Yarrowia lipolytica secreting human lactoferrin and construction method and application thereof

Abstract

The present application relates to the field of biosynthesis and microbial fermentation engineering technology, in particular to a kind of secretion expression human lactoferrin Yarrowia lipolytica and its construction method and application.The present application provides a kind of secretion expression human lactoferrin recombinant Yarrowia lipolytica, the secretion expression of human lactoferrin in the recombinant Yarrowia lipolytica is guided by secretion signal peptide;The expression of chaperone HAC1, Cne and Pdi1 is simultaneously enhanced.The present application realizes the secretion expression of human lactoferrin in Yarrowia lipolytica, effectively promotes the folding and secretion expression of human lactoferrin by the cooperation of chaperone, significantly improves the yield of human lactoferrin, and has important application value in the industrial scale production of human lactoferrin.

Description

Technical Field

[0001] This invention relates to the fields of biosynthesis and microbial fermentation engineering technology, specifically to a lipothermic yeast that secretes and expresses human lactoferrin, its construction method, and its application. Background Technology

[0002] Human lactoferrin (hLF) is a multifunctional iron-binding glycoprotein naturally found in various secretions, such as breast milk, saliva, tears, intestinal secretions, exocrine secretions, and neutrophils. Mature human lactoferrin is a single-chain glycoprotein composed of approximately 691 amino acids, with a molecular weight of approximately 80 kDa. Structurally, human lactoferrin mainly comprises two highly similar globular domains (N-lobes and C-lobes), each capable of independently chelating one iron ion (Fe2+). 3+ These two structural domains are connected by an α-helical hinge structure.

[0003] Human lactoferrin possesses a wide range of biological functions. It exerts broad-spectrum antimicrobial activity by efficiently chelating iron ions, depriving pathogens such as bacteria and fungi of essential iron for growth. Simultaneously, human lactoferrin also exhibits immunomodulatory functions, regulating the release of inflammatory factors by activating macrophages, neutrophils, and natural killer cells (NK cells), thereby exerting anti-inflammatory effects. In terms of nutritional metabolism, human lactoferrin acts as a highly efficient carrier, participating in the regulation of iron concentration in the human body. Through small intestinal-specific receptors, it mediates iron absorption and transport, significantly improving iron bioavailability and preventing oxidative damage caused by free iron, thus mitigating oxidative stress-induced cell damage. Furthermore, human lactoferrin maintains intestinal microecological balance and barrier function by promoting the proliferation of beneficial bacteria and inhibiting the colonization of harmful bacteria. Studies have shown that it also participates in promoting cell proliferation and tissue repair processes, potentially regulating wound healing and bone development. Human lactoferrin's broad biological activity endows it with core biological value in many aspects, including immune defense, pathogen clearance, iron metabolism, gut health, oxidative protection, and tissue repair, making it widely applicable in industries such as infant formula, healthcare products, and cosmetics.

[0004] Currently, commercially available human lactoferrin is mainly produced through extraction from human milk or recombinant expression in systems such as prokaryotic cells, yeast, mammalian cells, and plant cells. However, these systems all have significant limitations. The average concentration of lactoferrin in human milk can reach 1-2 g / L, but extraction from human milk has low yields, high costs, and is easily affected by differences in biological origin. Prokaryotic systems, such as those using *E. coli* (…),… Escherichia coliWhile expression methods are low-cost and fast-growing, they often form inclusion bodies, requiring complex refolding steps, and lack glycosylation modification capabilities, affecting protein stability and activity. Mammalian and plant cell culture systems can synthesize proteins with glycosylation similar to those in humans, exhibiting high biological activity; however, their production cycles are long, culture costs are high, and they require stringent aseptic and controlled conditions, making them unsuitable for large-scale, low-cost industrial production. Yeast, as a eukaryotic microorganism, possesses both post-translational modification capabilities and industrial scalability, making it a highly promising expression system. However, commonly used yeasts such as Pichia pastoris (… Pichia pastoris Although it has high expression levels and a strong promoter, its reliance on methanol induction poses safety risks, and high-glucose modification can easily lead to changes in protein function; Kluyveromyces martensii ( Kluyveromyces marxianus It possesses the ability to withstand high temperatures and utilize lactose, but its gene manipulation tools are not yet perfect, and the secretion efficiency of complex proteins remains limited.

[0005] Yarrowia lipolytica ( Yarrowia lipolytica Yeast extract is a non-traditional yeast with a strong protein secretion capacity. It has received GRAS (Generally Recognized as Safe) certification and has mature genetic modification tools. Compared with other yeasts, it has a lower degree of glycosylation modification, can tolerate industrial fermentation conditions, and supports the secretory expression of complex heterologous proteins. Despite its many advantages, Yeast extract has not yet been used for the expression and production of human lactoferrin. Summary of the Invention

[0006] This invention provides a lipophilic yeast strain that secretes and expresses human lactoferrin, its construction method, and its application.

[0007] Specifically, the present invention provides the following technical solutions.

[0008] In a first aspect, the present invention provides a recombinant Yersinia lipolytica that secretes and expresses human lactoferrin, wherein the recombinant Yersinia lipolytica contains a human lactoferrin gene carrying a secretion signal peptide; and in the recombinant Yersinia lipolytica, the expression of molecular chaperone proteins HAC1, Cne and Pdi1 is enhanced.

[0009] This invention discovers that in Yersinia lipolyticis, the expression of molecular chaperones HAC1, Cne, and Pdi1 can be enhanced simultaneously. These molecular chaperones HAC1, Cne, and Pdi1 can work synergistically to significantly increase the secretory expression of human lactoferrin. However, replacing one of these molecular chaperones with another disrupts this synergistic effect, and the secretory expression of human lactoferrin is significantly reduced.

[0010] In this invention, the molecular chaperone proteins HAC1, Cne, and Pdi1 are preferably derived from Yersinia lipolyticis.

[0011] Preferably, the amino acid sequences of the molecular chaperone proteins HAC1, Cne and Pdi1 are as shown in SEQ ID NO1, 2 and 3, respectively.

[0012] In this invention, the human lactoferrin is preferably truncated human lactoferrin.

[0013] Preferably, the amino acid sequence of the human lactoferrin is shown as positions 20-710 of SEQ ID NO. 6.

[0014] In this invention, the secretion signal peptide includes any one selected from human lactoferrinogen secretion signal peptide hLFSP, Yeast lipolyticis secretion signal peptides XPR2prepro and Lip2prepro, and recombinant secretion signal peptide Lip2pre3xLA.

[0015] Preferably, the amino acid sequences of Lip2pre3xLA, hLFSP, Lip2prepro, and XPR2prepro are as shown in SEQ ID NO.7, 8, 9, and 10, respectively.

[0016] The role of secretion signal peptides is to guide the transmembrane transport and secretion of human lactoferrin into the extracellular space. Different signal peptides may have different effects on the secretory expression of different proteins, and their effectiveness in guiding secretory expression also varies in different microorganisms. It has been verified that the above-mentioned secretion signal peptides can all guide the secretory expression of human lactoferrin in *Yarrowia lipolytica*. Furthermore, this invention also found that, compared with other signal peptides, the recombinant secretion signal peptide Lip2pre3xLA has the best promoting effect on the secretory expression of human lactoferrin in *Yarrowia lipolytica*.

[0017] Preferably, the secretion signal peptide is Lip2pre3xLA.

[0018] The recombinant Yersinia lipolytica described above has at least one copy of the human lactoferrin gene carrying the secretion signal peptide integrated into its genome.

[0019] Preferably, the human lactoferrin gene is a coding gene sequence obtained by codon optimization of the human lactoferrin amino acid sequence based on the codon preference of Yersinia lipolyticis.

[0020] Preferably, the nucleotide sequence of the human lactoferrin gene is shown as positions 58 to 2130 of SEQ ID NO.11.

[0021] Preferably, the nucleotide sequence of the human lactoferrin gene carrying the secretion signal peptide Lip2pre3xLA is shown in SEQ ID NO.11.

[0022] In some embodiments of the present invention, a copy of the human lactoferrin gene carrying a secretion signal peptide is integrated into the genome of the recombinant Yersinia lipolytica.

[0023] To increase expression levels, more copies of the human lactoferrin gene can be integrated into the genome of the recombinant Yersinia lipophila.

[0024] In some embodiments of the present invention, the genome of the recombinant Yersinia lipolyticis is integrated with at least one copy of a human lactoferrin gene expression cassette, the expression cassette comprising a promoter, a secretion signal peptide, and a human lactoferrin gene.

[0025] Optionally, a protein purification tag may be added to the C-terminus of the human lactoferrin to facilitate downstream protein purification. There are no specific limitations on the protein purification tag, including but not limited to His-tags.

[0026] In this invention, the expression enhancement can be achieved through any one or more of the following methods:

[0027] (1) Increase the copy number of the target gene;

[0028] (2) Replace the transcriptional and / or translational regulatory elements of the target gene with elements with stronger activity.

[0029] In (1) above, increasing the copy number can be achieved by introducing an expression plasmid carrying the target gene or by integrating the target gene into the genome.

[0030] In (2) above, the transcriptional regulatory elements include promoters, etc.; for example, replacing the promoter of the target gene with a stronger promoter.

[0031] In some embodiments of the present invention, the expression of the molecular chaperones HAC1, Cne and Pdi1 is enhanced by increasing the copy number of the HAC1, Cne and Pdi1 genes in the genome of Yersinia lipolyticis.

[0032] Increasing the copy number of HAC1, Cne, and Pdi1 genes in the genome of Yersinia lipolyticis can be achieved using conventional genetic engineering techniques, such as homologous recombination.

[0033] In some embodiments of the present invention, increasing the copy number of the HAC1, Cne, and Pdi1 genes in the genome of *Yarrowia lipolytica* is achieved by integrating expression cassettes of the HAC1, Cne, and Pdi1 genes into the genome of *Yarrowia lipolytica*. The expression cassette includes a promoter, the HAC1 gene, the Cne gene, and the Pdi1 gene, and may also include a terminator. The HAC1, Cne, and Pdi1 genes may share a promoter or each gene may contain an upstream promoter for driving its transcription.

[0034] In some embodiments of the present invention, the recombinant Yersinia lipolyticis genome integrates a lactoferrin secretion expression cassette; the lactoferrin secretion expression cassette includes a promoter, a human lactoferrin gene carrying a secretion signal peptide, and the HAC1 gene, Cne gene, and Pdi1 gene.

[0035] Preferably, each of the human lactoferrin gene, HAC1 gene, Cne gene, and Pdi1 gene carrying the secretion signal peptide contains a promoter for transcription.

[0036] Preferably, the transcription of the human lactoferrin gene carrying the secretion signal peptide is driven by the hp4d promoter; the transcription of the HAC1 gene, Cne gene, and Pdi1 gene is driven by the TDH1 promoter.

[0037] Preferably, the lactoferrin secretion expression cassette further includes terminators for terminating transcription of each gene downstream of the human lactoferrin gene, HAC1 gene, Cne gene, and Pdi1 gene carrying the secretion signal peptide. The present invention does not have any particular limitations on the terminator; commonly used terminators such as the XPR2 terminator and the MIG1 terminator can be selected.

[0038] In some embodiments of the present invention, the recombinant Yersinia lipolyticis genome integrates a lactoferrin secretion expression cassette; the lactoferrin secretion expression cassette comprises, from the 5' to the 3' end, the following components in sequence: hp4d promoter, secretion signal peptide, human lactoferrin gene, XPR2 terminator, TDH1 promoter, HAC1 gene, MIG1 terminator, TDH1 promoter, Cne gene, MIG1 terminator, TDH1 promoter, Pdi1 gene, and MIG1 terminator.

[0039] The sequence of the hp4d promoter is shown in SEQ ID NO.13. The sequence of the TDH1 promoter is shown in SEQ ID NO.14. The sequence of the XPR2 terminator is shown in SEQ ID NO.15. The sequence of the MIG1 terminator is shown in SEQ ID NO.16.

[0040] Secondly, the present invention also provides a method for constructing recombinant Yersinia lipolytica that secretes and expresses human lactoferrin as described above, the method comprising: introducing a human lactoferrin gene carrying a secretion signal peptide into Yersinia lipolytica; and enhancing the expression of molecular chaperones HAC1, Cne and Pdi1.

[0041] In some embodiments of the present invention, the method includes: integrating the lactoferrin secretion expression cassette into the genome of *Yersinia lipolytica*. Optionally, the method includes: integrating a recombinant vector containing the lactoferrin secretion expression cassette or the lactoferrin secretion expression cassette into a *Yersinia lipolytica* genomic locus via homologous recombination. Optionally, the lactoferrin secretion expression cassette is ligated into the pYLSC3' vector (this plasmid is modified from the commercial plasmid pYLSC1 (https: / / yeastern.com / ?page_id=1390), the modification method of which can be found in the literature: https: / / doi.org / 10.1016 / j.synbio.2025.07.008) to obtain a recombinant vector, the recombinant vector is introduced into *Yersinia lipolytica*, and the recombinant vector containing the lactoferrin secretion expression cassette is integrated into a *Yersinia lipolytica* genomic locus via homologous recombination.

[0042] In principle, this invention does not impose any particular restrictions on the starting strain for constructing recombinant Yersinia lipolytica that secretes and expresses human lactoferrin; it can be any Yersinia lipolytica suitable as a protein expression host.

[0043] Optionally, the starting strain is Yersinia lipolytica Po1g or a derivative thereof, including Yersinia lipolytica strains with pBR322 site and LEU2, AEP, and AXP knocked out.

[0044] Optionally, the starting strain is Yersinia lipolyticis Po1f or a derivative thereof, including Yersinia lipolyticis strains with URA3, LEU2, AEP, and AXP knocked out.

[0045] Optionally, the starting strain is Yersinia lipolyticis Po1k or a derivative thereof, including Yersinia lipolyticis strains with KU70, URA3, LEU2, AEP, and AXP knocked out.

[0046] Thirdly, the present invention provides any one of the following applications of the recombinant Yersinia lipolyticis yeast that secretes and expresses human lactoferrin as described above:

[0047] (1) Preparation of human lactoferrin;

[0048] (2) Construct a production strain of human lactoferrin.

[0049] Fourthly, the present invention provides a method for preparing human lactoferrin, the method comprising: culturing recombinant Yersinia lipolytica that secretes and expresses human lactoferrin as described above, and isolating human lactoferrin from the culture.

[0050] Preferably, the culture includes fermentation culture. The temperature of the fermentation culture is 25-33°C.

[0051] Fifthly, the present invention provides a method for promoting the secretory expression of human lactoferrin in Yersinia lipolyticis, the method comprising: guiding the secretory expression of human lactoferrin with a secretion signal peptide, while enhancing the expression of molecular chaperone proteins HAC1, Cne and Pdi1 in Yersinia lipolyticis.

[0052] In some embodiments of the present invention, the method includes: integrating a lactoferrin secretion expression cassette into the genome of Yersinia lipolytica, the lactoferrin secretion expression cassette comprising a human lactoferrin gene carrying a secretion signal peptide, a HAC1 gene, a Cne gene, and a Pdi1 gene.

[0053] The molecular chaperone proteins HAC1, Cne, and Pdi1 mentioned above are derived from Yersinia lipolytica.

[0054] Preferably, the amino acid sequences of the molecular chaperone proteins HAC1, Cne and Pdi1 are as shown in SEQ ID NO.1, 2 and 3 respectively.

[0055] In this invention, the human lactoferrin is preferably truncated human lactoferrin.

[0056] Preferably, the amino acid sequence of the human lactoferrin is shown as positions 20-710 of SEQ ID NO. 6.

[0057] The beneficial effects of this invention include at least the following: This invention provides a recombinant *Yersinia lipolytica* yeast that secretes and expresses human lactoferrin. In this recombinant *Yersinia lipolytica* yeast, the secretory expression of human lactoferrin is guided by a secretion signal peptide; simultaneously, the expression of molecular chaperone proteins HAC1, Cne, and Pdi1 is enhanced. This invention achieves, for the first time, the secretory expression of human lactoferrin in *Yersinia lipolytica* yeast. Through the synergistic effect of molecular chaperones, the folding and secretory expression of human lactoferrin are effectively promoted, significantly increasing the yield of human lactoferrin, and has important application value in the industrial-scale production of human lactoferrin. Attached Figure Description

[0058] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0059] Figure 1 This is the plasmid map of pYLSC3-Lip2pre3xLA_hLF in Example 1 of the present invention.

[0060] Figure 2 This is an SDS-PAGE image of the fermentation supernatant of recombinant Yersinia lipolytica in Example 1 of this invention. Lane 1 is the positive standard of human lactoferrin; lane 2 is the supernatant protein of the negative control Po1g-pYLSC3'; lanes 3, 5, 7, and 8 are the supernatant proteins of Po1g-hLFSP_hLF, Po1g-XPR2prepro_hLF, Po1g-Lip2prepro_hLF, and Po1g-Lip2pre3xLA_hLF, respectively; lanes 4 and 6 are control samples that did not perform well during the optimization process (the scheme was abandoned); the arrows indicate human lactoferrin.

[0061] Figure 3 The secretion levels of recombinant human lactoferrin using different secretion signal peptides in Example 1 of this invention are shown.

[0062] Figure 4 This is a Western blot image of the fermentation supernatant from the Po1g-Lip2pre3xLA_hLF strain after 5 days of shake-flask fermentation in Example 1 of this invention. The lanes from left to right represent the fermentation supernatant at hours 24, 48, 72, 96, and 120.

[0063] Figure 5 This is the plasmid map of pYLSC3-Lip2pre3xLA_hLF-HAC1sp-Cne-Pdi1 in Example 2 of the present invention.

[0064] Figure 6 This represents the secretion level of recombinant human lactoferrin after co-expression of different combinations of molecular chaperone proteins in Example 3 of the present invention.

[0065] Figure 7 This refers to the fermentation process data of the 1L fermenter in Embodiment 4 of the present invention. Detailed Implementation

[0066] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0067] Example 1: Construction of recombinant Yersinia lipophila expressing human lactoferrin

[0068] 1. Construct a plasmid containing the human lactoferrin gene carrying the secretion signal peptide.

[0069] The selected plasmid vector was pYLSC3', and it was constructed using the Gibson seamless cloning method. The pYLSC3' vector was double-digested with HindIII and KpnI, and the linearized vector fragment was obtained by gel recovery.

[0070] Taking Lip2pre3xLA as an example, partially complementary primers lip2pre3xLA-F and lip2pre3xLA-R were designed. After mixing the two primers in equal amounts, the mixture was denatured at 95°C for 30 seconds and annealed at 25°C for 30 seconds, causing the two single-stranded DNAs to partially complement each other to form a double-stranded DNA fragment with a 3' overhang. This fragment encodes the secretion signal peptide Lip2pre3xLA.

[0071] Using Beyotime's Seamless Cloning Kit, 50 ng of linearized vector, 10 ng of Lip2pre3xLA primer mixture, and 10 μL of 2× Seamless Cloning Mix were added. The recombinant volume was brought to 20 μL with ddH2O. After incubation at 50°C for 30 min, DH5α was chemically transformed. The next day, a single colony was sent for sequencing using SC3-seq-F primers. The plasmid pYLSC3-Lip2pre3xLA was constructed, with an XbaI restriction site added downstream of the Lip2pre3xLA secretory peptide.

[0072] The amino acid sequence of human lactoferrin was retrieved from Uniprot (Uniprot ID: P02788). Based on the codon preference of Yeast lipolyticis, the gene was codon optimized and chemically synthesized by Genewiz.

[0073] The pYLSC3-Lip2pre3xLA vector was double-digested with XbaI and KpnI, and the linearized vector fragment was obtained by gel recovery.

[0074] Primers lip2pre3xLA-hLF-F and lip2pre3xLA-hLF-R were designed, and the human lactoferrin gene with homologous arms was amplified by PCR using hLF as a template.

[0075] The PCR amplification system consisted of: 25 μL Phanta Flash Master Mix (Novizan), 1 μL template, 1 μL each of forward and reverse primers, and 22 μL ddH2O.

[0076] The PCR amplification program was as follows: 95℃ pre-denaturation for 60s, 95℃ denaturation for 15s, 55℃ annealing for 15s, 72℃ extension for 20s, for 28 cycles; 72℃ extension for 2min. A hLF gene fragment of approximately 2100bp was obtained.

[0077] Using Beyotime's seamless cloning kit, 50 ng of linearized vector, 100 ng of PCR purified product, and 10 μL of 2× Seamless Cloning Mix were added. The recombinant system was brought to a final volume of 20 μL with ddH2O. After incubation at 50°C for 30 min, DH5α was chemically transformed. The next day, single colonies were sent for sequencing using SC3-seq-F and SC3-seq-R primers. The constructed plasmid was pYLSC3-Lip2pre3xLA_hLF, which is a pYLSC3 plasmid carrying the human lactoferrin gene linked to the signal peptide Lip2pre3xLA. Figure 1 ).

[0078] This method can be used to construct expression plasmids carrying other secretion signal peptides of human lactoferrin genes: pYLSC3-hLFSP_hLF, pYLSC3-Lip2prepro_hLF, and pYLSC3-XPR2prepro_hLF.

[0079] 2. Construction of recombinant lipophilic Yersinia

[0080] Using the pYLSC3-Lip2pre3xLA_hLF plasmid as an example, a *Yersinia lipophilia* strain expressing human lactoferrin was constructed. The pYLSC3-Lip2pre3xLA_hLF plasmid was digested with NotI, and the linearized vector fragment was obtained by gel extraction.

[0081] With Po1g yeast ( Yarrowia lipolyticaUsing strain Po1g as the expression host, the strain was cultured in YPD medium (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose), with 20 g / L agar added when preparing the solid medium. The Po1g strain was streaked onto YPD solid medium and incubated at 30°C for 24 hours. Single colonies were picked and added to yeast transformation buffer (80 μL 50% PEG4000, 5 μL 2M lithium acetate, 5 μL boiled single-stranded salmon sperm DNA), along with 200 ng of the linearized vector fragment, and brought to a final volume of 100 μL with ddH2O. After vortexing and incubation at 39°C for 60 min, the bacterial culture was plated onto leucine-free selection plates CSM-Leu (20 g / L glucose, 6.7 g / L yeast nitrogen basal source, 5 g / L ammonium sulfate, 0.7 g / L CSM-Leu, 20 g / L agar). Incubate at 30°C for at least 2-3 days until visible colonies form.

[0082] The recombinant Yersinia lipolyticis strain Po1g-Lip2pre3xLA_hLF was obtained by selective screening using the LEU2 gene marker. This strain is an hLF containing the secretion signal peptide Lip2pre3xLA integrated into the Po1g genome.

[0083] This method can be used to construct recombinant Yersinia lipophila strains that integrate human lactoferrin genes carrying other secretion signal peptides: Po1g-hLFSP_hLF, Po1g-Lip2prepro_hLF, and Po1g-XPR2prepro_hLF.

[0084] The obtained recombinant Yersinia lipolytica strain was inoculated into CSM-Leu liquid medium (20 g / L glucose, 6.7 g / L yeast nitrogen basal source, 5 g / L ammonium sulfate, 0.7 g / L CSM-Leu), and cultured at 30°C for 24 hours. Then, the seed culture was inoculated into fermentation medium in shake flasks (10 g / L yeast extract, 20 g / L peptone, 40 g / L glucose), and the initial OD was determined. 600 After fermentation at 0.1 mg / L and 30°C for 5 days, the supernatant was collected by centrifugation, and the products cultured at the shake-flask level were detected by SDS-PAGE and HPLC. The secretion effects of each secretion signal peptide are as follows: Figure 2 and Figure 3 As shown in the figure. All seven validated secretion signal peptides can guide the secretion and expression of human lactoferrin, with Lip2pre3xLA showing the best effect, achieving a recombinant human lactoferrin secretion level of 67 mg / L. The results of Western blot analysis of the fermentation supernatant from Po1g-Lip2pre3xLA_hLF for 5 consecutive days are shown in the figure. Figure 4 As shown.

[0085] Example 2: Molecular chaperone-enhanced expression strategy to improve the secretory expression of human lactoferrin

[0086] This embodiment further enhances the expression of different molecular chaperone proteins and their different combinations to analyze their effects on the secretory expression of human lactoferrin. The main components include: constructing a recombinant plasmid containing a human lactoferrin gene carrying a signal peptide and a molecular chaperone protein gene, transforming it into yeast and integrating it into the yeast genome to achieve co-expression of human lactoferrin and molecular chaperones.

[0087] 1. Construct a recombinant plasmid containing a human lactoferrin gene with a linker signal peptide and a single molecular chaperone protein gene.

[0088] Using the plasmid pYLSC3-Lip2pre3xLA_hLF-Cne, which enhances the expression of human lactoferrin and the molecular chaperone Cne (amino acid sequence shown in SEQ ID NO.2), as an example, this paper illustrates how to construct a recombinant plasmid for co-expression of human lactoferrin and a molecular chaperone. pYL24 was used as the vector plasmid, and the pYL24 vector was double-digested with XbaI and KpnI. The linearized vector fragment was obtained by gel recovery.

[0089] Primer pairs yl24-Cne-F and yl24-Cne-R were designed, and the Po1g genome was used as a template to amplify the 1800bp calcium-linked protein gene (Cne) with homologous arms by PCR.

[0090] The PCR amplification system and amplification procedure are the same as described in Example 1.

[0091] Using Beyotime's Seamless Cloning Kit, 50 ng of linearized vector, 100 ng of PCR purified product, and 10 μL of 2× Seamless Cloning Mix were added. The recombinant system was then brought to a final volume of 20 μL with ddH2O. After incubation at 50°C for 30 min, the cells were chemically transformed into DH5α. The following day, single colonies were sent for sequencing using pYL24-seqFw and pYL24-seqRv primers. The constructed plasmid was pYL24-Cne.

[0092] This method can be used to construct expression plasmids for other molecular chaperone proteins: pYL24-Pdi1 (the amino acid sequence of Pdi1 is shown in SEQ ID NO.3), pYL24-Pdi2 (the amino acid sequence of Pdi2 is shown in SEQ ID NO.4), pYL24-Ero1 (the amino acid sequence of Ero1 is shown in SEQ ID NO.5), and pYL24-HAC1sp (HAC1sp is the spliced ​​form of the HAC1 gene with introns removed, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.1). The pYLSC3-Lip2pre3xLA_hLF plasmid was double-digested with NheI and SalI, and the linearized vector fragment was obtained by gel recovery.

[0093] Primer pairs SC3-yl24-sub-F and SC3-yl24-sub-R were designed, and pYL24-Cne was used as a template to amplify a gene fragment of about 3800 bp containing the expression cassette of the calcium-linked protein gene (Cne).

[0094] The PCR amplification system and amplification procedure are the same as described in Example 1.

[0095] Using Beyotime's Seamless Cloning Kit, 50 ng of linearized vector, 100 ng of PCR purified product, and 10 μL of 2× Seamless Cloning Mix were added. The recombinant system was then brought to a final volume of 20 μL with ddH2O. After incubation at 50°C for 30 min, DH5α was chemically transformed. The following day, single colonies were picked, and plasmids were extracted and verified by enzyme digestion. The constructed plasmid was pYLSC3-Lip2pre3xLA_hLF-Cne.

[0096] This method can be used to construct co-expression plasmids of human lactoferrin and other chaperone proteins: pYLSC3-Lip2pre3xLA_hLF-Pdi1, pYLSC3-Lip2pre3xLA_hLF-Pdi2, pYLSC3-Lip2pre3xLA_hLF-Ero1, and pYLSC3-Lip2pre3xLA_hLF-HAC1sp.

[0097] 2. Construct a recombinant plasmid containing a human lactoferrin gene carrying a secretion signal peptide and a molecular chaperone combination.

[0098] By constructing a recombinant plasmid containing the human lactoferrin gene carrying the secretion signal peptide and multiple molecular chaperone protein genes, and then transforming and integrating it into the yeast genome, co-expression of lactoferrin and multiple molecular chaperone combinations can be achieved.

[0099] Taking pYLSC3-Lip2pre3xLA_hLF-HAC1sp-Cne as an example, this paper illustrates how to construct a recombinant plasmid for co-expression of lactoferrin and multiple molecular chaperones. The pYLSC3-Lip2pre3xLA_hLF-HAC1sp vector was linearized by XbaI digestion, and the linearized vector fragment was obtained by gel extraction. Primer pairs SC3-yl24-sub-F2 and SC3-yl24-sub-R were designed, and using the pYL24-Cne genome as a template, PCR amplification was performed to obtain a gene fragment of approximately 3800 bp containing the expression cassette of the calcium-linking protein gene (Cne). The PCR amplification system and procedure are the same as described in Example 1.

[0100] Using Beyotime's Seamless Cloning Kit, 50 ng of the linearized vector pYLSC3-Lip2pre3xLA_hLF-HAC1sp, 100 ng of the purified PCR product containing the calcified protein gene (Cne) expression cassette, and 10 μL of 2×Seamless Cloning Mix were added. The recombinant system was then brought to a final volume of 20 μL with ddH2O. After incubation at 50°C for 30 min, the mixture was chemically transformed into DH5α. The following day, single colonies were picked, plasmids were extracted via shaking, and enzyme digestion was performed for verification. The constructed plasmid was pYLSC3-Lip2pre3xLA_hLF-HAC1sp-Cne. This plasmid contains the human lactoferrin gene carrying the signal peptide Lip2pre3xLA, as well as the HAC1sp and Cne genes. The human lactoferrin gene carrying the signal peptide Lip2pre3xLA is transcribed using php4d (SEQ ID NO.13) as the promoter and XPR2 terminator (SEQ ID NO.15) as the terminator. The HAC1sp gene is transcribed using pTDH1 (SEQ ID NO.14) as the promoter and MIG1 terminator (SEQ ID NO.16) as the terminator. The Cne gene is transcribed using pTDH1 as the promoter and MIG1 terminator as the terminator.

[0101] Following the above method, by inserting a gene fragment containing a Pdi1 expression cassette (containing a TDH1 promoter, the Pdi1 gene, and a MIG1 terminator) downstream of the Cne expression cassette, a recombinant plasmid pYLSC3-Lip2pre3xLA_hLF-HAC1sp-Cne-Pdi1, a combination of three molecular chaperone proteins, can be constructed. Figure 5 ).

[0102] This method allows for the construction of co-expression plasmids for human lactoferrin and other molecular chaperone combinations, including pYLSC3-Lip2pre3xLA_hLF-HAC1sp-Pdi1, pYLSC3-Lip2pre3xLA_hLF-HAC1sp-Pdi2, pYLSC3-Lip2pre3xLA_hLF-HAC1sp-Ero1, pYLSC3-Lip2pre3xLA_hLF-HAC1sp-Cne-Pdi2, and pYLSC3-Lip2pre3xLA_hLF-HAC1sp-Cne-Ero1. The promoters and terminators used for the expression of each molecular chaperone protein are both the TDH1 promoter and the MIG1 terminator.

[0103] 3. Construction of recombinant Yersinia lipolyticis

[0104] The plasmid was digested with NotI and the linearized vector fragment was obtained by gel recovery.

[0105] Use Yersinia lipolytica (Yersinia lipolytica) Yarrowia lipolytica Po1g was used as the expression host and cultured in YPD medium (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose), with 20 g / L agar added when preparing the solid medium. The Po1g strain was streaked onto YPD solid medium and incubated at 30°C for 24 hours. Single colonies were picked and transferred to yeast transformation buffer (80 μL 50% PEG4000, 5 μL 2M lithium acetate, 5 μL boiled single-stranded salmon sperm DNA), and 200 ng of the linearized vector fragment pYLSC3-Lip2pre3xLA_hLF-HAC1sp-Cne-Pdi1 was added. The volume was then brought to 100 μL with ddH2O. After shaking and mixing, the culture was incubated at 39°C for 60 min. The bacterial culture was then spread onto a leucine-free selection plate, CSM-Leu (20 g / L glucose, 6.7 g / L yeast nitrogen basal source, 5 g / L ammonium sulfate, 0.7 g / L CSM-Leu, 20 g / L agar). The culture was incubated at 30°C for at least 2-3 days until visible colonies formed. Selection was performed using the LEU2 gene marker to obtain the recombinant *Yarrowia lipolytica* strain Po1g-Lip2pre3xLA_hLF-HAC1sp-Cne-Pdi1, which secretes the signal peptide Lip2pre3xLA and co-expresses the molecular chaperones HAC1, Cne, and Pdi1.

[0106] This method can be used to construct recombinant Yersinia lipolyticis strains co-expressing human lactoferrin and other different molecular chaperones or combinations thereof, including Po1g-Lip2pre3xLA_hLF-Cne, Po1g-Lip2pre3xLA_hLF-Pdi1, Po1g-Lip2pre3xLA_hLF-Pdi2, Po1g-Lip2pre3xLA_hLF-Ero1, Po1g-Lip2pre3xLA_hLF-HAC1sp, Po1g-Lip2pre3xLA_hLF-HAC1sp-Cne, Po1g-Lip2pre3xLA_hLF-HAC1sp-Pdi1, Po1g-Lip2pre3xLA_hLF-HAC1sp-Pdi2, Po1g-Lip2pre3xLA_hLF-HAC1sp-Ero1, and Po1g-Lip2pre3xLA_hLF-HAC1sp-Cne. HAC1sp-Cne-Pdi2, Po1g-Lip2pre3xLA_hLF- HAC1sp-Cne-Ero1. This invention involved extensive screening of secretory signal peptides and molecular chaperones, as well as their different combinations. Due to the large number of strains involved, they are not listed individually here; only some of the strains involved in the screening process are listed.

[0107] In the above embodiments, the amino acid sequences of the secretion signal peptides Lip2pre3xLA, hLFSP, Lip2prepro, and XPR2prepro are shown in SEQ ID NO. 7, 8, 9, and 10, respectively. The amino acid sequences of the molecular chaperone proteins HAC1, Cne, Pdi1, Pdi2, and Ero1 are shown in SEQ ID NO. 1, 2, 3, 4, and 5, respectively. The human lactoferrin is a truncated human lactoferrin, and its amino acid sequence is shown in SEQ ID NO. 6, lines 20-710aa. A histidine purification tag (His-tag) is added to the C-terminal amino acid sequence of the human lactoferrin, and the amino acid sequence of the histidine purification tag (His-tag) is HHHHHH (SEQ ID NO. 12). The nucleotide sequence of the human lactoferrin gene carrying the recombinant secretion signal peptide Lip2pre3xLA is shown in SEQ ID NO. 11.

[0108] The primer sequences used in the above embodiments are shown in Table 1.

[0109] Table 1: Primer sequences used in this embodiment

[0110]

[0111] Example 3 Performance verification of recombinant Yersinia lipolyticis secretion expressing human lactoferrin

[0112] The recombinant *Yersinia lipolyticis* strains obtained in Examples 1 and 2 were inoculated into CSM-Leu liquid medium (20 g / L glucose, 6.7 g / L yeast nitrogen basal source, 5 g / L ammonium sulfate, 0.7 g / L CSM-Leu) and cultured at 30°C for 24 hours to obtain seed culture. The seed culture was then inoculated into shake flasks with fermentation medium (YPD medium, 10 g / L yeast extract, 20 g / L peptone, 40 g / L glucose) for shake flask fermentation, with the initial OD... 600 After fermentation at 0.1 g / L and 30°C for 5 days, the supernatant was collected by centrifugation, and the product was verified and detected by SDS-PAGE and HPLC.

[0113] The results showed that ( Figure 6 The human lactoferrin secreted by the Po1g-Lip2pre3xLA_hLF strain carrying the Lip2pre3xLA secretion signal peptide can reach 72 mg / L. Different chaperone proteins and their combinations have different degrees of promoting effect on the secretion of the target protein. Among them, the extracellular secretion of the Po1g-Lip2pre3xLA_hLF-HAC1sp strain can reach 115 mg / L at the shake flask level, the extracellular secretion of the Po1g-Lip2pre3xLA_hLF-Cne strain can reach 88 mg / L at the shake flask level, and the extracellular secretion of the Po1g-Lip2pre3xLA_hLF-Pdi1 strain can reach 74 mg / L at the shake flask level. Combining HAC1sp, Cne, and Pdi1 can significantly increase the secretion of recombinant human lactoferrin, with a significantly better effect than enhancing the expression of these three molecular chaperones alone. The extracellular secretion of the Po1g-Lip2pre3xLA_hLF-HAC1sp-Cne-Pdi1 strain can reach 188 mg / L at the shake flask level, which is significantly higher than the combination of other molecular chaperone proteins.

[0114] Example 4: Application of recombinant Yersinia lipolyticis in the fermentation production of human lactoferrin

[0115] The strain Po1g-Lip2pre3xLA_hLF-HAC1sp-Cne-Pdi1 was inoculated into CSM-Leu liquid medium and cultured at 30℃ for 24 hours to obtain seed culture. The seed culture was then inoculated into a 1L fermenter at a rate of 5%. The initial fermentation medium formulation is shown in Table 2, with carbon and nitrogen sources supplemented. The fermentation pH was maintained at 4.6-7.8, and fermentation lasted for 10 days at 30℃. After fermentation, the supernatant was collected by centrifugation, and the content of recombinant human lactoferrin in the fermentation broth was detected by SDS-PAGE and HPLC. The fermentation results are shown below. Figure 7 As shown, the secretion of recombinant human lactoferrin reached 1187.7 mg / L on day 10.

[0116] Table 2: Initial Fermentation Medium Formulation

[0117]

[0118] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant Yersinia lipolyticis yeast that secretes and expresses human lactoferrin, characterized in that, The recombinant Yersinia lipolytica contains a human lactoferrin gene carrying a secretion signal peptide; In the recombinant Yersinia lipolyticis, the expression of molecular chaperone proteins HAC1, Cne, and Pdi1 was enhanced; The molecular chaperone proteins HAC1, Cne, and Pdi1 are all derived from Yersinia lipolyticis; the amino acid sequences of the molecular chaperone proteins HAC1, Cne, and Pdi1 are shown in SEQ ID NO.1, 2, and 3, respectively. The amino acid sequence of the human lactoferrin is shown in positions 20-710 of SEQ ID NO. 6; The secretion signal peptide is a recombinant secretion signal peptide Lip2pre3xLA, and the amino acid sequence of Lip2pre3xLA is shown in SEQ ID NO.

7.

2. The recombinant Yersinia lipolyticis yeast expressing human lactoferrin according to claim 1, characterized in that, The recombinant Yersinia lipolyticis genome integrates at least one copy of the human lactoferrin gene carrying a secretion signal peptide.

3. The recombinant Yersinia lipophila expressing human lactoferrin according to claim 1, characterized in that, The expression of the molecular chaperone proteins HAC1, Cne, and Pdi1 was enhanced by increasing the copy number of the HAC1, Cne, and Pdi1 genes in the genome of Yersinia lipolytica.

4. The recombinant Yersinia lipolyticis yeast expressing human lactoferrin according to any one of claims 1 to 3, characterized in that, The recombinant Yeast lipolyticis genome integrates a lactoferrin secretion expression cassette; the lactoferrin secretion expression cassette includes a promoter, a human lactoferrin gene carrying a secretion signal peptide, and the HAC1, Cne, and Pdi1 genes.

5. The method for constructing recombinant Yersinia lipolyticis expressing human lactoferrin according to any one of claims 1 to 4, characterized in that, The method includes: introducing a human lactoferrin gene carrying a secretion signal peptide into Yersinia lipophila; and enhancing the expression of molecular chaperones HAC1, Cne, and Pdi1.

6. Any one of the following applications of the recombinant Yersinia lipolyticis expressing human lactoferrin as described in any one of claims 1 to 4: (1) Preparation of human lactoferrin; (2) Construct a production strain of human lactoferrin.

7. A method for preparing human lactoferrin, characterized in that, The method comprises: culturing recombinant Yersinia lipophila that secretes and expresses human lactoferrin as described in any one of claims 1 to 4, and isolating human lactoferrin from the culture.

8. A method for promoting the secretory expression of human lactoferrin in Yersinia lipolyticis, characterized in that, The method includes: guiding the secretory expression of human lactoferrin with a secretion signal peptide, while enhancing the expression of endogenous molecular chaperone proteins HAC1, Cne and Pdi1 in Yeastromyces lipolyticus; The Yersinia lipophila is as described in claim 1.

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