CN114634946B

CN114634946B - Construction of Pichia pastoris genetically engineered bacteria and application of pichia pastoris genetically engineered bacteria in improving methanol assimilation rate and fixing carbon dioxide

Info

Publication number: CN114634946B
Application number: CN202210181077.8A
Authority: CN
Inventors: 王昕�; 郭元柯; 王静; 马琛; 陈可泉
Original assignee: Nanjing Tech University
Current assignee: Nanjing Tech University
Priority date: 2022-02-26
Filing date: 2022-02-26
Publication date: 2023-12-22
Anticipated expiration: 2042-02-26
Also published as: CN114634946A

Abstract

The invention discloses construction of pichia pastoris genetic engineering bacteria and application thereof in improving methanol assimilation rate and fixing carbon dioxide, wherein a methanol metabolism path of pichia pastoris is modified, and a reducing glycine path is reconstructed by knocking out a dissimilation path, so that carbon atom loss caused by carbon dioxide generated by dissimilation is eliminated, and the utilization efficiency of carbon atoms is improved. In order to further improve the assimilation of methanol and carbon dioxide, the preferred compartmentalization rational design is carried out on the methanol catabolism path of the pichia pastoris, the biomass of the metabolizing modified strain can be improved by 8.7 percent, compared with the original pichia pastoris GS115, the genetically engineered strain has the same methanol consumption rate, but obvious formic acid accumulation of about 0.66g/L, and further proves that the pichia pastoris genetically engineered strain uses more carbon for central metabolism.

Description

Construction of Pichia pastoris genetically engineered bacteria and application of pichia pastoris genetically engineered bacteria in improving methanol assimilation rate and fixing carbon dioxide

Technical Field

The invention belongs to the field of strain metabolism transformation, and particularly relates to construction of a Pichia pastoris genetically engineered bacterium and application of the Pichia pastoris genetically engineered bacterium in improving methanol assimilation rate and fixing carbon dioxide.

Background

Global food shortage has prompted the search for alternative sources of raw materials for biofuel or chemical production. Methanol is a non-food-derived C1 feedstock and is becoming an attractive alternative to industrial bio-production due to its low price and high energy content. Methanol can be readily produced from coal, natural gas or other renewable resources with annual production capacity exceeding 1 million tons worldwide. In recent years, by CO ₂ The technology of synthesizing methanol by hydrogenation is broken through. The development of methanol bio-production is a key factor in establishing sustainable cycle carbon economy.

Methylotrophic bacteria are microorganisms that are capable of growing on methanol and providing a carbon source and an energy source with methanol, both of which are necessary for methanol bio-production. Many natural methylotrophic bacteria, such as methanobacteria and methanobacteria, have recently been identified and used to produce single cell proteins, PHB or amino acids. In addition, some model organisms, such as E.coli, saccharomyces cerevisiae and Corynebacterium glutamicum, are also designed for methanol assimilation. Wherein Pichia pastoris is preparedP.pastoris）Is reclassified asKomagatella phaffiiIs representative of methylotrophic yeasts. Pichia pastoris grows faster on methanol medium than the bacterial methylotrophic bacteria. The high cell density fermentation of Pasteurella in minimal media is easy to achieve and various fermentation strategies have been established with strict control by methanol.

All these characteristics mean that pichia pastoris has a greater potential for large-scale industrial use than other representative methylotrophic organisms. Some biopharmaceuticals or proteins produced by pichia pastoris have been marketed. However, the methanol utilization rate of Pichia pastoris is low, and most of carbon flow generates CO through a dissimilation path ₂ Loss, furthermore, the energy in the methanol assimilation pathway is limited to the XuMP cycle and cannot be fully used for product production. Pichia pastoris is a chassis cell with lower methanol utilization efficiency.

Disclosure of Invention

Aiming at the problem of low methanol utilization efficiency of pichia pastoris, the invention provides the construction of the pichia pastoris genetic engineering bacterium and the application of the pichia pastoris genetic engineering bacterium in improving the methanol assimilation rate and fixing carbon dioxide, which not only can solve the problem of carbon atom waste, but also can endow pichia pastoris with carbon fixation and carbon atom reutilization capability to further improve the utilization efficiency of carbon atoms.

The construction of the Pichia pastoris genetic engineering bacteria comprises the following steps:

step 1, respectively designing primers corresponding to a reductive glycine pathway related gene MIS1, a reductive glycine pathway related gene GCV2 and a reductive glycine pathway related gene GCV3, using pichia pastoris GS115 genome as a template, and obtaining MIS1, GCV2 and GCV3 gene fragments corresponding to the primers respectively by PCR amplification, wherein the nucleic acid sequence of an upstream primer MIS1-F used by MIS1 is shown as SEQ ID No. 1, and the nucleic acid sequence of a downstream primer MIS1-R is shown as SEQ ID No. 2; the nucleic acid sequence of the upstream primer GCV1-F used by the GCV1 is shown as SEQ ID No. 3, the nucleic acid sequence of the downstream primer GCV1-R is shown as SEQ ID No. 4, the nucleic acid sequence of the upstream primer GVC2-F used by the GCV2 is shown as SEQ ID No. 5, the nucleic acid sequence of the downstream primer GVC 2-R is shown as SEQ ID No. 6, the nucleic acid sequence of the upstream primer used by the GCV3 is shown as SEQ ID No. 7, and the nucleic acid sequence of the downstream primer GCV3-R is shown as SEQ ID No. 8;

step 2, knocking out FDH genes in a dissimilation pathway in Pichia pastoris GS115 by using a CRISPR-Cas9 gene knockout technology to obtain a recombinant strain GS 115-delta FDH;

step 3, constructing recombinant plasmids pGAP-MIS1-P2A-GCV1 and pGAP-GCV2-P2A-GCV3, and a knockout plasmid pET28a-His for knocking out DAS genes, wherein the recombinant plasmid pGAP-MIS1-P2A-GCV1 is a recombinant plasmid containing the MIS1 gene fragment and the GCV1 gene fragment amplified in the step 1, the recombinant plasmid pGAP-GCV2-P2A-GCV3 is a recombinant plasmid containing the GCV2 gene fragment and the GCV3 gene fragment amplified in the step 1, the amino acid sequence coded by the MIS1 gene fragment is shown as SEQ ID No. 9, and the amino acid sequence coded by the GCV1 gene fragment is shown as SEQ ID No. 10; the amino acid sequence of the GCV2 gene fragment is shown as SEQ ID No. 11, and the amino acid sequence of the GCV3 gene fragment is shown as SEQ ID No. 12.

Step 4, recombinant plasmids pGAP-MIS1-P2A-GCV1 and pGAP-GCV2-P2A-GCV3 are introduced into pichia pastoris GS 115-delta FDH, and double-crossover homologous recombination is carried out by using knockout plasmid pET28a-His to knockout DAS genes in pichia pastoris, so that the original methanol assimilation way is replaced by a reductive glycine way;

step 5, constructing a recombinant plasmid pIC9K-FLD-P2A-FGH-PTS1, wherein the recombinant plasmid pIC9K-FLD-P2A-FGH-PTS1 comprises FLD gene fragments, FGH gene fragments and PTS1 signal peptide, carrying out PCR amplification on Pichia pastoris GS115 methanol catabolism pathway FLD and FGH genes by using specific primers, and adding PTS1 signal peptide at the sequence C end to position the catabolism pathway in a peroxisome, wherein the amino acid sequence coded by the FGH gene fragments is shown as SEQ ID No. 13, and the amino acid sequence coded by the FLD gene fragments is shown as SEQ ID No. 14;

and 6, introducing the recombinant plasmid pIC9K-FLD-P2A-FGH-PTS1 into the recombinant pichia pastoris constructed in the step 4, and realizing compartmentalization positioning expression of a pichia pastoris methanol catabolism pathway to obtain the pichia pastoris genetic engineering bacteria.

As an improvement, the reaction conditions of the PCR described in step 1 are: 95 ℃ for 2 min, 95 ℃ for 20 s,55 ℃ for 20 s,72 ℃ for 10 s, and 30 cycles in total; and at 72℃for 5 min.

SEQ ID No .1:

TATTTCGAAACGAGGAATTGAAACGATGCTTTCAAGAATTGGATCTAGAAATAAAG；

SEQ ID No .2：

ACGTCTCCTGCTTGCTTTAACAGAGAGAAGTTCGTGGCAAATAA；

SEQ ID No .3：

TAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCTGAAACGA；

SEQ ID No .4：

AGATGAGTTTTTGTTCTAGTCACTCCGGCTTATAAAATTTGGGGGC；

SEQ ID No .5:

TATTTCGAAACGAGGAATTGAAACGATGTTAAGAACCCAGTTCTCCA；

SEQ ID No .6：

TCTCCTGCTTGCTTTAACAGAGAGAAGTTCGTGGCTTGACTAGCA；

SEQ ID No .7：

TGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCTGAAACGA；

SEQ ID No .8：

ATGATGATGATGATGGTCGATTAGCTTTCCTCCTGCAAAAACTTGAC。

SEQ ID No .9

MLSRIGSRSKATSTVRRLAGDLLLGSRKLHASAVFSQAQILSGTKLAKSIRSKVFEEINVFKATHPEFQPAVTIIQVGDRPDSSAYVRMKLKAASESGIICNVIKLPEDTAEPTLINRIHKLNNDNSVHGILVQLPLPSHIDETHITNSVSILKDVDGFDRFNVGELAKKGGNPTFLPCTPNGCMKLLEASGVELSGKSAVVLGRSDIVGTPVAQLLNKANCTVTVCHSRTPNIAEIVKNSDIVVAAIGKANFVKGEWLKPGAVVIDVGINYVPDPTKKSGQRLVGDVEFESAKEKASVITPVPGGVGPMTVAMLVSNIAHAAKLQLENDLKPAKIYANPLELKTPVPSDIEISRAQEPRYIKDIATDLGIKDKELELYGHYKAKVSLDVYDRLAKNRENGNYVLVAGITPTPLGEGKSTTTMGLVQALGAHLNLAAVANVRQPSMGPTFGVKGGAAGGGYAQVIPMDEFNMHLTGDIHAIGAANNLLAAALDTRIFHETTQRDAIFYKRLVPAKKGVRKFTPSMLKRLEKLGINKTEPDSLTPEEATKFAKLNIDPQTITVKRVLDVNDRFVRQVTIGEAPTEKGHIRKTGFDITVASEVMAILALSKDLKDLRSRVGAIVVASSYEGVPITAEDLGVAGALTALLKDAVKPNLMQTLEGTPVFVHAGPFANISIGASSVIADRVALKLIGASKSDVVAKSEKGFVVTEAGFDFTMGGERFYNIKCRASGLKPNTVVLVATSRALKLHGGAPDVKPGQALPEEYTTENVELVRRGCANLAKQISNASQYGAPVVVAINQFETDSPAELSVIREEALKAGAFDAVPSNHWAEGGKGAVELAKAVVSSCASCPEPNAEKAELYSLENNTIEDRLNTIVQKMYNGDGIELSELAKEQIARYEAQGFGNLPICIAKTQYSLSHDPSLKNVPAGFTVPIREVRLSAGAGYLSCLAAEIMTIPGLPTHPGYLNVEVNDDGEIEGLF

SEQ ID No .10

MIRSCRRYSTGVEALIKTPLYSVHVEHGATLVPYANFAMPVLYKGQSHIESHNWTRSKVGIFDVSHMLQHRVKGNSAAEFLQKITPSDLKALKPFTSTLSVLLNDKGGVIDDCIITKHDENEFYIVTNAGCRDKDTSFIKEEISQFGNASHENFEGTLLAIQGPQAAETLQKFTNLDLTKLYFGNSAFAKLEGFTSDVIHIARSGYTGEDGFELSIPVDSEGLEFTKALLELEQVKPIGLAARDSLRLEAGMCLYGHELNEQTTPVESSLNWLIAKSRRTPKTAGFNGSENILSQLENPKSVTFKRVGIQSKGPSPREGNKVYSFEEPDKQIGVVCSGSPSPTLGGNVGQAFIHKPHQKAGTKILIEIRNKKREAHVAKLPFVAPKFYKPE

SEQ ID No .11

MLRTQLSKALLKSHSVKAWARVGTATTSRFVSDTTSATFRSIYEKGSSTPSPYDGTDSFPRRHLGPTPNNVDEMLQDLKESDLDSFIAKVVPDNVLIRRSLKVEPLKGFTESEMLAELARIAEKNDLVKPLIGKGFYGTIIPPVILRNLLENPGWYTSYTPYQPEVSQGRLESLLNFQTVISDLTGLPVANASLLDESSAAAEAMILSFTSLKSKKPKYFVDSNIHEQTLAVLKSRAHTLSIEVVVADLTTDEGFAALQENKDQLCGAMVQYPATDGSIETFKAYSRIAELVHSVKGLYAVASDLMALTLLHAPSKFGADIVFGSSQRFGVPLGYGGPHAAFFSVVESLQRKIPGRIVGVSKDRLGNNALRLALQTREQHIKRERATSNICTAQALLANIAANYVVYHGIEGLRNISKRIHGFTTLLANSINESSSHKVLNEKWFDTLSVDVGSADEFIAKALSKNFNVFRVNNSTIQLSLDETVTKNELTTLISLFTGSSEVNLPETLPSFYEEWTRQDDILTNEVFRTHRSETSMLRYLTHLQKKDISLADSMISLGSCTMKLNATVEMMPISWPKFANIHPFAPKDQVKGYTELIVELEKDLADITGFHSTTLQPNSGAQGEYTGLSVIAKYFESKGEAHRNIVLIPVSAHGTNPASAAMAGLKVVPIKCLKDGSLDLQDLESKASKHAKNLAAMMVTYPSTYGLFEPGIIDAIKIIHNHGGQVYLDGANMNAQVGLTSPGDLGADVCHLNLHKTFAIPHGGGGPGVGPICVKEHLTPFLPSHPIIATTNQTEQSINPVVAAPFGSASILPISYAYIKMMGGSNLQYSSIIAILNANYMVAKLKNHFEILFTGGDAKYCGHEFIIDLRPFKKFGIEAIDVAKRLQDYGFHAPTMSFPIPGTLMVEPTESEYKEELDRFIDSMISIREEIRKVENGETDGAILHNSPHNLKDIISTSDAEWSQRGYTREEAAYPLPFLKEQKTWPTVSRVDDTYGDMNLLCTCPSVEEVAASQ

SEQ ID No .12

SEQ ID No .13

MSSITTSIFKVTAEIQSFGGKLVKLQHKSDETKTDMDVNVYLPAQFFANGAKGKSLPVLLYLSGLTCTPNNASEKAFWQPYANKYGFAVVFPDTSPRGLNIEGEHDSYDFGSGAGFYVDATTEKWKDNYRMYSYVNSELLPKLQADFPILNFDNISITGHSMGGYGALQLFLRNPGKFKSVSAFSPISNPTKAPWGEKCFSGYLGQDKSTWTQYDPTELIGKYQGPSDSSILIHVGKSDSFYFKDHQLLPENFLKASENSVFKGKVDLNLVDGYDHSYYFISSFTDVHAAHHAKYLGLN

SEQ ID No .14

MSTEGQIIKCKAAVAWEAGKDLSIEEIEVLPPRAHEVRVKVEFTGVCHTDAYTLSGADAEGSFPVVFGHEGAGVVESVGEGVESVKVGDSVVLLYTPECRECKFCLSGKTNLCGKIRATQGKGLLPDGTSRFRCKGKDLFHYMGCSSFSQYTVVADISVVKVQDEAPKDKTCLLGCGVTTGYGAAINTAKISKGDKIGVFGAGCIGLSVIQGAVSKGASEIIVIDINDSKKAWADQFGATKFVNPTTLPEGTNIVDYLIDITDGGFDYTFDCTGNVQVMRNALESCHKGWGESIIIGVAAAGKEISTRPFQLVTGRVWRGCAFGGIKGRTQMPSLVQDYLDGKIKVDEFITHRHDLDNINKAFHDMHAGNCIRAVITMH

SEQ ID No .15：

CAACTAATTATTCGAAGGATCCGAAACGATGTCATCAATTACTACTTCAATCTTCAAG

SEQ ID No .16：

CACGTCTCCTGCTTGCTTTAACAGAGAGAAGTTCGTGGCCAACTTAGAGTTTAACCCCAAATACTTTGCATGGTG

SEQ ID No .17：

TGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCTGAAACGATGTCTACCGAAGGTCAAGTAAGTTCAAT

SEQ ID No .18：

AAGGCGAATTAATTCGCGGCCGCTTACAACTTAGAGTGCATAGTAATCACAGCAC

The application of the pichia pastoris genetically engineered bacteria in improving the methanol efficiency and fixing the carbon dioxide comprises the following specific steps: firstly, selecting positive clone of pichia pastoris genetic engineering bacteria, transferring to an M9 culture medium after shaking overnight culture, and culturing by taking methanol as a unique carbon source; second, sampling every 24 hours and drawing a growth curve in the process of culturing cells, and using OD ₆₀₀ The measurement is carried out, and the biomass is finally measured.

The beneficial effects are that:

compared with the prior art, the construction of the pichia pastoris genetic engineering bacteria and the application thereof in improving the methanol assimilation rate and fixing the carbon dioxide are realized, the methanol metabolism pathway of pichia pastoris is modified, the carbon atom loss caused by carbon dioxide generated by dissimilation is eliminated by knocking out the dissimilation pathway and reconstructing the reductive glycine pathway, the carbon atom utilization efficiency is improved, the utilization efficiency of methanol by the bacteria strain subjected to metabolic modification and compartmentalization design is improved, and the carbon dioxide can be fixed by utilizing the reductive glycine pathway for improving the biomass.

The method has the specific advantages that:

1. the overexpressed gene related to the glycine pathway is cloned from pichia pastoris for the first time, and has low cost, simple molecular level operation, easy cloning and expression and better repeatability;

2. the modified strain constructed in the invention knocks out the dissimilation path thereof, thereby avoiding carbon flow from using CO ₂ The form loss of the pichia pastoris improves the utilization efficiency of the carbon atoms of the methanol;

3. the modified strain constructed in the invention has the capability of fixing carbon dioxide on the overexpressed reductive glycine path, thereby enabling pichia pastoris to be modified into autotrophic strain;

4. the biomass of the compartmentalized recombinant strain constructed in the invention is improved by 8.7%, and the methanol assimilation efficiency is further improved.

Drawings

FIG. 1 is a diagram of a related plasmid of the present invention wherein (a) pGAP-MIS1-P2A-GCV1; (b) pGAP-GCV2-P2A-GCV3, (c) pET28a-His; (d) pIC9K-FLD-P2A-FGH-PTS1

FIG. 2 is a recombinant strain fermentation characterization wherein (a) the DAS gene knockout strain is fermentation verified; (b) Green fluorescent positioning of FGH-TPS1, (c) red fluorescent positioning of FDH-TPS1, (d) fermentation verification of recombinant strain; (e) Methanol consumption of fermentation Strain (f) accumulation of fermentation fungus formic acid

FIG. 3 shows the result of related gene amplification;

FIG. 4 is a diagram of recombinant strains ¹³ C carbon dioxide labeling.

Detailed Description

The invention is further illustrated by the following description of specific embodiments, which are not intended to be limiting, and various modifications or improvements can be made by those skilled in the art in light of the basic idea of the invention, but are within the scope of the invention without departing from the basic idea of the invention.

The techniques not mentioned in the examples are conventional in the art, and in addition, materials such as the Pichia pastoris GS115 source ATCC, trans1-T1, pXY3, top10 and the like are all commercial products and can be purchased directly.

Example 1

And knocking out the FDH gene in the catabolism pathway in pichia pastoris by using a CRISPR-Cas9 gene knockout technology to obtain a recombinant strain GS 115-delta FDH.

Example 2

1. Construction of pGAPZA-MIS1-P2A-GCV1 recombinant plasmid

The nucleotide coding sequence of the corresponding gene was replicated by conventional PCR amplification using Pichia pastoris GS115 genome with MIS1, GVC1 genes as a template.

The MIS1 upstream primer used has a homology arm and has the sequence:

MIS1-F:TATTTCGAAACGAGGAATTGAAACGATGCTTTCAAGAATTGGATCTAGAAATAAAG

the MIS1 downstream primer has a P2A sequence as a homology arm, and the sequence is:

MIS1-R：

ACGTCTCCTGCTTGCTTTAACAGAGAGAAGTTCGTGGCAAATAA。

the GCV1 upstream primer used has a P2A sequence as a homology arm, and the sequence is:

GCV1-F：TAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCTGAAACGA

the GCV1 downstream primer has a homology arm and has the sequence:

GCV1-R：

AGATGAGTTTTTGTTCTAGTCACTCCGGCTTATAAAATTTGGGGGC。

the reaction conditions are as follows: 95 ℃ for 2 min, 95 ℃ for 20 s,55 ℃ for 20 s,72 ℃ for 10 s, and 30 cycles in total; the obtained sequence was subjected to 1% agarose gel electrophoresis at 72℃for 5 min, and the corresponding fragment was recovered.

The recovered fragments were added as templates to the upper primer MIS1-F and the lower primer GCV1-R was used to join the two fragments together using OVERLAP PCR. The reaction conditions are as follows: 95 ℃ for 2 min, 95 ℃ for 20 s,55 ℃ for 20 s,72 ℃ for 10 s, and 30 cycles in total; the obtained sequence was subjected to 1% agarose gel electrophoresis at 72℃for 5 min, and the corresponding fragment was recovered.

The expression vector pGAP was digested with Sal I and EcoR I of Takara, the digestion reaction system was: 10 Xbuffer 1. Mu.L, sal I1. Mu.L, ecoR I1. Mu.L, pGAP vector 7. Mu.L. The cleavage system was reacted at 37℃for 2 hours. The 15-20 bp sequence at the end of the linearized vector after cleavage was used as homology arm and added to the 5' end of the gene-specific upstream and downstream primer sequences (MIS 1-F, GCV 1-R), respectively. The recombination reaction system is as follows: 5X CE II buffer 4. Mu.L of Vazyme, 2. Mu.L of Exnase II, 10. Mu.L of gene fragment and 2. Mu.L of vector were used. The ligation was carried out at 37℃for 1 hour. The ligation product was transformed into E.coli Trans1-T1. The positive strain Trans 1-T1-pGAP-MIS 1-P2A-GCV1 was screened by PCR and DNA sequencing was performed to verify that the recombinant plasmid was constructed correctly (FIG. 1 (a)).

The positive strain was inoculated into 5 mL of LB/KanR liquid medium consisting of peptone 10 g/L, yeast powder 5 g/L, sodium chloride 5 g/L, and cultured overnight at 37℃under shaking at 200 rpm. The plasmid pGAP-MIS1-P2A-GCV1 was extracted 24 hours later according to the instructions of the Tiangen plasmid extraction kit. mu.L of pGAP-MIS1-P2A-GCV1 plasmid was electrotransformed into Pichia pastoris which had knocked out the catabolism pathway. The positive strain GS115-MIS1-P2A-GCV1 was screened by PCR.

2. Construction of pGAP-GCV2-P2A-GCV3 recombinant plasmid

The nucleotide coding sequence of the GCV2 and GCV3 genes is amplified and copied by conventional PCR by taking pichia pastoris genome as a template.

The GCV2 upstream primer used has a homology arm and has the sequence:

GCV2-F:TATTTCGAAACGAGGAATTGAAACGATGTTAAGAACCCAGTTCTCCA

the GCV2 downstream primer has a P2A sequence as a homology arm, and the sequence is:

GCV2-R：

TCTCCTGCTTGCTTTAACAGAGAGAAGTTCGTGGCTTGACTAGCA。

the GCV3 upstream primer used had the P2A sequence as homology arm and the sequence was:

GCV3-F：

TGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCTGAAACGA

the GCV3 downstream primer has a homology arm and has the sequence:

GCV3-R：

ATGATGATGATGATGGTCGATTAGCTTTCCTCCTGCAAAAACTTGAC。

the reaction conditions are as follows: 95 ℃ for 2 min, 95 ℃ for 20 s,55 ℃ for 20 s,72 ℃ for 10 s, and 30 cycles in total; and at 72℃for 5 min. The resulting sequence was subjected to 1% agarose gel electrophoresis and the corresponding fragment was recovered.

The recovered fragments were added as templates to the upper primer GCV2-F and the lower primer GCV3-R was used to join the two fragments together using OVERLAP PCR. The reaction conditions are as follows: 95 ℃ for 2 min, 95 ℃ for 20 s,55 ℃ for 20 s,72 ℃ for 10 s, and 30 cycles in total; the obtained sequence was subjected to 1% agarose gel electrophoresis at 72℃for 5 min, and the corresponding fragment was recovered.

The expression vector pGAP was digested with Sal I and EcoR I of Takara, the digestion reaction system was: 10 Xbuffer 1. Mu.L, sal I1. Mu.L, ecoR I1. Mu.L, pGAP vector 7. Mu.L. The cleavage system was reacted at 37℃for 2 hours. The 15-20 bp sequence at the end of the linearized vector after cleavage was used as homology arm and added to the 5' end of the gene specific upstream and downstream primer sequences (GVC 2-F, GVC 3-R), respectively. The recombination reaction system is as follows: 5X CE II buffer 4. Mu.L of Vazyme, 2. Mu.L of Exnase II, 10. Mu.L of gene fragment and 2. Mu.L of vector were used. The ligation was carried out at 37℃for 1 hour. The ligation product was transformed into E.coli Trans1-T1. The colony PCR was used to screen the positive strain Trans 1-T1-pGAP-GCV 2-P2A-GCV3 and to verify that the recombinant plasmid was constructed correctly (FIG. 1 (b)).

The positive strain was inoculated into 5 mL of LB/KanR liquid medium consisting of peptone 10 g/L, yeast powder 5 g/L, sodium chloride 5 g/L, and cultured overnight at 37℃under shaking at 200 rpm. Extracting plasmid pGAP-GCV2-P2A-GCV3 according to the operation instruction of a root plasmid extraction kit after 24 hours, taking 2 mu L of pGAP-GCV2-P2A-GCV plasmid, electrically transforming into pGAP-GCV2-P2A-GCV3 pichia pastoris, and carrying out PCR screening on positive strain GS115-MIS1-P2A-GCV1-GCV21-P2A-GCV3, wherein the obtained recombinant strain is named as follows: GS115- ΔFDH-rGly

3. Construction of pET28a-His recombinant plasmid

The nucleotide coding sequence of the homology arm was amplified by conventional PCR using Pichia pastoris genome with the upper and lower homology arms of the DAS gene as a template.

The upper homology arm upstream primer is provided with a homology arm, and the sequence is as follows:

S-F:

GTGGTGGTGGTGGTGCTCGAGGGTACTCTTTGACATAGCTATCAACTAACTTTTTCC

the downstream primer of the upper homology arm is provided with a homology arm, and the sequence is as follows:

S-R：

GAAACTGGGACTTATTTAAATGGCTAGAATTCCAAAAGCAGTATCG。

the upper stream of the lower homology arm is provided with a homology arm, and the sequence is as follows:

X-F:

ACGGAGCTCGAATTCGGATCCATGGCTAGAATTCCCAAAGCAGTTT

the downstream primer of the lower homology arm is provided with a homology arm, and the sequence is as follows:

X-R:

GGTGCCGCGCGGCAGCCATATGGATAGTTCTTAACGTACTCATCCAAAAGTTTCTTCC。

The nucleotide coding sequence of the histidine-screening tag was amplified by conventional PCR using the pPIC9k plasmid with the histidine-screening tag gene as a template.

The upstream primer of the histidine screening tag gene is provided with a homology arm, and the sequence is as follows:

H-F: TTTTGGAATTCTAGCCATTTAAATAAGTCCCAGTTTCTCCATACGAACC

the downstream primer of the histidine screening tag gene is provided with a homologous arm, and the sequence is as follows:

H-R:

CCGAATTCGAGCTCCGTCGACTCAGAATTGGTTAATTGGTTGTAACACTGGC。

The recovered fragments were used as templates for addition to the upper primer S-F and the lower primer X-R, which were used to join the three fragments together using OVERLAP PCR. The reaction conditions are as follows: 95 ℃ for 2 min, 95 ℃ for 20 s,55 ℃ for 20 s,72 ℃ for 10 s, and 30 cycles in total; and at 72℃for 5 min. The resulting sequence was subjected to 1% agarose gel electrophoresis and the corresponding fragment was recovered.

The expression vector pET28a was digested with Nde I and Xho I from Takara, and the digestion reaction system was: 10 Xbuffer 1. Mu.L, nde I. Mu.L, xho I1. Mu.L, pET28a vector 7. Mu.L. The cleavage system was reacted at 37℃for 2 hours. The 15-20 bp sequence at the end of the linearized vector after cleavage was used as homology arm and added to the 5' end of the gene-specific upstream and downstream primer sequences (S-F, H-R), respectively. The recombination reaction system is as follows: 5X CE II buffer 4. Mu.L of Vazyme, 2. Mu.L of Exnase II, 10. Mu.L of gene fragment and 2. Mu.L of vector were used. The ligation was carried out at 37℃for 1 hour. The ligation product was transformed into E.coli Trans1-T1. The positive strain Trans 1-T1-pET 28a-His was screened by PCR and DNA sequencing was performed to verify that the recombinant plasmid was constructed correctly (FIG. 1 (c)).

The positive strain was inoculated into 5 mL of LB/KanR liquid medium consisting of peptone 10 g/L, yeast powder 5 g/L, sodium chloride 5 g/L, and cultured overnight at 37℃under shaking at 200 rpm. After 24 hours, 2. Mu.L of pET28a-His plasmid was electrotransformed into GS 115-. DELTA.FDH-rGly Pichia pastoris according to the instructions of the day root plasmid extraction kit. The positive strain GS 115-DeltaFDH-rGly-DeltaDAS was screened by PCR.

Similarly, 2. Mu.L of pET28a-His plasmid was electrotransformed into Pichia pastoris GS 115. Positive strain GS115- Δdas was screened by PCR.

4. Construction of pIC9K-FLD-P2A-FGH-PTS1 recombinant plasmid

The Pichia genome with FGH and FLD genes is used as template to amplify and copy the nucleotide coding sequence of the gene by conventional PCR.

The FGH upstream primer used had a homology arm with the sequence:

FGH-F:

TATTTCGAAACGAGGAATTGAAACGATGTTAAGAACCCAGTTCTCCA

FGH downstream primer has P2A sequence as homology arm, and the sequence is:

FGH-R：

CACGTCTCCTGCTTGCTTTAACAGAGAGAAGTTCGTGGCCAACTTAGAGTTTAACCCCAAATACTTTGCATGGTG。

the FLD upstream primer used has a P2A sequence as a homology arm, and the sequence is:

FLD-F：

TGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCTGAAACGATGTCTACCGAAGGTCAAGTAAGTTCAAT。

the FLD downstream primer has a homology arm and has the sequence:

FLD-R：

AAGGCGAATTAATTCGCGGCCGCTTACAACTTAGAGTGCATAGTAATCACAGCAC。

The recovered fragment was used as template to add to the upper primer FGH-F and the lower primer FLD-R to join the two fragments together using OVERLAP PCR. The reaction conditions are as follows: 95 ℃ for 2 min, 95 ℃ for 20 s,55 ℃ for 20 s,72 ℃ for 10 s, and 30 cycles in total; the obtained sequence was subjected to 1% agarose gel electrophoresis at 72℃for 5 min, and the corresponding fragment was recovered.

The expression vector PIC9K was digested with BamH I and Not I from Takara, and the digestion reaction system was: 10 Xbuffer 1. Mu.L, bamH 1. Mu.L, not I1. Mu.L, PIC9K vector 7. Mu.L. The enzyme digestion system is reacted for 1 hour at 30 ℃ before the reaction for 1 hour at 37 ℃. The 15-20 bp sequence at the end of the linearized vector after cleavage was used as homology arm and added to the 5' end of the gene-specific upstream and downstream primer sequence (FGH-F, FLD-R), respectively. The recombination reaction system is as follows: 5X CE II buffer 4. Mu.L of Vazyme, 2. Mu.L of Exnase II, 10. Mu.L of gene fragment and 2. Mu.L of vector were used. The ligation was carried out at 37℃for 1 hour. The ligation product was transformed into E.coli Trans1-T1. The colony PCR was used to screen the positive strain Trans 1-T1-pIC 9K-FLD-P2A-FGH-PTS1 and to verify that the recombinant plasmid was constructed correctly (FIG. 1 d).

The positive strain was inoculated into 5 mL of LB/KanR liquid medium consisting of peptone 10 g/L, yeast powder 5 g/L, sodium chloride 5 g/L, and cultured overnight at 37℃under shaking at 200 rpm. The plasmid pIC9K-FLD-P2A-FGH-PTS1 was extracted 24 hours later according to the instructions of the day root plasmid extraction kit. mu.L of pIC9K-FLD-P2A-FGH-PTS1 plasmid was electrotransformed into GS115- ΔFDH-rGly- ΔDAS Pichia pastoris, and positive strains were screened by colony PCR, and the obtained recombinant strains were named as follows: GS115- ΔFDH-rGly- ΔDAS-FGH-FLD-TPS1.

5. Construction of compartmentalized characterization Strain GS115-FGH-PTSI-GFP, GS115-FLD-PTS1-RFP

The FGH upstream primer used had a homology arm with the sequence:

FGH-F:

AACTAATTATTCGAAGGATCCATGTCATCAATTACTACTTCAATCTTCAAGGTAACAG

FGH downstream primer has linker sequence as homology arm, and the sequence is:

FGH-R：

CACCGCCAGAGCCACCTCCGCCGTTTAACCCCAAATACTTTGCATGGTGAG。

the GFP upstream primer used carries a linker sequence as a homology arm, and the sequence is:

GFP-F：

GGTGGCTCTGGCGGTGGCGGATCGATGGTGAGCAAGGGCGAG。

the GFP downstream primer has a PTS1 sequence homology arm, and the sequence is:

GFP-R：

AAGGCGAATTAATTCGCGGCCGCTTACAACTTAGACTTGTACAGCTCGTCCAT。

The recovered fragments were added as templates to the upper primer FGH-F and the lower primer GFP-R was used to join the two fragments together using OVERLAP PCR. The reaction conditions are as follows: 95 ℃ for 2 min, 95 ℃ for 20 s,55 ℃ for 20 s,72 ℃ for 10 s, and 30 cycles in total; the obtained sequence was subjected to 1% agarose gel electrophoresis at 72℃for 5 min, and the corresponding fragment was recovered.

The expression vector PIC9K was digested with BamH I and Not I from Takara, and the digestion reaction system was: 10 Xbuffer 1. Mu.L, bamH 1. Mu.L, not I1. Mu.L, PIC9K vector 7. Mu.L. The enzyme digestion system is reacted for 1 hour at 30 ℃ before the reaction for 1 hour at 37 ℃. The 15-20 bp sequence at the end of the linearized vector after cleavage was used as homology arm and added to the 5' end of the gene-specific upstream and downstream primer sequences (FGH 2-F, GFP-R), respectively. The recombination reaction system is as follows: 5X CE II buffer 4. Mu.L of Vazyme, 2. Mu.L of Exnase II, 10. Mu.L of gene fragment and 2. Mu.L of vector were used. The ligation was carried out at 37℃for 1 hour. The ligation product was transformed into E.coli Trans1-T1. The colony PCR is used for screening a positive strain Trans 1-T1-pIC 9K-FGH-GFP-PTS 1, and the construction of a recombinant plasmid is verified to be correct.

The positive strain was inoculated into 5 mL of LB/KanR liquid medium consisting of peptone 10 g/L, yeast powder 5 g/L, sodium chloride 5 g/L, and cultured overnight at 37℃under shaking at 200 rpm. The plasmid pIC 9K-FGH-GFP-PTS 1 was extracted 24 hours later according to the instructions of the day root plasmid extraction kit. mu.L of pIC 9K-FGH-GFP-PTS 1 plasmid was electrotransformed into GS115 in Pichia pastoris, and positive strains were screened by colony PCR, and the obtained recombinant strains were named as follows: GS 115-FGH-GFP-PTS 1.

The FLD upstream primer has a homology arm and has the sequence:

FLD-F:

TTATTCGAAGGATCCGAAACGATGTCTACCGAAGGTCAAGTAAGTTCAAT。

the FLD2 downstream primer has a linker sequence as a homology arm, and the sequence is:

FLD2-R：

CACCGCCAGAGCCACCTCCGCCGTGCATAGTAATCACAGCACGAATACAG。

the RFP upstream primer is provided with a linker sequence as a homology arm, and the sequence is as follows:

RFP-F：

GGTGGCTCTGGCGGTGGCGGATCGATGGTGCGCTCCTCCAAG。

the RFP downstream primer has a PTS1 sequence homology arm, and the sequence is as follows:

RFP-R：

AGTTTTTGTTCTAGGCGGCCGCTTACAACTTAGACAGGAACAGGTGGTG。

The recovered fragment was used as a template to add to the upper primer FLD-F, and the lower primer RFP-R was used to join the two fragments together using OVERLAP PCR. The reaction conditions are as follows: 95 ℃ for 2 min, 95 ℃ for 20 s,55 ℃ for 20 s,72 ℃ for 10 s, and 30 cycles in total; the obtained sequence was subjected to 1% agarose gel electrophoresis at 72℃for 5 min, and the corresponding fragment was recovered.

The expression vector PGAPZA was digested with BamH I and Not I from Takara, and the digestion reaction system was: 10 Xbuffer 1. Mu.L, bamH 1. Mu.L, not I1. Mu.L, PIC9K vector 7. Mu.L. The enzyme digestion system is reacted for 1 hour at 30 ℃ before the reaction for 1 hour at 37 ℃. The 15-20 bp sequence at the end of the linearized vector after cleavage was used as homology arm and added to the 5' end of the gene specific upstream and downstream primer sequence (FLD-F, RFP-R), respectively. The recombination reaction system is as follows: 5X CE II buffer 4. Mu.L of Vazyme, 2. Mu.L of Exnase II, 10. Mu.L of gene fragment and 2. Mu.L of vector were used. The ligation was carried out at 37℃for 1 hour. The ligation product was transformed into E.coli Trans1-T1. The colony PCR is used for screening a positive strain Trans 1-T1-pGAPZA-FLD-RFP-PTS 1, and the construction of a recombinant plasmid is verified to be correct.

The positive strain was inoculated into 5 mL of LB/KanR liquid medium consisting of peptone 10 g/L, yeast powder 5 g/L, sodium chloride 5 g/L, and cultured overnight at 37℃under shaking at 200 rpm. The plasmid pGAPZA-FLD-RFP-PTS 1 was extracted 24 hours later according to the procedure of the day root plasmid extraction kit. mu.L of pGAPZA-FLD-RFP-PTS 1 plasmid was electrotransformed into GS115 in Pichia pastoris, and positive strains were selected by colony PCR, and the obtained recombinant strains were named as follows: GS 115-FLD-RFP-PTS 1.

EXAMPLE 3 phenotypic verification of recombinant strains

1. Knock-out strain fermentation verification

After the DAS gene knockout is verified, the pichia pastoris assimilation effect is verified, and pichia pastoris GS115 and GS 115-delta DAS strains are respectively inoculated into 5 mL YPD liquid culture medium and are subjected to shake culture at 30 ℃ and 200 rpm until OD600 is approximately equal to 5. The cultured bacterial solutions are all according to the initial OD ₆₀₀ After centrifugation and resuspension of =0.2, the samples were inoculated into 100mL of fresh M9 liquid medium and 1% methanol was added thereto, and the samples were taken at 24-hour intervals by shaking culture at 30 ℃ and 200 rpm to measure the OD ₆₀₀ . Strains after knockout of the DAS gene were unable to grow in medium with methanol as the sole carbon source from the fermentation-verifying structure (fig. 2 (a)).

2. Compartmentalized positioned recombinant strain fermentation characterization

And verifying the positioning effect of the signal peptide PTS1, respectively inoculating the constructed characterization strains GS115-FGH-PTSI-GFP and GS115-FLD-PTS1-RFP to 100mL YPD liquid culture medium, adding 1% methanol in volume for induction at 30 ℃ and under 200 rpm, carrying out shaking culture for 24 hours, collecting thalli, and observing fluorescent expression at excitation wavelengths of 487nm and 554nm by using a fluorescent microscope. From the fluorescent pictures it can be seen that the signal peptide PTS1 is able to localize the protein into the peroxisome (FIG. 2 (b), FIG. 2 (c)).

3. Recombinant strain fermentation verification

Recombinant strains GS 115-DeltaFDH, original Pichia pastoris GS115, GS 115-DeltaFDH-rGly-DeltaDAS-FGH-FLD-TPS 1 are respectively inoculated into 5 mL YPD liquid culture medium, and are subjected to shaking culture at 30 ℃ and 200 rpm until OD600 is approximately equal to 5. The cultured bacterial solutions are all according to the initial OD ₆₀₀ After centrifugation and resuspension of =0.2, the samples were inoculated into 100mL of fresh M9 liquid medium and 1% methanol was added thereto, and the samples were taken at 24-hour intervals by shaking culture at 30 ℃ and 200 rpm to measure the OD ₆₀₀ 。

From the growth curve, it can be seen that the reconstruction of the reductive glycine pathway in pichia pastoris can replace the original methanol metabolic pathway, so that the DAS gene knockout strain can normally utilize methanol, and the growth condition of the recombinant strain with the compartmentalization design is improved to a certain extent compared with that of the original strain (fig. 2 (d)).

Wherein, M9 medium (g/L): na (Na) ₂ PO ₄ ·7H ₂ O 12.8、KH ₂ PO ₄ 3.0、NaCl 0.5、NH ₄ Cl 1、MgSO ₄ ·7H ₂ O 0.492、CaCl ₂ ·6H ₂ O 0.02191。

YPD Medium (g/L): peptone 2, yeast powder 1 and glucose 2.

4. Recombinant strain fermentation methanol consumption and formic acid accumulation

Recombinant strains GS 115-DeltaFDH, original Pichia pastoris GS115, GS 115-DeltaFDH-rGly-DeltaDAS-FGH-FLD-TPS 1 are respectively inoculated into 5 mL YPD liquid culture medium, and are subjected to shaking culture at 30 ℃ and 200 rpm until OD600 is approximately equal to 5. The cultured bacterial solutions are all according to the initial OD ₆₀₀ After centrifugation and resuspension of =0.2, the culture medium was inoculated into 100mL of fresh M9 liquid medium, 1% methanol was added thereto, the culture was performed at 30 ℃ under 200 rpm with shaking, samples were taken every 24 hours, and the supernatant was collected by centrifugation and monitored for methanol consumption and formic acid accumulation during fermentation by HPLC high performance liquid chromatography. The recombinant strain and the original strain were the same in methanol consumption rate from the data, but the recombinant strain had a significant accumulation of formic acid (FIG. 2 (e), FIG. 2 (f)). The modified strain is proved to avoid the generation of CO by the differentiation of formic acid ₂ The formate is preferably taken away to reconstitute the reductive glycine pathway into central metabolism.

EXAMPLE 4 recombinant Strain ¹³ C carbon dioxide labelling experiment

To further demonstrate the disruption of the reductive glycine pathway, one would proceed ¹³ C carbon dioxide labelling experiments. Recombinant strains the recombinant strains GS115- ΔFDH, pichia pastoris GS115, GS115- ΔFDH-rGly- ΔDAS-FGH-FLD-TPS1 were inoculated into 5 mL YPD liquid medium and cultured overnight at 30℃and 200 rpm. Then the cultured bacterial liquid is processed according to the initial OD ₆₀₀ After centrifugation and resuspension =0.2, the cells were inoculated into 10mL of fresh M9 liquid medium and 1% by volume of methanol, 20Mm, was added ¹³ C-NaHCO ₃ The culture was carried out at 30℃and 200 rpm for about 144 hours to carry out gas detection.

From FIG. 4 ¹³ C- NaHCO ₃ From the calibration results, CO ₂ Can be immobilized by the reconstituted reductive glycine pathway, and further indicates successful opening of the pathway (fig. 4).

In conclusion, the invention provides a compartmentalization theoretical design of the reductive glycine pathway and the catabolism pathway by manually constructing in pichia pastoris for the first time, thereby improving the methanol and CO assimilation of the pichia pastoris ₂ The efficiency is further improved, a foundation is laid for producing the compound by utilizing pichia pastoris in the future, and the method has profound significance.

In the foregoing, the protection scope of the present invention is not limited to the preferred embodiments of the present invention, and any simple changes or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention disclosed in the present invention fall within the protection scope of the present invention.

Sequence listing

<110> university of Nanjing Industrial science

<120> construction of Pichia pastoris genetically engineered bacterium and application thereof in improving methanol assimilation rate and fixing carbon dioxide

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<213> Artificial sequence (Artificial Sequence)

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<213> Artificial sequence (Artificial Sequence)

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agatgagttt ttgttctagt cactccggct tataaaattt gggggc 46

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<213> Artificial sequence (Artificial Sequence)

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tatttcgaaa cgaggaattg aaacgatgtt aagaacccag ttctcca 47

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<213> Artificial sequence (Artificial Sequence)

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tctcctgctt gctttaacag agagaagttc gtggcttgac tagca 45

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<213> Artificial sequence (Artificial Sequence)

<400> 7

tgttaaagca agcaggagac gtggaagaaa accccggtcc tgaaacga 48

<210> 8

<211> 47

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

atgatgatga tgatggtcga ttagctttcc tcctgcaaaa acttgac 47

<210> 9

<211> 981

<212> PRT

<213> amino acid sequence (Amino Acid Sequence)

<400> 9

Met Leu Ser Arg Ile Gly Ser Arg Ser Lys Ala Thr Ser Thr Val Arg

1 5 10 15

Arg Leu Ala Gly Asp Leu Leu Leu Gly Ser Arg Lys Leu His Ala Ser

20 25 30

Ala Val Phe Ser Gln Ala Gln Ile Leu Ser Gly Thr Lys Leu Ala Lys

35 40 45

Ser Ile Arg Ser Lys Val Phe Glu Glu Ile Asn Val Phe Lys Ala Thr

50 55 60

His Pro Glu Phe Gln Pro Ala Val Thr Ile Ile Gln Val Gly Asp Arg

65 70 75 80

Pro Asp Ser Ser Ala Tyr Val Arg Met Lys Leu Lys Ala Ala Ser Glu

85 90 95

Ser Gly Ile Ile Cys Asn Val Ile Lys Leu Pro Glu Asp Thr Ala Glu

100 105 110

Pro Thr Leu Ile Asn Arg Ile His Lys Leu Asn Asn Asp Asn Ser Val

115 120 125

His Gly Ile Leu Val Gln Leu Pro Leu Pro Ser His Ile Asp Glu Thr

130 135 140

His Ile Thr Asn Ser Val Ser Ile Leu Lys Asp Val Asp Gly Phe Asp

145 150 155 160

Arg Phe Asn Val Gly Glu Leu Ala Lys Lys Gly Gly Asn Pro Thr Phe

165 170 175

Leu Pro Cys Thr Pro Asn Gly Cys Met Lys Leu Leu Glu Ala Ser Gly

180 185 190

Val Glu Leu Ser Gly Lys Ser Ala Val Val Leu Gly Arg Ser Asp Ile

195 200 205

Val Gly Thr Pro Val Ala Gln Leu Leu Asn Lys Ala Asn Cys Thr Val

210 215 220

Thr Val Cys His Ser Arg Thr Pro Asn Ile Ala Glu Ile Val Lys Asn

225 230 235 240

Ser Asp Ile Val Val Ala Ala Ile Gly Lys Ala Asn Phe Val Lys Gly

245 250 255

Glu Trp Leu Lys Pro Gly Ala Val Val Ile Asp Val Gly Ile Asn Tyr

260 265 270

Val Pro Asp Pro Thr Lys Lys Ser Gly Gln Arg Leu Val Gly Asp Val

275 280 285

Glu Phe Glu Ser Ala Lys Glu Lys Ala Ser Val Ile Thr Pro Val Pro

290 295 300

Gly Gly Val Gly Pro Met Thr Val Ala Met Leu Val Ser Asn Ile Ala

305 310 315 320

His Ala Ala Lys Leu Gln Leu Glu Asn Asp Leu Lys Pro Ala Lys Ile

325 330 335

Tyr Ala Asn Pro Leu Glu Leu Lys Thr Pro Val Pro Ser Asp Ile Glu

340 345 350

Ile Ser Arg Ala Gln Glu Pro Arg Tyr Ile Lys Asp Ile Ala Thr Asp

355 360 365

Leu Gly Ile Lys Asp Lys Glu Leu Glu Leu Tyr Gly His Tyr Lys Ala

370 375 380

Lys Val Ser Leu Asp Val Tyr Asp Arg Leu Ala Lys Asn Arg Glu Asn

385 390 395 400

Gly Asn Tyr Val Leu Val Ala Gly Ile Thr Pro Thr Pro Leu Gly Glu

405 410 415

Gly Lys Ser Thr Thr Thr Met Gly Leu Val Gln Ala Leu Gly Ala His

420 425 430

Leu Asn Leu Ala Ala Val Ala Asn Val Arg Gln Pro Ser Met Gly Pro

435 440 445

Thr Phe Gly Val Lys Gly Gly Ala Ala Gly Gly Gly Tyr Ala Gln Val

450 455 460

Ile Pro Met Asp Glu Phe Asn Met His Leu Thr Gly Asp Ile His Ala

465 470 475 480

Ile Gly Ala Ala Asn Asn Leu Leu Ala Ala Ala Leu Asp Thr Arg Ile

485 490 495

Phe His Glu Thr Thr Gln Arg Asp Ala Ile Phe Tyr Lys Arg Leu Val

500 505 510

Pro Ala Lys Lys Gly Val Arg Lys Phe Thr Pro Ser Met Leu Lys Arg

515 520 525

Leu Glu Lys Leu Gly Ile Asn Lys Thr Glu Pro Asp Ser Leu Thr Pro

530 535 540

Glu Glu Ala Thr Lys Phe Ala Lys Leu Asn Ile Asp Pro Gln Thr Ile

545 550 555 560

Thr Val Lys Arg Val Leu Asp Val Asn Asp Arg Phe Val Arg Gln Val

565 570 575

Thr Ile Gly Glu Ala Pro Thr Glu Lys Gly His Ile Arg Lys Thr Gly

580 585 590

Phe Asp Ile Thr Val Ala Ser Glu Val Met Ala Ile Leu Ala Leu Ser

595 600 605

Lys Asp Leu Lys Asp Leu Arg Ser Arg Val Gly Ala Ile Val Val Ala

610 615 620

Ser Ser Tyr Glu Gly Val Pro Ile Thr Ala Glu Asp Leu Gly Val Ala

625 630 635 640

Gly Ala Leu Thr Ala Leu Leu Lys Asp Ala Val Lys Pro Asn Leu Met

645 650 655

Gln Thr Leu Glu Gly Thr Pro Val Phe Val His Ala Gly Pro Phe Ala

660 665 670

Asn Ile Ser Ile Gly Ala Ser Ser Val Ile Ala Asp Arg Val Ala Leu

675 680 685

Lys Leu Ile Gly Ala Ser Lys Ser Asp Val Val Ala Lys Ser Glu Lys

690 695 700

Gly Phe Val Val Thr Glu Ala Gly Phe Asp Phe Thr Met Gly Gly Glu

705 710 715 720

Arg Phe Tyr Asn Ile Lys Cys Arg Ala Ser Gly Leu Lys Pro Asn Thr

725 730 735

Val Val Leu Val Ala Thr Ser Arg Ala Leu Lys Leu His Gly Gly Ala

740 745 750

Pro Asp Val Lys Pro Gly Gln Ala Leu Pro Glu Glu Tyr Thr Thr Glu

755 760 765

Asn Val Glu Leu Val Arg Arg Gly Cys Ala Asn Leu Ala Lys Gln Ile

770 775 780

Ser Asn Ala Ser Gln Tyr Gly Ala Pro Val Val Val Ala Ile Asn Gln

785 790 795 800

Phe Glu Thr Asp Ser Pro Ala Glu Leu Ser Val Ile Arg Glu Glu Ala

805 810 815

Leu Lys Ala Gly Ala Phe Asp Ala Val Pro Ser Asn His Trp Ala Glu

820 825 830

Gly Gly Lys Gly Ala Val Glu Leu Ala Lys Ala Val Val Ser Ser Cys

835 840 845

Ala Ser Cys Pro Glu Pro Asn Ala Glu Lys Ala Glu Leu Tyr Ser Leu

850 855 860

Glu Asn Asn Thr Ile Glu Asp Arg Leu Asn Thr Ile Val Gln Lys Met

865 870 875 880

Tyr Asn Gly Asp Gly Ile Glu Leu Ser Glu Leu Ala Lys Glu Gln Ile

885 890 895

Ala Arg Tyr Glu Ala Gln Gly Phe Gly Asn Leu Pro Ile Cys Ile Ala

900 905 910

Lys Thr Gln Tyr Ser Leu Ser His Asp Pro Ser Leu Lys Asn Val Pro

915 920 925

Ala Gly Phe Thr Val Pro Ile Arg Glu Val Arg Leu Ser Ala Gly Ala

930 935 940

Gly Tyr Leu Ser Cys Leu Ala Ala Glu Ile Met Thr Ile Pro Gly Leu

945 950 955 960

Pro Thr His Pro Gly Tyr Leu Asn Val Glu Val Asn Asp Asp Gly Glu

965 970 975

Ile Glu Gly Leu Phe

980

<210> 10

<211> 391

<212> PRT

<213> amino acid sequence (Amino Acid Sequence)

<400> 10

Met Ile Arg Ser Cys Arg Arg Tyr Ser Thr Gly Val Glu Ala Leu Ile

1 5 10 15

Lys Thr Pro Leu Tyr Ser Val His Val Glu His Gly Ala Thr Leu Val

20 25 30

Pro Tyr Ala Asn Phe Ala Met Pro Val Leu Tyr Lys Gly Gln Ser His

35 40 45

Ile Glu Ser His Asn Trp Thr Arg Ser Lys Val Gly Ile Phe Asp Val

50 55 60

Ser His Met Leu Gln His Arg Val Lys Gly Asn Ser Ala Ala Glu Phe

65 70 75 80

Leu Gln Lys Ile Thr Pro Ser Asp Leu Lys Ala Leu Lys Pro Phe Thr

85 90 95

Ser Thr Leu Ser Val Leu Leu Asn Asp Lys Gly Gly Val Ile Asp Asp

100 105 110

Cys Ile Ile Thr Lys His Asp Glu Asn Glu Phe Tyr Ile Val Thr Asn

115 120 125

Ala Gly Cys Arg Asp Lys Asp Thr Ser Phe Ile Lys Glu Glu Ile Ser

130 135 140

Gln Phe Gly Asn Ala Ser His Glu Asn Phe Glu Gly Thr Leu Leu Ala

145 150 155 160

Ile Gln Gly Pro Gln Ala Ala Glu Thr Leu Gln Lys Phe Thr Asn Leu

165 170 175

Asp Leu Thr Lys Leu Tyr Phe Gly Asn Ser Ala Phe Ala Lys Leu Glu

180 185 190

Gly Phe Thr Ser Asp Val Ile His Ile Ala Arg Ser Gly Tyr Thr Gly

195 200 205

Glu Asp Gly Phe Glu Leu Ser Ile Pro Val Asp Ser Glu Gly Leu Glu

210 215 220

Phe Thr Lys Ala Leu Leu Glu Leu Glu Gln Val Lys Pro Ile Gly Leu

225 230 235 240

Ala Ala Arg Asp Ser Leu Arg Leu Glu Ala Gly Met Cys Leu Tyr Gly

245 250 255

His Glu Leu Asn Glu Gln Thr Thr Pro Val Glu Ser Ser Leu Asn Trp

260 265 270

Leu Ile Ala Lys Ser Arg Arg Thr Pro Lys Thr Ala Gly Phe Asn Gly

275 280 285

Ser Glu Asn Ile Leu Ser Gln Leu Glu Asn Pro Lys Ser Val Thr Phe

290 295 300

Lys Arg Val Gly Ile Gln Ser Lys Gly Pro Ser Pro Arg Glu Gly Asn

305 310 315 320

Lys Val Tyr Ser Phe Glu Glu Pro Asp Lys Gln Ile Gly Val Val Cys

325 330 335

Ser Gly Ser Pro Ser Pro Thr Leu Gly Gly Asn Val Gly Gln Ala Phe

340 345 350

Ile His Lys Pro His Gln Lys Ala Gly Thr Lys Ile Leu Ile Glu Ile

355 360 365

Arg Asn Lys Lys Arg Glu Ala His Val Ala Lys Leu Pro Phe Val Ala

370 375 380

Pro Lys Phe Tyr Lys Pro Glu

385 390

<210> 11

<211> 1015

<212> PRT

<213> amino acid sequence (Amino Acid Sequence)

<400> 11

Met Leu Arg Thr Gln Leu Ser Lys Ala Leu Leu Lys Ser His Ser Val

1 5 10 15

Lys Ala Trp Ala Arg Val Gly Thr Ala Thr Thr Ser Arg Phe Val Ser

20 25 30

Asp Thr Thr Ser Ala Thr Phe Arg Ser Ile Tyr Glu Lys Gly Ser Ser

35 40 45

Thr Pro Ser Pro Tyr Asp Gly Thr Asp Ser Phe Pro Arg Arg His Leu

50 55 60

Gly Pro Thr Pro Asn Asn Val Asp Glu Met Leu Gln Asp Leu Lys Glu

65 70 75 80

Ser Asp Leu Asp Ser Phe Ile Ala Lys Val Val Pro Asp Asn Val Leu

85 90 95

Ile Arg Arg Ser Leu Lys Val Glu Pro Leu Lys Gly Phe Thr Glu Ser

100 105 110

Glu Met Leu Ala Glu Leu Ala Arg Ile Ala Glu Lys Asn Asp Leu Val

115 120 125

Lys Pro Leu Ile Gly Lys Gly Phe Tyr Gly Thr Ile Ile Pro Pro Val

130 135 140

Ile Leu Arg Asn Leu Leu Glu Asn Pro Gly Trp Tyr Thr Ser Tyr Thr

145 150 155 160

Pro Tyr Gln Pro Glu Val Ser Gln Gly Arg Leu Glu Ser Leu Leu Asn

165 170 175

Phe Gln Thr Val Ile Ser Asp Leu Thr Gly Leu Pro Val Ala Asn Ala

180 185 190

Ser Leu Leu Asp Glu Ser Ser Ala Ala Ala Glu Ala Met Ile Leu Ser

195 200 205

Phe Thr Ser Leu Lys Ser Lys Lys Pro Lys Tyr Phe Val Asp Ser Asn

210 215 220

Ile His Glu Gln Thr Leu Ala Val Leu Lys Ser Arg Ala His Thr Leu

225 230 235 240

Ser Ile Glu Val Val Val Ala Asp Leu Thr Thr Asp Glu Gly Phe Ala

245 250 255

Ala Leu Gln Glu Asn Lys Asp Gln Leu Cys Gly Ala Met Val Gln Tyr

260 265 270

Pro Ala Thr Asp Gly Ser Ile Glu Thr Phe Lys Ala Tyr Ser Arg Ile

275 280 285

Ala Glu Leu Val His Ser Val Lys Gly Leu Tyr Ala Val Ala Ser Asp

290 295 300

Leu Met Ala Leu Thr Leu Leu His Ala Pro Ser Lys Phe Gly Ala Asp

305 310 315 320

Ile Val Phe Gly Ser Ser Gln Arg Phe Gly Val Pro Leu Gly Tyr Gly

325 330 335

Gly Pro His Ala Ala Phe Phe Ser Val Val Glu Ser Leu Gln Arg Lys

340 345 350

Ile Pro Gly Arg Ile Val Gly Val Ser Lys Asp Arg Leu Gly Asn Asn

355 360 365

Ala Leu Arg Leu Ala Leu Gln Thr Arg Glu Gln His Ile Lys Arg Glu

370 375 380

Arg Ala Thr Ser Asn Ile Cys Thr Ala Gln Ala Leu Leu Ala Asn Ile

385 390 395 400

Ala Ala Asn Tyr Val Val Tyr His Gly Ile Glu Gly Leu Arg Asn Ile

405 410 415

Ser Lys Arg Ile His Gly Phe Thr Thr Leu Leu Ala Asn Ser Ile Asn

420 425 430

Glu Ser Ser Ser His Lys Val Leu Asn Glu Lys Trp Phe Asp Thr Leu

435 440 445

Ser Val Asp Val Gly Ser Ala Asp Glu Phe Ile Ala Lys Ala Leu Ser

450 455 460

Lys Asn Phe Asn Val Phe Arg Val Asn Asn Ser Thr Ile Gln Leu Ser

465 470 475 480

Leu Asp Glu Thr Val Thr Lys Asn Glu Leu Thr Thr Leu Ile Ser Leu

485 490 495

Phe Thr Gly Ser Ser Glu Val Asn Leu Pro Glu Thr Leu Pro Ser Phe

500 505 510

Tyr Glu Glu Trp Thr Arg Gln Asp Asp Ile Leu Thr Asn Glu Val Phe

515 520 525

Arg Thr His Arg Ser Glu Thr Ser Met Leu Arg Tyr Leu Thr His Leu

530 535 540

Gln Lys Lys Asp Ile Ser Leu Ala Asp Ser Met Ile Ser Leu Gly Ser

545 550 555 560

Cys Thr Met Lys Leu Asn Ala Thr Val Glu Met Met Pro Ile Ser Trp

565 570 575

Pro Lys Phe Ala Asn Ile His Pro Phe Ala Pro Lys Asp Gln Val Lys

580 585 590

Gly Tyr Thr Glu Leu Ile Val Glu Leu Glu Lys Asp Leu Ala Asp Ile

595 600 605

Thr Gly Phe His Ser Thr Thr Leu Gln Pro Asn Ser Gly Ala Gln Gly

610 615 620

Glu Tyr Thr Gly Leu Ser Val Ile Ala Lys Tyr Phe Glu Ser Lys Gly

625 630 635 640

Glu Ala His Arg Asn Ile Val Leu Ile Pro Val Ser Ala His Gly Thr

645 650 655

Asn Pro Ala Ser Ala Ala Met Ala Gly Leu Lys Val Val Pro Ile Lys

660 665 670

Cys Leu Lys Asp Gly Ser Leu Asp Leu Gln Asp Leu Glu Ser Lys Ala

675 680 685

Ser Lys His Ala Lys Asn Leu Ala Ala Met Met Val Thr Tyr Pro Ser

690 695 700

Thr Tyr Gly Leu Phe Glu Pro Gly Ile Ile Asp Ala Ile Lys Ile Ile

705 710 715 720

His Asn His Gly Gly Gln Val Tyr Leu Asp Gly Ala Asn Met Asn Ala

725 730 735

Gln Val Gly Leu Thr Ser Pro Gly Asp Leu Gly Ala Asp Val Cys His

740 745 750

Leu Asn Leu His Lys Thr Phe Ala Ile Pro His Gly Gly Gly Gly Pro

755 760 765

Gly Val Gly Pro Ile Cys Val Lys Glu His Leu Thr Pro Phe Leu Pro

770 775 780

Ser His Pro Ile Ile Ala Thr Thr Asn Gln Thr Glu Gln Ser Ile Asn

785 790 795 800

Pro Val Val Ala Ala Pro Phe Gly Ser Ala Ser Ile Leu Pro Ile Ser

805 810 815

Tyr Ala Tyr Ile Lys Met Met Gly Gly Ser Asn Leu Gln Tyr Ser Ser

820 825 830

Ile Ile Ala Ile Leu Asn Ala Asn Tyr Met Val Ala Lys Leu Lys Asn

835 840 845

His Phe Glu Ile Leu Phe Thr Gly Gly Asp Ala Lys Tyr Cys Gly His

850 855 860

Glu Phe Ile Ile Asp Leu Arg Pro Phe Lys Lys Phe Gly Ile Glu Ala

865 870 875 880

Ile Asp Val Ala Lys Arg Leu Gln Asp Tyr Gly Phe His Ala Pro Thr

885 890 895

Met Ser Phe Pro Ile Pro Gly Thr Leu Met Val Glu Pro Thr Glu Ser

900 905 910

Glu Tyr Lys Glu Glu Leu Asp Arg Phe Ile Asp Ser Met Ile Ser Ile

915 920 925

Arg Glu Glu Ile Arg Lys Val Glu Asn Gly Glu Thr Asp Gly Ala Ile

930 935 940

Leu His Asn Ser Pro His Asn Leu Lys Asp Ile Ile Ser Thr Ser Asp

945 950 955 960

Ala Glu Trp Ser Gln Arg Gly Tyr Thr Arg Glu Glu Ala Ala Tyr Pro

965 970 975

Leu Pro Phe Leu Lys Glu Gln Lys Thr Trp Pro Thr Val Ser Arg Val

980 985 990

Asp Asp Thr Tyr Gly Asp Met Asn Leu Leu Cys Thr Cys Pro Ser Val

995 1000 1005

Glu Glu Val Ala Ala Ser Gln

1010 1015

<210> 12

<211> 1015

<212> PRT

<213> amino acid sequence (Amino Acid Sequence)

<400> 12

Met Leu Arg Thr Gln Leu Ser Lys Ala Leu Leu Lys Ser His Ser Val

1 5 10 15

Lys Ala Trp Ala Arg Val Gly Thr Ala Thr Thr Ser Arg Phe Val Ser

20 25 30

Asp Thr Thr Ser Ala Thr Phe Arg Ser Ile Tyr Glu Lys Gly Ser Ser

35 40 45

Thr Pro Ser Pro Tyr Asp Gly Thr Asp Ser Phe Pro Arg Arg His Leu

50 55 60

Gly Pro Thr Pro Asn Asn Val Asp Glu Met Leu Gln Asp Leu Lys Glu

65 70 75 80

Ser Asp Leu Asp Ser Phe Ile Ala Lys Val Val Pro Asp Asn Val Leu

85 90 95

Ile Arg Arg Ser Leu Lys Val Glu Pro Leu Lys Gly Phe Thr Glu Ser

100 105 110

Glu Met Leu Ala Glu Leu Ala Arg Ile Ala Glu Lys Asn Asp Leu Val

115 120 125

Lys Pro Leu Ile Gly Lys Gly Phe Tyr Gly Thr Ile Ile Pro Pro Val

130 135 140

Ile Leu Arg Asn Leu Leu Glu Asn Pro Gly Trp Tyr Thr Ser Tyr Thr

145 150 155 160

Pro Tyr Gln Pro Glu Val Ser Gln Gly Arg Leu Glu Ser Leu Leu Asn

165 170 175

Phe Gln Thr Val Ile Ser Asp Leu Thr Gly Leu Pro Val Ala Asn Ala

180 185 190

Ser Leu Leu Asp Glu Ser Ser Ala Ala Ala Glu Ala Met Ile Leu Ser

195 200 205

Phe Thr Ser Leu Lys Ser Lys Lys Pro Lys Tyr Phe Val Asp Ser Asn

210 215 220

Ile His Glu Gln Thr Leu Ala Val Leu Lys Ser Arg Ala His Thr Leu

225 230 235 240

Ser Ile Glu Val Val Val Ala Asp Leu Thr Thr Asp Glu Gly Phe Ala

245 250 255

Ala Leu Gln Glu Asn Lys Asp Gln Leu Cys Gly Ala Met Val Gln Tyr

260 265 270

Pro Ala Thr Asp Gly Ser Ile Glu Thr Phe Lys Ala Tyr Ser Arg Ile

275 280 285

Ala Glu Leu Val His Ser Val Lys Gly Leu Tyr Ala Val Ala Ser Asp

290 295 300

Leu Met Ala Leu Thr Leu Leu His Ala Pro Ser Lys Phe Gly Ala Asp

305 310 315 320

Ile Val Phe Gly Ser Ser Gln Arg Phe Gly Val Pro Leu Gly Tyr Gly

325 330 335

Gly Pro His Ala Ala Phe Phe Ser Val Val Glu Ser Leu Gln Arg Lys

340 345 350

Ile Pro Gly Arg Ile Val Gly Val Ser Lys Asp Arg Leu Gly Asn Asn

355 360 365

Ala Leu Arg Leu Ala Leu Gln Thr Arg Glu Gln His Ile Lys Arg Glu

370 375 380

Arg Ala Thr Ser Asn Ile Cys Thr Ala Gln Ala Leu Leu Ala Asn Ile

385 390 395 400

Ala Ala Asn Tyr Val Val Tyr His Gly Ile Glu Gly Leu Arg Asn Ile

405 410 415

Ser Lys Arg Ile His Gly Phe Thr Thr Leu Leu Ala Asn Ser Ile Asn

420 425 430

Glu Ser Ser Ser His Lys Val Leu Asn Glu Lys Trp Phe Asp Thr Leu

435 440 445

Ser Val Asp Val Gly Ser Ala Asp Glu Phe Ile Ala Lys Ala Leu Ser

450 455 460

Lys Asn Phe Asn Val Phe Arg Val Asn Asn Ser Thr Ile Gln Leu Ser

465 470 475 480

Leu Asp Glu Thr Val Thr Lys Asn Glu Leu Thr Thr Leu Ile Ser Leu

485 490 495

Phe Thr Gly Ser Ser Glu Val Asn Leu Pro Glu Thr Leu Pro Ser Phe

500 505 510

Tyr Glu Glu Trp Thr Arg Gln Asp Asp Ile Leu Thr Asn Glu Val Phe

515 520 525

Arg Thr His Arg Ser Glu Thr Ser Met Leu Arg Tyr Leu Thr His Leu

530 535 540

Gln Lys Lys Asp Ile Ser Leu Ala Asp Ser Met Ile Ser Leu Gly Ser

545 550 555 560

Cys Thr Met Lys Leu Asn Ala Thr Val Glu Met Met Pro Ile Ser Trp

565 570 575

Pro Lys Phe Ala Asn Ile His Pro Phe Ala Pro Lys Asp Gln Val Lys

580 585 590

Gly Tyr Thr Glu Leu Ile Val Glu Leu Glu Lys Asp Leu Ala Asp Ile

595 600 605

Thr Gly Phe His Ser Thr Thr Leu Gln Pro Asn Ser Gly Ala Gln Gly

610 615 620

Glu Tyr Thr Gly Leu Ser Val Ile Ala Lys Tyr Phe Glu Ser Lys Gly

625 630 635 640

Glu Ala His Arg Asn Ile Val Leu Ile Pro Val Ser Ala His Gly Thr

645 650 655

Asn Pro Ala Ser Ala Ala Met Ala Gly Leu Lys Val Val Pro Ile Lys

660 665 670

Cys Leu Lys Asp Gly Ser Leu Asp Leu Gln Asp Leu Glu Ser Lys Ala

675 680 685

Ser Lys His Ala Lys Asn Leu Ala Ala Met Met Val Thr Tyr Pro Ser

690 695 700

Thr Tyr Gly Leu Phe Glu Pro Gly Ile Ile Asp Ala Ile Lys Ile Ile

705 710 715 720

His Asn His Gly Gly Gln Val Tyr Leu Asp Gly Ala Asn Met Asn Ala

725 730 735

Gln Val Gly Leu Thr Ser Pro Gly Asp Leu Gly Ala Asp Val Cys His

740 745 750

Leu Asn Leu His Lys Thr Phe Ala Ile Pro His Gly Gly Gly Gly Pro

755 760 765

Gly Val Gly Pro Ile Cys Val Lys Glu His Leu Thr Pro Phe Leu Pro

770 775 780

Ser His Pro Ile Ile Ala Thr Thr Asn Gln Thr Glu Gln Ser Ile Asn

785 790 795 800

Pro Val Val Ala Ala Pro Phe Gly Ser Ala Ser Ile Leu Pro Ile Ser

805 810 815

Tyr Ala Tyr Ile Lys Met Met Gly Gly Ser Asn Leu Gln Tyr Ser Ser

820 825 830

Ile Ile Ala Ile Leu Asn Ala Asn Tyr Met Val Ala Lys Leu Lys Asn

835 840 845

His Phe Glu Ile Leu Phe Thr Gly Gly Asp Ala Lys Tyr Cys Gly His

850 855 860

Glu Phe Ile Ile Asp Leu Arg Pro Phe Lys Lys Phe Gly Ile Glu Ala

865 870 875 880

Ile Asp Val Ala Lys Arg Leu Gln Asp Tyr Gly Phe His Ala Pro Thr

885 890 895

Met Ser Phe Pro Ile Pro Gly Thr Leu Met Val Glu Pro Thr Glu Ser

900 905 910

Glu Tyr Lys Glu Glu Leu Asp Arg Phe Ile Asp Ser Met Ile Ser Ile

915 920 925

Arg Glu Glu Ile Arg Lys Val Glu Asn Gly Glu Thr Asp Gly Ala Ile

930 935 940

Leu His Asn Ser Pro His Asn Leu Lys Asp Ile Ile Ser Thr Ser Asp

945 950 955 960

Ala Glu Trp Ser Gln Arg Gly Tyr Thr Arg Glu Glu Ala Ala Tyr Pro

965 970 975

Leu Pro Phe Leu Lys Glu Gln Lys Thr Trp Pro Thr Val Ser Arg Val

980 985 990

Asp Asp Thr Tyr Gly Asp Met Asn Leu Leu Cys Thr Cys Pro Ser Val

995 1000 1005

Glu Glu Val Ala Ala Ser Gln

1010 1015

<210> 13

<211> 299

<212> PRT

<213> amino acid sequence (Amino Acid Sequence)

<400> 13

Met Ser Ser Ile Thr Thr Ser Ile Phe Lys Val Thr Ala Glu Ile Gln

1 5 10 15

Ser Phe Gly Gly Lys Leu Val Lys Leu Gln His Lys Ser Asp Glu Thr

20 25 30

Lys Thr Asp Met Asp Val Asn Val Tyr Leu Pro Ala Gln Phe Phe Ala

35 40 45

Asn Gly Ala Lys Gly Lys Ser Leu Pro Val Leu Leu Tyr Leu Ser Gly

50 55 60

Leu Thr Cys Thr Pro Asn Asn Ala Ser Glu Lys Ala Phe Trp Gln Pro

65 70 75 80

Tyr Ala Asn Lys Tyr Gly Phe Ala Val Val Phe Pro Asp Thr Ser Pro

85 90 95

Arg Gly Leu Asn Ile Glu Gly Glu His Asp Ser Tyr Asp Phe Gly Ser

100 105 110

Gly Ala Gly Phe Tyr Val Asp Ala Thr Thr Glu Lys Trp Lys Asp Asn

115 120 125

Tyr Arg Met Tyr Ser Tyr Val Asn Ser Glu Leu Leu Pro Lys Leu Gln

130 135 140

Ala Asp Phe Pro Ile Leu Asn Phe Asp Asn Ile Ser Ile Thr Gly His

145 150 155 160

Ser Met Gly Gly Tyr Gly Ala Leu Gln Leu Phe Leu Arg Asn Pro Gly

165 170 175

Lys Phe Lys Ser Val Ser Ala Phe Ser Pro Ile Ser Asn Pro Thr Lys

180 185 190

Ala Pro Trp Gly Glu Lys Cys Phe Ser Gly Tyr Leu Gly Gln Asp Lys

195 200 205

Ser Thr Trp Thr Gln Tyr Asp Pro Thr Glu Leu Ile Gly Lys Tyr Gln

210 215 220

Gly Pro Ser Asp Ser Ser Ile Leu Ile His Val Gly Lys Ser Asp Ser

225 230 235 240

Phe Tyr Phe Lys Asp His Gln Leu Leu Pro Glu Asn Phe Leu Lys Ala

245 250 255

Ser Glu Asn Ser Val Phe Lys Gly Lys Val Asp Leu Asn Leu Val Asp

260 265 270

Gly Tyr Asp His Ser Tyr Tyr Phe Ile Ser Ser Phe Thr Asp Val His

275 280 285

Ala Ala His His Ala Lys Tyr Leu Gly Leu Asn

290 295

<210> 14

<211> 379

<212> PRT

<213> amino acid sequence (Amino Acid Sequence)

<400> 14

Met Ser Thr Glu Gly Gln Ile Ile Lys Cys Lys Ala Ala Val Ala Trp

1 5 10 15

Glu Ala Gly Lys Asp Leu Ser Ile Glu Glu Ile Glu Val Leu Pro Pro

20 25 30

Arg Ala His Glu Val Arg Val Lys Val Glu Phe Thr Gly Val Cys His

35 40 45

Thr Asp Ala Tyr Thr Leu Ser Gly Ala Asp Ala Glu Gly Ser Phe Pro

50 55 60

Val Val Phe Gly His Glu Gly Ala Gly Val Val Glu Ser Val Gly Glu

65 70 75 80

Gly Val Glu Ser Val Lys Val Gly Asp Ser Val Val Leu Leu Tyr Thr

85 90 95

Pro Glu Cys Arg Glu Cys Lys Phe Cys Leu Ser Gly Lys Thr Asn Leu

100 105 110

Cys Gly Lys Ile Arg Ala Thr Gln Gly Lys Gly Leu Leu Pro Asp Gly

115 120 125

Thr Ser Arg Phe Arg Cys Lys Gly Lys Asp Leu Phe His Tyr Met Gly

130 135 140

Cys Ser Ser Phe Ser Gln Tyr Thr Val Val Ala Asp Ile Ser Val Val

145 150 155 160

Lys Val Gln Asp Glu Ala Pro Lys Asp Lys Thr Cys Leu Leu Gly Cys

165 170 175

Gly Val Thr Thr Gly Tyr Gly Ala Ala Ile Asn Thr Ala Lys Ile Ser

180 185 190

Lys Gly Asp Lys Ile Gly Val Phe Gly Ala Gly Cys Ile Gly Leu Ser

195 200 205

Val Ile Gln Gly Ala Val Ser Lys Gly Ala Ser Glu Ile Ile Val Ile

210 215 220

Asp Ile Asn Asp Ser Lys Lys Ala Trp Ala Asp Gln Phe Gly Ala Thr

225 230 235 240

Lys Phe Val Asn Pro Thr Thr Leu Pro Glu Gly Thr Asn Ile Val Asp

245 250 255

Tyr Leu Ile Asp Ile Thr Asp Gly Gly Phe Asp Tyr Thr Phe Asp Cys

260 265 270

Thr Gly Asn Val Gln Val Met Arg Asn Ala Leu Glu Ser Cys His Lys

275 280 285

Gly Trp Gly Glu Ser Ile Ile Ile Gly Val Ala Ala Ala Gly Lys Glu

290 295 300

Ile Ser Thr Arg Pro Phe Gln Leu Val Thr Gly Arg Val Trp Arg Gly

305 310 315 320

Cys Ala Phe Gly Gly Ile Lys Gly Arg Thr Gln Met Pro Ser Leu Val

325 330 335

Gln Asp Tyr Leu Asp Gly Lys Ile Lys Val Asp Glu Phe Ile Thr His

340 345 350

Arg His Asp Leu Asp Asn Ile Asn Lys Ala Phe His Asp Met His Ala

355 360 365

Gly Asn Cys Ile Arg Ala Val Ile Thr Met His

370 375

<210> 15

<211> 58

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

caactaatta ttcgaaggat ccgaaacgat gtcatcaatt actacttcaa tcttcaag 58

<210> 16

<211> 75

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

cacgtctcct gcttgcttta acagagagaa gttcgtggcc aacttagagt ttaaccccaa 60

atactttgca tggtg 75

<210> 17

<211> 76

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

tgttaaagca agcaggagac gtggaagaaa accccggtcc tgaaacgatg tctaccgaag 60

gtcaagtaag ttcaat 76

<210> 18

<211> 55

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

aaggcgaatt aattcgcggc cgcttacaac ttagagtgca tagtaatcac agcac 55

Claims

1. The construction method of the Pichia pastoris genetic engineering bacteria is characterized by comprising the following steps:

step 3, constructing recombinant plasmids pGAP-MIS1-P2A-GCV1 and pGAP-GCV2-P2A-GCV3, and a knockout plasmid pET28a-His for knocking out DAS genes, wherein the recombinant plasmid pGAP-MIS1-P2A-GCV1 is a recombinant plasmid containing the MIS1 gene fragment and the GCV1 gene fragment amplified in the step 1, the recombinant plasmid pGAP-GCV2-P2A-GCV3 is a recombinant plasmid containing the GCV2 gene fragment and the GCV3 gene fragment amplified in the step 1, the amino acid sequence coded by the MIS1 gene fragment is shown as SEQ ID No. 9, and the amino acid sequence coded by the GCV1 gene fragment is shown as SEQ ID No. 10; the amino acid sequence of the GCV2 gene fragment is shown as SEQ ID No. 11, and the amino acid sequence of the GCV3 gene fragment is shown as SEQ ID No. 12;

2. The method for constructing a genetically engineered pichia pastoris of claim 1, wherein the reaction conditions for the PCR in step 1 are: 95 ℃ for 2 min, 95 ℃ for 20 s,55 ℃ for 20 s,72 ℃ for 10 s, and 30 cycles in total; and at 72℃for 5 min.

3. The application of the Pichia pastoris engineering bacteria constructed by the construction method of claim 1 in improving methanol assimilation rate of Pichia pastoris and fixing carbon dioxide.