspended in resuscitation medium (0.5 mL 1 M sorbitol, 0.5 mL YPD) and then incubated at 30°C for 3 h in a shaker. After cultivation, the bacterial culture was spread onto 0.2 g / L HYG-YPD plates and incubated at 30°C inverted for 3-4 days. Transformants were then observed and verified by colony PCR. The transformation status of each knockout gene is shown below. Figure 28 As shown in Table 15, a certain number of transformants were obtained from each strain. Then, 24 transformants were selected for yeast colony PCR verification. The primer sequences are shown in Table 15, and the PCR results are as follows: Figure 29As shown in Table 16, the theoretical band lengths after gene knockout are as follows. PCR results show that knockout genes 1, 4, and 6 successfully amplified single bands of approximately 2413 bp, 2258 bp, and 2218 bp, respectively, indicating that gene knockout of genes 1, 4, and 6 was successful. The corresponding knockout bacteria were named SMD1168HΔ1 / pPICZH-panARS-DASTT1-cas9-HTX-HH-gRNA1-1-HDV, SMD1168HΔ4 / pPICZH-panARS-DASTT1-cas9-HTX-HH-gRNA1-4-HDV, and SMD1168HΔ6 / pPICZH-panARS-DASTT1-cas9-HTX-HH-gRNA1-6-HDV, respectively.

[0050] After confirming that all genes were successfully knocked out, the Cas9 editing gene plasmids were lost according to the method described in Example 4. The plasmid loss results for the above three protease gene knockout bacteria are as follows: Figure 30 As shown in the figure, all three knockout bacteria were able to grow on YPD-free substrates, but at 0.1 g·L⁻¹... −1 The fact that colonies could not grow on the hygromycin plate meant that the plasmids in these 10 bacterial strains were successfully lost, and the recombinant bacteria obtained were named SMD1168HΔ1, SMD1168HΔ4 and SMD1168HΔ6, respectively.

[0051] Example 8: Construction and screening of SMD1168H knockout bacteria containing recombinant collagen gene The recombinant plasmid pPIC9KM-C3A1 was linearized by single enzyme digestion with Sal I. The digestion system (50 μL) consisted of 5 μL of 10×Q-cut buffer, 1 μL of Sac I or Sal, 1~2.0 μg of plasmid, and ddH2O to a final volume of 50 μL. The digestion reaction was carried out at 37℃ for 2~4 h. After digestion, the digested product was purified and recovered using the Tiangen PCR product recovery kit for later use.

[0052] After obtaining transformants, single colonies of *Pichia pastoris* SMD1168H and *Pichia pastoris* SMD1168H Δ1, Δ4, and Δ6 were picked and inoculated into sterile shake tubes containing 3 mL of YPD medium. The colonies were incubated overnight at 30°C and 250 rpm. The next day, 1 mL of the seed culture was transferred to a 500 mL Erlenmeyer flask containing 50 mL of YPD medium and incubated at 30°C and 250 rpm until OD500 was reached. 600The bacterial culture was initially set to 1.0. The bacterial suspension was then collected in 50 mL round-bottom spiral centrifuge tubes and centrifuged at 1500 g for 5 min at 4°C, discarding the supernatant. The cells were resuspended in 40 mL of pre-chilled sterile water and centrifuged at 1500 g for 5 min at 4°C, discarding the supernatant. This sterile water washing step was repeated once. The cells were then resuspended in 10 mL of pre-chilled 1 M sorbitol and centrifuged at 1500 g for 5 min at 4°C, discarding the supernatant. 300 μL of pre-chilled 1 M sorbitol was then added, and the mixture was gently pipetted to mix. Competent cells of each bacterial species were aliquoted into 1.5 mL centrifuge tubes at 100 μL each for later use.

[0053] Add 10 μL of the purified and recovered product to the prepared competent cells, incubate on ice for 5 min, then transfer the mixture to a 0.2 cm electroporation cuvette. Set the electroporator to SC2 program for electroporation. After electroporation, add 1 mL of pre-chilled 1 M sorbitol, then incubate on a shaker at 30°C and 150 rpm for 1 h. Spread the mixture to a concentration of 0.3 mg / mL. −1 On the G418 YPD plate, after the transformants have grown ( Figure 31 Colony PCR verification was performed, and the results of the colony PCR were as follows: Figure 32 As shown in the figure, the PCR successfully amplified a C3A1 band of approximately 3087 bp, indicating that all linearized products were successfully integrated into the yeast genome.

[0054] Positive colonies grown on the plates were selected for expression strain verification and screening. The specific steps are as follows: (1) Pick the colonies that can grow on high concentration G418 plates and put them into a 96-well deep plate containing 300 μL YPD medium. Incubate at 30℃ and 250 rpm for 12~24 h.

[0055] (2) Transfer 10-100 μL of bacterial culture to two 48-well deep-well plates containing 500 μL of BMGY medium and incubate at 30°C and 250 rpm for 12-18 h.

[0056] (3) Centrifuge at 4000 rpm for 10 min, discard the supernatant, add 500 μL of BMMY medium, induce at 28℃ and 250 rpm for 24~48 h, and add 1% methanol at 24 h.

[0057] (4) After induction, centrifuge at 4000 rpm for 10 min, and use a multi-channel pipette to aspirate 20 μL of supernatant into each 96-well PCR tube. Add 5 μL of 5×Sample Loading Buffer and denature at room temperature for 5 min, then centrifuge at 4000 rpm for 10 min. Aspirate 10 μL of supernatant for SDS-PAGE analysis. After Coomassie Brilliant Blue staining and destaining, the tubes are ready for photographing.

[0058] SDS-PAGE screening results are as follows Figure 33 As shown in the figure, the target band was observed in all knockout bacteria between 95 and 140 kDa. The strains were selected for shake-flask fermentation to compare the effects. These strains were named SMD1168HΔ1 / pPIC9KM-C3A1, SMD1168HΔ4 / pPIC9KM-C3A1 and KM71HΔ6 / pPIC9KM-C3A1, respectively.

[0059] Example 9: Validation of the degradation mitigation effect of knockout bacteria (SMD 1168H) The above-mentioned strains were sequentially inoculated into 3 mL of YPD medium and cultured overnight, then transferred to 50 mL of BMGY medium and cultured until OD. 600 The OD values ​​reached 4.0–6.0, and the culture was transferred to 100 mL of BMMY medium with an initial OD of 2.0 for induction of protein expression. The results are as follows: Figure 34 As shown in the figure, C3A1 was well expressed in all knockout bacteria. However, the target band expression in knockout bacteria SMD1168HΔ1 / pPIC9KM-C3A1, SMD1168HΔ4 / pPIC9KM-C3A1, and SMD1168HΔ6 / pPIC9KM-C3A1 was not significantly improved compared to the control group. Given that the growth of the cells would be affected after knockout, we will conduct on-tank fermentation comparison and verification again. Meanwhile, we extracted the genomes of these three knockout bacteria (…). Figure 35 Analysis of the relative copy number of the C3A1 gene, such as Figure 36 As shown, the relative copy number of the C3A1 gene in these three knockout bacteria is around 1.0, ruling out differences in expression levels due to differences in integration copy number.

[0060] To further verify the effects of knockout strains *Pichia pastoris* SMD1168H / pPIC9KM-C3A1, *Pichia pastoris* SMD1168HΔ1 / pPIC9KM-C3A1, *Pichia pastoris* SMD1168HΔ4 / pPIC9KM-C3A1, and *Pichia pastoris* SMD1168HΔ6 / pPIC9KM-C3A1 on alleviating C3A1 degradation compared to the control strain, we performed on-tank fermentation expression verification according to the method described in Example 6. The protein expression during the fermentation process of the four strains is as follows: Figure 37 As shown in the figure, the expression level of C3A1 protein increased significantly with the extension of fermentation time. Specifically, the expression levels of the knockout strains *Pichia pastoris* SMD1168HΔ1 / pPIC9KM-C3A1, *Pichia pastoris* SMD1168HΔ4 / pPIC9KM-C3A1, and *Pichia pastoris* SMD1168HΔ6 / pPIC9KM-C3A1 all exceeded those of the control strain (3.9 g·L⁻¹). −1 The concentrations reached 10.8, 11.0, and 4.4 g·L⁻¹ at 72 h. −1 To rule out whether the difference in expression levels was due to variations in bacterial count, we also measured the wet weight of these knockout bacteria at 72 h, which was only 257 g·L⁻¹. −1 301 g·L −1 and 274 g·L −1 The control strain, Pichia pastoris SMD1168H / pPIC9KM-C3A1, had a wet weight of 304 g·L⁻¹. −1 The protein production of 1, 4, and 6 units of knockout bacteria was calculated to be 0.042 g·g⁻¹. −1 0.036 g·g −1 and 0.016 g·g −1 The amounts were wild mushrooms (0.012 g·g⁻¹). −1 The total protein expression levels of the knockout bacteria were 3.50, 3.00, and 1.33 times higher than those of the control bacteria, respectively. Therefore, the total protein expression level and the amount of protein expressed per unit cell were significantly better than those of the control bacteria, which further verified the effect of knocking out genes 1, 4, and 6 in alleviating C3A1 protein degradation. Moreover, we found that compared with KM71H, the host SMD1168H had fewer miscellaneous protein bands, with more than 70% being target protein bands. This means that the proteinase A gene can also play a role in alleviating collagen degradation to some extent.

[0061] Table 18 Comparison of expression levels of knockout bacteria in a 5L fermenter Example 10: Validation of the degradation mitigation effect of dual-gene knockout bacteria (KM71H) The above examples all demonstrate that knocking out gene 1 and gene 4 can alleviate collagen degradation to some extent. To further verify whether dual gene knockout has a better effect, we constructed a dual gene knockout bacterium: Since the KM71H host has fewer knocked-out genes than SMD1168H, its growth rate is faster at the shake-flask level (under normal nutrient and dissolved oxygen conditions), making it convenient to quickly determine the effect of gene knockout on alleviating protein degradation. Therefore, we preferentially chose to construct a knockout bacterium based on KM71H that simultaneously knocks out genes 1 and 4, and electroporated the collagen gene C3A1 into the dual knockout bacterium to observe its protein expression. The colony PCR results are as follows. Figure 38 As shown, the results of protein expression are as follows: Figure 39 As shown in the figure, C3A1 bands were observed between 95 and 140 kDa, and... Figure 21 Compared with the single-knockout strains KM71HΔ1 / pPIC9KM-C3A1 and KM71HΔ4 / pPIC9KM-C3A1, the number of heterogeneous bands was significantly reduced, indicating that the dual-gene knockout strain can further alleviate protein degradation compared with the single-gene knockout. Subsequently, strains with high expression levels were selected for gene copy number identification. The results showed that the relative copy number of the C3A1 gene was 1.0. The strain was preserved and named KM71HΔ1Δ4 / pPIC9KM-C3A1. It was then compared with the previously constructed knockout strains KM71HΔ1 / pPIC9KM-C3A1, KM71HΔ4 / pPIC9KM-C3A1, and KM71H / pPIC9KM-C3A1(con) in shake-flask fermentation. The results are as follows. Figure 40 As shown in the figure, the target bands of all knockout bacteria are significantly thicker and the number of impurity bands is significantly reduced compared to the control. Furthermore, the target bands of the double gene knockout bacteria (Δ1 and Δ4) are also significantly thicker than those of the single knockout bacteria (Δ1 or Δ4), which further demonstrates that the simultaneous knockout of the two genes chr1-4_0611 and chr1-4_0362 is more effective in alleviating protein degradation.

[0062] In summary, this invention, utilizing genomic annotation information and through classification and statistical analysis, screened and validated three protease genes—chr1-4_0611, chr1-4_0362, and chr2-2_0380—that can alleviate the degradation of collagen C3A1. Knocking out any one of these three genes resulted in a 6-350% increase in the accumulation of the target protein. Furthermore, these three genes were found to belong to the aminopeptidase and metalloproteinase families, with chr1-4_0611 being metal-dependent and belonging to the metalloproteinase family. This will provide valuable theoretical guidance for subsequent research on the degradation of other recombinant proteins in Pichia pastoris. It should be noted that this invention uses type III collagen as an example, but the method and the engineered bacteria obtained after modification are also applicable to alleviating the degradation of other types of collagen in the Pichia pastoris host.

[0063] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in methods and compositions, will be apparent to those skilled in the art without departing from the scope and spirit of this invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A type of bioengineered bacterium, characterized in that, The bioengineered bacteria are Pichia pastoris strains in which one or more of the proteases chr1-4_0611, chr1-4_0362 or chr2-2_0380 in the genome have been knocked out.

2. The bioengineered bacteria according to claim 1, characterized in that, The Pichia pastoris is any one of GS115, KM71H, SMD1168H, or SMD1163.

3. The bioengineered bacteria according to claim 1, characterized in that, The nucleotide sequences of the genomes chr1-4_0611, chr1-4_0362, or chr2-2_0380 are shown in Seq ID NO.1, Seq ID NO.4, and Seq ID NO.6, respectively.

4. The use of the bioengineered bacteria as described in any one of claims 1-3 for the preparation of recombinant collagen.

5. The use according to claim 4, characterized in that, The collagen is any one of type I, type II, type III or type XVII collagen.

6. A method for preparing recombinant collagen, wherein the method comprises using the bioengineered bacteria as described in any one of claims 1-3 as the fermentation strain, culturing and fermenting, and collecting the fermentation products to obtain recombinant collagen.

7. The method according to claim 6, characterized in that, The Pichia pastoris is any one of GS115, KM71H, SMD1168H, or SMD1163.

8. The method according to claim 6, characterized in that, The collagen is any one of type I, type II, type III or type XVII collagen.

9. The method according to claim 6, characterized in that, The method specifically involves: constructing gene-knockout Pichia pastoris, screening out successfully knocked-out strains, transforming plasmids containing collagen gene expression fragments into the knockout strains, inoculating them into a culture medium, and collecting the fermentation products to obtain collagen.

10. The method according to claim 9, characterized in that, The gene knockout method is to use the CRISPR gene editing system, Cre / loxp, or Flp / FRT for knockout.

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