Lactoferrin expression strains and methods of use in saccharomyces marxianus

Abstract

The present application relates to the field of biotechnology, in particular to a lactoferrin expression strain of Kluyveromyces marxianus and application and method. The construction method of the lactoferrin expression strain of the present application comprises: replacing the coding gene of inulinase in the genome of the recipient strain of Kluyveromyces marxianus with the coding gene of lactoferrin, and replacing the Sga1 gene of glucose amylase in the genome of the recipient strain of Kluyveromyces marxianus with the coding gene expression frame of EIF4A protein, so as to promote the secretion and expression of lactoferrin and lactoferrin peptide in Kluyveromyces marxianus. The present application has important significance for improving the yield of Kluyveromyces marxianus in the fermentation production of lactoferrin and lactoferrin peptide.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to lactoferrin expression strains in Kluyveromyces martensii, their applications, and methods. Background Technology

[0002] Lactoferrin (LF) is an iron-binding glycoprotein belonging to the transferrin family. It contains approximately 703 amino acids and has a molecular weight of about 80 kDa, making it a single-chain protein. Its secondary structure is mainly composed of alternating α-helices and β-sheets, with α-helices significantly outnumbering β-sheets. Lactoferrin possesses various physiological activities, participating in the regulation of immune function, antibacterial activity, regulation of inflammatory responses, regulation of iron absorption, and promotion of intestinal cell proliferation and differentiation, among other physiological and pathological processes. It has wide applications in foods, health products, and daily chemical products for infants, pregnant women, and the elderly.

[0003] Traditionally, lactoferrin is primarily obtained through extraction from cow's milk. However, cow's milk contains very low levels of lactoferrin, only 0.03-0.49 g / L, leading to high extraction costs. Furthermore, consuming heterologous proteins derived from cow's milk may pose an antigenic risk, limiting its application in certain populations. To achieve large-scale production of lactoferrin while avoiding the potential risks associated with heterologous proteins, methods for producing human lactoferrin using genetic engineering techniques have been developed. Host cells for lactoferrin production mainly include *E. coli*, yeast, mammalian cells, and plant cells. *E. coli* expression systems lack glycosylation modification mechanisms, preventing the production of complete, biologically active lactoferrin. While mammalian and plant cells can undergo appropriate glycosylation modification, their long growth cycles and complex culture processes hinder large-scale production.

[0004] Yeast expression systems are characterized by rapid growth, ease of operation, and post-translational processing and modification capabilities, making yeast cells a significant advantage for heterologous expression of lactoferrin. However, the yeast cell secretion system has certain defects, preventing the protein from being smoothly secreted extracellularly, making it difficult to achieve efficient secretory expression of large human proteins in yeast. When heterologous proteins accumulate in large quantities intracellularly, they not only easily form aggregates and enter degradation pathways, but also put significant stress on the cellular secretion system, potentially leading to cell collapse and severely impacting protein yield. Summary of the Invention

[0005] To address the aforementioned technical problems and promote the correct folding of lactoferrin and lactoferrin peptides in Kluyveromyces martensii, thereby enhancing the efficient expression of lactoferrin and lactoferrin peptides, this invention provides lactoferrin expression strains in Kluyveromyces martensii, along with their applications and methods.

[0006] The technical solution of this invention is as follows: On one hand, the present invention provides a lactoferrin expression strain based on Kluyveromyces martensii. The method for constructing the lactoferrin expression strain includes: replacing the inulinase coding gene in the genome of the Kluyveromyces martensii recipient strain with the lactoferrin coding gene, and replacing the glucoamylase gene Sga1 in the genome of the Kluyveromyces martensii recipient strain with the EIF4A protein coding gene expression cassette, thereby promoting the secretory expression of lactoferrin and lactoferrin peptide in Kluyveromyces martensii.

[0007] Specifically, the amino acid sequence of the EIF4A protein is shown in SEQ ID NO.1.

[0008] SEQ ID NO.1: MSDGITDIDEGQIQSTYDKVVYSFDDLKLKEELLRGIFGYGFVEPSAIQQRAILPIIEEKDVLAQAQSGTGKTGTFAIAALQNIDEKVKAPQALILAPT RELALQIQKVVMALAIHMDVKVHACIGGTSLQEDSEALRGGAQIVVGTPGRVYDMIDRRIFKTDNIKMFILDEADEMLSTGFKEQIYNIFTMLPPTSQV VLLSATMPGDVLEVTTKFMKDPVRILVKKDELTLEGIGQYYVNVEEEQYKYDCLTDLYDSISVTQAVIFCNTRRKVEELTERLRENNFTVSAIYSDLQQQQRDTIMKEFRSGSSRILIATDLLARGIDIQQVSLVINYDLPSNKENYIHRIRGGGRFGRKGIAINFVTNKDIGAMRELERFYSTQIEELPSSINELFD.

[0009] Preferably, the nucleotide sequence of the gene encoding the EIF4A protein is shown in SEQ ID NO.12.

[0010] SEQ ID NO.12:

[0011] Specifically, in the gene expression frame of the EIF4A protein, the promoter that initiates the expression of the gene encoding the EIF4A protein is the endogenous Hsp26 promoter of Kluyveromyces martensii.

[0012] Preferably, the nucleotide sequence of the Hsp26 promoter is shown in SEQ ID NO.9.

[0013] SEQ ID NO.9: .

[0014] More preferably, the gene expression cassette encoding the EIF4A protein is composed of SEQ ID NO.9 (Hsp26 promoter) and SEQ ID NO.12 (the gene encoding the EIF4A protein) from the 5' end to the 3' end.

[0015] Specifically, the replacement of the Sga1 glucoamylase gene with the EIF4A protein encoding gene expression cassette in the genome of *Kluyveromyces martensii* recipient strain is achieved through homologous recombination.

[0016] Preferably, the nucleotide sequence of the upstream homologous arm used in the homologous recombination is shown in SEQ ID NO.10.

[0017] SEQ ID NO.10: gaaacgcgaagaggtagggagaaagacgactaacccaggggaaatagccaagaaattacacgtatattgccccccaagtaacgtgggaacgcgtgctgcttggtaggagactttttttttagcaggaaagagaagggcatccatctatcacaaacagcataatatagaatagtatagtatattcgcgaaaggttgattgatctattgatttattgcttggttaatacattttgatcgacaaagcgaacacaaaaacgctagccaggagactagatagatcaggttttttttttatttgcggggggttgatgaaaggtgtggggaagcagaaggatacatacacacacacacaagaatcttattgtgtcagtctgtctgtcattgatacagggaggtttgtcgcaaaaacggaaaaaataaataaataaaaaggtgaaaataaaaccaataaaaggcgtttgaagaagatctggtttgacttagattttgctgttgtctaaccaaaataatattttgttttgtctggggacccaggtatagtaacaacttggaaattctttttgtgacgtttttttcttttagtggtttatctttttgtgtgttaccgtttgagcacttacaggtaggtctgtctgttcccagcctttttttttttatttgatctgaaagagatagatatatacgcttgtgttgttggtaaaggcaatataagagagtgaaaagtttttaaagataaggac。

[0018] Preferably, the nucleotide sequence of the downstream homologous arm used in the homologous recombination is as shown in SEQ ID NO.11.

[0019] SEQ ID NO.11: .

[0020] In the method, the step of "replacing the glucoamylase gene Sga1 in the genome of *Kluyveromyces martensii* recipient strain with the gene encoding the EIF4A protein" means replacing the portion of the genome of *Kluyveromyces martensii* recipient strain located between SEQ ID NO. 10 and SEQ ID NO. 11 with the expression cassette of the gene encoding the EIF4A protein, while keeping everything else unchanged.

[0021] In the method, the replacement of the glucoamylase gene Sga1 in the genome of *Kluyveromyces martensii* recipient bacteria with the gene encoding the EIF4A protein can be achieved through homologous recombination.

[0022] In one embodiment of the present invention, the method is specifically achieved through CRISPR / Cas9 combined homologous recombination. Specifically, this involves introducing a recombinant vector and a homologous recombination fragment into the *Kluyveromyces martensii* recipient strain, which expresses the Cas9 protein and targets the glucosylamylase gene Sga1 in the *Kluyveromyces martensii* recipient genome. The homologous recombination fragment consists, from the 5' end to the 3' end, an upstream homologous arm, an expression cassette of the gene encoding the EIF4A protein, and a downstream homologous arm. Furthermore, in the recombinant vector, the coding sequence for the spacer region used to express the gRNA is TGAGGCTGGTATCGATAAGG. Further, the nucleotide sequence of the upstream homologous arm is shown in SEQ ID NO. 10; the nucleotide sequence of the downstream homologous arm is shown in SEQ ID NO. 11.

[0023] Specifically, the lactoferrin includes any one or more of human lactoferrin, bovine lactoferrin, feline lactoferrin, canine lactoferrin, and ovine lactoferrin; the lactoferrin peptides include any one or more of human lactoferrin peptides, bovine lactoferrin peptides, feline lactoferrin peptides, canine lactoferrin peptides, and ovine lactoferrin peptides.

[0024] Preferably, the amino acid sequence of the human lactoferrin is shown in SEQ ID NO.2: MRQVWFSWIVGLFLCFFNVSSAALGLCLAKRGRRRRSVQWCAVSQPEATKCFQWQRNMRKVRGPPVSCIKRDSPIQCIQAIAENRADAVTLDGGFIYEAGLAPYKLRPVAAEVYGTERQPRTHYYAVAVVKKGGSFQLNELQGLKSCHTGLRRTAGWNVPIGTLRPFLNWTGPPEPIEAAVARFFSASCVPGADKGQFPNLCRLCAGTGENKCAFSSQEPYFSYSGAFKCLRDGAGDVAFIRESTVFEDLSDEAERDEYELLCPDNTRKPVDKFKDCHLARVPSHAVVARSVNGKEDAIWNLLRQAQEKFGKDKSPKFQLFGSPSGQKDLLFKDSAIGFSRVPPRIDSGLYLGSGYFTAIQNLRKSEEEVAARRARVVWCAVGEQELRKCNQWSGLSEGSVTCSSASTTEDCIALVLKGEADAMSLDEGYVYTAGKCGLVPVLAENYKSQQSSDPDPNCVDRPVEGYLAVAVVRRSDTSLTWNSVKGKKSCHTAVDRTAGWNIPMGLLFNQTGSCKFDEYFSQSCAPGSDPRSNLCALCIGDEQGENKCVPNSNERYYGYTGAFRCLAENAGDVAFVKDVTVLQNTDGNNNEAWAKDLKLADFALLCLDGKRKPVTEARSCHLAMAPNHAVVSRMDKVERLKQVLLHQQAKFGRNGSDCPDKFCLFQSETKNLLFNDNTECLARLHGKTTYEKYLGPQYVAGITNLKKCSTSPLLEACEFLRK。

[0025] Preferably, the amino acid sequence of the bovine lactoferrin is as shown in SEQ ID NO.3: MRQVWFSWIVGLFLCFFNVSSAALGLCLAKRAPRKNVRWCTISQPEWFKCRRWQWRMKKLGAPSITCVRRAFALECIPGIAEKKADAVTLDGGMVFEAGRDPYKLRPVAAEIYGTKESPQTHYYAVAVVKKGSNFQLDQLQGRKSCHTGLGRSAGWIIPMGILRPYLSWTESLEPLQGAVAKFFSASCVPCIDRQAYPNLCQLCKGEGENQCACSSREPYFGYSGAFKCLQDGAGDVAFVKETTVFENLPEKADRDQYELLCLNNSRAPVDAFKECHLAQVPSHAVVARSVDGKEDLIWKLLSKAQEKSGKNKSRSFQLFGSPPGQRDLLFKDSALGFLRIPSKVDSALYLGSRYLTTLKNLRETAEEVKARYTRVVWCAVGPEEQKKCQQWSQQSGQNVTCATASTTDDCIVLVLKGEADALNLDGGYIYTAGKCGLVPVLAENRKSSKHSSLDCVLRPTEGYLAVAVVKKANEGLTWNSLKDKKSCHTAVDRTAGWNIPMGLIVNQTGSCAFDEFFSQSCAPGADPKSRLCALCAGDDQGLDKCVPNSKEKYYGYTGAFRCLAEDVGDVAFVKNDTVWENTNGESTADWAKNLNREDFRLLCLDGTRKPVTEAQSCHLAVAPNHAVVSRSDRAAHVKQVLLHQQALFGKNGKNCPDKFCLFKSETKNLLFNDNTECLAKLGGRPTYEEYLGTEYVTAIANLKKCSTSPLLEACAFLTR。

[0026] Preferably, the amino acid sequence of the feline lactoferrin is as shown in SEQ ID NO.4: MRQVWFSWIVGLFLCFFNVSSAALGLCLAKRTPGRRVRWCTISQAEETKCTKFQQNMREVGGPHVSCTRKNSRQECIQAIKANEADAVTLDGGLVFEAGLDPNKLRPVAAEVYGTQAEPQIHYYAVAIAKKGTNFQLNQLQGRKSCHTGLGRSAGWNIPMGLLRPFLNWAGPPEPLEKAAANFFSASCVPCADGKEYPNLCRLCAGTGENKCACSSREPYFGYAGAFKCLHEGGGDVAFVRHTTVFESLPDKADQDKYELLCLNNTRKPVDAFKECHLARIPSHAVVARSVDGKEDLIWELLNKAQEKFGKDKSPAFQLFGSPKDEKDLLFKDSALGFLRVPSKVDSGLYLGFNYLTAIQGLRETAAEVAERRARVAWCAVGREEERKCQQWSRASLGNVTCVSAPTGEDCIALVLKGEADALSLDGGLIYVAGKCGLVPVLAENQKPQIPNNERCEDRRVEGYLAVAVVRKSDADITWNSLRGRKSCHTAVDRTAGWNIPMGLLFSQTGSCKFGEFFSKSCAPGSDPNSNLCALCIGDERGENKCVPNSSERYFGYTGAFRCLAEKAGDVAFVKDSTVLQNTNGGSSEAWAKDLRLEDFELLCLDGTRKPVKEAERCHLARAPNHGVVSREDKAQHLEQVLLQQQTHFGTNGDKCPSEFCLFRSKTKNLLFNDNTECLAKLQGKTTYEEYLGSAYVTAVGNLRQCSTSPLLEACAFLRRSLIYSIRLFRKYVYGVPGVCPAPSWVNKVNINERGRRTS。

[0027] Preferably, the amino acid sequence of the dog lactoferrin is as shown in SEQ ID NO.5: MRQVWFSWIVGLFLCFFNVSSAALGLCLAKRAPRKNVRWCTTSKAEAKKCSKFQVNMKKVGGPIVSCTRKASRQECIQAIKANKADAVTLDGGLVFEAGLEPNKLRPIAAEVYGTQTKQQIHYYAVAIAKKGTNFQLNQLQGVRSCHTGLGRSAGWNIPIGTLRPFLNWTGPPEPLEEAVAKFFSASCVPCADGKQYPNLCRLCAGTEQNKCACSSQEPYFGYSGAFKCLQDGAGDVAFVRDSTVFENLPDKADQDKYELLCLNNTRKPVDAFKDCHLARVPSHAVVARSVGGKEDLIWRLLQKAQENFGKDKSSAFQLFGSPSGEKDLLFKDSAIGFLRIPSNIDSELYLGFNYINAIQSLKEKDSDSAERRARVAWCAVGEAEQRKCERWVRASGGSVSCVSAPSGEDCIALVLKGKADALSLDGGLIYVAGKCGLVPVLAENQKSQNPSDVDCVDRLAEGYLAVAVVRKSDADLTWNTLRGRKSCHTAVGRTAGWNIPMGLLFNQLGSCKFDEFFSQSCAPGADPKSSLCALCIGDEKGENKCAPNNSERYFGYTGAFRCLAEKAGDVAFVRDSTILQNTNGGNPEPWARDLKLEDFELLCLDGTRQPVTKARRCHLAMAPNHAVVSREEKAEHLKQVLLLQQTRFGRNGTKCPSEFCLFQSETKNLLFNDNTECLAKLQGKNTYEEYLGSAYVTAVANLRQCSASPLLEACAFLRR。

[0028] Preferably, the amino acid sequence of the sheep lactoferrin is as shown in SEQ ID NO.6: MRQVWFSWIVGLFLCFFNVSSAALGLCLAKRAPRKNVRWCAISLPEWSKCYQWQRRMRKLGAPSITCIRRTSALECIRAIAGKNADAVTLDSGMVFEAGLDPYKLRPVAAEIYGTEKSPQTHYYAVAVVKKGSNFQLDQLQGQKSCHMGLGRSAGWNIPVGILRPFLSWTESAEPLQGAVARFFSASCVPCVDGKAYPNLCQLCKGVGENKCACSSQEPYFGYSGAFKCLQDGAGDVAFVKETTVFENLPEKADRDQYELLCLNNTRAPVDAFKECHLAQVPSHAVVARSVDGKENLIWELLRKAQEKFGKNKSQSFQLFGSPEGRRDLLFKDSALGFVRIPSKVDSALYLGSRYLTALKNLRETAEELKARCTRVVWCAVGPEEQSKCQQWSEQSGQNVTCATASTTDDCIALVLKGEADALSLDGGYIYTAGKCGLVPVMAENRKSSKYSSLDCVLRPTEGYLAVAVVKKANEGLTWNSLKGKKSCHTAVDRTAGWNIPMGLIANQTGSCAFDEFFSQSCAPGADPKSSLCALCAGDDQGLDKCVPNSKEKYYGYTGAFRCLAEDVGDVAFVKNDTVWENTNGESSADWAKNLNREDFRLLCLDGTTKPVTEAQSCYLAVAPNHAVVSRSDRAAHVEQVLLHQQALFGKNGKNCPDQFCLFKSETKNLLFNDNTECLAKLGGRPTYEKYLGTEYVTAIANLKKCSTSPLLEACAFLTR。

[0029] Preferably, the nucleotide sequence of the encoding gene of the human lactoferrin is as shown in SEQ ID NO.13:

[0030] Preferably, the nucleotide sequence of the gene encoding bovine lactoferrin is shown in SEQ ID NO.14:

[0031] Preferably, the nucleotide sequence of the gene encoding feline lactoferrin is shown in SEQ ID NO.15:

[0032] Preferably, the nucleotide sequence of the gene encoding canine lactoferrin is shown in SEQ ID NO.16:

[0033] Preferably, the nucleotide sequence of the gene encoding sheep lactoferrin is shown in SEQ ID NO.17:

[0034] Specifically, the replacement of the inulinase coding gene in the genome of *Kluyveromyces martensii* recipient bacteria with the lactoferrin coding gene is achieved through homologous recombination.

[0035] Preferably, the nucleotide sequence of the upstream homologous arm used in the homologous recombination is shown in SEQ ID NO.7.

[0036] SEQ ID NO.7: .

[0037] Preferably, the nucleotide sequence of the downstream homologous arm used in the homologous recombination is shown in SEQ ID NO.8.

[0038] SEQ ID NO.8: .

[0039] In the method, the step of "replacing the inulinase coding gene in the genome of *Kluyveromyces martensii* recipient strain with the lactoferrin coding gene" specifically means replacing the portion of the genome of *Kluyveromyces martensii* recipient strain located between SEQ ID NO. 7 and SEQ ID NO. 8 with the lactoferrin coding gene, while keeping the rest unchanged.

[0040] In this method, knocking out and replacing the inulinase gene is to enable lactoferrin to mimic the constitutive expression pattern of the inulinase gene in Max Kluwer, so as to effectively utilize the inulinase protein to express the secretion pathway in the original expression system, thereby increasing the expression and secretion.

[0041] In the method, the step of "replacing the inulinase-coding gene in the genome of *Kluyveromyces martensii* recipient bacteria with the lactoferrin-coding gene" can be achieved through homologous recombination.

[0042] In one embodiment of the present invention, the method is specifically achieved through CRISPR / Cas9 combined homologous recombination. Specifically, this involves introducing a recombinant vector and a homologous recombination fragment into the *Kluyveromyces martensii* recipient strain, which expresses the Cas9 protein and a gRNA encoding the inulinase gene targeting the *Kluyveromyces martensii* recipient genome. The homologous recombination fragment consists, from the 5' end to the 3' end, an upstream homologous arm, the lactoferrin encoding gene, and a downstream homologous arm. Furthermore, in the recombinant vector, the coding sequence for the spacer region used to express the gRNA is GAATCCAACTTCACCCACCA. Further, the nucleotide sequence of the upstream homologous arm is shown in SEQ ID NO. 7; the nucleotide sequence of the downstream homologous arm is shown in SEQ ID NO. 8.

[0043] Specifically, the Kluyveromyces recipient strain is Kluyveromyces masculinum with the Ura3 gene missing.

[0044] Preferably, the recipient strain of *Kluyveromyces martensii* is *Kluyveromyces martensii* (…). Kluyveromyces marxianus The CS-01 was obtained by knocking out the Ura3 gene in the genome; the Kluyveromyces Marcius CS-01 has the accession number CGMCC No.30573 and was deposited at the China General Microbiological Culture Collection Center on May 9, 2024. The Kluyveromyces Marcius CS-01 has been disclosed in patent CN120424972A.

[0045] Preferably, the GenBank address of the Ura3 gene is KX453285.1.

[0046] Preferably, the 18S rDNA sequence of the Kluyveromyces Marcius CS-01 is shown in SEQ ID NO.19.

[0047] SEQ ID NO.19: .

[0048] On the other hand, the present invention provides a method for constructing the lactoferrin expression strain described in any of the above claims.

[0049] In another aspect, the present invention provides the application of the lactoferrin expression strain described in any of the above claims in the fermentation production of lactoferrin and lactoferrin peptides.

[0050] In another aspect, the present invention provides a method for fermenting to produce lactoferrin and lactoferrin peptides, the method comprising using the lactoferrin expression strain described in any of the preceding claims.

[0051] Specifically, the method includes inoculating a lactoferrin-expressing strain into a culture medium for fermentation.

[0052] The beneficial effects of this invention are as follows: This invention expresses lactoferrin intracellularly using constitutive expression sites in *Kluyveromyces martensii* and verifies the effect of the EIF4A protein in the genome on the expression levels of lactoferrin and lactoferrin peptides. Through shake-flask fermentation, the protein in the supernatant was obtained for verification, revealing that EIF4A protein promotes the expression levels of lactoferrin and lactoferrin peptides. This invention not only achieves the goal of increasing the yield of lactoferrin and lactoferrin peptides produced by fermentation in *Kluyveromyces martensii*, but also provides a method for increasing the secretory expression levels of lactoferrin and lactoferrin peptides in *Kluyveromyces martensii*. Attached Figure Description

[0053] Figure 1 For Max Kluyveromyces ( Kluyveromyces marxianus Microscopic examination (40x) results of CS-01.

[0054] Figure 2 The results of colony PCR verification for the selected lactoferrin clones are as follows: Nos. 1-3 are human lactoferrin strain KM cs-01-hLF (ura3-), with an amplification product of 2317bp; Nos. 5-6 are bovine lactoferrin strain KM cs-01-bLF (ura3-), with an amplification product of 2308bp; No. 7 is feline lactoferrin strain KM cs-01-cLF (ura3-), with an amplification product of 2425bp; Nos. 11 and 12 are canine lactoferrin strain KM cs-01-dLF (ura3-), with an amplification product of 2317bp.

[0055] Figure 3 To screen for EIF4A protein clones, colony PCR was used to verify the results. The target band size of the positive transformants was 3350 bp. Transformants 1-2 were human lactoferrin strains overexpressing EIF4A, and the resulting positive transformants were named KM cs-01-hLF-EIF4A (ura3-). Transformants 4-6 were bovine lactoferrin strains overexpressing EIF4A, and the resulting positive transformants were named KMcs-01-bLF-EIF4A (ura3-). Transformants 7-9 were feline lactoferrin strains overexpressing EIF4A, and the resulting positive transformants were named KM cs-01-cLF-EIF4A (ura3-). Transformants 10-12 were canine lactoferrin strains overexpressing EIF4A, and the resulting positive transformants were named KM cs-01-dLF-EIF4A (ura3-).

[0056] Figure 4This is an SDS-PAGE plot showing the expression levels of human lactoferrin and bovine lactoferrin. The leftmost sample is the bovine lactoferrin positive specimen. Sample 1 is a human lactoferrin clone strain KM cs-01-hLF(ura3-), sample 2 is a bovine lactoferrin clone strain KM cs-01-bLF(ura3-), sample 3 is a human lactoferrin clone strain KMcs-01-cLF-EIF4A(ura3-) overexpressing EIF4A protein, and sample 4 is a bovine lactoferrin clone strain KM cs-01-bLF-EIF4A(ura3-) overexpressing EIF4A protein. Arrow 1 represents the intact lactoferrin band, and arrow 2 represents the lactoferrin peptide.

[0057] Figure 5 SDS-PAGE plots showing the expression levels of feline and canine lactoferrin; the leftmost sample is the positive standard for bovine lactoferrin, samples 5 and 6 are feline lactoferrin strain KM cs-01-cLF(ura3-), sample 7 is canine lactoferrin strain KM cs-01-dLF(ura3-), samples 1 and 2 are feline lactoferrin strain KM cs-01-cLF-EIF4A(ura3-) overexpressing EIF4A protein, and samples 3 and 4 are canine lactoferrin strain KM cs-01-dLF-EIF4A(ura3-) overexpressing EIF4A protein. Arrow 2 indicates lactoferrin peptide. Detailed Implementation

[0058] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0059] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0060] The quantitative data involved in the following examples are all results obtained from more than three repeated experiments.

[0061] Example 1: Kluyveromycin ( Kluyveromyces marxianus Separation and identification of CS-01 1. Strains Isolation The Kluyveromyces marxianus CS-01 strain was isolated from yak yogurt samples from Madoi County, Qinghai Province in August 2022.

[0062] Take 1g of yogurt sample, dilute it with 50mL of sterile water, then dilute it 10-10000 times with sterile water, take 100μL of the diluted solution and spread it on yeast solid culture medium YNB (amino-free yeast nitrogen source) plate, incubate at 30℃ for 3-4 days, and pick single colonies.

[0063] 2. Strain identification The bacterial strain was identified using the 18S rDNA method. Using the genome of strain CS-01 as a template, PCR amplification and sequencing were performed using the universal 18S rDNA primers ITS1 and ITS4. The resulting 18S rDNA sequence is shown in SEQ ID NO.19. After comparison with known sequences in GenBank, it was found that the 18S rDNA sequence of CS-01 (SEQ ID NO.19) is consistent with... Kluyveromyces marxianus strain XJ-50 and Kluyveromyces marxianus isolate The sequence similarity of ilia21 was 99.71%, therefore strain CS-01 was identified as *Kluyveromyces martensii*. Kluyveromyces marxianus ).

[0064] ITS1 (SEQ ID NO. 21):TCCGTAGGTGAACCTGCG; ITS4 (SEQ ID NO. 22):TCCTCCGCTTATTTATGG.

[0065] Cell morphological observation revealed that the colonies of strain CS-01 were round, milky white, with neat edges. Microscopic observation showed that the yeast cells were oval, approximately 4-8 μm in size, and reproduced by budding, with single, two, or multiple lines adhering together. Figure 1 ), which is consistent with the cell morphology of Max Kluyveromyces.

[0066] Based on the above identification, strain CS-01 was identified as *Kluyveromyces martensii* (Kluyveromyces). Kluyveromyces marxianus This strain was deposited at the China General Microbiological Culture Collection Center on May 9, 2024, with the registration number CGMCC No. 30573. The Kluyveromyces Marcius CS-01 has been disclosed in patent CN120424972A.

[0067] Example 2: Amplification of donor DNA containing the lactoferrin encoding gene The inulinase site in the *Kluyveromyces martensii* genome was selected to express the encoding genes for human lactoferrin (SEQ ID NO. 13), bovine lactoferrin (SEQ ID NO. 14), feline lactoferrin (SEQ ID NO. 15), and canine lactoferrin (SEQ ID NO. 16), and the inulinase gene in the genome was completely replaced. The inulinase site is a constitutive expression site, and it is not affected by carbon source or temporal and spatial factors during fermentation, which is beneficial for improving the transcription level of LF.

[0068] Using primers Up-R and Up-F respectively, the *Kluyveromyces martensii* obtained in Example 1 (… Kluyveromyces marxianus Using the CS-01 genome as a template, PCR amplification was performed to obtain the upstream homologous arm SEQ ID NO.7 of the lactoferrin expression cassette containing the knock-in inulinase site.

[0069] Using primers LF-F and hLF-R, a fragment containing the encoding gene of human lactoferrin (SEQ ID NO. 13) was amplified using the recombinant plasmid pUC57-hLF as a template. The recombinant plasmid pUC57-hLF was obtained by inserting the DNA fragment shown in "SEQ ID NO. 13" into the EcoRV site of the pUC57 plasmid.

[0070] Using primers LF-F and bLF-R, a fragment containing the gene encoding bovine lactoferrin (SEQ ID NO. 14) was amplified using the recombinant plasmid pUC57-bLF as a template. The recombinant plasmid pUC57-bLF was obtained by inserting the DNA fragment shown in SEQ ID NO. 14 into the EcoRV site of the pUC57 plasmid.

[0071] Using primers LF-F and cLF-R, a fragment containing the gene encoding feline lactoferrin (SEQ ID NO. 15) was amplified using the recombinant plasmid pUC57-cLF as a template. The recombinant plasmid pUC57-cLF was obtained by inserting the DNA fragment shown in "SEQ ID NO. 15" into the EcoRV site of the pUC57 plasmid.

[0072] Using primers LF-F and dLF-R, a fragment containing the gene encoding canine lactoferrin (SEQ ID NO. 16) was amplified using the recombinant plasmid pUC57-dLF as a template. The recombinant plasmid pUC57-dLF was obtained by inserting the DNA fragment shown in "SEQ ID NO. 16" into the EcoRV site of the pUC57 plasmid.

[0073] Using primers Down-Fh, Down-Fb, Down-Fc, Down-Fd, and Down-R, respectively, the genome of Kluyveromyces marxianus CS-01 obtained in Example 1 was used as a template for PCR amplification to obtain a fragment containing the downstream homologous arm sequence (SEQ ID NO.8) of the lactoferrin expression cassette containing the inulinase site knocked in.

[0074] Furthermore, using the amplification products obtained above as templates, PCR amplification was performed using primers Up-F and Down-R to obtain Overlap PCR product 1 to Overlap PCR product 4, respectively. These products are donor DNA fragments containing lactoferrin-encoding genes (human lactoferrin-encoding gene, bovine lactoferrin-encoding gene, feline lactoferrin-encoding gene, or canine lactoferrin-encoding gene).

[0075] Overlap PCR product 1: From the 5' end to the 3' end, it contains the Up homologous arm of the inulinase site (SEQ ID NO. 7), the gene encoding human lactoferrin (SEQ ID NO. 13), and the Down homologous arm of the knock-in inulinase site (SEQ ID NO. 8). Overlap PCR product 2: From the 5' end to the 3' end, it contains the Up homologous arm of the inulinase site (SEQ ID NO. 7), the gene encoding bovine lactoferrin (SEQ ID NO. 14), and the Down homologous arm of the knock-in inulinase site (SEQ ID NO. 8). Overlap PCR product 3: From the 5' end to the 3' end, it contains the Up homologous arm of the inulinase site (SEQ ID NO. 7), the gene encoding feline lactoferrin (SEQ ID NO. 15), and the Down homologous arm of the knock-in inulinase site (SEQ ID NO. 8). Overlap PCR product 3: From the 5' end to the 3' end, it contains the Up homologous arm of the inulinase site (SEQ ID NO. 7), the gene encoding canine lactoferrin (SEQ ID NO. 16), and the Down homologous arm of the knock-in inulinase site (SEQ ID NO. 8).

[0076] The primers used are shown in Table 1: Table 1

[0077] Example 3 Construction of inulinase site gene editing plasmid First, circular PCR was used to reverse-pair the pK-Cas9 plasmid (which contains a prokaryotic origin of replication, an antibiotic resistance marker gene, a Cas9 gene expression cassette, a gRNA gene expression cassette, and an autonomous replication sequence; the Cas9 gene expression cassette, from upstream to downstream, includes the Cas9 gene promoter, the Cas9 gene, and a terminator; the gRNA gene expression cassette, from upstream to downstream, includes the RNA polⅢ promoter, tRNA, and tRNA). Leu The sequence was amplified using guide sequences, scaffold RNA sequences, and polyT sequences, and large fragments were recovered.

[0078] Then, the *Max Kluyveromyces* obtained in Example 1 (… Kluyveromyces marxianus The inulinase editing site was found in the CS-01 genome. Based on the N20 sequence design requirements, the 20 bases preceding the NGG target sequence were located within the inulinase gene. Circular PCR primers were designed using N20 as the homologous arm, as shown in Table 2. Amplification was performed using pK-Cas9 as a template. The PCR reaction mixture consisted of: 25 μL of 2×KOD Buffer, 1.5 μL each of primers N20-F and N20-R (10 μmol / L), 1 μL of DNA template (90 ng / μL), 10 μL of dNTPs (2 mM each), 1 μL of KOD polymerase (TOYOBO, 1 U / μL), and water to a final volume of 50 μL. The product was recovered and transformed into DH5α competent cells, then plated onto ampicillin LB agar plates. Verification was performed the following day using primers N20YZ-F and N20YZ-R. A PCR product of 1000 bp was considered a successful plasmid construction; no product indicated construction failure. The primer sequences mentioned above are shown in Table 2.

[0079] Table 2

[0080] The final gene-edited plasmid, named pK-Cas9-N20, was obtained by replacing the original N20 fragment of the pK-Cas9 plasmid with GAATCCAACTTCACCCACCA. The full sequence of the pK-Cas9-N20 plasmid is shown in SEQ ID NO. 18, where the N20 sequence at positions 9350-9369 is GAATCCAACTTCACCCACCA.

[0081] SEQ ID NO.18:

[0082] Example 4: Transformation of inulinase site gene editing plasmid pK-Cas9-N20 and donor DNA The transformation recipient was *Kluyveromyces martensii* KM cs-01 (ura3-). *Kluyveromyces martensii* KM cs-01 (ura3-) was obtained by knocking out the Ura3 gene (GenBank: KX453285.1) in the genome of *Kluyveromyces martensii* CS-01 (CGMCC No. 30573) obtained in Example 1 using CRISPR technology. The Ura3 gene was knocked out because production requirements prohibit the introduction of resistance selection markers.

[0083] A single colony of *Kluyveromyces martensii* KM cs-01 (ura3-) was picked and cultured overnight at 30°C and 200 rpm in 5 mL YPD medium (formulation: 20 g / L tryptone, 10 g / L yeast extract, 10 g / L glucose). The overnight culture was then inoculated into 50 mL YPD medium at a 1% (v / v) inoculum and cultured at 30°C and 200 rpm for expansion.

[0084] When the OD600 of the yeast culture to be expanded reaches 1.3-1.5, the culture is placed in a 50mL centrifuge tube, incubated at 5000g, 4℃, for 5min, and the supernatant is discarded. The precipitated cells are washed twice with 50mL of sterile ddH2O water. 20mL of pretreatment solution (formulation: 100mM lithium acetate, 10mM DTT, 0.6M sorbitol, 10mM Tris-HCl, pH 7.5) is added, and the culture is incubated at 30℃, 100rpm for 30min. The supernatant is then discarded by centrifugation. The cells are washed twice with 20mL of sorbitol (1mol / L). Finally, the cells are resuspended in 200μL of sorbitol (1mol / L) to obtain competent yeast cells.

[0085] Mix 80 μL of competent yeast cells with 500 ng of pK-Cas9-N20 plasmid prepared in Example 3. Then add 1 μg of donor DNA containing the human lactoferrin-encoding gene (Overlap PCR product 1), bovine lactoferrin-encoding gene (Overlap PCR product 2), feline lactoferrin-encoding gene (Overlap PCR product 3), or canine lactoferrin-encoding gene (Overlap PCR product 4) prepared in Example 2, mix well, and add to a pre-cooled 2 mm electroporation cuvette. After incubating on ice for 5 min, perform electroporation (2.5 kV, 5 ms, 25 μF). Immediately after electroporation, add 1 mL of sorbitol and 1 mL of YPD, and incubate at 30 °C for 2 h. Finally, take 200 μL and plate it on a selection plate (formulation: Minimal SD Base 26.7 g / L, uracil 100 μg / mL, 5-FOA 1 mg / mL, zeocin). Incubate at 30°C for 2-3 days using 100 μg / mL primers. After transformants grow on the plates, perform PCR and phenotypic verification. The verification primers are shown in Table 3. Table 3

[0086] The amplification product of human lactoferrin was 2317 bp, that of bovine lactoferrin was 2308 bp, that of feline lactoferrin was 2425 bp, and that of canine lactoferrin was 2308 bp. The resulting positive transformant was named KM cs-01-hLF(ura3-). Figure 2 (No. 1-3 in the middle), KM cs-01-bLF (ura3-) ( Figure 2 (No. 5-6 in the middle), KM cs-01-cLF (ura3-) ( Figure 2 (No. 7 in the middle), KM cs-01-dLF (ura3-) ( Figure 2(Numbers 11-12 in the list). The KM cs-01-hLF(ura3-) strain is obtained by replacing the inulinase-encoding gene in the KM cs-01(ura3-) genome of *Kluyveromyces martensii* with the human lactoferrin-encoding gene; the KM cs-01-bLF(ura3-) strain is obtained by replacing the inulinase-encoding gene in the KM cs-01(ura3-) genome of *Kluyveromyces martensii* with the bovine lactoferrin-encoding gene; the KM cs-01-cLF(ura3-) strain is obtained by replacing the inulinase-encoding gene in the KM cs-01(ura3-) genome of *Kluyveromyces martensii* with the feline lactoferrin-encoding gene; and the KM cs-01-dLF(ura3-) strain is obtained by replacing the inulinase-encoding gene in the KM cs-01(ura3-) genome of *Kluyveromyces martensii* with the canine lactoferrin-encoding gene. In other words, the original inulinase-encoding gene was knocked out at the inulinase site, and a mammalian lactoferrin-encoding gene was knocked in at that site.

[0087] For the amount of competent yeast cells, plasmid, and donor DNA, refer to the general yeast electroporation transformation procedure. The total volume of pK-Cas9-N20 plasmid and donor DNA should not exceed 1 / 10 of the volume of competent cells. For electroporation and recovery conditions, refer to the general yeast electroporation and recovery procedure.

[0088] Example 5: Amplification of donor DNA containing the EIF4A expression cassette The EIF4A gene was knocked into the genome of *Kluyveromyces martensii*, completely replacing the glucosidase gene Sga1 (GenBank: CP015060.1, positions 304353-306850). This example will construct donor DNA containing the EIF4A expression cassette, as detailed below: Using primers EIF4A-1 and EIF4A-2, the *Kluyveromyces martensii* obtained in Example 1 (… Kluyveromyces marxianus The genome of CS-01 was used as a template for PCR amplification to obtain a fragment containing the Up homologous arm (SEQ ID NO.10) with a knock-in glucosylamylase site.

[0089] Using primers EIF4A-3 and EIF4A-4, the *Kluyveromyces martensii* obtained in Example 1 (… Kluyveromyces marxianus The genome of CS-01 was used as a template to amplify a fragment containing the Hsp26 promoter (SEQ ID NO. 9).

[0090] Using primers EIF4A-5 and EIF4A-6, the *Kluyveromyces martensii* obtained in Example 1 (… Kluyveromyces marxianusThe genome of CS-01 was used as a template to amplify a fragment containing the EIF4A gene (SEQ ID NO. 12).

[0091] Using primers EIF4A-7 and EIF4A-8, the *Kluyveromyces martensii* obtained in Example 1 (… Kluyveromyces marxianus The genome of CS-01 was used as a template for PCR amplification to obtain a fragment containing a Down homologous arm (SEQ ID NO.11) with a knock-in glucosylamylase site.

[0092] Furthermore, using the four amplification products obtained above as templates, PCR amplification was performed using primers EIF4A-1 and EIF4A-8 to obtain the overlap PCR product Up-Hsp26-EIF4A-Down. This product is the donor DNA fragment containing the EIF4A expression cassette, consisting, from the 5' end to the 3' end, of the Up homologous arm of the glucosylase site (SEQ ID NO.10), the Hsp26 promoter (SEQ ID NO.9), the EIF4A gene (SEQ ID NO.12), and the Down homologous arm of the knock-in glucosylase site (SEQ ID NO.11). The primers used are shown in Table 4: Table 4

[0093] Example 6 Construction of a gene-editing plasmid containing the SGA1 glucoamylase site First, circular PCR was used to reverse-pair the pK-Cas9 plasmid (this plasmid contains a prokaryotic origin of replication, an antibiotic resistance marker gene, a Cas9 gene expression cassette, a gRNA gene expression cassette, and an autonomous replication sequence; the Cas9 gene expression cassette, from upstream to downstream, includes the Cas9 gene promoter, the Cas9 gene, and a terminator; the gRNA gene expression cassette, from upstream to downstream, includes the RNA polⅢ promoter, tRNA, and tRNA). Leu The sequence was amplified using guide sequences, scaffold RNA sequences, and polyT sequences, and large fragments were recovered.

[0094] The glucoamylase editing site was then located in the genome of *Kluyveromyces martensii* KM cs-01 (ura3-). Following the N20 sequence design requirements, the 20 bases preceding the NGG target sequence within the glucoamylase gene were identified. Circular PCR primers were designed using N20 as a homologous arm, as shown in Table 5. Amplification was performed using pK-Cas9 as a template. The PCR reaction mixture consisted of: 25 μL of 2×KOD Buffer, 1.5 μL each of primers N20-F and N20-R (10 μmol / L), 1 μL of DNA template (90 ng / μL), 10 μL of dNTPs (2 mM each), 1 μL of KOD polymerase (TOYOBO, 1 U / µL), and water added to a final volume of 50 μL. The recovered product was transformed into DH5α competent cells and plated onto ampicillin LB agar plates. The results will be verified the following day. Primers used are N20YZ-F and N20YZ-R. If a PCR product of 1000 bp is produced, plasmid construction is successful; otherwise, construction fails. The specific primer sequences involved are shown in Table 5.

[0095] Table 5

[0096] The final gene-edited plasmid, named pK-Cas9-SGA1, was obtained by replacing the original N20 fragment of the pK-Cas9 plasmid with TGAGGCTGGTATCGATAAGG. The full sequence of the pK-Cas9-SGA1 plasmid is shown in SEQ ID NO. 20, where the N20 sequence at positions 9350-9369 is TGAGGCTGGTATCGATAAGG. SEQ ID NO. 20:

[0097] Example 7: Transformation of glucosylamylase site editing plasmid pK-Cas9-SGA1 and donor DNA Single colonies of *Kluyveromyces martensii* KM cs-01-hLF(ura3-), KM cs-01-hLF(ura3-), KM cs-01-cLF(ura3-), and KM cs-01-dLF(ura3-) constructed in Example 4 were respectively inoculated into 5 mL of YPD medium (formulation: 20 g / L tryptone, 10 g / L yeast extract, 10 g / L glucose) and cultured overnight at 30°C and 200 rpm. The overnight culture was then inoculated into 50 mL of YPD medium at a 1% volume percentage and cultured at 30°C and 200 rpm for expansion.

[0098] When the OD600 of the yeast culture to be expanded reaches 1.3-1.5, the culture is placed in a 50mL centrifuge tube, centrifuged at 5000g, 4℃ for 5min, and the supernatant is discarded. The precipitated cells are washed twice with 50mL of sterile ddH2O water. 20mL of pretreatment solution (formulation: 100mM lithium acetate, 10mM DTT, 0.6M sorbitol, 10mM Tris-HCl, pH 7.5) is added, and the culture is carried out at 30℃, 100rpm for 30min. The supernatant is then discarded by centrifugation. The cells are washed twice with 20mL of sorbitol (1mol / L). Finally, the cells are resuspended in 200μL (1mol / L) of sorbitol to obtain KM cs-01-hLF(ura3-), KM cs-01-hLF(ura3-), KM cs-01-cLF(ura3-), or KM cs-01-dLF(ura3-) yeast competent cells.

[0099] 80 μL of competent yeast cells were mixed with 500 ng of pK-Cas9-SGA1 plasmid prepared in Example 6 and 1 μg of donor DNA containing the EIF4A expression cassette prepared in Example 5. The mixture was added to a pre-cooled 2 mm electroporation cuvette, incubated on ice for 5 min, and then electroporated (2.5 kV, 5 ms, 25 μF). Immediately after electroporation, 1 mL of sorbitol and 1 mL of YPD were added, and the mixture was incubated at 30 °C for 2 h. Finally, 200 μL of the mixture was plated on a selection plate (formulation: Minimal SD Base 26.7 g / L, uracil 100 μg / mL, 5-FOA 1 mg / mL, zeocin 100 μg / mL) and incubated at 30 °C for 2-3 days. After transformants appeared on the plate, PCR and phenotypic verification were performed. EIF4A-1 and EIF4A-8 were used as verification primers (sequences are shown in Table 4). The target band size of the positive transformant was 3350 bp. Figure 3 ).

[0100] For the amount of competent yeast cells, plasmid, and donor DNA, refer to the general yeast electroporation transformation procedure. The total volume of pK-Cas9-SGA1 plasmid and donor DNA should not exceed 1 / 10 of the competent cell volume. For electroporation and recovery conditions, refer to the general yeast electroporation and recovery procedure.

[0101] The resulting positive transformant was named KM cs-01-hLF-EIF4A(ura3-) Figure 3 (No. 1-2), KM cs-01-bLF-EIF4A (ura3-) Figure 3 (No. 4-6), KM cs-01-cLF-EIF4A (ura3-) Figure 3 (No. 7-9), KM cs-01-dLF-EIF4A (ura3-) Figure 3 (Nos. 10-12). The KM cs-01-hLF-EIF4A(ura3-) strain is obtained by replacing the Sga1 glucosylase gene in the genome of *Kluyveromyces martensii* KM cs-01-hLF(ura3-) with a DNA fragment consisting of SEQ ID NO. 9 (Hsp promoter) and SEQ ID NO. 12 (encoding gene for EIF4A protein) from the 5' to 3' end, while keeping other sequences unchanged. The KM cs-01-bLF-EIF4A(ura3-) strain is obtained by replacing the Sga1 glucosylase gene in the genome of *Kluyveromyces martensii* KM cs-01-bLF(ura3-) with a DNA fragment consisting of SEQ ID NO. 9 (Hsp promoter) and SEQ ID NO. 12 (encoding gene for EIF4A protein) from the 5' to 3' end, while keeping other sequences unchanged. The KM cs-01-cLF-EIF4A(ura3-) strain is obtained by replacing the Sga1 glucosylase gene in the genome of *Kluyveromyces martensii* KM cs-01-cLF(ura3-) with a DNA fragment consisting of SEQ ID NO.9 (Hsp promoter) and SEQ ID NO.12 (encoding gene for EIF4A protein) from the 5' to 3' end, while keeping other sequences unchanged. The KM cs-01-dLF-EIF4A(ura3-) strain is obtained by replacing the Sga1 glucosylase gene in the genome of *Kluyveromyces martensii* KM cs-01-dLF(ura3-) with a DNA fragment consisting of SEQ ID NO.9 (Hsp promoter) and SEQ ID NO.12 (encoding gene for EIF4A protein) from the 5' to 3' end, while keeping other sequences unchanged.

[0102] Example 8: Detection of translational levels of lactoferrin in recombinant Kluyveromyces martensii. The positive transformant single colonies KM cs-01-hLF(ura3-), KM cs-01-bLF(ura3-), KM cs-01-cLF(ura3-), and KM cs-01-dLF(ura3-) obtained in Example 4, and the positive transformant single colonies KM cs-01-hLF-EIF4A(ura3-), KM cs-01-bLF-EIF4A(ura3-), and KM cs-01-cLF-EIF4A(ura3-) obtained in Example 7, were compared. cs-01-dLF-EIF4A (ura3-) was picked and fermented in YG medium (formulation: yeast extract 20 g / L, glucose 40 g / L) at 30°C and 200 rpm for 3 days. Then, the supernatant was collected by centrifugation at 8000 rpm for 15 min using a benchtop refrigerated centrifuge. 10% of the supernatant volume of 1M trichloroacetic acid was added to precipitate the secretory proteins in the supernatant. The mixture was incubated overnight at 4°C. The next day, it was centrifuged at 12000 rpm for 20 min at 4°C. The supernatant was discarded, and the precipitate was collected. The precipitate was washed twice with 2 mL of pre-cooled anhydrous ethanol, the supernatant was discarded, and the container was then placed on ice to evaporate the ethanol. Finally, 50 mM Tris-HCl (pH 8.5) was added to dissolve the precipitate, yielding the secretory proteins. Lactoferrin was quantitatively analyzed using SDS-PAGE.

[0103] The SDS-PAGE graph of lactoferrin expression level is shown below. Figures 4-5 As shown.

[0104] Figure 4 Sample 1 is KM cs-01-hLF (ura3-), sample 2 is KM cs-01-bLF (ura3-), sample 3 is KM cs-01-hLF-EIF4A (ura3-), and sample 4 is KM cs-01-bLF-EIF4A (ura3-). As shown in the figure, the bands of intact lactoferrin expressed by KMcs-01-hLF-EIF4A(ura3-) and KMcs-01-bLF-EIF4A(ura3-) (arrow 1) are thicker than those of intact lactoferrin expressed by KMcs-01-hLF(ura3-) and KMcs-01-bLF(ura3-). At the same time, the bands of lactoferrin peptides expressed by KMcs-01-hLF-EIF4A(ura3-) and KMcs-01-bLF-EIF4A(ura3-) (arrow 2) are thicker than those of lactoferrin peptides expressed by KMcs-01-hLF(ura3-) and KMcs-01-bLF(ura3-).

[0105] Figure 5Samples 5 and 6 were KM cs-01-cLF(ura3-), sample 7 was KM cs-01-dLF(ura3-), samples 1 and 2 were KM cs-01-cLF-EIF4A(ura3-), and samples 3 and 4 were KM cs-01-dLF-EIF4A(ura3-). As shown in the figure, the bands of lactoferrin peptides expressed by KM cs-01-cLF-EIF4A(ura3-) and KM cs-01-dLF-EIF4A(ura3-) (arrow 2) are thicker than those expressed by KM cs-01-cLF(ura3-) and KM cs-01-dLF(ura3-).

[0106] Experiments showed that after integrating the human lactoferrin coding gene into the inulinase site of the Kluyveromyces martensii host genome, the expression level of human lactoferrin was 0.51 mg / L, as quantified by Image J. Simultaneously, knocking in the EIF4A coding gene (replacing the glucoamylase gene Sga1) significantly improved human lactoferrin secretion, reaching 1.05 mg / L. Figure 4 After integrating the gene encoding bovine lactoferrin into the inulinase site of the Kluyveromyces martensii host genome, quantification using ImageJ showed that bovine lactoferrin expression was 0.48 mg / L. Simultaneously, knocking in the gene encoding EIF4A (replacing the glucoamylase gene Sga1) significantly improved human lactoferrin secretion, reaching 1.12 mg / L. Figure 4 Simultaneously, overexpression of EIF4A can promote the secretion of human lactoferrin peptide, bovine lactoferrin peptide, feline lactoferrin peptide, and canine lactoferrin peptide. Figure 4 and Figure 5 Arrow 2). This invention is of great significance for increasing the yield of lactoferrin and lactoferrin peptides produced by Kluyveromyces fermentation.

[0107] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A lactoferrin expression strain based on Kluyveromyces martensii, characterized in that, The method for constructing the lactoferrin expression strain includes: replacing the inulinase coding gene in the genome of *Kluyveromyces martensii* recipient strain with the lactoferrin coding gene, and replacing the glucoamylase gene Sga1 in the genome of *Kluyveromyces martensii* recipient strain with the EIF4A protein coding gene expression cassette, thereby promoting the secretory expression of lactoferrin and lactoferrin peptide in *Kluyveromyces martensii*.

2. The lactoferrin expression strain according to claim 1, characterized in that, The amino acid sequence of the EIF4A protein is shown in SEQ ID NO.

1.

3. The lactoferrin expression strain according to claim 1, characterized in that, The nucleotide sequence of the gene encoding the EIF4A protein is shown in SEQ ID NO.

12.

4. The lactoferrin expression strain according to claim 1, characterized in that, In the gene expression frame for the EIF4A protein, the promoter for initiating the expression of the gene encoding the EIF4A protein is the endogenous Hsp26 promoter of Kluyveromyces martensii.

5. The lactoferrin expression strain according to claim 4, characterized in that, The nucleotide sequence of the Hsp26 promoter is shown in SEQ ID NO.

9.

6. The lactoferrin expression strain according to claim 1, characterized in that, The gene expression cassette encoding the EIF4A protein, which replaces the Sga1 glucoamylase gene in the genome of Kluyveromyces martensii recipient, is achieved through homologous recombination; the nucleotide sequence of the upstream homologous arm used in the homologous recombination is shown in SEQ ID NO.10, and the nucleotide sequence of the downstream homologous arm is shown in SEQ ID NO.

11.

7. The lactoferrin expression strain according to claim 1, characterized in that, The lactoferrin includes any one or more of human lactoferrin, bovine lactoferrin, feline lactoferrin, canine lactoferrin, and ovine lactoferrin; the lactoferrin peptides include any one or more of human lactoferrin peptides, bovine lactoferrin peptides, feline lactoferrin peptides, canine lactoferrin peptides, and ovine lactoferrin peptides.

8. The lactoferrin expression strain according to claim 7, characterized in that, The amino acid sequence of the human lactoferrin is shown in SEQ ID NO.2; the amino acid sequence of the bovine lactoferrin is shown in SEQ ID NO.3; the amino acid sequence of the feline lactoferrin is shown in SEQ ID NO.4; the amino acid sequence of the canine lactoferrin is shown in SEQ ID NO.5; and the amino acid sequence of the sheep lactoferrin is shown in SEQ ID NO.

6.

9. The lactoferrin expression strain according to claim 7, characterized in that, The nucleotide sequence of the gene encoding human lactoferrin is shown in SEQ ID NO.13; the nucleotide sequence of the gene encoding bovine lactoferrin is shown in SEQ ID NO.14; the nucleotide sequence of the gene encoding feline lactoferrin is shown in SEQ ID NO.15; the nucleotide sequence of the gene encoding canine lactoferrin is shown in SEQ ID NO.16; and the nucleotide sequence of the gene encoding sheep lactoferrin is shown in SEQ ID NO.

17.

10. The lactoferrin expression strain according to claim 1, characterized in that, The replacement of the inulinase coding gene in the genome of *Kluyveromyces martensii* recipient bacteria with the lactoferrin coding gene is achieved through homologous recombination; the nucleotide sequence of the upstream homologous arm used in the homologous recombination is shown in SEQ ID NO.7, and the nucleotide sequence of the downstream homologous arm is shown in SEQ ID NO.

8.

11. The lactoferrin expression strain according to claim 1, characterized in that, The recipient strain of *Kluyveromyces martensii* is *Kluyveromyces martensii* with the Ura3 gene missing.

12. The lactoferrin expression strain according to claim 11, characterized in that, The Kluyveromyces martensii recipient strain was obtained by knocking out the Ura3 gene in the genome of Kluyveromyces martensii CS-01; the preservation number of Kluyveromyces martensii CS-01 is CGMCC No. 30573.

13. The method for constructing the lactoferrin expression strain according to any one of claims 1-12.

14. The use of the lactoferrin expression strain according to any one of claims 1-12 in the fermentation production of lactoferrin and lactoferrin peptides.

15. A method for fermenting to produce lactoferrin and lactoferrin peptides, characterized in that, The method includes using the lactoferrin-expressing strain according to any one of claims 1-12.

Images