CN117777276A - Method for promoting secretion expression of human lactoferrin by kluyveromyces marxianus - Google Patents
Method for promoting secretion expression of human lactoferrin by kluyveromyces marxianus Download PDFInfo
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Abstract
The invention discloses a method for promoting secretion expression of human lactoferrin by kluyveromyces marxianus. The invention provides a method for promoting secretory expression of human lactoferrin in kluyveromyces marxianus, the method comprises the following steps: while expressing human lactoferrin in kluyveromyces marxianus recipients, the endogenous Gap3 promoter of kluyveromyces marxianus is adopted to increase the expression of EmP protein, so that the secretory expression of the human lactoferrin in kluyveromyces marxianus is promoted. The invention has important significance for improving the yield of human lactoferrin produced by fermenting Kluyveromyces marxianus.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for promoting secretion of kluyveromyces marxianus to express human lactoferrin.
Background
Lactoferrin (LF) is a glycoprotein capable of binding iron and has a molecular weight of about 80 kDa and is one of the members of the transferrin family. Its isoelectric point (pI) is 8.0-8.5. Lactoferrin is widely found in many mammalian secretions, including mammalian milk, saliva, tears, bronchial and intestinal secretions, and secondary particles of neutrophils. Lactoferrin is approximately 700 amino acids, consists of two spherical leaflets, and is linked by an alpha-helix, they are called the amino-terminal region and the carboxy-terminal region, and denature at two different temperatures: about 60 ℃ and about 90 ℃. The secondary structure of lactoferrin is mainly alternating arrangement of alpha-helix and beta-sheet, and its higher structure is formed by folding polypeptide chain based on the secondary structure.
Lactoferrin has a wide range of biological properties including antibacterial, antiviral, anti-inflammatory, antioxidant, anticancer, immunomodulating and enzymatic activities, etc. Meanwhile, lactoferrin also plays a role in transporting iron ions in vivo. It is known as a natural antibiotic and is an important component connecting the innate and adaptive immune systems of mammals. Lactoferrin can exert its cell-protecting effect at various stages of life, and thus it is considered as a novel antibacterial, anticancer drug. Currently, lactoferrin is added in many commercial products, such as infant formulas, nutritional supplements, toothpastes, cosmetics, and the like. Lactoferrin has a variety of health promoting functions and is widely used in real life, and more researchers turn their eyes towards this functional protein.
Currently, lactoferrin is obtained mainly by separation and extraction from cow's milk, however, cow's milk contains only 0.03-0.49g/L lactoferrin. In the extraction process, the price is high, and the human body can bring a certain negative influence to eat the heterologous protein, so that antigen reaction is generated. In order to obtain a large amount of lactoferrin and avoid side effects caused by the lactoferrin, scientific researchers construct engineering strains in a genetic engineering mode, and then ferment to obtain a large amount of lactoferrin. Currently, host cells producing lactoferrin mainly include E.coli, yeast, mammalian cells, and plant cells. Coli expression systems lack glycosylation modification mechanisms, resulting in the inability to produce bioactive lactoferrin. Mammalian cells and plant cells can be appropriately glycosylation-modified, but large-scale production is difficult due to long cell growth cycle and complicated culture.
Yeast cells can substantially avoid the disadvantages of the cells, and have basic production conditions as the simplest eukaryotic organisms. In the last decade, researchers have performed heterologous expression of human lactoferrin in yeast cells as hosts. Although the yeast expression system has the characteristics of fast growth, simple operation and post-translational processing and modification functions, some inherent defects of the system, such as that human protein molecules and cytokines cannot be efficiently expressed in yeast, and protein products are easy to form polymers so as to cause protein degradation.
Researchers have enhanced the expression levels of foreign proteins, mainly by optimizing key expression elements (promoters, terminators, enhancers, and silencers) in yeast expression systems. However, when a large amount of exogenous protein is accumulated in the cell, a great pressure is caused to a cell secretion system, so that the cell is easy to collapse, the exogenous protein is degraded, and the yield of the protein is reduced. In order to enhance the expression level of the foreign gene, it is necessary to solve the problem of smooth secretion of the foreign protein, thereby realizing mass production of the target protein.
Disclosure of Invention
In order to solve the technical problems, the invention promotes the correct folding of human lactoferrin in kluyveromyces marxianus, can be secreted out of cells rapidly, and realizes the enhancement of the high-efficiency expression of the human lactoferrin. The invention not only realizes the purpose of improving the yield of human lactoferrin produced by fermenting Kluyveromyces marxianus, but also provides a method for improving the secretion expression level of human lactoferrin in Kluyveromyces marxianus.
In a first aspect, the invention claims a method for promoting secretory expression of human lactoferrin in kluyveromyces marxianus.
The method for promoting the secretory expression of human lactoferrin in kluyveromyces marxianus provided by the invention can comprise the following steps: while expressing human lactoferrin in kluyveromyces marxianus recipients, the endogenous Gap3 promoter of kluyveromyces marxianus is adopted to increase the expression of EmP protein, so that the secretory expression of the human lactoferrin in kluyveromyces marxianus is promoted.
In this method, promoting secretory expression of the human lactoferrin in kluyveromyces marxianus is achieved by increasing expression of the EmP47 protein.
Wherein the nucleotide sequence of the Emp47 protein is derived from Trichoderma reesei QM6a. Further, the amino acid sequence of the EmP47 protein is shown as SEQ ID No. 1. The amino acid sequence of the human lactoferrin is shown as SEQ ID No. 4.
Further, the expression of the human lactoferrin in the kluyveromyces marxianus recipient strain may be achieved by introducing a gene encoding the human lactoferrin into the kluyveromyces marxianus recipient strain.
Further, increasing the expression of the EmP protein using the Gap3 promoter endogenous to kluyveromyces marxianus can be accomplished by introducing the EmP protein gene expression cassette into the kluyveromyces marxianus recipient. In the EmP protein gene expression cassette, the GAP3 promoter endogenous to Kluyveromyces marxianus initiates expression of the EmP protein encoding gene.
Further, the nucleotide sequence of the encoding gene of the human lactoferrin is shown as SEQ ID No. 5.
Further, in the EmP protein gene expression frame, the nucleotide sequence of the Gap3 promoter endogenous to kluyveromyces marxianus is shown in SEQ ID No. 3; the nucleotide sequence of the coding gene of the EmP protein is shown as SEQ ID No. 2.
In a specific embodiment of the present invention, the gene expression cassette of the EmP protein is inserted into and replaces the genome of the Kluyveromyces marxianus recipient strainOch1And (3) a gene. N-glycosylation modifications in the endoplasmic reticulum of eukaryotes are highly conserved, differingThe point is that after transferring to the Golgi apparatus, the yeast passes throughOch1The gene is added with an alpha-1, 6-mannose on the alpha-1, 3-mannose, then other mannose transferases and phosphomannose transferases continue to add mannose upwards based on the sugar chain structure, and finally a high mannose structure is formed. Lactoferrin is a glycosylated protein, and yeast expressed glycosylated proteins have the disadvantage that they are glycosylated in a different manner from mammalian cells and that high mannose glycosylated proteins are easily formed. Knock-outOch1The gene is used for blocking high mannose glycosylation modification of yeast and preparing for subsequent glycosylation modification.
Specifically, the gene expression cassette of the EmP protein inserts and replaces the Kluyveromyces marxianus receptor in the genomeOch1The gene is realized by homologous recombination. The sequence of the upstream homology arm for carrying out the homologous recombination is shown as SEQ ID No.6, and the sequence of the downstream homology arm is shown as SEQ ID No. 7.
In a specific embodiment of the present invention, the Kluyveromyces marxianus recipient is Kluyveromyces marxianusKM::△ura3The method comprises the steps of carrying out a first treatment on the surface of the The Kluyveromyces marxianusKM::△ura3To mix Kluyveromyces marxianus FIM1 genomeUra3The strain obtained after gene knockout. Because the production requirement does not require the introduction of a resistance selection marker, the genome is knocked outUra3And (3) a gene.
In a second aspect, the invention claims a method of constructing a kluyveromyces marxianus engineered strain for the fermentative production of human lactoferrin.
The method of constructing a kluyveromyces marxianus engineered strain for the fermentative production of human lactoferrin as claimed in the present invention may comprise the steps as described in the first aspect hereinbefore.
In a third aspect, the invention claims a kluyveromyces marxianus engineered strain for the fermentative production of human lactoferrin.
The Kluyveromyces marxianus engineering strain for producing human lactoferrin by fermentation is constructed by adopting the method in the second aspect.
In a fourth aspect, the invention claims the use of a kluyveromyces marxianus engineered strain as described in the third aspect above for the fermentative production of human lactoferrin.
Experiments prove that when compared with the original expression systemEmp47After the gene is integrated into the Kluyveromyces marxianus host, the secretion amount of human lactoferrin is obviously increased by about 3 times, and 378 mug/L is reached. The invention can effectively improve the secretion expression quantity of human lactoferrin by enhancing the expression of Emp 47. The invention has important significance for improving the yield of human lactoferrin produced by fermenting Kluyveromyces marxianus.
Drawings
FIG. 1 shows the results of colony PCR verification of the selected clone strain of interest. The arrow marks the homozygote strain obtainedKM::△Ura3::Emp47The rest is wild strainOch1Genes still exist.
FIG. 2 is a SDS-PAGE map of the expression level of human lactoferrin. Wherein 1 is a controlKM::△Ura3/ pUKDN119-LFIs a negative control; 2 is a lactoferrin positive standard; 3 and 4 areKM::△Ura3::Emp47/ pUKDN119-LFAnd (3) supernatant protein. The arrow shows lactoferrin.
FIG. 3 is a Western Blot chart showing expression levels of human lactoferrin. Wherein 1 is a lactoferrin positive standard; 2 is used as a controlKM::△ura3/pUKDN119-LFIs a negative control; 3 and 4 areKM::△ura3::Emp47/ pUKDN119-LFAnd (3) supernatant protein. The arrow shows lactoferrin.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Kluyveromyces marxianus FIM1: (CGMCC No. 10621): described in "Liu Y, mo WJ, shi TF., mutioal Mtc6p attenuates autophagy and improves secretory expression of heterologous proteins inKluyveromyces marxianusMicrob Cell face.2018 Sep 14;17 (1): 144. Doi: 10.1186/s12934-018-0993-9. PMID: 30217195; PMCID: PMC6138896, "one text, available to the public from applicant, as far as possible for repeated use of the experiments of the present invention. Kluyveromyces marxianusKM::△ura3To introduce Kluyveromyces marxianus FIM1 into the genomeUra3The strain obtained after gene knockout is also described in the above-mentioned documents.
Example 1, emp47 optimization strategy
Emp47 used in the present invention is derived from Trichoderma reesei QM6a, and its amino acid sequence is shown in SEQ ID No. 1. Under the condition of unchanged amino acid sequence, the nucleotide sequence of the Kluyveromyces marxianus is optimized according to a high-use frequency table (table 1), and the optimized nucleotide sequence is shown as SEQ ID No. 2.
Example 2 amplification of donor DNA
The Kluyveromyces marxianus endogenous Gap3 promoter is selected to express Emp47, the GAP3 promoter is a glucose carbon source promoter, and the carbon source used in the fermentation process is glucose, which is favorable for the promotion of the GAP3 promoterEmp47Transcription level of the gene.
Kluyveromyces marxianus was performed with primers Up-F and Up-R (Table 2)KM::△ura3PCR amplification was performed using the genome of (C) as a template to obtain the Up homology arm (SEQ ID No. 6) knocked in the Och1 site.
Primers Emp47-F and Emp47-R (Table 2) were used to recombine plasmid pUC57-P Gap3 Emp47 as template to obtain the "Gap3 promoterEmp47Gene fragment, gap3 promoter andEmp47the gene fragment is a sequence obtained by combining SEQ ID No.3 (Gap 3 promoter) and SEQ ID No.2 (optimized)Emp47Genes) are sequentially connected end to end. Wherein, recombinant plasmid pUC57-P Gap3 Emp47 is in pUC57 plasmidEcoRV site is inserted into the DNA fragment shown in SEQ ID No.3+SEQ ID No. 2.
Kluyveromyces marxianus was performed with primers Down-F and Down-R (Table 2)KM::△ura3PCR amplification was performed using the genome of (C) as a template to obtain a Down homology arm (SEQ ID No. 7) knocked in the Och1 site.
Further, the 3 amplification products obtained above are used as templates, and primers Up-F and Down-R are used for PCR amplification, so that the overlay PCR product can be obtained. The product is a donor DNA fragment, which sequentially comprises an Up homology arm (SEQ ID No. 6), a Gap3 promoter (SEQ ID No. 3) of an Och1 site from the 5 'end to the 3' end,Emp47A gene (SEQ ID No. 2) and a Down homology arm (SEQ ID No. 7) knocked in to the Och1 site.
Example 3 construction of Gene editing plasmid
The LHZ531 plasmid (the full sequence of which is shown in SEQ ID No.8, having the TEF1 promoter therein, was first of all digested with SapI endonuclease (Takara, cat. No. 1631),Cas9The gene, CYC1 terminator, ura3 promoter and genes thereof, ARS1 terminator, ampicillin gene, guide RNA, tRNA-gly) are digested, and large fragment products are recovered. Then in Kluyveromyces marxianusKM::△ura3Find Och1 editing site in genome of (2) and according to N20 sequence design requirementOch1The first 20 bases of the NGG target sequence are found inside the gene. The ligation-cohesive ends of the SapI cleavage site were added to the 5' end of the N20 primer (table 3), TCA and AAC, respectively. Two N20 primers (N20 in Table 3) were mixed 10. Mu.L each after dilution at 95℃for 3min, followed by 10 min at room temperature. Finally, the N20 sequence was ligated to LHZ531 large fragment using T4 ligase (Biolab, cat. B0202S) and transformed into DH 5. Alpha. Competent, ampicillin resistant LB solid was coatedA culture medium. After the next day verification, the primers YY161F and N20-R were used, if there was a PCR product of 1000bp size, the plasmid construction was successful, and if there was no product, the construction failed. The primer sequences involved are shown in Table 3.
Note that: v in SEQ ID No.18 represents A or C or G.
Finally obtaining the gene editing plasmid which is named LHZ531-N20. Namely, a small fragment between two cleavage sites SapI of the LHZ531 plasmid is replaced with accggcgtataacatgtcag (i.e., positions 4 to 23 of SEQ ID No. 16), thereby obtaining a recombinant gene editing plasmid.
Example 4 Yeast chemical transformation of the editing plasmid LHZ531-N20 and donor DNA
Selecting Kluyveromyces marxianusKM::△ura3Single colonies were cultured to OD in 30mL YPD medium 600 And (3) centrifuging the bacterial solution in a 2mL Ep tube to obtain a precipitate, adding sterile water to 30mL, and uniformly mixing 3mL of 1M LiAc and 3mL of 10 xTE (formula: 100mM Tris-HCl,10mM EDTA is used for adjusting pH=8.0), thereby obtaining the 1 xLiAc/TE buffer solution. Washing the pellet twice with 1 XLIAC/TE buffer, removing the supernatant, adding 5 mu L of Carrier DNA (Takara, cat. No. 630440) in the center of the pellet, adding 1 mu g of the donor DNA fragment obtained in example 2 and 500 ng of the edited plasmid LHZ531-N20 constructed in example 3, gently mixing with the gun head, finally adding 300 mu L of PEG/LiAc/TE solution (preparation method: 40g of PEG is dissolved in water until the scale mark 80mL,115 ℃ C., 20min for sterilization, taking sterile 1M LiAC and 10 XTE, respectively 10mL of each, adding 10mM of each, mixing, gently mixing, adding a proper amount of 1M DTT in the system until the final concentration of DTT is 10mM, mixing again, then carrying out water bath 15min at 30 ℃ C., water bath 15min at 47 ℃ C., finally centrifuging, obtaining the pellet, adding 150 mu L of sterile water, coating SD plate, culturing in a 30 ℃ C. Incubator for 2-4 days until single colony is grown, and the screened clone is obtainedKM::△ura3::Emp47As shown in FIG. 1, the size of the destination band is 2832bp.
Example 5 Chassis cell transformation of expression plasmid pUKDN119-LF
UsingSacII (NEB, R0157S) andSpe the I (NEB, R3133S) endonuclease was directed against the pUKDN119 plasmid (see SEQ ID No.9 for the complete sequence, which contains the elements: PKD1 fragment, inulase promoter and signal peptide, PMD18-T, inulase terminator, ura3 promoter,Ura3Gene) and then enzyme-cutting to recover large fragments. Then using primer LF-F/R to amplify human lactoferrin encoding gene by taking the plasmid containing human lactoferrin encoding gene as template, adding corresponding enzyme cutting sites at two ends of the primer, cutting at the same time, mixing with pUKDN119 carrier large fragment according to mole ratio of 1:4 after recovery treatment, using T4 ligase to connect, and the obtained recombinant plasmid is named pUKDN119-LF. The structure of the recombinant plasmid is described as follows: restriction enzyme site of pUKDN119 plasmidSacII and IISpe The small fragments between I are replaced by recombinant plasmids obtained after the human lactoferrin encoding gene (SEQ ID No. 5). The amino acid sequence of human lactoferrin is shown as SEQ ID No.4, and the corresponding coding gene sequence is shown as SEQ ID No. 5.
Finally, the recombinant plasmid pUKDN119-LF was transformed into the chassis cell obtained in example 4KM::△ura3:: Emp47In the course of operation, reference is made to example 4. After single colonies were grown, they were verified using primers PINU-F and LF-R, and single colonies with a band of 2200 bp were amplified to be correct, followed by sequencing. The primers according to this example are shown in Table 4. The positive transformants obtained finally were designated asKM::△ura3::Emp47/pUKDN119-LF。
Simultaneously, the recombinant plasmid pUKDN119-LF is transformed into chassis cellsKM::△ura3As a negative control, the resulting transformant was designated asKM::△ura3/pUKDN119-LF。
Example 6 detection of translation level of human lactoferrin in recombinant KM Yeast
Single colonies of the positive transformants obtained in example 5 aboveKM::△ura3::Emp47/pUKDN119-LFPicking up YG medium (formula: yeast)20g/L of powder and 40g/L of glucose) and continuously fermenting for 3 days at 30 ℃ in a shaking table at 200 rpm. Then, the supernatant was collected by centrifugation using a table-type refrigerated centrifuge at 8000rpm for 15min, 1M trichloroacetic acid 10% of the volume of the supernatant was added to precipitate secreted proteins in the supernatant, the supernatant was collected by centrifugation at 4℃overnight at 12000rpm for 20min at the next day, the supernatant was discarded, the precipitate was collected, washed twice with 2mL of pre-cooled absolute ethanol, the supernatant was discarded and then placed on ice with an opening to volatilize ethanol, and finally 50mM Tris-HCl solution precipitate at pH8.5 was added to obtain secreted proteins. Human lactoferrin was quantitatively analyzed using SDS-PAGE and Western blot. Simultaneous setting of the strain obtained in example 5KM::△ura3/pUKDN119-LFAs a negative control.
SDS-PAGE of human lactoferrin expression levels is shown in FIG. 2, and Western Blot is shown in FIG. 3. From the figure, it can be seen thatKM::△ura3::Emp47/pUKDN119-LFIs capable of expressing intact human lactoferrin, and controlsKM::△ura3 / pUKDN119-LFThe absence of a full-size human lactoferrin fragment in the supernatant suggests that Emp47 has a promoting effect on the formation of the correct spatial structure of human lactoferrin, increasing human lactoferrin stability and secretion.
EXAMPLE 7 Elisa detection of secretion of human lactoferrin in recombinant KM Yeast
The supernatant obtained by initial centrifugation in example 6 was 5-fold diluted with a coating solution as an antigen, while lactoferrin (Saputo, 1001102) was used as a standard curve at an initial concentration of 10 mg/L, followed by stepwise 5-fold dilution to 1.28X10 -4 mg/L, 100. Mu.L of coating solution (formulation: 1.59g Na) 2 CO 3 ，2.93g NaHCO 3 Distilled water diluted to 1000 mL), refrigerator at 4 ℃ for 2h, pour out the liquid in the wells, add 150 μl of wash (formulation: 0.2g KH 2 PO 4 ，2.9g Na 2 HPO 4 •12H 2 O,0.5 mL Tween-20; distilled water is added to 1000 mL), the mixture is left for 3min, and the washing is repeated for 3 times, and the washing liquid is removed. 100. Mu.L of a blocking solution (formula: 5g of skim milk was dissolved in 100mL of a washing solution), left at 37℃for 2 hours, washed 3 times, diluted 2000-fold with 0.1. 0.1 mL of primary antibody-rabbit anti-lactoferrin (Bioss, bs-5810R) was added to each reaction well, incubated at 37℃for 1 h, and washed. Adding0.1 An enzyme-labeled secondary antibody-goat anti-rabbit (Industry, SA 00001-2) diluted 6000 times in mL was placed in each reaction well, incubated at 37℃for 1 h, and then washed. Adding TMB substrate solution 0.1-mL into each reaction well, reacting at room temperature for 3-10 min, adding 50 μl of 2 mol/L H into each well 2 SO 4 The reaction was terminated, and the values were read on an ELISA detector at 450 nm, and the constructed chassis cells (i.e., single colonies of positive transformants obtained in example 5)KM::△ura3::Emp47/pUKDN119-LF) The expressed human lactoferrin was tested and after the OD450 value was measured, the expression was measured according to the LF standard curve y= 0.0556 x+ 0.0758 (y is OD450 reading, x is LF concentration, R 2 =0.9575) the yield of human lactoferrin was calculated, and the average of the human lactoferrin secretion yields measured in triplicate was 378 μg/L. Specifically, the results are shown in Table 5.
Note that: p <0.05 indicates a significant difference between the two.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.
Claims (10)
1. A method for promoting secretory expression of human lactoferrin in kluyveromyces marxianus comprising the steps of: while expressing human lactoferrin in kluyveromyces marxianus recipients, the endogenous Gap3 promoter of kluyveromyces marxianus is adopted to increase the expression of EmP protein, so that the secretory expression of the human lactoferrin in kluyveromyces marxianus is promoted.
2. The method according to claim 1, characterized in that: the amino acid sequence of the EmP protein is shown as SEQ ID No. 1;
the amino acid sequence of the human lactoferrin is shown as SEQ ID No. 4.
3. The method according to claim 1, characterized in that: expression of the human lactoferrin in the kluyveromyces marxianus recipient is achieved by introducing a gene encoding the human lactoferrin into the kluyveromyces marxianus recipient;
increasing the expression of the EmP protein using the Gap3 promoter endogenous to kluyveromyces marxianus is achieved by introducing the EmP protein gene expression cassette into the kluyveromyces marxianus recipient; in the EmP protein gene expression cassette, the GAP3 promoter endogenous to Kluyveromyces marxianus initiates expression of the EmP protein encoding gene.
4. A method according to claim 3, characterized in that: the nucleotide sequence of the coding gene of the human lactoferrin is shown as SEQ ID No. 5.
5. A method according to claim 3, characterized in that: in the EmP protein gene expression frame, the nucleotide sequence of the Gap3 promoter endogenous to Kluyveromyces marxianus is shown in SEQ ID No. 3; the nucleotide sequence of the coding gene of the EmP protein is shown as SEQ ID No. 2.
6. A method according to claim 3, characterized in that: the EmP protein gene expression frame is inserted into and replaces the Kluyveromyces marxianus receptor strain genomeOch1And (3) a gene.
7. The method according to any one of claims 1-6, wherein: the Kluyveromyces marxianus recipient is Kluyveromyces marxianusKM::△ura3The method comprises the steps of carrying out a first treatment on the surface of the The Kluyveromyces marxianusKM::△ura3To use wild Kluyveromyces marxianus genomeUra3The strain obtained after gene knockout.
8. A method for constructing a kluyveromyces marxianus engineering strain for producing human lactoferrin by fermentation, which is characterized in that: the method comprising the steps of any one of claims 1-7.
9. A kluyveromyces marxianus engineering strain for producing human lactoferrin by fermentation, which is characterized in that: the Kluyveromyces marxianus engineering strain is constructed by the method of claim 8.
10. The use of the kluyveromyces marxianus engineered strain of claim 9 in the fermentative production of human lactoferrin.
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