Preparation method for yeast-expressed type i recombinant human collagen with tri-helical structure and use thereof

Abstract

Provided are a preparation method for a yeast-expressed type I recombinant human collagen with a tri-helical structure and use thereof. The preparation method comprises the steps of performing fermentation using an engineered bacterium and performing purification to obtain the type I recombinant human collagen with a tri-helical structure. A method for constructing the engineered bacterium comprises the step of transforming a host bacterium with plasmids pMChZ-α1(I), pMCrN-α2(I), pPIC9K-P4H(DP)-1, and pMCeH-[α1(I)] 2α2(I) to construct the engineered bacterium. The type I recombinant human collagen with a tri-helical structure prepared using the preparation method comprises hydroxyproline, and hydroxyproline accounts for 30% or more of total proline.

Description

A method for preparing recombinant human collagen of type I with triple helix structure expressed in yeast and its application. Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for preparing recombinant human collagen of type I triple helix structure expressed in yeast and its application. Background Technology

[0002] Collagen is a vital protein in living organisms, consisting of three left-handed alpha peptide chains intertwined to form a right-handed triple helix. Type I collagen is one of the most abundant triple-helix collagens in the human body, playing a crucial role in maintaining skin health and the integrity of bone structure. In the human body, Type I collagen is primarily a heterotrimer composed of two alpha1 chains and one alpha2 chain. This triple helix structure endows collagen with excellent biomechanical properties and biocompatibility, enabling it to play a key role in maintaining skin elasticity, bone strength, and tendon resilience.

[0003] Recombinant human type I collagen, as a substitute for natural human collagen, is widely used to inhibit and alleviate dermatitis, improve sensitive skin, and care for postoperative wounds, playing a significant role in functional biomaterials, cosmetics, and food. Currently, most commercially available recombinant human type I collagen products are recombinant human collagen or recombinant collagen-like proteins, which can be expressed in large quantities in microorganisms at low cost. However, these collagens lack a triple-helix structure. The mechanical properties of single-chain collagen are also far inferior to those of triple-helix collagen, making it unsuitable for applications requiring high mechanical strength. Therefore, there is an urgent need to develop a method for preparing triple-helix type I recombinant human collagen. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing type I recombinant human collagen with a triple-helix structure expressed in yeast and its application, thereby solving the problems existing in the prior art. The type I recombinant human collagen prepared using this method has a triple-helix structure, which allows it to acquire good biomechanical properties and biocompatibility.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a method for constructing an engineered bacterium expressing a triple-helix type I recombinant human collagen, comprising the steps of transforming pMChZ-α1(Ⅰ) plasmid, pMCrN-α2(Ⅰ) plasmid, pPIC9K-P4H(DP)-1 plasmid and pMCeH-[α1(Ⅰ)]2α2(Ⅰ) plasmid into a host bacterium to construct the engineered bacterium;

[0007] The pMChZ-α1(Ⅰ) plasmid was obtained by cloning α1(Ⅰ) into the pMChZ-AOX vector;

[0008] The pMCrN-α2(Ⅰ) plasmid was obtained by cloning α2(Ⅰ) into the pMCrN-AOX vector;

[0009] The pPIC9K-P4H(DP)-1 plasmid is obtained by replacing the KanR in the P4H 9K DP plasmid with a DNA molecule whose nucleotide sequence is shown in SEQ ID NO.12;

[0010] The pMCeH-[α1(Ⅰ)]2α2(Ⅰ) plasmid was obtained by cloning the [α1(Ⅰ)]2α2(Ⅰ) transcription unit into the pMCeH-AOX vector;

[0011] The nucleotide sequence of α1(Ⅰ) is shown in SEQ ID NO.3;

[0012] The nucleotide sequence of α2(Ⅰ) is shown in SEQ ID NO.6;

[0013] The nucleotide sequence of the [α1(Ⅰ)]2α2(Ⅰ) transcription unit is shown in SEQ ID NO.13;

[0014] The pMChZ-AOX vector is obtained by modifying the pPIC9K plasmid as follows: replacing 4648-9221 bp of the pPIC9K plasmid with a DNA molecule whose nucleotide sequence is shown in SEQ ID NO.7; deleting BamHI located upstream of αMF in the pPIC9K plasmid; and introducing a BamHI recognition site between 1583-1584 bp of the pPIC9K plasmid through point mutation.

[0015] The pMCrN-AOX vector was constructed by replacing the PpHIS4 and Zeocin selection markers on the pMChZ-AOX plasmid with DNA molecules whose nucleotide sequences are shown in SEQ ID NO.8 and SEQ ID NO.9, respectively.

[0016] The pMCeH-AOX vector was constructed by replacing PpHIS4 and PEM7-BleoR-CYC1 tt on the pMChZ-AOX plasmid with DNA molecules whose nucleotide sequences are shown in SEQ ID NO.10 and SEQ ID NO.11, respectively.

[0017] Furthermore, the construction method specifically includes the following steps:

[0018] The pMChZ-α1(Ⅰ) plasmid was transformed into the host bacteria to obtain the KM71H / pMChZ-α1(Ⅰ) strain;

[0019] The pMCrN-α2(Ⅰ) plasmid was transformed into the KM71H / pMChZ-α1(Ⅰ) strain to obtain the KM71H / α1(Ⅰ)-α2(Ⅰ) strain;

[0020] The pPIC9K-P4H(DP)-1 plasmid was transformed into the KM71H / α1(Ⅰ)-α2(Ⅰ) strain to obtain the KM71H / α1(Ⅰ)-α2(Ⅰ)-P4H strain;

[0021] The pMCeH-[α1(Ⅰ)]2α2(Ⅰ) plasmid was transformed into the KM71H / α1(Ⅰ)-α2(Ⅰ)-P4H strain to obtain the engineered strain.

[0022] Furthermore, the host bacteria is Pichia pastoris X33 or Pichia pastoris KM71.

[0023] Furthermore, the BamHⅠ located upstream of αMF in the pPIC9K plasmid is deleted by point mutation.

[0024] The present invention also provides an engineered bacterium expressing type I recombinant human collagen with a triple helix structure, constructed according to the above-described construction method.

[0025] The present invention also provides the application of the above-mentioned engineered bacteria in the preparation of triple-helix type I recombinant human collagen.

[0026] The present invention also provides a method for preparing recombinant human collagen of triple helix type I expressed by yeast, including the step of obtaining the recombinant human collagen of triple helix type I by fermentation and purification using the above-mentioned engineered bacteria.

[0027] The present invention also provides a triple-helix type I recombinant human collagen prepared according to the above preparation method.

[0028] The present invention also provides the application of the above-mentioned triple-helix type I recombinant human collagen in the preparation of functional biomaterials, cosmetics or medical devices.

[0029] The present invention discloses the following technical effects:

[0030] This invention provides a method for preparing type I recombinant human collagen with a triple-helix structure expressed in yeast. The type I recombinant human collagen prepared using this method has a triple-helix structure, thereby enabling it to acquire excellent biomechanical properties and biocompatibility. The triple-helix type I recombinant human collagen prepared by this invention can be widely used in cosmetics, medical aesthetics, medical devices, biomedical materials, and other fields, and has significant market application value. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 shows the plasmid map of pPIC9K;

[0033] Figure 2 shows the plasmid map of pMChZ-AOX;

[0034] Figure 3 shows the plasmid map of pMCrN-AOX;

[0035] Figure 4 shows the plasmid map of pMCeH-AOX;

[0036] Figure 5 shows the plasmid map of pPIC9K-P4H(DP)-1;

[0037] Figure 6 shows the results of collagen detection using COL1A1 and COL1A2 antibodies, respectively;

[0038] Figure 7 shows the results of collagen detection using P4HA1 and P4HB antibodies, respectively.

[0039] Figure 8 shows the results of circular dichroism spectroscopy detection;

[0040] Figure 9 shows the results of transmission electron microscopy. Detailed Implementation

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0043] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0044] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0045] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0046] The Pichia pastoris KM71 used in the following examples was purchased from Invitrogen; the pPIC9K vector was purchased from Invitrogen.

[0047] Example 1

[0048] 1. Construct the sequence encoding the α1(Ⅰ) chain of type I recombinant human collagen:

[0049] A His Tag was added between the signal peptide and the N-terminal peptide of sequence (SEQ ID NO.1); a TEV restriction sequence was added between the N-terminal peptide and the triple helix region; a TEV restriction sequence was added between the triple helix region and the C-terminal peptide; and a His Tag was added to the C-terminus to obtain α1(Ⅰ). The amino acid sequence of α1(Ⅰ) is shown in SEQ ID NO.2. The amino acid sequence of α1(Ⅰ) was optimized according to the expression preferences of Pichia pastoris to obtain the coding gene, and the nucleic acid sequence is shown in SEQ ID NO.3.

[0050] 2. Construct the sequence encoding the α2(Ⅰ) chain of type I recombinant human collagen:

[0051] A His Tag was added between the signal peptide and the N-terminal peptide in sequence (SEQ ID NO.4); a TEV restriction sequence was added between the N-terminal peptide and the triple helix region; a TEV restriction sequence was added between the triple helix region and the C-terminal peptide; and a His Tag was added to the C-terminus to obtain α2(Ⅰ). The amino acid sequence of α2(Ⅰ) is shown in SEQ ID NO.5. The amino acid sequence of α2(Ⅰ) was optimized according to the expression preferences of Pichia pastoris to obtain the coding gene, and the nucleic acid sequence is shown in SEQ ID NO.6.

[0052] 3. Constructing plasmids:

[0053] 3.1 Construction of plasmid pMChZ-AOX

[0054] The substituted nucleotide sequence shown in SEQ ID NO.7 was synthesized to replace 4648-9221 bp of the pPIC9K vector sequence, i.e., the KanR resistance gene, Bom, and AOX13' fragment element in pPIC9K were replaced with the DNA molecule shown in SEQ ID NO.1. BamHI (938-943 bp) and αMF in pPIC9K were deleted by point mutation. A BamHI recognition site was introduced downstream of AOX1tt (also known as AOX1 transcription termination or AOX1termination) (between 1583-1584 bp) by point mutation. Other vector element sequences remained unchanged, resulting in the plasmid pMChZ-AOX (where h represents the recombination site PpHIS4 and Z represents the selection marker BleoR). This plasmid can express Cre recombinase upon methanol induction, catalyzing the recombination of lox71 and lox66 to recover the resistance gene. The BamHI located after AOX1tt can be used for isosigmazyme construction of in vitro multicopy plasmids. The plasmid maps of the original plasmid pPIC9K and the pMChZ-AOX constructed in this invention are shown in Figure 1 and Figure 2.

[0055] Among them, the nucleotide sequence shown in SEQ ID NO.7 contains lox71 and lox66 sequences at both ends. upstream of lox71 is ori, and inside is the Cre transcription unit. The Cre gene is started by the AOX1 promoter with a mutation at the SacⅠ restriction site. upstream of BleoR are prokaryotic and eukaryotic promoters, which can be used for screening prokaryotes and eukaryotes. upstream of AmpR is a prokaryotic promoter, which can be used for screening prokaryotes.

[0056] The meanings of each part in SEQ ID NO.7 are explained as follows:

[0057] The lowercase letter is shown as ori, 5' end The image shows the 3' end of a lox71. The value shown is 66. The image shown is of AmpR. The image shows pAmpR (AmpR promoter). The image shows the pAOX1 promoter with a mutated SacⅠ site. The image shows the Cre recombinase gene. The image shows AOX1tt. The image shows pTEF1 (corresponding to the TEF1 promoter in the diagram). Meanwhile, the lowercase part is pEM7 (corresponding to EM7promoter in the image). The image shown is of BleoR. Meanwhile, the lowercase part is CYC1tt (corresponding to CYC1terminator in the figure).

[0058] SEQ ID NO.7:

[0059] 3.2 Construction of plasmid pMCrN-AOX

[0060] The SEQ ID NO:8 sequence (PpRGI2) and SEQ ID NO:9 sequence were synthesized and replaced the PpHIS4 and Zeocin selection marker sequences on pMChZ-AOX obtained in step 3.1, respectively, to obtain plasmid pMCrN-AOX (where r represents the recombination site as PpRGI2 and N represents the selection marker as NrsR), and the spectrum is shown in Figure 3.

[0061] 3.3 Construction of plasmid pMCeH-AOX

[0062] The sequences SEQ ID NO.10 (PpENO) and SEQ ID NO.11 were synthesized and replaced PpHIS4 and PEM7-BleoR-CYC1 tt on pMChZ-AOX, respectively, to obtain plasmid pMCeH-AOX (e represents the recombination site as PpENO, and H represents the selection marker as HygR), as shown in Figure 4.

[0063] SEQ ID NO.11:

[0064]

[0065] 4. Constructing recombinant expression vectors:

[0066] 4.1 α1(Ⅰ) was cloned into the pMChZ-AOX vector constructed in 3.1 above to obtain the recombinant expression plasmid pMChZ-α1(Ⅰ).

[0067] 4.2 α2(Ⅰ) was cloned into the pMCrN-AOX vector constructed in 3.2 above to obtain the recombinant expression plasmid pMCrN-α2(Ⅰ).

[0068] 4.3 Using pMChZ-α1(Ⅰ) and pMCrN-α2(Ⅰ) as templates, the target genes α1(Ⅰ) and α2(Ⅰ) were amplified respectively, and the [α1(Ⅰ)]2α2(Ⅰ) transcription unit (SEQ ID NO.13) was constructed. The [α1(Ⅰ)]2α2(Ⅰ) transcription unit was cloned into the pMCeH-AOX vector constructed in 3.3 above using BsaⅠ endonuclease to obtain pMCeH-[α1(Ⅰ)]2α2(Ⅰ).

[0069] 4.4 Construction of the P4H expression vector pPIC9K-P4H(DP)-1

[0070] The sequence SEQ ID NO.12 was used to replace KanR in the P4H 9K DP plasmid (disclosed in Chinese patent CN114480471A). Specifically, PTEF and PEM7 were added to the 5' end of the selection marker (in the direction of the pPIC9K plasmid), and CYC1tt was added to the 3' end to obtain the P4H expression vector pPIC9K-P4H(DP)-1, whose plasmid spectrum is shown in Figure 5.

[0071] SEQ ID NO.12: in, The image shown is PTEF. The image shows PEM7. The image shown is of KANR. The image shows CYC1TT.

[0072] 5. Construction of recombinant engineered strains, induction of expression, and strain screening:

[0073] Using Pichia pastoris KM71 (or X33) as the starting strain, competent Pichia pastoris cells were prepared according to the competent cell preparation method described by Lin-Cereghino (Lin-Cereghino et al., 2005). The pMChZ-α1(Ⅰ) obtained in 4.1 above was linearized using SalⅠ to obtain the pMChZ-α1(Ⅰ) linearized plasmid. The pMChZ-α1(Ⅰ) linearized plasmid was electroporated into competent Pichia pastoris cells according to the method in the Invitrogen Pichia Expression Kit USER GUIDE, and then plated on YPDZ (bleomycin, 300 μg / mL) plates to obtain strain KM71H / pMChZ-α1(Ⅰ).

[0074] Starting with strain KM71H / pMChZ-α1(Ⅰ), pMCrN-α2(Ⅰ) obtained in 4.2 above was linearized using SalⅠ and transformed into competent cells of the starting strain to obtain strain KM71H / α1(Ⅰ)-α2(Ⅰ).

[0075] Using KM71H / α1(Ⅰ)-α2(Ⅰ) as the starting strain, pPIC9K-P4H(DP)-1 obtained in 4.4 above was linearized using BspEⅠ and transformed into competent cells of the starting strain to obtain strain KM71H / α1(Ⅰ)-α2(Ⅰ)-P4H.

[0076] Using KM71H / α1(Ⅰ)-α2(Ⅰ)-P4H as the starting strain, pMCeH-[α1(Ⅰ)]2α2(Ⅰ) obtained in 4.3 above was linearized using SalⅠ and transformed into competent cells of the starting strain to obtain strain KM71H / [α1(Ⅰ)]2α2(Ⅰ)-P4H.

[0077] 6. Inoculate the engineered strain obtained above into a 100mL Erlenmeyer flask containing 10mL of BMGY medium and incubate at 28℃ and 220rpm until OD reaches 100mL. 600 The bacterial cells were centrifuged at 3000g for 5 min at room temperature, collected, and resuspended in BMMY medium to adjust the OD value. 600The culture medium was incubated at approximately 2°C on a shaker at 220 rpm for 3 days, with 100% methanol added every 24 hours until the final concentration reached 1.0%. After methanol induction, bacterial culture samples were collected every 24 hours, with a sample volume of 1 mL placed in a 1.5 mL EP tube. The tubes were centrifuged at 12000 g for 5 min at 4°C, and the supernatant and bacterial cells were collected separately. 80 μL of the supernatant was added to 20 μL of loading buffer and heated at 80°C for 5 min to prepare the sample. 20 μL of this sample was then subjected to SDS-PAGE. Add 200 μL of cell disruption buffer and 40% liquid volume of 0.5 mm glass beads to the bacterial cells; vortex for 1 min, place on ice for 1 min, and repeat 6 times; centrifuge at 12000 g for 30 min at 4 °C, take 80 μL of the supernatant, add 20 μL of loading buffer, heat at 99 °C for 5 min to prepare the sample, dissolve the precipitate in 8M urea, and prepare the sample according to the supernatant procedure, take 20 μL for SDS-PAGE, and then perform Western blotting. Collagen was detected using COL1A1 and COL1A2 antibodies, and the detection results are shown in Figure 6. The results show that KM71H / α1(Ⅰ)-α2(Ⅰ) and KM71H / [α1(Ⅰ)]2α2(Ⅰ)-P4H can detect full-length COL1A1 and COL1A2. The α subunit of P4H was detected using P4HA1 antibody, and the β subunit of P4H was detected using P4HB antibody. The results are shown in Figure 7. The results show that KM71H / α1(Ⅰ)-α2(Ⅰ) and KM71H / [α1(Ⅰ)]2α2(Ⅰ)-P4H can normally express the P4HA and P4HB subunits.

[0078] Cell wall disruption solution formulation: 50mM sodium phosphate (pH 7.4), 1mM EDTA, 5% glycerol and 1mM benzyl sulfonyl fluoride (PMSF) are dissolved in anhydrous ethanol to prepare a 100× stock solution, which is added before cell wall disruption.

[0079] 7. High-density fermentation culture and protein purification: The engineered bacteria with high protein expression levels identified through protein expression are used for high-density fermentation culture and protein purification in a fermenter.

[0080] Inoculate KM71H / [α1(Ⅰ)]2α2(Ⅰ)-P4H into a 1L shake flask containing 200mL of seed culture medium YPG, and incubate at 220rpm and 30℃ for 18h until OD600=2~10.

[0081] A 5L fermenter (Baoxing Biotechnology) was used, filled with 2L of fermentation medium. Before inoculation, the fermentation speed was adjusted to 300 rpm, the aeration rate to 4L / min, and the temperature to 30℃. The pH was adjusted to 6.0 using a concentrated ammonia solution. First, 0.9mL of PTM1 was added, followed by 200mL of the prepared seed culture (flame ring inoculation). The dissolved oxygen electrode was then calibrated, and fermentation began. When the dissolved oxygen level first dropped to 30%, the dissolved oxygen cascade speed function was used to maintain 30%. The fermentation continued until the glycerol was depleted, the dissolved oxygen rebounded, and the dissolved oxygen level exceeded 70% (OD). 600 (Value approximately 20), cancel dissolved oxygen cascade stirring speed, increase stirring speed to 650 rpm, and use 30% continuous feeding of glycerol, adding 150 mL at a time. Stop feeding glycerol, and after dissolved oxygen rebounds to above 70%, induce culture with methanol at a constant feeding rate of 4 mL / h. After 80 h of induction, OD... 600 Once the change is not significant or decreases, the mixture can be placed in a tank to obtain the fermentation broth.

[0082] After fermentation, the bacterial culture was centrifuged at 5000 rpm for 30 min to collect the bacterial cells. The cells were resuspended in the purified cell-wall-breaking solution at a ratio of 1:10 (W:V) and homogenized at 1000 bar. After homogenization, the supernatant and precipitate were separated by centrifugation at 12000 rpm and 4℃ for 30 min. The precipitate was washed, renatured, digested with pepsin, desalted by ultrafiltration, and then lyophilized to obtain α1(Ⅰ)α2(Ⅰ) lyophilized powder.

[0083] The purification and cell wall disruption solution formula consists of 10 mM Tris-HCl (pH 7.4), 100 mM NaCl, and 100 mM glycine.

[0084] 8. Structural characterization of α1(Ⅰ) and α2(Ⅰ) lyophilized powders:

[0085] After reconstitution of α1(Ⅰ) and α2(Ⅰ) lyophilized powders, acid hydrolysis was performed. The peak area of ​​the target data was calculated using MassLynx quantitative software and the identification results were obtained by standard curve method. The results showed that the hydroxyproline content reached more than 30% of the total proline.

[0086] 9. The lyophilized powders α1(Ⅰ) and α2(Ⅰ) were dissolved in water and detected by a circular dichroism spectrometer. The test results are shown in Figure 8, which are consistent with the circular dichroism spectral characteristics of triple helix collagen.

[0087] 10. After negative staining, collagen samples were examined by transmission electron microscopy. The test results are shown in Figure 9, which shows the typical structural features of collagen microfibers with alternating light and dark characteristics.

[0088] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for constructing an engineered bacterium expressing a triple helix structure type I recombinant human collagen, characterized in that, The step of constructing the engineering bacteria includes transforming pMChZ-alpha1(I) plasmid, pMCrN-alpha2(I) plasmid, pPIC9K-P4H(DP)-1 plasmid and pMCeH-[alpha1(I)]2alpha2(I) plasmid into host bacteria. The pMChZ-alpha1(I) plasmid is obtained by cloning alpha1(I) into pMChZ-AOX vector. The pMCrN-alpha2(I) plasmid is obtained by cloning alpha2(I) into pMCrN-AOX vector. The pPIC9K-P4H(DP)-1 plasmid is obtained by replacing KanR in P4H 9K DP plasmid with a sequence shown in SEQ ID NO.

12. The pMCeH-[alpha1(I)]2alpha2(I) plasmid is obtained by cloning [alpha1(I)]2alpha2(I) transcription unit into pMCeH-AOX vector. The nucleotide sequence of alpha1(I) is shown in SEQ ID NO.

3. The nucleotide sequence of alpha2(I) is shown in SEQ ID NO.

6. The nucleotide sequence of [alpha1(I)]2alpha2(I) transcription unit is shown in SEQ ID NO.

13. The pMChZ-AOX vector is obtained by modifying pPIC9K plasmid as follows: replacing 4648-9221 bp of the pPIC9K plasmid with a DNA molecule having a nucleotide sequence shown in SEQ ID NO. 7; deleting BamH I upstream of alphaMF in the pPIC9K plasmid; introducing a BamH I recognition site by point mutation between 1583-1584 bp of the pPIC9K plasmid. The pMCrN-AOX vector is obtained by replacing PpHIS4 and Zeocin screening marker of the pMChZ-AOX plasmid with DNA molecules having nucleotide sequences shown in SEQ ID NO. 8 and SEQ ID NO. 9, respectively. The pMCeH-AOX vector is obtained by replacing PpHIS4 and PEM7-BleoR-CYC1 tt of the pMChZ-AOX plasmid with DNA molecules having nucleotide sequences shown in SEQ ID NO. 10 and SEQ ID NO. 11, respectively.

2. The construction method of claim 1, wherein, The construction method specifically includes the following steps: The pMChZ-alpha1(I) plasmid is transformed into the host bacteria to obtain KM71H / pMChZ-alpha1(I) strain; The pMCrN-alpha2(I) plasmid is transformed into the KM71H / pMChZ-alpha1(I) strain to obtain KM71H / alpha1(I)-alpha2(I) strain; The pPIC9K-P4H(DP)-1 plasmid is transformed into the KM71H / alpha1(I)-alpha2(I) strain to obtain KM71H / alpha1(I)-alpha2(I)-P4H strain; The pMCeH-[alpha1(I)]2alpha2(I) plasmid is transformed into the KM71H / alpha1(I)-alpha2(I)-P4H strain to obtain the engineering bacteria.

3. The construction method of claim 1, wherein, The host strain is Pichia pastoris X33 or Pichia pastoris KM71.

4. The construction method of claim 1, wherein, The point mutation deletion of the BamH Ⅰ site upstream of the αMF in the pPIC9K plasmid.

5. The engineered bacteria expressing the triple helix structure type I recombinant human collagen prepared by the construction method according to any one of claims 1-4.

6. The use of the engineered bacteria according to claim 5 in the preparation of the triple helix structure type I recombinant human collagen.

7. A method for preparing a yeast-expressed triple-helical structure type I recombinant human collagen protein, characterized by, The steps of fermenting and purifying the triple helix structure type I recombinant human collagen by using the engineered bacteria according to claim 5.

8. The triple helix structure type I recombinant human collagen prepared by the preparation method according to claim 7.

9. The use of the triple helix structure type I recombinant human collagen according to claim 8 in the preparation of functional biomaterials, cosmetics or medical devices.

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