CN113249237A

CN113249237A - Pichia pastoris engineering strain KM71H _ pCHIT and construction method and application thereof

Info

Publication number: CN113249237A
Application number: CN202110356267.4A
Authority: CN
Inventors: 肖萌; 米哈尔·卡茨马雷克; 蒋莹; 毛睿慈
Original assignee: Peking University Shenzhen Graduate School
Current assignee: Peking University Shenzhen Graduate School
Priority date: 2021-04-01
Filing date: 2021-04-01
Publication date: 2021-08-13

Abstract

The invention relates to the technical field of genetic engineering, in particular to a Pichia pastoris engineering strain and a construction method and application thereof. After being induced by methanol, the engineered strain constructed by the present invention can simultaneously secrete chitinase protein (ChdaII), chitosanase protein (ChiA) and chitin deacetylase protein (ChoA) in the extracellular space. Its derivatives were enzymatically degraded, and their corresponding enzymatic activities were 1.99U/mL, 3.46U/mL, and 0.25U/mL, respectively. Compared with the traditional thermochemical degradation method, the engineering strain of the present invention is efficient, environmentally friendly, and has a specific product, and is suitable for industrial application.

Description

Pichia pastoris engineering strain KM71H _ pCHIT and construction method and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, and particularly relates to a pichia pastoris engineering strain, and a construction method and application thereof.

Background

Chitin is one of the most abundant natural biopolymers in nature, second only to cellulose. Chitin is found in nature mainly in crustacean shells, insect shells and the cell walls of filamentous fungi, whereas in the marine and biotechnological industries, these natural chitins are often regarded as waste pollutants. The chitin and the degradation products chitosan and chitosan oligosaccharide thereof have wide application value in the fields of food, cosmetics, medicine, agriculture, biofuel and the like. However, chitin is very limited in its industrial application due to its highly crystalline structure and its insolubility in common organic substances.

At present, chitin is mainly degraded by a thermochemical method, and the method has low production efficiency, more byproducts and serious environmental pollution. Enzymatic modification of chitin and its derivatives can effectively overcome the defects of the traditional methods. Chitin deacetylases (EC 3.5.1.41) biologically convert chitin to chitosan through a deacetylation enzymatic reaction. This enzymatic reaction has many advantages over traditional chemical degradation, the main advantage of which is the higher degree of polymerization and higher degree of deacetylation of the resulting chitosan. The other two hydrolases involved in the conversion of chitin and chitosan are chitosanase (EC 3.2.1.132) and chitinase (EC 3.2.1.14), both of which act to catalyze the hydrolysis of glycosidic bonds, but differ in their specific substrates, the former being the hydrolytic bond of chitosan and the latter being the hydrolytic bond of chitin. Because the catalytic substrate protein has high specificity, the catalytic reaction is easier to control and the purity of the product is higher. However, these three catalytic reactions often need to be performed in multiple stages without intersecting, which reduces the efficiency of the overall reaction, increases the cost, and is not suitable for large-scale industrial production. Therefore, it is important to develop a system capable of simultaneously performing three catalytic reactions, and the synthesis of a eukaryotic polycistronic expression vector capable of simultaneously encoding chitinase, chitosanase and chitin deacetylase genes can effectively achieve the purpose, so that the economic cost of chitin degradation can be greatly reduced, and the large-scale application of the chitin in industrial production is facilitated.

Disclosure of Invention

The invention aims to construct a pichia pastoris engineered strain KM71H _ pCHIT capable of producing chitinase, chitosanase and chitin deacetylase simultaneously. The genes chdaiII, chia and choa are connected and inserted into a pichia integrated vector pPICZ alpha C by a 2A peptide and LCR technology to obtain a polycistronic expression vector of an engineering strain KM71H _ pCHIT. And the vector is introduced into a pichia pastoris KM71H competent cell through electrotransformation, so as to obtain a KM71H _ pCHIT engineering strain. After fermentation culture in YPD culture medium and methanol induction expression, chitinase, chitosanase and chitin deacetylase can be produced simultaneously.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a construction method of a pichia pastoris engineering strain comprises the following specific steps:

(1) the chdaII gene was amplified by PCR from an existing pJET transformant, and its nucleotide sequence was shown in SEQ ID NO. 1.

(2) The chia and choa genes are obtained by synthesis, and the nucleotide sequences are respectively shown as SEQ ID NO.2 and SEQ ID NO. 3.

(3) The vectors for the co-expression of chdaII, chia and choa genes were linked by 2A peptide, 2A peptide bridging oligonucleotides were synthesized based on the amino acid sequences P2A (ATNFSLLKQAGDVEENPGP) and T2A (EGRGSLLTCGDVEENPGP), respectively, the bridging oligonucleotide sequences are shown in SEQ ID No.4 and SEQ ID No.5, respectively, and DNA shuffling was performed by the program with the following program parameters: 95-2 min; 70-10 min; 72-5 min; 4 ℃ to end.

(4) Connecting each part of DNA fragments of an expression vector of the pichia pastoris engineering bacteria by an LCR method, connecting the obtained synthetic fragment with a pichia pastoris integrated vector pPICZ alpha C, then converting the synthetic fragment into E.coli DH5 alpha competent cells For amplification, after resuscitating and shaking the bacteria and plating the cells, selecting a single clone, carrying out PCR positive identification (AOX1_ For5 '-GACTGGTTCCAATTGACAAGC-3' and AOX1_ Rev 5'-GCAAATGGCATTCTGA CATCC-3') by using a universal primer, sending the single clone to sequencing identification, extracting recombinant plasmids from the successfully constructed strain, extracting the recombinant plasmids from the E.coli DH5 alpha, and obtaining the expression vector of the pichia pastoris engineering bacteria.

(5) The expression vector of the pichia pastoris engineered strain obtained in the step (4) is digested by restriction enzyme SacI overnight, the obtained linear plasmid (100ng) is added into 100ul of pichia pastoris KM71H electric transformation competent cells, then the mixture is carefully transferred into a frozen and sterile electric transfer tank slide for electric transfer (voltage is 1.5 kilovolt, resistance is 125 ohms, pulse length is 3 milliseconds), the cells are resuspended in 500 ul of 1M cold sorbitol immediately after the electric transfer, and then 500 ul of YPD culture medium with pH value of 7.5 is added into the cell mixture. The cells were shake-cultured at 30 ℃ for 3-4 hours, after completion of transformation, the cells were pelleted by centrifugation, 700. mu.l of supernatant was removed, the cells were resuspended in the remaining medium, plated onto YPDS agar plates containing 100. mu.g/mL Zeocin, and cultured at 30 ℃ for 48 hours.

(6) A single colony was picked and colony PCR was performed using a series of primers, as shown in Table 1, to confirm successful transformation of the plasmid into the host, thereby obtaining a Pichia pastoris engineered strain KM71H _ pCHIT.

The invention also provides an application of the pichia pastoris engineering strain KM71H _ pCHIT. The method comprises the following steps:

(1) the Pichia pastoris KM71H single colony with the target plasmid is inoculated into 25mLBMGY culture medium containing 100 mu g/mL bleomycin (placed in a 250mL conical flask with a baffle), and is subjected to shake culture at the rotating speed of 250-300 rpm at the temperature of 28-30 ℃ for 18h until the OD is reached₆₀₀Is 10-12.

(2) Taking the bacterial liquid obtained in the step (1) into a 1000mL conical flask with a baffle and 100mL BMGY to the final OD₆₀₀After 0.2, the mixture is cultured at 28-30 ℃ with shaking at the rotation speed of 250-300 rpm to OD₆₀₀Is 2-3.

(3) And (3) centrifuging the bacterial liquid obtained in the step (2) at the rotating speed of 1,500-3,000 Xg for 5min at room temperature to obtain thalli, then suspending the thalli in 20mL of BMMY culture medium (placing the BMMY culture medium in a 100mL conical flask with a baffle, sealing the conical flask with two layers of sterile gauze), adding pure methanol to the concentration of 0.5%, and carrying out induced expression on the engineering strain KM71H _ pCHIT.

The main benefit of the invention is to solve the economic burden brought by multi-stage catalysis in chitin biotransformation, the invention connects the genes coding chitinase, chitosanase and chitin deacetylase on a carrier through 2A peptide, and expresses in pichia pastoris, so as to achieve the purpose of catalyzing a plurality of chemical reactions simultaneously. The invention provides technical support for the biological transformation industrialization of chitin.

Drawings

FIG. 1 recombinant plasmid map of Pichia pastoris engineering strain KM71H _ pCHIT

FIG. 2 is a schematic diagram of colony PCR result and expression system of Pichia pastoris engineering strain KM71H _ pCHIT

FIG. 3SDS-PAGE analysis of KM 71H-pCHIT expression results induced by methanol

Detailed Description

In the examples described below, the chitinase-encoding gene and the chitosanase-encoding gene, alpha-factor and 2A peptide-bridged oligonucleotide were synthesized by Genewiz (Suzhou, China), but are not limited thereto. pJET transformant was the existing sample, and chdaII gene was derived from Mucor circinelloides (IBT-8 strain).

Example 1 Synthesis of KM71H _ pCHIT expression System

1.1 full Length cloning of the chitin deacetylase (chdaiII) Gene

Amplification of the chdaII gene was performed by high fidelity PCR from known pJET transformants, and also by high fidelity PCR using the synthetic chia and choa genes as templates.

1.2 joining the target gene fragment of each part of the engineered strain KM71H _ pCHIT vector by LCR assembling method

The specific reaction system (20 μ l) of the assembly method is as follows: 2nM of each DNA fragment of the gene of interest, 10nM bridging oligonucleotide, 1xTaq ligase buffer, 5% DMSO and 40U Taq ligase. The assembly method comprises the following specific program parameters: first, initial denaturation at 95 ℃ for 2 min; then denaturation at 95 ℃ for 30sec, annealing at 60 ℃ for 2min, and performing 30 cycles; finally, the extension is carried out for 10min at 55 ℃.

1.3 construction of recombinant plasmids containing genes of chdaII, chia and choa

And (2) connecting the target gene fragment obtained in the step (1.2) with an insertion vector pPICZ alpha C to obtain a recombinant plasmid, completing the step by using a ClonExpress II one step cloning kit of Vazyme, transforming the recombinant plasmid into E.coli DH5 alpha competent cells, recovering, coating the recovered recombinant plasmid on a bleomycin LB plate containing 25 mu g/mL, culturing at 37 ℃ for 12h, selecting a single clone to perform colony PCR positive identification by using a universal primer, sending the colony PCR positive identification to sequencing identification, and extracting the recombinant plasmid from the successfully constructed strain.

1.5 construction of Pichia pastoris engineering Strain KM71H _ pCHIT

The recombinant plasmid obtained in step 1.3 was digested overnight with the restriction enzyme SacI to obtain linearized DNA. Linear plasmid (100ng) was added to 100uL of Pichia pastoris KM71H electroporation competent cells, followed by careful transfer of the mixture to a frozen and sterile electroporation cuvette slide for electroporation (voltage 1.5 kV, resistance 125 ohm, pulse length 3 ms), immediately followed by resuspension of the cells in 500. mu.l of 1M cold sorbitol, followed by addition of 500. mu.l of YPD medium pH 7.5 to the cell mixture. The cells were cultured at 30 ℃ for 3-4 hours with shaking horizontally, after completion of transformation, the cells were pelleted by centrifugation, 700. mu.l of supernatant was removed, the cells were resuspended in the remaining medium, plated onto YPDS agar plates containing 100. mu.g/mL bleomycin, and cultured at 30 ℃ for 2 days. A eukaryotic polycistronic expression system strain KM71H _ pCHIT encoding chitinase, chitosanase and chitin deacetylase genes was obtained by confirming the successful transformation of the plasmid into the host by a series of colony PCR, and the primers used in the process are shown in Table 1. The theoretical DNA size and colony PCR results are shown in FIG. 3, in which the action sites of the primers have been marked, and five DNA bands distributed in 600bp-3500bp can be seen through DNA agarose gel, and the sizes correspond to the theoretical values.

TABLE 1 colony PCR primers for engineering strain KM71H _ pCHIT

Example 2 application of KM71H _ pCHIT engineered Strain

2.1 KM71H _ pCHIT fermentation and detection of protein expression after fermentation

The engineered strain KM71H _ pCHIT single colony prepared in example 1 was inoculated into 25mLBMGY medium containing 100. mu.g/mL bleomycin (placed in a 250mL baffled Erlenmeyer flask), and shake-cultured at 250-300 rpm at 28-30 ℃ for 18h until OD is reached₆₀₀Is 10-12. Taking a proper amount of the bacterial liquid into a 1000mL conical flask with a baffle and 100mL of BMGY until the final OD of the bacterial liquid is reached₆₀₀After 0.2, the mixture is cultured at 28-30 ℃ with shaking at the rotation speed of 250-300 rpm to OD₆₀₀Is 2-3. The obtained bacterial liquid is centrifuged for 5min at the rotating speed of 1,500-3,000 Xg at room temperature to obtain thalli, and then the thalli are suspended in 20mL BMMY culture medium (placed in a 100mL conical flask with a baffle and sealed by two layers of sterile gauze), and pure methanol is added to the concentration of 0.5 percent for induced expression. Samples were taken from the 0, 24, 48, 72 and 96h cultures, respectively, and stored to-80 ℃ until use for SDS-PAGE analysis and detection of the enzyme activity of the recombinant protein.

2.2 SDS-PAGE analysis

And (3) taking all the bacterial liquid obtained in the step (2.1), centrifuging at low temperature, collecting supernatant, and detecting the protein content by using a Thermo scientific PierceBCA protein quantitative analysis kit. 20ul of the supernatant of the resulting bacterial suspension was diluted to 100ul, and SDS-PAGE protein electrophoresis was carried out using the diluted bacterial suspension, and the results are shown in FIG. 3. The gel clearly shows the three gene expression products, with a mass range of 46 to 49kDa (1 and 2) and a mass of about 27kDa for product 3. The theoretical mass of the protein encoded by the fragment of the chdaII gene with the P2A sequence was 49kDa, while the theoretical mass of the protein encoded by the recombinant gene of chitinase with the T2A sequence was 45.9kDa, and

bands

1 and 2 of the electrophoresis gel could correspond to the theoretical values. The weight of the third product in the gel was different from the theoretical weight of the chitosanase, resulting in 23.6kDa, while the theoretical weight was 28 kDa. This may be a result of the potential for high glycosylation of proteins.

2.3 determination of enzyme Activity of three enzymes

The enzyme activities of the three enzymes were measured, and the final results are shown in Table 2, which finally yielded a chdaII enzyme activity of 0.25U/mL, a CHIA enzyme activity of 1.99U/mL and a ChoA enzyme activity of 3.46U/mL.

2.3.1 assay of enzyme Activity of CHDAII

The activity of ChdaII enzyme was determined by measuring the amount of acetic acid released during the deacetylation reaction of chitosan. 1mL of 0.5% chitosan standard solution and 100. mu.l of the supernatant obtained during protein expression were placed in sterile 1.5mL microcentrifuge tubes and preheated at 50 ℃ for 2 minutes, respectively. The reaction was then initiated by adding ChdaII to the polymer solution and shaking the reaction at 300rpm for 120 minutes at 50 ℃. Thereafter, the sample was left to react at 80 ℃ for 20 minutes and then cooled. The acetic acid content released during the reaction was determined by gas chromatography-mass spectrometry using columns:

30m；0,18 mm ID；0,18μm。

2.3.2 chitinase Activity assay

The chitin standard was dissolved in citric acid buffer (PH 4.5) to obtain a 1% chitin standard solution, and 250 μ l of the chitin standard solution d and the supernatant obtained during protein expression were placed in a sterile 1.5mL microcentrifuge tube and preheated at 50 ℃ for 2 minutes, respectively. The reaction was then triggered by the addition of chitinase and was shaken at 550rpm for 60 minutes at 50 ℃. Subsequently, the sample was reacted at 100 ℃ for5 minutes, and then cooled. The content of reducing sugar obtained by the reaction is determined by using a Somogyi-Nelson colorimetric method.

2.3.3 measurement of Chitosan enzyme Activity

The enzymatic activity of the glycanase is determined by measuring the amount of acetate released during the chitosanase-catalyzed reaction. Chitosan was dissolved in 2% acetic acid to obtain a chitosan standard solution with a final concentration of 0.5%, 100. mu.l of the supernatant obtained during the protein expression and the chitosan standard solution were placed in sterile 1.5mL microcentrifuge tubes, and preheated at 50 ℃ for 2 minutes, respectively. The chitosanase is subsequently reactedThe reaction was started by adding to the polymer solution and shaking the reaction at 300rpm for 120 minutes at 50 ℃. Thereafter, the sample was left to react at 80 ℃ for 20 minutes and then cooled. The acetic acid content released during the reaction was determined by gas chromatography-mass spectrometry using columns:

30m；0,18mm ID；0,18μm。

table 2 KM71H _ pCHIT Strain expresses enzyme Activity

Dissolved in hydrochloric acid, dissolved in 2% acetic acid

2.4 determination of the degree of deacetylation of the chitinase

The degree of deacetylation under the action of chitin deacetylase was determined by potentiometric titration. Centrifuging the product after the catalytic reaction, taking a precipitate, freeze-drying the precipitate, dissolving the freeze-dried product in 0.02M hydrochloric acid to a final concentration of 0.2% (W/V), determining two titration end Points (PK) by a first derivative method, and establishing a dependence legend of a relation of delta pH/V ═ f (V), wherein delta pH and delta V respectively represent the increment of pH change and the increment of titrant volume, and the deacetylation degree is calculated by the following formula:

wherein: degree of DD-deacetylation [% ]]，c_NaOHTitrant concentration [ mol. dm^-3]，m_Ch-chitosan sample mass [ g [ ]]，V₁Titration volumes [ dm ] for neutralizing hydrochloric acid³]， V₂Titration volumes for neutralizing hydrochloric acid and protonated amine groups, M_GlcNAc(iii) molar mass of (E) -2-acetamido-2-deoxy-D-glucopyranose [ g.mol [)^-1]，M_GlcN(iii) molar mass of (E) -2-amino-2-deoxy-D-glucopyranose [ g.mol [)^-1] 。

SEQUENCE LISTING (SEQUENCE LISTING)

<110> Shenzhen institute of university of Beijing

<120> pichia pastoris engineering strain KM71H _ pCHIT and construction method and application thereof

<160> 8

<170> PatentIn version 3.3

<210> 1

<211> 1299

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gctacttcca ccaaatccgc cactacatca actgcatcaa cattggcaaa gcctcaagat 60

tactggaaga acttcaagtc tttagtcgat cctttgaaca ttaccattga agcaattcct 120

caaactacat ccaatgatcc ctctgttgaa tgtacttact atcaacctcc ttccaacttc 180

aagttcaatg aaaaggaatg gcctactctt tgggaaactg ctacctctaa cggtatgacc 240

aacactgccg agttcaaagc tctctacaac tctattgact ggaccaaggc tcccaagatt 300

gctgttcgca agttgaactc tgatggttcc atcaacatga ctggttacga tcaaaacaag 360

gatcccgatt gttggtggtc tgcttccact tgtaccaagc ccaagctcga gggcgtcaat 420

gaagatatct acacctgtcc tgagcccgag acctggggtc ttacctacga tgatggtccc 480

aactgttctc acaatgcttt ctacgactat ctcgaagctg aaaagatcaa ggcttccatg 540

ttctacattg gttccaacgt tgttaactgg ccctacggtg ctcaacgtgg tcaaaaggct 600

ggtcaccaca ttgctgatca cacctggtct catcaattga tgactactct taccaacact 660

gaagttttgg ctgaactcta ctatactcaa aaggctatca agatggttac tggtgtcacc 720

cctattcact ggcgtcccgc tttcggtgat gtcgatgatc gtgttcgttg gattgctact 780

caacttggct taactactat tctctgggat cttgatactg atgattgggc tgctggtact 840

tctgaaactt tggatactgt tcaaaagact tatgaagatt tcattgaaat gggttctaac 900

ggtactttca agaacagtgg tcaaattgtc ttgactcacg aaattgataa caccactatg 960

acccttgcca tggaattctt gcccaagatc agagctgcct acaagaacgt tcttgatgtc 1020

gccacctgta tgaacatcac caatccttac cacgaatcta ctgtcaccat tacccctttc 1080

aatgccactg gttctactag ctctgctgcc gcctcttcat caagtgcttc tgcttctgct 1140

gtttcatctg gtgcttctcc cgctgctgcc aaggctgcct ccagtgcttc ttcttctcct 1200

gccgctgttg tcaacaacgc tagtgccacc tccgcaggtg ttcaagttaa ccccaatgcc 1260

ttgctcatgg ctgcctttgc cgttgctgct tacattttc 1299

<210> 2

<211> 1200

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggctaccg gattcagaac tgtcgcctac ttcgtcaatt gggccatcta cggaagaaac 60

cacaaccctc aggacttgcc tgccgagaag ttgacccaca tcctttacgc tttcgccaac 120

gttcgtcctg actccggtga agtctacctt accgatacct ggtccgacac cgacaaacac 180

tacccttctg actcctggaa cgataccggt accaacgtct atggatgtat taagcagttg 240

tttcttttga agaagagaca tagaaagttg aaagtcttgt tgtctatcgg aggatggacc 300

tactcctcta actttgccca accagcttcc actgaggctg gtagagaaac cttcgcccgt 360

actgctacca gattggtctt ggacttgggt ttggacggtt tggacatcga ctgggagtac 420

ccacaggacg ataatcaggc cagagacttc gtcgccttgt tgagaaagtg cagagagcac 480

ttggactacg ccgctggtcc taatagacgt ttcttgttga ctatcgcctg tcctgccgga 540

ccaaacaact tcaccaaatt gcgtttgcca gagatgaccc catacttgga cttttacaat 600

ttgatgggtt acgactacgc tggttcttgg gaccagttgg ctggtcacca agccaacatc 660

ttcccatcct ccactaaccc agcttccact cctttttcta ccgacgccgc tttgcgtcat 720

tacatctccg tctccggtgt cccatcttct aaaatggttt tgggtatgcc attgtacggt 780

cgtgcctttc agaacaccaa cggtcctgga accccatttt ccggtgttgg tgaaggttct 840

tgggagcagg gtgtctggga ttacaaggct ttgccaagac ctggtgctac tgagcatgtc 900

gacccaaaca ttggtgcttc ctggtcctac gatccacaga cccgtaccat ggttacctac 960

gataacgtcg ctgttgccga gatcaaggcc aactttgttc gtggtgctgg attgggtggt 1020

ggtatgtggt gggaatcctc cgccgataga ggaggtaaaa ctgccaacaa ggccgatggt 1080

tccttgatcg gaaccttcgt tgacggattg ggtggtgtct tcgccttgga tcagtcccct 1140

aacaacttgg actacccaga gtccaagtat gacaatttgc gtgccggttt cccaggtgaa 1200

<210> 3

<211> 666

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tacaacttgc caaacaactt gaagcaaatt tatgataagc ataaaggtaa gtgttccaag 60

gtcttggcca agggtttcac caacggagac gcttcccaag gtaagtcctt ctcctactgc 120

ggtgacatcc caggtgctat tttcatttct tcttctaagg gatacaccaa catggacatc 180

gactgtgacg gtgctaacaa ctccgctggt aagtgtgcta acgatccatc cggacaagga 240

gagaccgctt tcaagtccga cgtcaagaag ttcggtattt ctgatcttga tgctaacatt 300

catccatacg tcgttttcgg taacgaggat cactctccaa agttcaagcc acagtcccac 360

ggaatgcagc ctttgtctgt tatggctgtc gtctgcaacg gtcagttgca ctacggtatt 420

tggggtgaca ctaacggtgg tgtctctact ggtgaggctt ccatctcctt ggctgacttg 480

tgctttccaa acgagcactt ggacggtaac cacggacacg acccaaacga tgttttgttc 540

attggtttca cttctaagga cgctgttcct ggtgctaccg ccaagtggaa ggccaagaac 600

gctaaggagt ttgaggactc tattaagtct atcggtgaca agttggttgc cggtttgaag 660

gcttaa 666

<210> 4

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gctactaact tttccttgtt gaagcaagct ggagatgtcg aagagaatcc aggtcca 57

<210> 5

<211> 54

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gaaggtagag gttctttgtt aacttgtggt gacgttgagg aaaaccctgg acca 54

<210> 6

<211> 267

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atgcgtttcc cttctatttt cactgccgtt cttttcgctg cctcttctgc tttggctgct 60

ccagttaata ccaccactga agatgagact gcccagattc ctgccgaagc tgtcatcgga 120

tactccgact tggaaggtga cttcgacgtc gccgttttgc ctttctctaa ctccactaac 180

aatggtttgt tgttcattaa caccaccatc gcttccattg ccgctaagga ggagggagtt 240

tctttggaaa agcgtgaggc cgaagct 267

<210> 7

<211> 267

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atgaggtttc catcaatatt caccgctgtc ttgtttgccg cttcctccgc cttggctgct 60

cctgttaaca ccaccaccga agatgagacc gctcaaattc cagctgaggc cgtcatcggt 120

tactctgacc ttgagggtga cttcgatgtc gccgtcttgc cattctccaa ctccactaac 180

aacggtcttt tgtttatcaa tactactatt gcttctattg ctgccaagga ggaaggtgtc 240

tctcttgaga agagagaggc tgaggct 267

<210> 8

<211> 267

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atgagattcc cttccatctt caccgctgtc ttgtttgccg cttcctctgc tttggctgct 60

cctgttaata ccaccactga ggacgaaacc gctcaaatcc cagctgaggc tgtcatcggt 120

tactccgatt tggagggaga cttcgacgtc gctgtcttgc cattctccaa ctccaccaac 180

aacggattgt tgttcattaa caccaccatc gcttccatcg ctgctaagga ggagggtgtc 240

tccttggaaa agagagaggc cgaggct 267

Claims

1. A pichia pastoris engineering strain is characterized in that the target pichia pastoris engineering strain can express chdaII, chia and choa genes simultaneously.

2. The construction method of the pichia pastoris engineering strain according to claim 1, comprising the following steps:

(2) The chia and choa genes are obtained by synthesis, and the nucleotide sequences of the genes are shown as SEQ ID NO.2 and SEQ ID NO. 3. (3) The vectors for the co-expression of chdaII, chia and choa genes were linked by 2A peptide, 2A peptide bridging oligonucleotides were synthesized based on the amino acid sequences P2A (ATNFSLLKQAGDVEENPGP) and T2A (EGRGSLLTCGDVEENPGP), respectively, the bridging oligonucleotide sequences are shown in SEQ ID No.4 and SEQ ID No.5, respectively, and DNA shuffling was performed by the program with the following program parameters: 95-2 min; 70-10 min; 72-5 min; 4 ℃ to end.

(4) And connecting each part of DNA fragments of an expression vector of the pichia pastoris engineering bacteria by an LCR method, connecting the obtained synthetic fragment with a pichia pastoris integrated vector pPICZ alpha C, transforming the synthetic fragment into E.coli DH5 alpha competent cells for replication, and extracting recombinant plasmids in E.coli DH5 alpha to obtain the expression vector of the pichia pastoris engineering bacteria.

(5) And (3) carrying out overnight enzyme digestion on the expression vector of the pichia pastoris engineering bacteria obtained in the step (4) by using a restriction enzyme SacI, and then transforming the linear plasmid obtained by enzyme digestion into a pichia pastoris KM71H electrotransformation competent cell, thereby obtaining a pichia pastoris engineering strain KM71H _ pCHIT.

3. The method for constructing the pichia pastoris engineering strain according to claim 2, wherein the DNA fragments of each part of the expression vector are connected by an LCR assembly method, and the specific reaction system (20 μ l) of the method is as follows: 2nM of each DNA fragment of the gene of interest, 10nM bridging oligonucleotide, 1xTaq ligase buffer, 5% DMSO and 40U Taq ligase. The assembly method comprises the following specific program parameters: first, initial denaturation at 95 ℃ for 2 min; then denaturation at 95 ℃ for 30sec, annealing at 60 ℃ for 2min, and performing 30 cycles; finally, the extension is carried out for 10min at 55 ℃, and the product is stored in an environment at 4 ℃.

4. The chia and choa gene according to claim 2, characterized in that: the alpha factor before the gene is optimized during synthesis, so that spontaneous homologous recombination in the cloning process is avoided, and the sequence of the alpha factor is shown as SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8.

5. The 2A peptide-bridged oligonucleotide according to claim 2, characterized in that: the target melting temperature (Tm) of the bridging oligonucleotide complementary to the target DNA terminus (half bridge oligonucleotide) was designed to be 50 ℃.

6. The pichia pastoris engineering strain KM71H _ pCHIT as well as the construction method and the application thereof according to claim 1, wherein the system can simultaneously express and produce chitinase, chitosanase and chitin deacetylase under the induction of methanol, and the specific steps of the induction expression are as follows:

(3) And (3) centrifuging the bacterial liquid obtained in the step (2) at the rotating speed of 1,500-3,000 Xg for 5min at room temperature to obtain thalli, then suspending the thalli in 20mL of BMMY culture medium (placing the BMMY culture medium in a 100mL conical flask with a baffle, sealing the BMMY culture medium with two layers of sterile gauze), and adding pure methanol to the concentration of 0.5% for induction expression.