A recombinant Pichia pastoris engineering bacterium and its construction method and application

Abstract

The invention provides a recombinant Pichia pastoris engineering bacterium and a construction method and application thereof, belonging to the technical field of biochemistry and genetic engineering. The recombinant Pichia pastoris engineering bacterium is a Pichia pastoris strain in which the FDH1 gene shown in SEQ ID NO.1 is targetedly knocked out, and the DAK2 gene shown in SEQ ID NO.3, the SHM2 gene shown in SEQ ID NO.5, the FBA1 gene shown in SEQ ID NO.7 and the MTD1 gene shown in SEQ ID NO.9 are inserted; the recombinant Pichia pastoris engineering bacterium provided by the invention can significantly improve the yield of recombinant proteins, especially has a large improvement in the yield of proteins such as recombinant human transferrin, recombinant human albumin, and recombinant human basic fibroblast growth factor.

Description

Technical Field

[0001] The invention relates to a recombinant Pichia pastoris engineering bacterium and a construction method and application thereof, belonging to the technical field of biochemistry and genetic engineering. Background Art

[0002] Pichia pastoris, also known as Komagataella phaffii, is a host bacteria widely used for industrial protein expression and metabolite production. It is widely adopted for its advantages in heterologous protein production, including food and feed enzymes, antibodies and vaccines, cosmetic ingredients, and biofuels and other chemicals. The characteristics of Pichia pastoris include: 1. Powerful promoters: Pichia pastoris uses strong promoters such as PAOX and PGAP, which ensures precise gene regulation. 2. Convenience of genetic manipulation: Pichia pastoris has user-friendly genetic manipulation capabilities, which makes it easier to manipulate the gene expression system. 3. Secretion system: Pichia pastoris has a mature secretion system that can efficiently release proteins into the external environment. 4. Post-translational modification: Pichia pastoris excels in post-translational modification of proteins, which is essential for the correct folding and function of proteins. 5. High-density fermentation: Pichia pastoris is able to achieve high-density fermentation of more than 150 g / L, which significantly increases the expression of proteins. 6. Gene editing technology: The emergence of CRISPR / Cas9 gene editing system makes the manipulation of gene expression system more efficient and greatly improves the efficiency of exploring gene functions. 7. Metabolic engineering: More metabolic engineering ideas for enhancing RP (recombinant protein) production have been proposed by integrating new pathways in microorganisms. 8. Mathematical models and high-throughput screening: The application of new mathematical models combined with high-throughput screening can better understand the bottlenecks in the biosynthesis process. 9. Product commercialization: The importance of Pichia pastoris expression systems in industrial applications is increasing, and many products heterologously expressed in Pichia pastoris have entered the market, such as FDA-approved biopharmaceuticals or industrial enzymes. 10. Development of new mutant strains: Two new Pichia chassis hosts have been developed, which are impaired in β-1,3-D-glucan biosynthesis and show enhanced performance in growth rate, protein expression and catalytic ability. These hosts have the potential to become attractive alternatives to Pichia pastoris GS115 in various bioproduction applications.

[0003] Pichia pastoris has been valued for its high efficiency and versatility in heterologous protein production, and with the progress of genetic engineering and metabolic engineering, its application prospects in bioproduction are broad. However, the protein yield of Pichia pastoris is low, and possible factors include: protein folding problems, endoplasmic reticulum burden, selection of secretion signals, metabolic burden, methanol utilization efficiency, temperature tolerance, proteolysis, and gene integration and mutation. For example, protein folding problems refer to the fact that, in some cases, overexpression of certain proteins such as folding enzyme Cpr5p and protein disulfide isomerase Pdi1p, which increase IgG production in Saccharomyces cerevisiae, has the opposite effect in Pichia pastoris, reducing protein titer and production. Endoplasmic reticulum (ER) burden refers to the fact that the reduction in IgG titer and production caused by overexpression of CPR5 may be caused by protein burden in the endoplasmic reticulum.

[0004] In summary, researchers are also working hard to try to cultivate recombinant Pichia pastoris through biotechnology to increase protein production. Summary of the invention

[0005] The present invention provides a recombinant Pichia pastoris engineered bacterium and a construction method and application thereof. The recombinant Pichia pastoris engineered bacterium can significantly increase the yield of recombinant proteins, especially the yield of recombinant human transferrin, recombinant human albumin, recombinant human basic fibroblast growth factor and the like.

[0006] To achieve this object, the present invention provides the following technical solutions:

[0007] The first aspect of the present invention provides a recombinant Pichia pastoris engineered bacterium, wherein the recombinant Pichia pastoris engineered bacterium is a Pichia pastoris strain in which the FDH1 gene shown in SEQ ID NO.1 is targetedly knocked out, and the DAK2 gene shown in SEQID NO.3, the SHM2 gene shown in SEQID NO.5, the FBA1 gene shown in SEQID NO.7 and the MTD1 gene shown in SEQ ID NO.9 are inserted.

[0008] The sequence of SEQ ID NO.1 is as follows:

[0009] 1 ATGAAAATCG TTCTCGTTTT GTACTCCGCT GGTAAGCACG CCGCCGATGAACCAAAGTTGTATGGTTGTA

[0010] 71 TCGAAAATGA ATTGGGTATT AGACAATGGC TTGAGAAGGG CGGCCATGAATTGGTTACTACATCAGACAA

[0011] 141 AGAGGGTGAA AACTCTGAGT TAGAAAAGCA CATTCCTGAC GCTGATGTGATTATTTCCACTCCATTCCAT

[0012] 211 CCAGCCTACA TCACGAAGGA GAGAATCCAA AAAGCCAAGA AGCTGAAGTTGTTGGTCGTTGCTGGTGTCG

[0013] 281 GTTCCGACCA CATTGACTTG GACTACATTG AAAAAAATGG CCTAGATATTTCGGTCCTAGAGGTTACTGG

[0014] 351 TTCCAACGTT GTTTCAGTGG CTGAGCATGT CGTTATGACT ATATTGAACTTGGTGAGAAACTTTGTTCCA

[0015] 421 GCTCACGAGC AAATTGTTAA CCACGGCTGG GACGTTGCTG CCATCGCCAAGGACGCCTACGATATCGAAG

[0016] 491 GTAAGACCAT CGCAACAATT GGTGCTGGAA GAATTGGTTA CAGAGTCTTAGAGAGACTTGTGGCTTTCAA

[0017] 561 CCCTAAGGAA TTGTTGTACT ACGACTACCA AGGTCTTCCA AAAGAGGCCGAGGAAAAAGTTGGTGCCAGA

[0018] 631 AGAGTCGACA CTGTCGAGGA GCTGGTTGCT CAAGCCGATG TTGTTACCGTCAATGCCCCACTGCACGCAG

[0019] 701 GTACTAAGGG TTTAGTTAAC AAGGAGCTTC TGTCCAAGTT CAAGAAGGGTGCTTGGTTGGTTAACACAGC

[0020] 771 CAGAGGTGCC ATCTGCAATG CTCAAGATGT CGCTGATGCC GTTGCATCTGGTCAATTGAGAGGTTACGGT

[0021] 841 GGTGACGTCT GGTTCCCTCA GCCAGCTCCA AAGGACCATC CATGGAGAGATATGAGAAACAAGTACGGAT

[0022] 911 ACGGAAACGC CATGACTCCT CATTACTCAG GTACCACTTT GGACGCCCAGGTCAGATATGCCGAAGGTAC

[0023] 981 CAAGAACATC TTGAACTCAT TCCTTACCAA GAAGTTTGAC TACAGACCTCAAGATGTCATTCTTTTGAAC

[0024] 1051 GGTAAGTACA AGACCAAGGC TTATGGTAAT GACAAAAAGG TCGCATAA

[0025] SEQ ID NO.3:

[0026] 1 ATGTCTAGTA AGCATTGGGA TTACAAAAAA GATTTAGTAC TATCACACTTGGCCGGCTTATGCCAATCCA

[0027] 71 ACCCACACGT TAGATTGATT GAATCCGAGA GGGTGGTCAT TAGTGCTGAGAATCAAGAGGACAAGATCAC

[0028] 141 ACTTATTTCT GGAGGTGGTT CTGGACACGA ACCTTTGCAT GCTGGATTCGTCACCAAAGACGGATTGTTG

[0029] 211 GATGCAGCTG TGGCAGGATT TATCTTTGCA TCTCCTTCAA CCAAACAAATATTCTCTGCTATCAAGGCAA

[0030] 281 AACCATCTAA AAAGGGAACT CTGATTATCG TTAAGAACTA CACGGGTGATATTCTGCATTTTGGACTAGC

[0031] 351 TGCTGAAAAA GCTAAAGCTG AGGGTTTGAA TGCCGAATTG TTGATCGTCCAGGATGATGTTTCCGTAGGA

[0032] 421 AAAGCCAAGA ACGGATTGGT TGGTCGTCGT GGTTTGGCTG GTACATCTTTGGTCCACAAGATTTTGGGAG

[0033] 491 CTAAAGCTTA CCTGCAAAAG GATAATCTGG AGCTGCATCA ATTAGTCACATTTGGTGAGAAGGTTGTTGC

[0034] 561 CAACTTAGTT ACCATCGGCG CCAGTTTGGA TCACGTAACC ATACCTGCCCGTGCTAACAAGCAAGAGGAG

[0035] 631 GACGATTCAG ATGATGAGCA TGGATATGAA GTGTTAAAGC ACGATGAGTTTGAGATAGGTATGGGAATTC

[0036] 701 ATAACGAGCC TGGCATCAAA AAATCATCCC CCATTCCAAC CGTTGATGAACTTGTCGCAGAACTGTTGGA

[0037] 771 ATATTTGTTG TCCACTACTG ACAAAGACCG TAACTACGTT CAATTTGACAAGAATGACGAGGTCGTCCTT

[0038] 841 CTTATCAATA ACCTTGGCGG AACTTCCGTG CTTGAATTAT ATGCTATACAAAATATCGTTGTTGATCAGT

[0039] 911 TGGCTTCAAA GTACTCAATC AAACCTGTCC GTATTTTTAC AGGTACCTTTACCACGTCTTTGGACGGACC

[0040] 981 CGGTTTTAGT ATCACCTTGT TGAATGCCAC CAAAACTGGT GACAAGGACATCTTAAAATT CTTGGACCAT

[0041] 1051 AAAACATCAG CACCAGGTTG GAATTCAAAC ATTTCCGACT GGTCTGGTAGAGTGGATAATTTTATTGTCG

[0042] 1121 CAGCCCCCGA GATTGACGAG GGAGACTCTTT CTTCCAAGGT TTCTGTGGATGCAAAGCTGTATGCTGATTT

[0043] 1191 GCTGGAATCC GGAGTCAAAA AGGTTATTTC TAAAGAACCA AAGATTACTTTGTATGATACTGTCGCTGGT

[0044] 1261 GATGGTGATT GCGGAGAAAC CTTAGCTAAT GGTTCTAATG CCATCTTGAAAGCTTTAGCCGAAGGAAAAC

[0045] 1331 TAGATTTGAA GGACGGTGTC AAGTCACTGG TTCAGATTAC GGACATTGTGGAAACCGCCATGGGAGGTAC

[0046] 1401 CAGTGGTGGA TTGTATTCAA TTTTCATCTC AGCTTTGGCT AAATCTCTGAAAAGAGAAGGAATTGAGTGAA

[0047] 1471 GGTGCCTATA CTTTAACATT GGAAACTATC TCCGGTTCCT TGCAAGCTGCTCTGCAGAGTCTGTTTAAAT

[0048] 1541 ACACACGTGC TCGTACTGGC GATAGAACAC TGATCGATGC TTTGGAGCCTTTCGTCAAGGAATTCGCAAA

[0049] 1611 GTCTAAGGAC CTGAAGTTGG CAAACAAAGC TGCTCATGAT GGTGCTGAAGCTACGAGAAAGCTAGAAGCT

[0050] 1681 AAGTTTGGAA GAGCTTCTTA CGTTGCTGAG GAGGAATTTA AACAATTTGAGTCCGAGGGTGGTCTGCCTG

[0051] 1751 ATCCAGGTGC TATCGGACTA GCTGCTCTTA TTTCCGGTAT AACCGACGCATACTTTAAGTCAGAGACTAA

[0052] 1821 ATTG

[0053] SEQ ID NO.5:

[0054] 1 ATGTCACACT ATGCTCTGTC TCCTGAACAT AAACAAATGG TTGAAGGTCATCTAGCTGAA ACAGACCCTG

[0055] 71 AAGTAAACCA GATTATCAAA GATGAGGTTG ACAGACAGAA ACATTCAATTGTTCTGATTG CTAGTGAAAA

[0056] 141 CTTTACTTCA ACCAGTGTCT TTGACGCTCT TGGTACTCCA ATGTGCAATAAGTACTCTGA AGGTTACCCA

[0057] 211 GGAGCTAGAT ATTACGGTGG AAACGAACAT ATTGATAGAA TGGAAATTTTGTGTCAACAG AGGGCTCTGA

[0058] 281 AAGCATTTCA CCTAGATGGT AGTAGATGGG GAGTTAATGT TCAGACTCTGTCCGGCAGTC CTGCCAATCT

[0059] 351 TCAAGTCTAC CAAGCAATAA TGAAGCCACA TGATAGGTTG ATGGGACTAGATTTGCCACA TGGTGGTCAT

[0060] 421 TTATCCCACG GTTATCAAAC TGATACAAGA AAAATTTCAG CTGTTTCTACCTATTTCGAA ACCATGCCAT

[0061] 491 ATAGGGTTGA CCTGGAGACC GGTATTATCG ACTATGATAT GTTGGAAAAGACTGCAGTGC TTTATAGGCC

[0062] 561 CAAAGTCTTG GTTGCCGGTA CGTCTGCCTA TTGTAGACTT ATTGACTACAAGAGGATGAG AGAGATCGCA

[0063] 631 GACAAGGTTG GTGCTTACCT GGTTGTTGAC ATGGCTCACA TTAGTGGCTTGATTGCTGCT GGAGTGATTC

[0064] 701 CCTCACCATT TGAATACGCT GACATTGTGA CCACAACTAC ACATAAATCCTTAAGAGGAC CTAGGGGAGC

[0065] 771 CATGATTTTC TTCAGGAAAG GTGTAAGAAG TGTTAATCCA AAGACTGGAAAAGAGATTTA CTATGATCTT

[0066] 841 GAGAACCCAA TCAACTTTTC AGTGTTCCCA GGACATCAGG GTGGTCCACATAACCATACA ATTGCTGCAT

[0067] 911 TAGCTACTGC CTTGAAACAA GCTGCCACAC CTGAATTTAA GCAGTATCAGGAACAAGTAT TGAAGAACGC

[0068] 981 TAAGGCCTTG GAAAATGAAT TCAAAAGATT GGGATATAAA CTGGTTAGTGACGGTACTGA TTCCCACATG

[0069] 1051 GTCTTGGTCA GTTTGAAGGA TAAAGATATC GATGGAGCCA GGATCGAGACAGTCTGTGAA AATATTACA

[0070] 1121 TCGCCCTTAA CAAGAATAGT ATACCCGGTG ATAGATCAGC ATTGGTGCCTGGAGGTGTTA GGATTGGAGC

[0071] 1191 TCCTGCTATG ACTACAAGAG GTGCCAGTGA GGAAGATTTT GTTAAAATCGCAAACTATAT TGACAGTCA

[0072] 1261 GTCCAGTACG CCAAAAAGGT TCAATCAGAA TTGCCTATTG AGGCTACAAGCTAAAGGAC TTCAAAGCCA

[0073] 1331 AGATAGCAGA GGGCTCTGAT GAAATTACAC AGTTGAAAAA TGAAATTTCTGCTTGGGCTG GAGAGTTCCC

[0074] 1401 CCTATCTGTA

[0075] SEQ ID NO.7 Associated:

[0076] 1 ATGCCAGACC AATTATCCCTT TTTGAAGAGA AAAACTGGAG TTATTGTCGGTGACGATGTG AGAAACTTGT

[0077] 71 TCTTACACGC TCAAAAAAAG GGTTTTGCTA TTCCTGCAAT CAACGTAACTAGTAGTTCAA CCGCTGTGGC

[0078] 141 TGCTTTGGAA GCCGCTCGTG ACAATAACTC ACCTATCATC TTACAGACCTCTCAAGGCGG TGCAGCCTAC

[0079] 211 TTTGCAGGCA AGGGAGTTAA AAATAGTAAC CAGGAAGCAA GTATTGGAGGTGCCGTTGCC GCTGCCCACT

[0080] 281 ACATAAGGTC AGTGGCCCCC TTGTACGGTA TTCCAGTCAT TCTGCATACGGATCACTGTG CCAAAAAATT

[0081] 351 GTTGCCATGG TTTGAGGGTT TTTTAGCTGC TGATGAAGCC TACTATAAAGAGCATGGAGA GCCATTATTT

[0082] 421 TCCTCCCATA TGTTGGATTT GTCTGAGGAG AGTGATGACG AAAACATTGCTACTTGCGTC AAGTACTTTA

[0083] 491 AGCGAATGGC TAAAATGAAC CAGTGGTTGG AGATGGAAAT TGGTATTACTGGTGGTGAGG AAGACGGTGT

[0084] 561 TAACAATGAG AACGCCGACA AAGAGTTGCT ATACACTAAG CCAGAAACTGTTTTCGCAGT CCACAAGGCC

[0085] 631 TTGGCTCCTA TTTCACCTAA CTTCTCAATT GCTGCCGCTT TCGGTAACGTTCATGGTGTC TATAAGCCAG

[0086] 701 GAAACGTCGT TCTGAGACCA TCACTTTTAG GTGACCACCA AAAGTACGCTAAGGAACAAC TGAAGTCAGA

[0087] 771 GTCTGACAAG CCACTTTTTT TGGTTTTTCA CGGAGGATCT GGTTCCACTCAGGAAGAATT TGACACGGGA

[0088] 841 ATTAGAAATG GAGTCGTTAA GGTTAATCTA GATACGGACT GCCAATATGCCTACTTGATT GGCATCAGGG

[0089] 911 ATTACGTCCT AAAAAAGAAA GACTACATCA TGTCTCAGGT CGGTAACCCTGAAGGTGATG ACAAACCCAA

[0090] 981 CAAGAAATAT TTCGACCCTC GTGTCTGGGT CAGAGAAGGT GAAAAAACAATGTCCGCTAG AGTCACGGAA

[0091] 1051 GCCTTAAATA TTTTCCATAC TGCTAATCAG TTG

[0092] SEQ ID NO.9:

[0093] 1 ATGTCATACA GAGCTGGTAA AACCATTTTA GCATCTACTA TAGCAAAAAAATACTCTGAG GAACTAACTA

[0094] 71 GTGTCACTTC CAAGATGGCT ACAAAGCCTA AGCTTGTTGG CCTATTGGGTAATCAAGACC CAGCTGCTAA

[0095] 141 GACTTATGCC GAATGGACCG CTCGAACTTG CGAAAGTCTT GGCTTCCAATACGAACTAAT ACAGTGTCCA

[0096] 211 AAAGAAAACT TAGAAGACGA GATTTACAAG TGTAACCAGG ATGATGCTGTTAATGGTATC ATGGTCTACT

[0097] 281 TCCCTGTTTA TAAGATGGA CAGGACCAGT ATCTGCAACA GTGTGTCTCTTATTGAAAAGG ATGTTGAAGG

[0098] 351 TCTAAACTTT AAATATGTCT CCAATATGTA CCACAACATC CGTTATCTAGACGCCCCAGA AAATACTAAG

[0099] 421 AAATCTATAT TACCATGTAC TCCATTGGCC ATGGTGAAGA TCCTTGAGTACTTGGGAGTT TATAATACTT

[0100] 491 TACTGCCATA CGGCAATCGT TTATTTGGCA AGACCATCAC CATTGTCAACAGATCAGAAA TCGTCGGTCG

[0101] 561 TCCTTTGGCT GCTCTGTTGG CAAACGACGG TGCAACTGTG TATTCTGTTGACATCAATAA CGTCCAGAAG

[0102] 631 TTTGAAAGAG GTGATGGTTT ACAACTGAAG TCACATAGGG TCGTTGACACCGAGTTCGAA CTTAAAGACG

[0103] 701 TTGCCCCCAA GAGTGACGTT ATAATTACAG GAGTACCATC TTCTAACTATAAGTTCCCCT TAGAGTACGT

[0104] 771 CAAAGATGGT GCTGTGGTTA TTAACTTCTC TTCTGAGAAG AACTTTGATGATAGAATCAA AGATATCGCC

[0105] 841 TCTCTGTATG TCCCTTCAAT AGGCAAAGTG ACCATTGCCA TGCTTCTACGAAATCTACTG CGTCTTATGA

[0106] 911 ATTACKAAAA GCCCCATAGT GTTGAAAAA TA

[0107] Preferably, the amino acid sequence encoded by the FDH1 gene is shown as SEQ ID NO.2; the amino acid sequence encoded by the DAK2 gene is shown as SEQ ID NO.4; the amino acid sequence encoded by the SHM2 gene is shown as SEQ ID NO.6; the amino acid sequence encoded by the FBA1 gene is shown as SEQ ID NO.8; and the amino acid sequence encoded by the MTD1 gene is shown as SEQ ID NO.10.

[0108] The sequence of SEQ ID NO.2 is as follows:

[0109] 1 MKIVLVLYSA GKHAADEPKL YGCIENELGI RQWLEKGGHE LVTTSDKEGENSELEKHIPDADVIISTPFH

[0110] 71 PAYITKERIQ KAKKLKLLVV AGVGSDHIDL DYIEQNGLDI SVLEVTGSNVVSVAEHVVMTILNLVRNFVP

[0111] 141 AHEQIVNHGW DVAAIAKDAY DIEGKTIATI GAGRIGYRVL ERLVAFNPKELLYYDYQGLPKEAEEKVGAR

[0112] 211 RVDTVEELVA QADVVTVNAP LHAGTKGLVN KELLSKFKKG AWLVNTARGAICNAQDVADAVASGQLRGYG

[0113] 281 GDVWFPQPAP KDHPWRDMRN KYGYGNAMTP HYSGTTLDAQ VRYAEGTKNILNSFLTKKFDYRPQDVILLN

[0114] 351 GKYKTKAYGN DKKVA

[0115] The sequence of SEQ ID NO.4 is as follows:

[0116] 1 MSSKHWDYKK DLVLSHLAGL CQSNPHVRLI ESERVVISAE NQEDKITLISGGGSGHEPLHAGFVTKDGLL

[0117] 71 DAAVAGFIFA SPSTKQIFSA IKAKPSKKGT LIIVKNYTGD ILHFGLAAEKAKAEGLNAELLIVQDDVSVG

[0118] 141 KAKNGLVGRR GLAGTSLVHK ILGAKAYLQK DNLELHQLVT FGEKVVANLVTIGASLDHVTIPARANKQEE

[0119] 211 DDSDDEHGYE VLKHDEFEIG MGIHNEPGIK KSSPIPTVDE LVAELLEYLLSTTDKDRNYVQFDKNDEVVL

[0120] 281 LINNLGGTSV LELYAIQNIV VDQLASKYSI KPVRIFTGTF TTSLDGPGFSITLLNATKTGDKDILKFLDH

[0121] 351 KTSAPGWNSN ISDWSGRVDN FIVAAPEIDE GDSSSKVSVD AKLYADLLESGVKKVISKEPKITLYDTVAG

[0122] 421 DGDCGETLAN GSNAILKALA EGKLDLKDGV KSLVQITDIV ETAMGGTSGGLYSIFISALAKSLKEKELSE

[0123] 491 GAYTLTLETI SGSLQAALQS LFKYTRARTG DRTLIDALEP FVKEFAKSKDLKLANKAAHDGAEATRKLEA

[0124] 561 KFGRASYVAE EEFKQFESEG GLPDPGAIGL AALISGITDA YFKSETKL

[0125] The sequence of SEQ ID NO.6 is as follows:

[0126] 1 MSHYALSPEH KQMVEGHLAE TDPEVNQIIK DEVDRQKHSI VLIASENFTS TSVFDALGTPMCNKYSEGYP

[0127] 71 GARYYGGNEH IDRMEILCQQ RALKAFHLDG SRWGVNVQTL SGSPANLQVY QAIMKPHDRLMGLDLPHGGH

[0128] 141 LSHGYQTDTR KISAVSTYFE TMPYRVDLET GIIDYDMLEK TAVLYRPKVL GUARD SYCRLIDYKRMREIA

[0129] 211 DKVGAYLVVD MAHISGLIAA GVIPSPFEYA DIVTTTTHKS LRGPRGAMIF FRKGVRSVNPKTGKEIYYDL

[0130] 281 ENPINFSVFP GHQGGPHNHT IAALATALKQ AATPEFKQYQ EQVLKNAKAL ENEFKRLGYKLVSDGTDSHM

[0131] 351 VLVSLKDKDI DGARIETVCE NINIALNKNS IPGDRSALVP GGVRIGAPAM TTRGASEEDFVKIANYIDKS

[0132] 421 VQYAKKVQSE LPIEANKLKD FKAKIAEGSD EITQLKNEIS AWAGEFPLSV

[0133] Sequence of SEQ ID NO.8:

[0134] 1 MPDQLSFLKR KTGVIVGDDV RNLFLHAQKK GFAIPAINVT SSSTAVAALE AARDNNSPIILQTSQGGAAY

[0135] 71 FAGKGVKNSN QEASIGGAVA AAHYIRSVAP LYGIPVILHT DHCAKKLLPW FEGFLAADEAYYKEHGEPLF

[0136] 141 SSHMLLDLSEE SDDENIATCV KYFKRMAKMN QWLEMEIGIT GGEEDGVNNE NADKELLYTKPETVFAVHKA

[0137] 211 LAPISPNFSI AAAFGNVHGV YKPGNVVLRP SLLGDHQKYA KEQLKSESDK PLFLVFHGGSGSTQEEFDTG

[0138] 281 IRNGVVKVNL DTDCQYAYLI GIRDYVLKKK DYIMSQVGNP EGDDKPNKKY FDPRVWVREGEKTMSARVTE

[0139] 351 ALNIFHTANQ L

[0140] The sequence of SEQ ID NO.10 is as follows:

[0141] 1 MSYRAGKTIL ASTIAKKYSE ELTSVTSKMA TKPKLVGLLG NQDPAAKTYA EWTARTCESLGFQYELIQCP

[0142] 71 KENLEDEIYK CNQDDAVNGI MVYFPVYNDG QDQYLQQCVS IEKDVEGLNF KYVSNMYHNIRYLDAPENTK

[0143] 141 KSILPCTPLA MVKILEYLGV YNTLLPYGNR LFGKTITIVN RSEIVGRPLA ALLANDGATVYSVDINNVQK

[0144] 211 FERGDGLQLK SHRVVDTEFE LKDVAPKSDV IITGVPSSNY KFPLEYVKDG AVVINFSSEKNFDDRIKDIA

[0145] 281 SLYVPSIGKV TIAMLLRNLL RLMNNKKPHS VEEI

[0146] Preferably, the recombinant expression cassettes encoding DAK2 and SHM2 genes are on the same nucleic acid construct of the selection marker; the recombinant expression cassettes encoding FBA1 and MTD1 genes are on the same nucleic acid construct of the selection marker.

[0147] Preferably, the recombinant Pichia pastoris is also inserted with a drug resistance gene.

[0148] Preferably, the drug resistance gene includes one or more of the blasticidin resistance gene, the geneticin G418 resistance gene, the hygromycin B resistance gene, and the zeocin resistance gene.

[0149] The nucleotide sequence of the blasticidin resistance gene is shown in SEQ ID NO.16 or a nucleotide sequence having at least 98% identity with SEQ ID NO.16.

[0150] The sequence of SEQ ID NO.16 is as follows:

[0151] 1 ATGGCCAAGC CTTTGTCTCA AGAAGAATCC ACCCTCATTG AAGAGCAAC GGCTACAATCAACAGCATCC

[0152] 71 CCATCTCTGA AGACTACAGC GTCGCCAGCG CAGCTCTCTC TAGCGACGGC CGCATCTTCACTGGTGTCAA

[0153] 141 TGTATATCAT TTTACTGGGG GACCTTGTGC AGAACTCGTG GTGCTGGGCA CTGCTGCTGCTGCGGCAGCT

[0154] 211 GGCAACCTGA CTTGTATCGT CGCGATCGGA AATGAGAACA GGGGCATCTT GAGCCCCTGCGGACGGTGCC

[0155] 281 GACAGGTGCT TCTCGATCTG CATCCTGGGA TCAAAGCCAT AGTGAAGGAC AGTGATGGACAGCCGACGGC

[0156] 351 AGTTGGGATT CGTGAATTGC TGCCCTCTGG TTATGTGTGG GAGGGCTAA

[0157] The nucleotide sequence of the geneticin G418 resistance gene is as shown in SEQ ID NO.17 or a nucleotide sequence having at least 98% identity with SEQ ID NO.17;

[0158] The sequence of SEQ ID NO.17 is as follows:

[0159] 1 ATGGGTAAGG AAAAGACTCA CGTTTCGAGG CCGCGATTAA ATTCCAACAT GGATGCTGATTTATATGGGT

[0160] 71 ATAAATGGGC TCGCGATAAT GTCGGGCAAT CAGGTGCGAC AATCTATCGA TTGTATGGGAAGCCCGATGC

[0161] 141 GCCAGAGTTG TTTCTGAAAC ATGGCAAAGG TAGCGTTGCC AATGATGTTA CAGATGAGATGGTCAGACTA

[0162] 211 AACTGGCTGA CGGAATTTAT GCCTCTTCCG ACCATCAAGC ATTTTATCCG TACTCCTGATGATGCATGGT

[0163] 281 TACTCACCAC TGCGATCCCC GGCAAAACAG CATTCCAGGT ATTAGAAGAA TATCCTGATTCAGGTGAAAA

[0164] 351 TATTGTTGAT GCGCTGGCAG TGTTCCTGCG CCGGTTGCAT TCGATTCCTG TTTGTAATTGTCCTTTTAAC

[0165] 421 AGCGATCGCG TATTTCGTCT GGCTCAGGCG CAATCACGAA TGAATAACGG TTTGGTTGATGCGAGTGATT

[0166] 491 TTGATGACGA GCGTAATGGC TGGCCTGTTG AACAAGTCTG GAAAGAAATG CATAAGCTTTTGCCATTCTC

[0167] 561 ACCGGATTCA GTCGTCACTC ATGGTGATTT CTCACTTGAT AACCTTATTT TTGACGAGGGGAAATTAATA

[0168] 631 GGTTGTATTG ATGTTGGACG AGTCGGAATC GCAGACCGAT ACCAGGATCT TGCCATCCTATGGAACTGCC

[0169] 701 TCGGTGAGTT TTCTCCTTCA TTACAGAAAC GGCTTTTTCA AAAATATGGT ATTGATAATCCTGATATGAA

[0170] 771 TAAATTGCAG TTTCATTTGA TGCTCGATGA GTTTTTCTAA

[0171] The nucleotide sequence of the hygromycin B resistance gene is as shown in SEQ ID NO.18 or a nucleotide sequence having at least 98% identity with SEQ ID NO.18;

[0172] The sequence of SEQ ID NO.18 is as follows:

[0173] 1 ATGGGTAAAA AGCCTGAACT CACCGCGACG TCTGTCGAGA AGTTTCTGAT CGAAAAGTTCGACAGCGTCT

[0174] 71 CCGACCTGAT GCAGCTCTCG GAGGGCGAAG AATCTCGTGC TTTCAGCTTC GATGTAGGAGGGCGTGGATA

[0175] 141 TGTCCTGCGG GTAAATAGCT GCGCCGATGG TTTCTACAAA GATCGTTATG TTTATCGGCACTTTGCATCG

[0176] 211 GCCGCGCTCC CGATTCCGGA AGTGCTTGAC ATTGGGGCAT TCAGCGAGAG CCTGACCTATTGCATCTCCC

[0177] 281 GCCGTGCACAGGGTGTCACG TTGCAAGACC TGCCTGAAAC CGAACTGCCC GCTGTTCTGCAACCGGTCGC

[0178] 351 GGAGGCGATG GATGCCATCG CTGCGGCCGA TCTTAGCCAG ACGAGCGGGT TCGGCCCATTCGGACCGCAA

[0179] 421 GGAATCGGTC AATACACTAC ATGGCGTGAT TTCATATGCG CGATTGCTGATCCCCATGTGTATCACTGGC

[0180] 491 AAACTGTGAT GGACGACACC GTCAGTGCGT CCGTCGCGCA GGCTCTCGAT GAGCTGATGCTTTGGGCCGA

[0181] 561 GGACTGCCCC GAAGTCCGGC ACCTCGTGCA CGCGGATTTC GGCTCCAACA ATGTCCTGACGGACAATGGC

[0182] 631 CGCATAACAG CGGTCATTGA CTGGAGCGAG GCGATGTTCG GGGATTCCCA ATACGAGGTCGCCAACATCT

[0183] 701 TCTTCTGGAG GCCGTGGTTG GCTTGTATGG AGCAGCAGAC GCGCTACTTC GAGCGGAGGCATCCGGAGCT

[0184] 771 TGCAGGATCG CCGCGGCTCC GGGCGTATAT GCTCCGCATT GGTCTTGACC AACTCTATCAGAGCTTGGTT

[0185] 841 GACGGCAATT TCGATGATGC AGCTTGGGCG CAGGGTCGAT GCGACGCAAT CGTCCGATCCGGAGCCGGGA

[0186] 911 CTGTCGGGCG TACACAAATC GCCCGCAGAA GCGCGGCCGT CTGGACCGAT GGCTGTGTAGAAGTACTCGC

[0187] 981 CGATAGTGGA AACCGACGCC CCAGCACTCG TCCGAGGGCA AAGGAATAG

[0188] The nucleotide sequence of the Zeocin resistance gene is shown in SEQ ID NO. 19 or a nucleotide sequence having at least 98% identity with SEQ ID NO. 19.

[0189] The sequence of SEQ ID NO.19 is as follows:

[0190] 1 ATGGCTAAAC TCACCTCTGC TGTTCCAGTC CTGACTGCTC GTGATGTTGC TGGTGCTGTTGAGTTCTGGA

[0191] 71 CTGATAGACT CGGTTTCTCC CGTGACTTCG TAGAGGACGA CTTTGCCGGT GTTGTACGTGACGACGTTAC

[0192] 141 CCTGTTCATC TCCGCAGTTC AGGACCAGGT TGTGCCAGAC AACACTCTGG CATGGGTATGGGTTCGTGGT

[0193] 211 CTGGACGAAC TGTACGCTGA GTGGTCTGAG GTCGTGTCTA CCAACTTCCG TGATGCATCTGGTCCAGCTA

[0194] 281 TGACCGAGAT CGGTGAACAG CCCTGGGGTC GTGAGTTTGC ACTGCGTGAT CCAGCTGGTAACTGCGTGCA

[0195] 351TTTCGTCGCA GAAGAACAGG ACTAA

[0196] Preferably, the recombinant Pichia pastoris engineered bacteria is also inserted with a functional vector.

[0197] Preferably, the functional vectors include a recombinant human transferrin expression vector, a recombinant human albumin expression vector, and a recombinant human basic fibroblast growth factor expression vector.

[0198] The nucleotide sequence of the nucleic acid molecule expressing recombinant human transferrin is shown in SEQ ID NO.12, and the amino acid sequence is shown in SEQ ID NO.11.

[0199] The sequence of SEQ ID NO.11 is as follows:

[0200] 1 VPDKTVRWCA VSEHEATKCQ SFRDHMKSVI PSDGPSVACV KKASYLDCIR AIAANEADAVTLDAGLVYDA

[0201] 71 YLAPNNLKPV VAEFYGSKED PQTFYYAVAV VKKDSGFQMN QLRGKKSCHT GLGRSAGWNIPIGLLYCDLP

[0202] 141 EPRKPLEKAV ANFFSGSCAP CADGTDFPQL CQLCPGCGCS TLNQYFGYSG AFKCLKDGAGDVAFVKHSTI

[0203] 211 FENLANKADR DQYELLCLDN TRKPVDEYKD CHLAQVPSHT VVARSMGGKE DLIWELLNQAQEHFGKDKSK

[0204] 281 EFQLFSSPHG KDLLFKDSAH GFLKVPPRMD AKMYLGYEYV TAIRNLREGT CPEAPTDECKPVKWCALSHH

[0205] 351 ERLKCDEWSV NSVGKIECVS AETTEDCIAK IMNGEADAMS LDGGFVYIAG KCGLVPVLAENYNKSDNCED

[0206] 421 TPEAGYFAIA VVKKSASDLT WDNLKGKKSC HTAVGRTAGW NIPMGLLYNK INHCRFDEFFSEGCAPGSKK

[0207] 491 DSSLCKLCMG SGLNLCEPNN KEGYYGYTGA FRCLVEKGDV AFVKHQTVPQ NTGGKNPDPWAKNLNEKDYE

[0208] 561 LLCLDGTRKP VEEYANCHLA RAPNHAVVTR KDKEACVHKI LRQQQHLFGS NVTDCSGNFCLFRSETKDLL

[0209] 631 FRDDTVCLAK LHDRNTYEKY LGEEYVKAVG NLRKCSTSSL LEACTFRRP

[0210] The sequence of SEQ ID NO.12 is as follows:

[0211] 1 GTACCAGATA AGACCGTCCG TTGGTGTGCC GTAAGTGAAC ACGAAGCCACCAAGTGTCAA AGTTTTCGTG

[0212] 71 ACCACATGAA ATCAGTAATT CCTTCAGACG GACCCTCTGT TGCTTGTGTTAAAAAGGCTT CTTACTTGGA

[0213] 141 TTGTATTAGA GCTATTGCTG CCAACGAAGC AGATGCTGTT ACACTGGACGCTGGTTTAGT TTACGATGCT

[0214] 211 TATCTAGCCC CCAACAACTT GAAACCAGTT GTGGCAGAAT TCTATGGTTCTAAAGAGGAC CCTCAAACAT

[0215] 281 TTTATTACGC TGTTGCTGTC GTGAAGAAAG ACTCAGGTTT TCAAATGAATCAGCTTCGTG GCAAGAAATC

[0216] 351 TTGTCACACT GGACTTGGTA GGTCCGCCGG TTGGAACATA CCAATCGGCCTTCTGTATTG TGACCTGCCA

[0217] 421 GAACCAAGAA AGCCCCTAGA GAAAGCTGTT GCCAACTTTT TTTCAGGCTCTTGCGCCCCC TGTGCCGATG

[0218] 491 GAACCGATTT CCCACAACTG TGTCAGTTGT GCCCTGGTTG TGGCTGCTCCACCTTGAACC AATACTTTGG

[0219] 561 ATATTCAGGA GCCTTTAAGT GCTTGAAAGA CGGCGCAGGT GACGTCGCTTTCGTTAAGCA TTCCACTATA

[0220] 631 TTTGAGAATT TGGCAAACAA GGCCGATAGA GATCAGTATG AGCTGTTATGTCTAGACAAC ACACGTAAAC

[0221] 701 CAGTAGACGA GTATAAGGAT TGTCATCTGG CCCAGGTCCC TTCACATACCGTAGTTGCAC GTTCTATGGG

[0222] 771 TGGTAAAGAG GATCTAATCT GGGAGCTGCT TAATCAAGCA CAGGAACACTTCGGTAAGGA TAAGTCTAAA

[0223] 841 GAGTTTCAAT TGTTTTCCTC ACCTCATGGT AAGGACCTTT TATTTAAAGACTCCGCACAT GGCTTTTTAA

[0224] 911 AAGTTCCACC TAGGATGGAT GCCAAAATGT ACTTGGGTTA TGAATATGTTACAGCAATTC GTAACCTTCG

[0225] 981 AGAGGGTACG TGTCCAGAAG CTCCCACTGA TGAATGTAAG CCTGTAAAGTGGTGTGCCCT TTCCCATCAC

[0226] 1051 GAAAGATTGA AGTGTGATGA GTGGAGTGTA AACTCAGTGG GAAAGATAGAGTGCGTTTCA GCTGAGACCA

[0227] 1121 CAGAAGATTG CATTGCAAAA ATCATGAACG GTGAGGCCGA TGCTATGAGTTTAGATGGTG GCTTTGTTTA

[0228] 1191 CATTGCTGGA AAGTGTGGAC TTGTTCCTGT ATTGGCAGAA AATTACAACAAGTCCGATAA TTGCGAGGAC

[0229] 1261 ACTCCCGAAG CAGGTTACTT CGCCATAGCA GTTGTAAAGA AATCTGCCTCCGATTTGACT TGGGACAATC

[0230] 1331 TAAAAGGTAA GAAAAGTTGT CATACAGCTG TTGGTAGAAC CGCAGGCTGGAACATTCCTA TGGGCCTGCT

[0231] 1401 TTACAACAAG ATAAACCATT GTAGATTCGA TGAGTTTTTT TCAGAGGGCTGTGCCCCTGG TTCTAAAAAG

[0232] 1471 GACAGTTCCT TGTGTAAATT GTGTATGGGA TCTGGTTTGA ACCTATGCGAGCCTAACAAC AAGGAAGGCT

[0233] 1541 ATTACGGATA CACTGGCGCT TTTAGATGTC TTGTCGAGAA GGGAGATGTTGCCTTTGTAA AACACCAAAC

[0234] 1611 CGTTCCACAA AACACTGGAG GAAAGAATCC TGACCCCTGG GCTAAAAACTTAAACGAGAA GGATTACGAG

[0235] 1681 CTACTGTGTC TTGATGGTAC ACGAAAACCA GTAGAAGAGT ATGCCAACTGCCATTTAGCT AGAGCACCTA

[0236] 1751 ACCATGCAGT AGTTACGCGA AAGGATAAGG AAGCTTGTGT GCACAAAATTTTGAGACAAC AGCAACACTT

[0237] 1821 GTTTGGCTCT AATGTAACAG ACTGTTCCGG AAATTTTTGT CTTTTCAGGTCCGAGACTAA GGACCTGCTT

[0238] 1891 TTTAGAGACG ATACCGTGTG CTTGGCTAAA TTGCATGATA GAAATACGTACGAGAAATAT TTAGGAGAAG

[0239] 1961 AATACGTGAA AGCAGTCGGT AACCTTAGAA AATGCTCAAC GTCATCATTGCTTGAAGCCT GTACATTCAG

[0240] 2031 GCGTCCA

[0241] The nucleotide sequence of the nucleic acid molecule expressing recombinant human albumin is such as SEQ ID NO.23, and the amino acid sequence is such as SEQ ID NO.22.

[0242] The sequence of SEQ ID NO.22 is as follows:

[0243] 1 DAHKSEVAHR FKDLGEENFK ALVLIAFAQY LQQCPFEDHV KLVNEVTEFA KTCVADESAENCDKSLHTLF

[0244] 71 GDKLCTVATL RETYGEMADC CAKQEPERNE CFLQHKDDNP NLPRLVRPEV DVMCTAFHDNEETFLKKYLY

[0245] 141 EIARRHPYFY APELLFFAKR YKAAFTECCQ AADKAACLLP KLDELRDEGK ASSAKQRLKCASLQKFGERA

[0246] 211 FKAWAVARLS QRFPKAEFAE VSKLVTDLTK VHTECCHGDL LECADDRADL AKYICENQDSISSKLKECCE

[0247] 281 KPLLEKSHCI AEVENDEMPA DLPSLAADFV ESKDVCKNYA EAKDVFLGMF LYEYARRHHDYSVVLLLRLA

[0248] 351 KTYETTLEKC CAAADPHECY AKVFDEFKPL VEEPQNLIKQ NCELFEQLGE YKFQNALLVRYTKKVPQVST

[0249] 421 PTLVEVSRNLGKVGSKCCKH PEAKRMPCAE DYLSVVLNQL CVLHEKTPVS DRVTKCCTESLVNRRPCFSA

[0250] 491 LEVDETYVPK EFNAETFTFH ADICTLSEKE RQIKKQTALV ELVKHKPKAT KEQLKAVMDDFAAFVEKCCK

[0251] 561 ADDKETCFAE EGKKLVAASQ AALGL

[0252] The sequence of SEQ ID NO.23 is as follows:

[0253] 1 GACGCACACA AATCCGAAGT AGCTCACCGT TTCAAAGACC TGGGCGAAGAAAATTTCAAA GCGCTGGTGC

[0254] 71 TGATCGCTTT TGCACAGTAT CTGCAACAGT GCCCATTTGA AGATCACGTGAAACTGGTAA ACGAAGTCAC

[0255] 141 CGAATTTGCG AAAACCTGTG TGGCTGACGA AAGCGCGGAA AATTGCGACAAATCCCTGCA CACTCTGTTC

[0256] 211 GGTGACAAAC TGTGTACCGT TGCAACTCTG CGCGAAACCT ACGGTGAAATGGCAGATTGT TGCGCGAAAC

[0257] 281 AGGAACCGGA ACGTAACGAA TGTTTCCTGC AGCATAAAGA TGATAACCCTAACCTGCCAC GTCTGGTTCG

[0258] 351 TCCGGAAGTG GACGTTATGT GCACGGCATT CCACGACAAC GAAGAAACCTTTCTGAAAAA ATACCTGTAC

[0259] 421 GAAATCGCTC GTCGTCACCC GTATTTCTAC GCACCGGAAC TGCTGTTCTTTGCGAAACGC TATAAAGCTG

[0260] 491 CGTTCACCGA ATGTTGCCAG GCGGCTGATA AAGCAGCCTG CCTGCTGCCGAAACTGGACG AACTGCGCGA

[0261] 561 TGAAGGCAAA GCTTCTTCTG CGAAACAACG TCTGAAATGC GCGAGCCTGCAGAAATTCGG TGAACGCGCG

[0262] 631 TTCAAAGCAT GGGCTGTCGC ACGCCTGTCC CAACGTTTCC CGAAAGCTGAATTTGCAGAA GTATCTAAAC

[0263] 701 TGGTCACCGA CCTGACGAAA GTACATACTG AATGCTGCCA TGGCGATCTGCTGGAATGCG CTGACGACCG

[0264] 771 TGCGGACCTG GCGAAATACA TTTGCGAGAA CCAAGACAGC ATCTCCTCTAAGCTGAAAGA ATGCTGCGAA

[0265] 841 AAGCCGCTGC TGGAAAAGTC CCATTGTATC GCAGAAGTAG AAAACGACGAAATGCCGGCG GATCTGCCTT

[0266] 911 CTCTGGCTGC GGATTTCGTT GAATCTAAGG ATGTTTGCAA AAACTACGCTGAGGCTAAAG ATGTTTTCCT

[0267] 981 GGGTATGTTC CTGTACGAAT ACGCTCGTCG CCACCCAGAT TACAGCGTTGTACTGCTGCT GCGTCTGGCG

[0268] 1051 AAGACCTACG AAACCACTCT GGAAAAATGT TGCGCTGCGG CAGACCCACACGAATGCTAC GCTAAAGTTT

[0269] 1121 TCGACGAGTT CAAGCCACTG GTAGAGGAAC CGCAGAATCT GATCAAACAGAACTGTGAGC TGTTCGAACA

[0270] 1191 GCTGGGTGAA TACAAGTTTC AGAACGCTCT GCTGGTTCGC TACACCAAAAAGTTCCTCCA GGTTAGCACC

[0271] 1261 CCAACTCTGG TAGAAGTTTC TCGTAACCTG GGCAAGGTTG GTTCCAAGTGCTGCAAACAC CCGGAGGCGA

[0272] 1331 AACGTATGCC GTGTGCAGAA GATTACCTGT CTGTGGTTCT GAACCAGCTGTGCGTGCTGC ACGAAAAGAC

[0273] 1401 TCCGGTGTCT GACCGTGTCA CCAAATGCTG TACTGAGTCT CTGGTCAATCGCCGCCCGTG TTTCAGCGCT

[0274] 1471 CTGGAAGTTG ACGAGACGTA CGTGCCGAAA GAATTTAATG CAGAAACCTTCACCTTCCAT GCGGACATCT

[0275] 1541 GTACTCTGAG CGAAAAAGAA CGTCAGATCAAAAACAGAC TGCGCTGGTTGAGCTGGTTA AGCACAAGCC

[0276] 1611 AAAGGCAACT AAAGAACAGC TGAAAGCGGT TATGGACGAC TTCGCGGCGTTCGTTGAAAA GTGCTGTAAG

[0277] 1681 GCTGACGACA AAGAAACGTG CTTTGCAGAA GAGGGCAAAA AACTGGTCGCGGCATCTCAG GCTGCTCTGG

[0278] 1751 GCCTG

[0279] The nucleotide sequence of the nucleic acid molecule expressing recombinant human basic fibroblast growth factor is as shown in SEQ ID NO.25, and the amino acid sequence is as shown in SEQ ID NO.24.

[0280] The sequence of SEQ ID NO.24 is as follows:

[0281] 1 PALPEDGGSG AFPPGHFKDP KRLYCKNGGF FLRIHPDGRV DGVREKSDPH IKLQLQAEERGVVSIKGVCA

[0282] 71 NRYLAMKEDG RLLASKCVTD ECFFFERLES NNYNTYRSRK YTSWYVALKR TGQYKLGSKTGPGQKAILFL

[0283] 141 PMSAKS

[0284] The sequence of SEQ ID NO.25 is as follows:

[0285] 1 CCAGCTTTGC CAGAAGACGG TGGAAGTGGT GCCTTCCCCC CAGGACACTT CAAAGATCCAAAAAGGTTAT

[0286] 71 ACGTAAAAAA CGGTGGTTTC TTCTTAAGAA TCCATCCTGA TGGTCGAGTA GATGGTGTTAGAGAAAAAATC

[0287] 141 AGACCCTCAC ATCAAGTTAC AACTTCAAGC TGAAGAAAGA GGATGGTCT CTATCAAGGGAGTTTGTGCC

[0288] 211 AATAGGTATC TAGCTATGAA GGAAGATGGA AGATTATTGG CTTCAAAGTG TGTGACTGATGAATGTTTTT

[0289] 281 TCTTCGAGAG GTTGGAGTCT AACAACTATA ACACGTACCG TTCTAGAAAA TACACCTCTTGGTATGTCGC

[0290] 351 CCTAAAGAGAACAGGACAAT ACAAGCTTGGTTCTAAAACAGGACCTGGTC AGAAAGCCATTTTGTTTCTA

[0291] 421 CCAATGTCAG CTAAGTCT

[0292] Preferably, the recombinant Pichia pastoris engineered bacteria further includes a signal peptide that can promote protein expression, and the nucleotide sequence of the signal peptide is shown in SEQ ID NO.20.

[0293] The sequence of SEQ ID NO.20 is as follows:

[0294] 1 ATGAGATTCC CCTCCATCTT CACCGCTGTT TTGTTCGCTG CTTCTTCTGC TTTGGCTGCTCCAGTTAACA

[0295] 71 CTACTACTGA GGACGAGACT GCTCAGATTC CAGCTGAAGC TGTTATTGGT TACTCCGACTTGGAAGGTGA

[0296] 141 CTTCGACGTT GCTGTTTTGC CATTCTCCAA CTCCACCAAC AACGGTCTGT TGTTCATCAACACCACTATC

[0297] 211 GCTTCCATTG CCGCTAAAGA AGAAGGCGTT TCCTTGGAGA AGAGAGAGGC TGAAGCT

[0298] The second aspect of the present invention provides a method for constructing the recombinant Pichia pastoris engineered bacteria of the present invention, comprising the following steps:

[0299] S1. The FDH1 gene was knocked out by using CRISPR-Cas9 gene editing technology. The strain with the FDH1 gene knocked out was named recombinant engineered strain 1.

[0300] S2, overexpressing DAK2 and SHM2 genes into recombinant engineering bacteria 1, named as recombinant engineering bacteria 2;

[0301] S3. Overexpressing the FBA1 and MTD1 genes and integrating them into the recombinant engineered bacteria 2, named as the recombinant engineered bacteria 3, which is the recombinant Pichia pastoris engineered bacteria.

[0302] The nucleotide sequence of the Cas9 gene is shown in SEQ ID NO.14, and the encoded amino acid sequence is shown in SEQ ID NO.15.

[0303] The sequence of SEQ ID NO.14 is as follows:

[0304] 1 ATGGACAAGA AGTACTCCAT CGGTCTGGAC ATCGGAACTA ACTCTGTTGGTTGGGCTGTT ATCACCGACG

[0305] 71 AGTACAAGGT TCCATCCAAG AAGTTCAAGG TCCTGGGTAA CACTGACAGACACTCCATCA AGAAGAACTT

[0306] 141 GATCGGAGCC TTGTTGTTCG ACTCTGGTGA AACTGCTGAG GCCACCAGATTGAAGAGAAC TGCCAGAAGA

[0307] 211 AGATACACCA GACGTAAGAA CAGAATCTGC TACCTGCAAG AGATTTTCTCCAACGAGATG GCCAAGGTCG

[0308] 281 ACGACTCATTTTCCACAGA TTGGAAGAGT CCTTCCTGGT CGAAGAGGATAAGAAGCACG AGAGACACCC

[0309] 351 AATCTTCGGT AACATCGTTG ACGAGGTTGC TTACCACGAG AAGTACCCAACTATCTACCA CCTGAGAAAG

[0310] 421 AAGTTGGTTG ACTCCACTGA CAAGGCCGAC TTGAGATTGA TCTACTTGGCTTTTGGCCCAC ATGATTAAGT

[0311] 491 TCAGAGGTCA CTTCTTGATC GAGGGTGACT TGAACCCAGA CAACTCTGACGTTGACAAGC TGTTCATCCA

[0312] 561 GTTGGTCCAG ACCTACAACC AGCTGTTCGA AGAGAACCCT ATTAACGCTTCTGGTGTTGA CGCTAAGGCT

[0313] 631 ATCTTGTCTG CCAGATTGTC CAAGTCCAGA AGATTGGAGA ACCTGATCGCTCAATTGCCA GGTGAGAGA

[0314] 701 AGAACGGTTT GTTCGGTAAC TTGATTGCCC TGTCCTTGGG TTTGACCCCAAACTTCAAGT CCAACTTCGA

[0315] 771 TTTGGCTGAG GACGCCAAGT TGCAGTTGTC TAAGGATACT TACGACGACGACCTGGACAA CTTGTTGGCT

[0316] 841 CAAATTGGTG ACCAGTACGC CGACTTGTTT TTGGCTGCTA AGAACTTGTCCGACGCCATC TTGTTGTCCG

[0317] 911 ACATCTTGAG AGTTAACACC GAGATCACTA AGGCTCCATT GTCCGCTTCCATGATCAAGA GATACGACGA

[0318] 981 ACACCACCAG GACTTGACTT TGTTGAAGGC CTTGGTTAGA CAGCAGCTGCCTGAGAAGTA AAAGAAATC

[0319] 1051 TTCTTCGATC AGTCCAAGAA CGGCTACGCT GGTTACATTG ATGGTGGTGCTTCTCAAGAA GAGTTCTACA

[0320] 1121 AGTTCATCAA GCCCATCTTG GAGAAGATGG ACGGTACTGA AGAGTTGCTGGTCAAGTTGA ACAGAGAGGA

[0321] 1191 CTTGCTGAGA AAGCAGAGAA CCTTCGACAA CGGTTCCATT CCACACCAGATTCACTTGGG TGAGTTGCAC

[0322] 1261 GCTATCTTGC GTAGACAAGA GGACTTCTAC CCATTCCTGA AGGACAACAGAGAGAAGATC GAAAAGATCC

[0323] 1331 TGACCTTCAG AATCCCCTAC TACGTTGGTC CATTGGCTAG AGGTAACTCTAGATTCGCTT GGATGACCAG

[0324] 1401 AAAGTCCGAA GAGACTATCA CCCCATGGAA CTTCGAAGAG GTTGTTGACAAGGGTGCTTC CGCTCAATCC

[0325] 1471 TTCATCGAGA GAATGACTAA CTTCGACAAG AACCTGCCAA ACGAGAAGGTCTTGCCAAAG CACTCTTTGC

[0326] 1541 TGTACGAGTA CTTCACCGTC TACAACGAGC TGACTAAGGT CAAGTACGTTACCGAGGGTA TGAGAAAGCC

[0327] 1611 AGCTTTCTTG TCTGGTGAGC AAAAGAAGGC TATCGTCGAC TTGCTGTTCAAGACCAACAG AAAGGTTACC

[0328] 1681 GTCAAGCAGC TGAAAGAGGA CTACTTCAAG AAAATCGAGT GCTTCGACTCCGTCGAGATT TCCGGTGTTG

[0329] 1751 TOTAL CAACGCCTCC TTGGGAACCT ACCACGACTT GTTGATCATCAAGGACA TOTALTCTT

[0330] 1821 GGACAACGAG GAAAACGAGG ACATTTTGGA GGACATCGTC TTGACCTTGACTCTGTTCGA GGACAGAG

[0331] 1891 ATGATTGAGG AAAGACTTAA GACTTACGCC CACCTGTTCG ACGACAAGGTTATGAAGCAA CTGAAGAGGC

[0332] 1961 GTAGATACAC CGGTTGGGGT AGATTGTCTA GAAAGCTGAT CAACGGTATCAGAGACAAGC AGTCCGGTAA

[0333] 2031 GACTATCCTG GACTTTTTGA AGTCCGACGG TTTCGCCAAC CGTAACTTCATGCAATTGAT CCACGACGAC

[0334] 2101 TCCCTGACTT TCAAAGAGGA CATTCAGAAG GCTCAGGTTT CCGGTCAAGGTGATTCCTTG CATGAGCACA

[0335] 2171 TTGCTAACTT GGCTGGTTCC CCAGCTATCA AGAAGGGTAT CTTGCAGACCGTTAAGGTTG TCGACGAGTT

[0336] 2241 GGTTAAGGTT ATGGGTAGAC ACAAGCCCGA GAACATCGTT ATCGAAATGGCTAGAGAGAA CCAGACTACC

[0337] 2311 CAGAAGGGTC AAAAGAACTC CAGAGAAAGA ATGAAGAGGA TCGAAGGGTATCAAAGAG CTGGGTTCCC

[0338] 2381 NATIONAL AGAGCACCCA GTTGAGACA CCCAGCTGCA GAACGAAAAGCTGTACTTGT ACTACTTGCA

[0339] 2451 GAACGGTAGA GACATGTACG TCGACCAAGA GCTGGACATT AACAGACTGTCTGACTACGA CGTTGACCAC

[0340] 2521 ATCGTTCCAC AGTCTTTTCT GAAGGACA TCCATCGACA ACAAGGTCTTGACTAGATCC GATAGACA

[0341] 2591 GGGGTAAGTC CGACAACGTT CCTTCTGAAG AGGTCGTCAA GAAGATGAACTACTGGC GTCAGTTGCT

[0342] 2661 GAACGCCAAG CTGATTACTC AGAGGAAGTT CGACAACTTG ACTAAGGCTGAGAGAGGTGG TTTGTCCGAA

[0343] 2731 TTGGATAAGG CCGGTTTCAT CAGACAAG CTGGTCGAGA CTAGACAGATCACAAAGCAC GTTGCCCAAA

[0344] 2801 TCTTGGACTC ACTAAGTACG ACTAAGTACG CAAGTTGATCCGTGAGGTTA AGGTCATCAC

[0345] 2871 CCTGAAGTCC AAGTTGGTGT CCGATTTCAG AAAGGACTTC CAATTCTACAAGGTCCGTGA GATCAACAAC

[0346] 2941 TACCACCATG CTCACGACGC TTACTTGAAC GCTGTTGTTG GTACTGCCCTGATCAAAAAG TACCCTAAGC

[0347] 3011 TGGAATCCGA GTTCGTCTAC GGTGACTACA AGGTTTACGA CGTCAGAAAGATGATCGCCA AGTCCGAACA

[0348] 3081 AGAGATCGGT AAGGCTACTG CCAAGTACTT TTTCTACTCC AACATCATGAACTTTTTCAA GACTGAGATC

[0349] 3151 ACCCTGGCCA ACGGTGAGAT CAGAAAAAGA CCACTGATCG AGACTAACGGTGAAACCGGT GAAATCGTTT

[0350] 3221 GGGACAAGGG TAGAGACTTC GCCACCGTTA GAAAGGTTTT GTCCATGCCACAGGTCAACA TCGTCAAAAA

[0351] 3291 GACTGAGGTT CAGACCGGTG GTTTCTCCAA AGAGTCCATC TTGCCTAAGAGAAACTCCGA CAAGCTGATC

[0352] 3361 GCCAGAAAGA AGGATTGGGA CCCAAAGAAG TACGGCGGTT TCGATTCTCCAACTGTTGCC TACTCCGTTT

[0353] 3431 TGGTTGTTGC TAAGGTCGAA AAGGGCAAGT CTAAGAAGCT GAAGTCCGTCAAAGAACTGC TGGGTATCAC

[0354] 3501 TATCATGGAA AGGTCCAGCT TTGAGAAGAA CCCAATCGAC TTTTTGGAGGCCAAGGGTTA CAAAGAGGTG

[0355] 3571 AAGAAGGACC TGATTATCAA GCTGCCAAAG TACTCCCTGT TCGAGTTGGAAAACGGTAGA AAGAGAATGT

[0356] 3641 TGGCTTCCGC CGGTGAATTG CAAAAGGGTA ACGAATTGGC TCTGCCATCCAAGTACGTCA ACTTTCTGTA

[0357] 3711 CTTGGCCTCT CACTACGAGA AGTTGAAGGG TTCTCCAGAG GACAACGAACAGAAGCAGTT GTTCGTTGAG

[0358] 3781 CAGCACAAGC ACTACTTGGA CGAGATCATT GAGCAGATTT CCGAGTTCTCCAAGCGTGTT ATTTTGGCTG

[0359] 3851 ATGCCAACCT GGATAAGGTC TTGTCCGCCT ACAACAAGCA CAGAGATAAGCCAATTAGAG AGCAGGCTGA

[0360] 3921 GAACATCATC CACTTGTTCA CTTTGACTAA CCTGGGTGCC CCAGCTGCTTTTAAGTACTT CGACACTACC

[0361] 3991 ATCGACAGAA AGAGATACAC CTCCACCAAA GAGGTTTTGG ACGCTACTTTGATCCACCAG TCCATCACTG

[0362] 4061 GTCTGTACGA AACCAGAATT GACTTGTCCC AGCTTGGTGG TGACCCTAAGAAGAAGAGAA AGGTCTAA

[0363] The sequence of SEQ ID NO.15 is as follows:

[0364] 1 MDKKYSIGLD IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGALLFDSGETAE ATRLKRTARR

[0365] 71 RYTRRKNRIC YLQEIFSNEM AKVDDSFFHR LEESFLVEED KKHERHPIFGNIVDEVAYHE KYPTIYHLRK

[0366] 141 KLVDSTDKAD LRLIYLALAH MIKFRGHFLI EGDLNPDNSD VDKLFIQLVQTYNQLFEENP INASGVDAKA

[0367] 211 ILSARLSKSR RLENLIAQLP GEKKNGLFGN LIALSLGLTP NFKSNFDLAEDAKLQLSKDT YDDDLDNLLA

[0368] 281 QIGDQYADLF LAAKNLSDAI LLSDILRVNT EITKAPLSAS MIKRYDEHHQDLTLLKALVR QQLPEKYKEI

[0369] 351 FFDQSKNGYA GYIDGGASQE EFYKFIKPIL EKMDGTEELL VKLNREDLLRKQRTFDNGSI PHQIHLGELH

[0370] 421 AILRRQEDFY PFLKDNREKI EKILTFRIPY YVGPLARGNS RFAWMTRKSEETITPWNFEE VVDKGASAQS

[0371] 491 FIERMTNFDK NLPNEKVLPK HSLLYEYFTV YNELTKVKYV TEGMRKPAFLSGEQKKAIVD LLFKTNRKVT

[0372] 561 VKQLKEDYFK KIECFDSVEI SGVEDRFNAS LGTYHDLLKI IKDKDFLDNEENEDILEDIV LTLTLFEDRE

[0373] 631 MIEERLKTYA HLFDDKVMKQ LKRRRYTGWG RLSRKLINGI RDKQSGKTILDFLKSDGFAN RNFMQLIHDD

[0374] 701 SLTFKEDIQK AQVSGQGDSL HEHIANLAGS PAIKKGILQT VKVVDELVKVMGRHKPENIV IEMARENQTT

[0375] 771 QKGQKNSRER MKRIEEGIKE LGSQILKEHP VENTQLQNEK LYLYYLQNGRDMYVDQELDI NRLSDYDVDH

[0376] 841 IVPQSFLKDD SIDNKVLTRS DKNRGKSDNV PSEEVVKKMK NYWRQLLNAKLITQRKFDNL TKAERGGLSE

[0377] 911 LDKAGFIKRQ LVETRQITKH VAQILDSRMN TKYDENDKLI REVKVITLKSKLVSDFRKDF QFYKVREINN

[0378] 981 YHHAHDAYLN AVVGTALIKK YPKLESEFVY GDYKVYDVRK MIAKSEQEIGKATAKYFFYS NIMNFFKTEI

[0379] 1051 TLANGEIRKR PLIETNGETG EIVWDKGRDF ATVRKVLSMP QVNIVKKTEVQTGGFSKESI LPKRNSDKLI

[0380] 1121 ARKKDWDPKK YGGFDSPTVA YSVLVVAKVE KGKSKKLKSV KELLGITIMERSSFEKNPID FLEAKGYKEV

[0381] 1191 KKDLIIKLPK YSLFELENGR KRMLASAGEL QKGNELALPS KYVNFLYLASHYEKLKGSPE DNEQKQLFVE

[0382] 1261 QHKHYLDEII EQISEFSKRV ILADANLDKV LSAYNKHRDK PIREQAENIIHLFTLTNLGA PAAFKYFDTT

[0383] 1331 IDRKRYTSTK EVLDATLIHQ SITGLYETRI DLSQLGGDPK KKRKV

[0384] Preferably, step S1 includes:

[0385] S11. Construct a co-expression vector of Cas9 and gRNA, named pART502;

[0386] S12: Preparation of donor DNA: Using Pichia pastoris genomic DNA as a template, FDH1-UP-F / FDH1-UP-R and FDH1-DN-F / FDH1-DN-R as primers, PCR was performed to obtain the left and right homology arm fragments of the FDH1 gene; then, using FDH1-UP-F and FDH1-DN-R as primers and the mixture of the left and right homology arm fragments as a DNA template, overlap extension PCR technology was used to obtain the FDH1 knockout donor DNA;

[0387] S13: Construction of a strain for knocking out the FDH1 gene: The Cas9 and gRNA co-expression vector pART502 and the donor DNA were co-transformed into Pichia pastoris CBS7435, spread on a plate, and cultured until colonies appeared. The positive transformants were identified as recombinant engineered bacteria 1 by colony PCR.

[0388] In the present invention, the primer sequences are as follows:

[0389] FDH1-UP-F (SEQ ID NO. 26)

[0390] AGGAATATCTCGTTGAAAGCTACTTG

[0391] FDH1-UP-R (SEQ ID NO. 27)

[0392] CATTTCAATGTTTAAGTGGGTGATGTTGGAGGTATTTGAG

[0393] FDH1-DN-F (SEQ ID NO. 28)

[0394] CCCACTTAACATTGAAATGTATTTAATTTG

[0395] FDH1-DN-R (SEQ ID NO. 29)

[0396] CCTTATCAAGATAGCTAGAAATAG

[0397] Preferably, the coating plate is a selection plate containing blasticidin antibiotic.

[0398] Preferably, step S2 comprises:

[0399] S21. Construction of a DAK2 and SHM2 co-expression vector: synthesize the recombinant expression cassettes encoding the DAK2 gene and the SHM2 gene, and connect them into the plasmid pGAPZA to form an expression vector named pART503;

[0400] S22. The expression vector pART503 was linearized using the DNA restriction endonuclease BglII, and the recombinant engineered bacteria 1 was transformed by electroporation. The cells were spread on a plate and cultured until colonies appeared. The positive transformants were identified as the recombinant engineered bacteria 2 by colony PCR.

[0401] Preferably, step S3 includes:

[0402] S31. Construction of a co-expression vector of FBA1 and MTD1: Synthesize recombinant expression cassettes encoding the FBA1 gene and the MTD1 gene, and connect them into plasmid pGAPZA to form an expression vector named pART504.

[0403] S32. The expression vector pART504 was linearized using the DNA restriction endonuclease BglII, and the recombinant engineered bacteria 2 was transformed by electroporation. The cells were spread on a plate and cultured until colonies appeared. The positive transformants were identified as the recombinant engineered bacteria 3 by colony PCR.

[0404] Preferably, step S21 or S31 further comprises: replacing the zeocin resistance gene with a drug resistance gene; and step S22 or S32 further comprises: the plate is a selection plate containing the antibiotic corresponding to the resistance gene.

[0405] Preferably, the drug resistance gene includes one or more of the blasticidin resistance gene, the geneticin G418 resistance gene, the hygromycin B resistance gene, and the zeocin resistance gene.

[0406] Preferably, after step S3, the following steps are further included:

[0407] S4. Prepare functional carriers.

[0408] S5. Use DNA restriction endonuclease BglII to linearize the functional vector, electroporate to transform the recombinant engineered bacteria 3, spread the cells on a plate, culture until colonies appear, and identify positive transformants by colony PCR.

[0409] Preferably, the functional vectors include a recombinant human transferrin expression vector, a recombinant human albumin expression vector, and a recombinant human basic fibroblast growth factor expression vector.

[0410] The third aspect of the present invention provides the recombinant Pichia pastoris engineered bacteria of the present invention, and the use of the recombinant Pichia pastoris engineered bacteria constructed by the method of the present invention in preparing engineered bacteria that highly express heterologous proteins.

[0411] Preferably, the heterologous protein includes recombinant human transferrin, recombinant human albumin, and recombinant human basic fibroblast growth factor.

[0412] The fourth aspect of the present invention provides a Pichia pastoris engineered bacterium that highly expresses recombinant human transferrin, wherein the recombinant Pichia pastoris engineered bacterium is a Pichia pastoris strain in which the FDH1 gene shown in SEQ ID NO.1 is targetedly knocked out, and the DAK2 gene shown in SEQ ID NO.3, the SHM2 gene shown in SEQ ID NO.5, the FBA1 gene shown in SEQ ID NO.7 and the MTD1 gene shown in SEQ ID NO.9 are inserted, and the nucleic acid molecule encoding the cerevisiae a mating factor signal peptide shown in SEQ ID NO.20 and the recombinant human transferrin expression vector shown in SEQ ID NO.12 are inserted.

[0413] The fifth aspect of the present invention provides a Pichia pastoris engineered bacterium that highly expresses recombinant human albumin, wherein the recombinant Pichia pastoris engineered bacterium is a Pichia pastoris strain in which the FDH1 gene shown in SEQ ID NO.1 is targetedly knocked out, and the DAK2 gene shown in SEQ ID NO.3, the SHM2 gene shown in SEQ ID NO.5, the FBA1 gene shown in SEQ ID NO.7 and the MTD1 gene shown in SEQ ID NO.9 are inserted, and the nucleic acid molecule encoding the cerevisiae a mating factor signal peptide shown in SEQ ID NO.20 and the recombinant human albumin expression vector shown in SEQ ID NO.23 are inserted.

[0414] The sixth aspect of the present invention provides a Pichia pastoris engineered bacterium producing a recombinant human basic fibroblast growth factor, wherein the recombinant Pichia pastoris engineered bacterium is a Pichia pastoris strain in which the FDH1 gene shown in SEQ ID NO.1 is targetedly knocked out, and the DAK2 gene shown in SEQ ID NO.3, the SHM2 gene shown in SEQ ID NO.5, the FBA1 gene shown in SEQ ID NO.7 and the MTD1 gene shown in SEQ ID NO.9 are inserted, and the nucleic acid molecule encoding the cerevisiae a mating factor signal peptide shown in SEQ ID NO.20 and the expression vector encoding the recombinant human basic fibroblast growth factor shown in SEQ ID NO.25 are inserted.

[0415] Compared with the prior art, the beneficial effects and significant progress of the technical solution of the present invention are: the present invention provides a recombinant Pichia pastoris engineered bacteria and its construction method and application, and the recombinant Pichia pastoris engineered bacteria can significantly increase the yield of recombinant protein. The recombinant Pichia pastoris engineered bacteria of the present invention uses CRISPR-Cas9 gene editing technology to target the knockout of the FDH1 gene; and the overexpression of the DAK2 and SHM2 genes is integrated into the strain, and the overexpression of the FBA1 and MTD1 genes is integrated into the strain, which significantly enhances the heterologous protein secretion ability of the engineered strain. The Pichia pastoris engineered bacteria of the present invention show strong potential in improving protein production and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0416] In order to more clearly illustrate the technical solution of the present invention, the drawings used in the embodiments of the present invention are briefly introduced below.

[0417] Figure 1 Schematic diagram of the structure of the expression vector pPICZA in Example 1;

[0418] Figure 2 This is a schematic diagram of the structure of the recombinant expression vector pART502 encoding the Cas9 recombinant nucleic acid molecule and the FDH1-gRNA recombinant nucleic acid molecule in Example 1;

[0419] Figure 3 It is a schematic diagram of the structure of the recombinant expression vector pART503 encoding DAK2 and SHM2 genes in Example 2;

[0420] Figure 4 This is a schematic diagram of the structure of the recombinant expression vector pART504 encoding the FBA1 and MTD1 genes in Example 3;

[0421] Figure 5 This is a schematic diagram of the results of expressing recombinant human transferrin by each group of recombinant engineering bacteria in Example 4;

[0422] Figure 6 This is a schematic diagram of the results of each group of recombinant engineering bacteria expressing recombinant human albumin in Example 5;

[0423] Figure 7 This is a schematic diagram of the results of each group of recombinant engineering bacteria expressing recombinant human basic fibroblast growth factor in Example 6. DETAILED DESCRIPTION

[0424] The present invention is further described below in conjunction with specific examples. The examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes and modifications to the present invention, and these equivalent forms also fall within the scope limited by the appended claims of the application.

[0425] In order to more fully understand the present invention, the professional terms in the present invention are explained below.

[0426] The "expression cassette" of the present invention refers to a gene expression system that contains all the necessary elements required for the expression of foreign proteins, including a promoter, a foreign gene cloning site, a signal peptide sequence, a mature peptide coding sequence of the target protein, a terminator, a screening marker, etc.

[0427] The "vector" of the present invention refers to autonomous DNA that can bring foreign DNA into host cells for replication or ultimately expression of foreign gene DNA. It is mainly divided into cloning vectors and expression vectors. The former is mainly used for gene replication and amplification, while the latter is mainly used for the expression of target genes.

[0428] The "host cell" of the present invention refers to a cell that receives foreign genes during transformation or transduction.

[0429] The "engineered bacteria" of the present invention refers to a fungal cell strain that uses genetic engineering methods to efficiently express exogenous genes.

[0430] If no specific conditions are specified in the examples, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer.

[0431] Unless otherwise specified, in the following embodiments, the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased from the market.

[0432] The molecular biology experimental methods not specifically described in the following examples were all carried out according to the kits and product instructions, or with reference to the methods described in the book Molecular Cloning Experiment Guide (3rd edition) by J. Sambrook.

[0433] The sources of experimental materials and reagents involved in the present invention are as follows:

[0434] Strain: Pichia pastoris CBS7435 was purchased from the American Type Culture Collection (ATCC) on behalf of Ningbo Mingzhou Biotechnology Co., Ltd. It is also known as Komagataella phaffii NRRL Y-11430, ATCC Number: 76273™.

[0435] Expression vector: The expression vector pPICZA was purchased from Hunan Fenghui Biotechnology Co., Ltd., with the catalog number JM006 and a specification of 20 μl; the expression vector pGAPZA was purchased from Hunan Fenghui Biotechnology Co., Ltd., with the catalog number ZT226 and a specification of 20 μl; the following expression cassettes were constructed in the laboratory: 1) Expression cassette, which encodes the Cas9 recombinant nucleic acid molecule and the FDH1-gRNA recombinant nucleic acid molecule, and its structural diagram is shown in the figure Figure 22) an expression cassette encoding a DAK2 recombinant nucleic acid molecule and a SHM2 recombinant nucleic acid molecule, the structure of which is shown in FIG. Figure 3 3) An expression cassette encoding a FBA1 recombinant nucleic acid molecule and a MTD1 recombinant nucleic acid molecule, the structure of which is shown in FIG. Figure 4 shown.

[0436] Enzymes and kits

[0437] Restriction endonuclease BglII (Cat. No. R0144S, specification 2000 units) was purchased from NEB; plasmid extraction kit (Cat. No. DP103-02, specification 50 preps), agarose gel recovery kit (Cat. No. DP209-02, specification 50 preps), DNA product purification kit (Cat. No. DP204-02, specification 50 preps), yeast genomic DNA extraction kit (Cat. No. DP307-02, specification 50 preps) were purchased from Tiangen Biotechnology Co., Ltd.; PrimeSTAR high-fidelity DNA polymerase (Cat. No. R047A, specification 100 Rxns) was purchased from Takara; Zeocin (Cat. No. R25001, specification 10 ml), Geneticin G418 (Cat. No. 10131035, specification 20 ml), Blasticidin (Cat. No. A1113902, specification 20 ml), Hygromycin B B (Cat. No. 10687010, specification 20 ml) was purchased from Thermo Fisher Scientific.

[0438] Culture medium

[0439] Escherichia coli culture medium LB (1% (w / v) tryptone, 0.5% (w / v) yeast extract, 1% (w / v) NaCl, pH 7.0).

[0440] Yeast medium YPD (1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose); yeast screening medium YPDZ (YPD + different concentrations of zeocin); YPDG (YPD + different concentrations of G418); YPDB (YPD + different concentrations of Blasticidin); YPDH (YPD + different concentrations of Hygromycin B).

[0441] Yeast medium BMGY (1% (w / v) yeast extract, 2% (w / v) peptone, 1.34% (w / v) YNB, 0.00004% (w / v) Biotin, 1% glycerol (V / V)), yeast induction medium BMMY (1% (w / v) yeast extract, 2% (w / v) peptone, 1.34% (w / v) YNB, 0.00004% (w / v) Biotin, 0.4% methanol (V / V)), Note: YNB is yeast nitrogen base (Yeast Nitrogen Base); Biotin is biotin.

[0442] Example 1 Construction of FDH1 gene targeted knockout strain (recombinant engineered strain 1)

[0443] 1.1. Construction of Cas9 and gRNA co-expression vector pART502

[0444] In this example, in order to establish a suitable genome editing CRISPR system, an attempt was made to use the dual promoter pHTX1 (nucleotide sequence as shown in SEQ ID NO.21) to construct a vector for co-expression of Cas9 and gRNA.

[0445] Wherein, SEQ ID NO.21 sequence is as follows:

[0446] 1 GTTGTAGTTT TAATATAGTT TGAGTATGAG ATGGAACTCA GAACGAAGGA ATTATCACCAGTTTATATAT

[0447] 71 TCTGAGGAAA GGGTGTGTCC TAAATTGGAC AGTCACGATG GCAATAAACG CTCAGCCAATCAGAATGCAG

[0448] 141 GAGCCATAAA TTGTTGTATT ATTGCTGCAA GATTTATGTG GGTTCACATT CCACTGAATGGTTTTCACTG

[0449] 211 TAGAATTGGT GTCCTAGTTG TTATGTTTCG AGATGTTTTC AAGAAAAACT AAAATGCACAAACTGACCAA

[0450] 281 TAATGTGCCG TCGCGCTTGG TACAAACGTC AGGATTGCCA CCACTTTTT CGCACTCTGGTACAAAAGTT

[0451] 351 CGCACTTCCC ACTCGTATGT AACGAAAAAC AGAGCAGTCT ATCCAGAACG AGACAAATTAGCGCGTACTG

[0452] 421 TCCCATTCCA TAAGGTATCA TAGGAAACGA GAGTCCTCCC CCCATCACGT ATATATAAACACACTGATAT

[0453] 491 CCCACATCCG CTTGTCACCA AACTAATACA TCCAGTTCAA GTTACCTAAA CAAATCAAA

[0454] The recombinant expression cassette encoding the Cas9 gene and the FDH1-gRNA (nucleotide sequence as shown in SEQ ID NO. 13) gene was directly synthesized by a biological company and then ligated into the plasmid pPICZA (such as Figure 1 ) and using gene homologous recombination technology, a biological company was commissioned to replace the zeocin resistance gene with the blasticidin resistance gene (nucleotide sequence as shown in SEQ ID NO.16) to form an expression vector named pART502 (as shown in Figure 2 as shown).

[0455] Among them, the sequence of SEQ ID NO.13 is: GGACGTTGCT GCCATCGCCA.

[0456] 1.2. Construction of FDH1 gene targeted knockout strain (recombinant engineered strain 1)

[0457] In order to prepare the donor DNA for FDH1 knockout, the following operations were performed: using Pichia pastoris genomic DNA as a template, FDH1-UP-F / FDH1-UP-R and FDH1-DN-F / FDH1-DN-R as primers, PCR was performed to obtain the left and right homologous arm fragments of the FDH1 gene; then, using FDH1-UP-F and FDH1-DN-R as primers and a mixture of the left and right homologous arm fragments as a DNA template, overlap extension PCR technology was used to obtain the donor DNA for FDH1 knockout.

[0458] The PCR reaction system is shown in Table 1, and the specific nucleotide sequences of the upstream primer and the downstream primer are shown in Table 2.

[0459] Table 1

[0460]

[0461] Table 2

[0462]

[0463] 2 μg of Cas9 and gRNA co-expression vector and 2 μg of donor DNA were co-transformed into Pichia pastoris CBS7435, respectively, spread on the selection plate YPDB containing blasticidin antibiotic, and cultured at 30°C until colonies appeared. The positive transformants were identified as FDH1 gene targeted knockout strains (recombinant engineered bacteria 1) by colony PCR.

[0464] Example 2 Construction of FDH1 gene targeted knockout strain (recombinant engineered strain 2) co-expressing DAK2 and SHM2

[0465] 2.1 Construction of DAK2 and SHM2 co-expression vector pART503

[0466] The recombinant expression cassettes encoding the DAK2 gene (nucleotide sequence such as SEQ ID NO.3, amino acid sequence such as SEQ ID NO.4) and the SHM2 gene (nucleotide sequence such as SEQ ID NO.5, amino acid sequence such as SEQ ID NO.6) were commissioned to be directly synthesized by a biological company, and then connected into the plasmid pGAPZA respectively. The zeocin resistance gene was replaced by the geneticin G418 resistance gene (nucleotide sequence such as SEQ ID NO.17) by the gene homologous recombination technology, and the expression vector was named pART503 (such as Figure 3 as shown).

[0467] 2.2 Construction of FDH1 gene targeted knockout strain (recombinant engineered strain 2) co-expressing DAK2 and SHM2

[0468] The expression vector pART503 was linearized with DNA restriction endonuclease BglII and transformed into the FDH1 gene targeted knockout strain (recombinant engineered bacteria 1) by electroporation. The cells were spread on the selection plate YPDG containing geneticin G418 antibiotic and cultured at 30°C until colonies appeared. The positive transformants were identified by colony PCR as the FDH1 gene targeted knockout strain (recombinant engineered bacteria 2) co-expressing DAK2 and SHM2.

[0469] Example 3 Construction of FDH1 gene targeted knockout strain (recombinant engineered strain 3) co-expressing FBA1, MTD1, DAK2 and SHM2

[0470] 3.1 Construction of FBA1 and MTD1 co-expression vector pART504

[0471] The recombinant expression cassettes encoding the FBA1 gene (nucleotide sequence such as SEQ ID NO.7, amino acid sequence such as SEQ ID NO.8) and the MTD1 gene (nucleotide sequence such as SEQ ID NO.9, amino acid sequence such as SEQ ID NO.10) were commissioned to be directly synthesized by a biological company, and then connected into the plasmid pGAPZA respectively. The zeocin resistance gene was replaced by the hygromycin B resistance gene (nucleotide sequence such as SEQ ID NO.18) by the gene homologous recombination technology, and the expression vector was named pART504 (such as Figure 4 as shown).

[0472] 3.2 Construction of FDH1 gene targeted knockout strain (recombinant engineered strain 3) co-expressing FBA1, MTD1, DAK2 and SHM2

[0473] The expression vector pART504 was linearized using DNA restriction endonuclease BglII, and the recombinant engineered bacteria 2 was transformed by electroporation. The cells were spread on a selection plate YPDH containing hygromycin B antibiotic and cultured at 30°C until colonies appeared. The positive transformants were identified as recombinant engineered bacteria 3 by colony PCR.

[0474] Example 4 Recombinant human transferrin Pichia pastoris strain and protein expression level detection

[0475] In this example, Pichia pastoris CBS7435 and the recombinant engineered bacteria 1-3 constructed in Examples 1-3 were used to further construct a recombinant human transferrin Pichia pastoris strain, and the protein expression levels of the constructed strains were detected respectively.

[0476] 4.1. Construction of recombinant engineered bacteria 4-7

[0477] The nucleic acid molecule encoding the signal peptide of the mating factor of Saccharomyces cerevisiae a (SEQ ID NO.20) and the nucleic acid molecule encoding the recombinant human transferrin (nucleotide sequence such as SEQ ID NO.12, amino acid sequence such as SEQ ID NO.11) were commissioned to a biological company for direct synthesis, and then ligated into the plasmid pPICZA digested with EcoRI and NotI to form a recombinant human transferrin expression vector.

[0478] The recombinant human transferrin expression vector was linearized using the DNA restriction endonuclease BglII, and electroporated to transform Pichia pastoris CBS7435, recombinant engineered bacteria 1, recombinant engineered bacteria 2, and recombinant engineered bacteria 3, respectively. The cells were spread on the selection plate YPDZ containing different high concentrations of zeocin antibiotics and cultured at 30°C until colonies appeared. The positive transformants identified by colony PCR were recombinant engineered bacteria 4, recombinant engineered bacteria 5, recombinant engineered bacteria 6, and recombinant engineered bacteria 7.

[0479] 4.2. Detection of expression level of recombinant human transferrin in recombinant engineering bacteria 4-7

[0480] Recombinant engineered bacteria 4-7 were inoculated into 40 mL of BMGY culture medium and cultured overnight at 220 rpm and 30°C. The culture medium was centrifuged and the precipitate was resuspended in 40 mL of BMMY culture medium. Induction was started at 220 rpm and 25°C. 0.4% methanol was added every 24 h. After 72 h, the supernatant was collected by centrifugation at 10,000 rpm for 5 min. The supernatant was detected by SDS-PAGE electrophoresis.

[0481] The experimental results are shown in Table 3 and Figure 5 shown.

[0482] Table 3

[0483]

[0484] The results showed that when the FDH1 gene was knocked out and the EXG1, SPR1, FBA1 and MTD1 genes were overexpressed at the same time, the expression level of transferrin was significantly increased, reaching 285%, taking the expression level of recombinant human transferrin of the recombinant engineered bacteria 4 as the control.

[0485] Example 5 Recombinant human albumin Pichia pastoris strain and protein expression level detection

[0486] In this example, Pichia pastoris CBS7435 and the recombinant engineered bacteria 1-3 constructed in Examples 1-3 were used to further construct a recombinant human albumin Pichia pastoris strain, and the protein expression levels of the constructed strains were detected respectively.

[0487] 5.1. Construction of recombinant engineered bacteria 8-11

[0488] The nucleic acid molecule encoding the signal peptide of the mating factor of Saccharomyces cerevisiae a (SEQ ID NO.20) and the nucleic acid molecule encoding the recombinant human albumin (nucleotide sequence such as SEQ ID NO.23, amino acid sequence such as SEQ ID NO.22) were commissioned to a biological company for direct synthesis, and then ligated into the plasmid pPICZA digested with EcoRI and NotI to form a recombinant human albumin expression vector.

[0489] The recombinant human albumin expression vector was linearized using the DNA restriction endonuclease BglII, and electroporated to transform Pichia pastoris CBS7435, recombinant engineered bacteria 1, recombinant engineered bacteria 2 and recombinant engineered bacteria 3, respectively. The cells were spread on the selection plate YPDZ containing different high concentrations of zeocin antibiotics and cultured at 30°C until colonies appeared. The positive transformants identified by colony PCR were recombinant engineered bacteria 8, recombinant engineered bacteria 9, recombinant engineered bacteria 10 and recombinant engineered bacteria 11.

[0490] 5.2. Detection of the expression level of recombinant human albumin in recombinant engineering bacteria 4-7

[0491] Recombinant engineered bacteria 8-11 were inoculated into 40 mL of BMGY culture medium at 220 rpm and 30°C for overnight culture. The culture medium was centrifuged and the precipitate was resuspended in 40 mL of BMMY culture medium. Induction was started at 220 rpm and 25°C. 0.4% methanol was added every 24 h. After 72 h, the supernatant was collected by centrifugation at 10,000 rpm for 5 min. The supernatant was subjected to SDS-PAGE electrophoresis. The specific results are shown in Table 4 and Figure 6 shown.

[0492] Table 4

[0493]

[0494] The results showed that when the FDH1 gene was knocked out and the EXG1, SPR1, FBA1 and MTD1 genes were overexpressed at the same time, the expression level of albumin was significantly increased, reaching 262%, taking the expression level of recombinant human albumin of the recombinant engineered bacteria 8 as the control.

[0495] Example 6 Detection of recombinant human basic fibroblast growth factor Pichia pastoris strain and protein expression level

[0496] In this example, Pichia pastoris CBS7435 and the recombinant engineering bacteria 1-3 constructed in Example 1-3 were used to further construct a recombinant human basic fibroblast growth factor Pichia pastoris strain, and the basic fibroblast growth factor expression level of the constructed strains was detected respectively.

[0497] 6.1. Construction of recombinant engineered bacteria 12-15

[0498] The nucleic acid molecule encoding the signal peptide of the mating factor of Saccharomyces cerevisiae a (SEQ ID NO.20) and the nucleic acid molecule encoding the recombinant human basic fibroblast growth factor (nucleotide sequence such as SEQ ID NO.25, amino acid sequence such as SEQ ID NO.24) were commissioned to a biological company for direct synthesis, and then ligated into the plasmid pPICZA digested with EcoRI and NotI to form a recombinant human basic fibroblast growth factor expression vector.

[0499] The recombinant human basic fibroblast growth factor expression vector was linearized using the DNA restriction endonuclease BglII, and electroporation was used to transform Pichia pastoris CBS7435, recombinant engineered bacteria 1, recombinant engineered bacteria 2 and recombinant engineered bacteria 3, respectively. The cells were spread on the selection plate YPDZ containing different high concentrations of zeocin antibiotics and cultured at 30°C until colonies appeared. The positive transformants identified by colony PCR were recombinant engineered bacteria 12, recombinant engineered bacteria 13, recombinant engineered bacteria 14 and recombinant engineered bacteria 15, respectively.

[0500] 6.2. Detection of the expression level of human basic fibroblast growth factor in recombinant engineered bacteria 12-15

[0501] Recombinant engineered bacteria 12-15 were inoculated into 40 mL BMGY culture medium at 220 rpm and 30°C overnight, the culture medium was centrifuged, the precipitate was resuspended in 40 mL BMMY culture medium, and induction was started at 220 rpm and 25°C. 0.4% methanol was added every 24 h, and centrifuged at 10000 rpm for 5 min after 72 h, and the supernatant was collected. The supernatant was subjected to SDS-PAGE electrophoresis. The specific results are shown in Table 5 and Figure 7 shown.

[0502] Table 5

[0503]

[0504] The results showed that when the FDH1 gene was knocked out and the EXG1, SPR1, FBA1 and MTD1 genes were overexpressed at the same time, the expression level of recombinant human basic fibroblast growth factor in the recombinant engineered bacteria 12 was used as a control, and the expression level of basic fibroblast growth factor was significantly increased to 167%.

[0505] The applicant declares that, in the description of the above specification:

[0506] The descriptions of the terms "this embodiment", "an embodiment of the present invention", "as shown in...", "further", "a further improved technical sub-scheme", etc., mean that the specific features, structures, materials or characteristics described in the embodiment or example are included in at least one embodiment or example of the present invention; in this specification, the schematic expressions of the above terms are not necessarily for the same embodiment or example, and the specific features, structures, materials or characteristics described may be combined or combined in a suitable manner in any one or more embodiments or examples; in addition, a person of ordinary skill in the art may combine or combine the different embodiments or examples and the features of the different embodiments or examples described in this specification without causing any contradiction.

[0507] Finally, it should be noted that:

[0508] The above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them.

[0509] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein, and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention. The non-essential improvements, adjustments or replacements made by those skilled in the art based on the contents of this specification are all within the scope of protection required by the present invention.

Claims

1. A recombinant Pichia pastoris engineered bacterium, characterized in that: The recombinant Pichia pastoris engineered bacteria is a Pichia pastoris strain in which the FDH1 gene shown in SEQ ID NO.1 is targetedly knocked out, and the DAK2 gene shown in SEQ ID NO.3, the SHM2 gene shown in SEQ ID NO.5, the FBA1 gene shown in SEQ ID NO.7, and the MTD1 gene shown in SEQ ID NO.9 are inserted; The recombinant Pichia pastoris is also inserted with a functional vector and a signal peptide that promotes protein expression. The functional vector is a recombinant human transferrin expression vector, a recombinant human albumin expression vector, and a recombinant human basic fibroblast growth factor expression vector; the nucleotide sequence of the signal peptide is shown in SEQ ID NO.20; The recombinant Pichia pastoris engineering bacteria can increase the production of recombinant human transferrin, recombinant human albumin and recombinant human basic fibroblast growth factor proteins.

2. A recombinant Pichia pastoris engineered bacterium according to claim 1, characterized in that: The recombinant Pichia pastoris engineered bacteria is also inserted with a drug resistance gene, which is one or more of a blasticidin resistance gene, a geneticin G418 resistance gene, a hygromycin B resistance gene, and a zeocin resistance gene.

3. The method for constructing a recombinant Pichia pastoris engineered bacterium according to any one of claims 1 to 2, characterized in that: The following steps are involved: S1. The FDH1 gene was knocked out by using CRISPR-Cas9 gene editing technology. The strain with the FDH1 gene knocked out was named recombinant engineered strain 1. S2, overexpressing DAK2 and SHM2 genes into recombinant engineering bacteria 1, named as recombinant engineering bacteria 2; S3. Overexpressing the FBA1 and MTD1 genes and integrating them into the recombinant engineered bacteria 2, named as the recombinant engineered bacteria 3, which is the recombinant Pichia pastoris engineered bacteria.

4. The method for constructing a recombinant Pichia pastoris engineering bacterium according to claim 3, characterized in that: Step S1 includes: S11, construct a Cas9 and gRNA co-expression vector named pART502; S12: Preparation of donor DNA: Using Pichia pastoris genomic DNA as a template, FDH1-UP-F / FDH1-UP-R and FDH1-DN-F / FDH1-DN-R as primers, PCR was performed to obtain the left and right homology arm fragments of the FDH1 gene; then, using FDH1-UP-F and FDH1-DN-R as primers and the mixture of the left and right homology arm fragments as a DNA template, overlap extension PCR technology was used to obtain the FDH1 knockout donor DNA; S13: Construction of a strain for knocking out the FDH1 gene: The Cas9 and gRNA co-expression vector pART502 and the donor DNA were co-transformed into Pichia pastoris, spread on a plate, and cultured until colonies appeared. The positive transformants were identified as recombinant engineered bacteria 1 by colony PCR.

5. The method for constructing a recombinant Pichia pastoris engineering bacterium according to claim 3, characterized in that: Step S2 includes: S21. Construction of a DAK2 and SHM2 co-expression vector: synthesize the recombinant expression cassettes encoding the DAK2 gene and the SHM2 gene, and connect them into the plasmid pGAPZA to form an expression vector named pART503; S22. The expression vector pART503 was linearized using the DNA restriction endonuclease BglII, and the recombinant engineered bacteria 1 was transformed by electroporation. The cells were spread on a plate and cultured until colonies appeared. The positive transformants were identified as the recombinant engineered bacteria 2 by colony PCR.

6. The method for constructing a recombinant Pichia pastoris engineering bacterium according to claim 3, characterized in that: Step S3 includes: S31, constructing a co-expression vector of FBA1 and MTD1: synthesizing recombinant expression cassettes encoding the FBA1 gene and the MTD1 gene, and then connecting them into the plasmid pGAPZA to form an expression vector, named pART504; S32. The expression vector pART504 was linearized using the DNA restriction endonuclease BglII, and the recombinant engineered bacteria 2 was transformed by electroporation. The cells were spread on a plate and cultured until colonies appeared. The positive transformants were identified as the recombinant engineered bacteria 3 by colony PCR.

7. The method for constructing a recombinant Pichia pastoris engineered bacterium according to claim 5 or 6, characterized in that: Step S21 or S31 further includes: replacing the zeocin resistance gene with a drug resistance gene; Step S22 or S32 also includes: the plate is a selection plate containing an antibiotic corresponding to the resistance gene; The drug resistance gene is one or more of the blasticidin resistance gene, the geneticin G418 resistance gene, the hygromycin B resistance gene, and the zeocin resistance gene.

8. The method for constructing a recombinant Pichia pastoris engineering bacterium according to claim 3, characterized in that: After step S3, the method further includes: S4, preparing a functional carrier; S5, using DNA restriction endonuclease BglII to linearize the functional vector, electroporating and transforming the recombinant engineered bacteria 3, spreading the cells on a plate, culturing until colonies appear, and identifying positive transformants by colony PCR; The functional vectors are a recombinant human transferrin expression vector, a recombinant human albumin expression vector, and a recombinant human basic fibroblast growth factor expression vector.

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