Genetically engineered Kluyveromyces marxianus bacteria capable of tolerating high concentration of chitosan oligosaccharides, preparation method and application thereof

Abstract

The present application discloses a genetically engineered strain of Kluyveromyces marxianus that can tolerate high concentrations of chitosan oligosaccharides, as well as a preparation method and application thereof. A chitosanase gene ChiA is cloned in the Kluyveromyces marxianus; the nucleotide sequence of the chitosanase gene is shown in SEQ ID NO.1. Using the genetically engineered strain of Kluyveromyces marxianus that tolerates chitosan oligosaccharides provided in the present application, chitosan oligosaccharides are prepared by a fermentation method. This method is not only simple and feasible, but also avoids the loss of enzyme activity and waste of costs during the separation and purification of chitosanase; it can also synthesize chitosan oligosaccharides with a specific degree of polymerization, and the conversion efficiency is relatively high; the engineered bacteria uses food-grade Kluyveromyces marxianus as a production strain, which is not only safe and reliable, but also provides an effective reference and reference for the industrial green production of chitosan oligosaccharides, and provides a new method and effective way for the efficient preparation of chitosan oligosaccharides.

Description

Technical Field

[0001] The present application belongs to the field of bioengineering technology, and specifically relates to a genetically engineered strain of Kluyveromyces marxianus that can tolerate high concentrations of chitosan oligosaccharides, and a preparation method and application thereof. Background Art

[0002] Chitosan is obtained by deacetylation of chitin, which is widely present in nature. It is the second most abundant polysaccharide in nature after cellulose and is widely used in food, medicine, and environmental protection. However, chitosan has the characteristics of large molecular weight and poor water solubility, which greatly limits its application. Chitooligosaccharides (COS) are low molecular weight polysaccharides composed of 2-10 glucosamines connected by β-1,4 glycosidic bonds. The structural formula is as follows:

[0003]

[0004] COS is a degradation product of chitosan. It not only retains some functional properties of chitosan, but also has higher water solubility and other characteristics, making it valuable for application. COS has antibacterial, antioxidant, anti-angiogenic, anti-tumor, anti-inflammatory and prebiotic properties. With the continuous expansion of the application field of chitosan oligosaccharides, seeking a stable and efficient green biosynthesis method for chitosan oligosaccharides has become a key issue that needs to be solved urgently.

[0005] At present, the preparation methods of chitosan oligosaccharides mainly include three categories: physical method, chemical method and enzymatic method. The physical and chemical methods have poor controllability for the preparation of chitosan oligosaccharides with specific molecular weights and are prone to environmental pollution. In recent years, enzymatic method has been a hot research topic in the research and development of the preparation of chitosan oligosaccharides industry due to its advantages such as mild reaction conditions, low by-product content and high product yield. Studies have reported that chitinase produced by Bacillus can hydrolyze chitin to form low molecular weight chitosan oligosaccharide products. The proportion of chitosan oligosaccharides with high polymerization degree (DP 5-10) in the degradation products of endo-chitosanase from Bacillus amyloliquefaciens reached 56.17%. Park et al. developed an effective system for producing chitosan oligosaccharides with different polymerization degrees. The chitosan oligosaccharides obtained by chitosanase from Bacillus sp. P21 were finally distributed in DP 3-7. In summary, the degradation of chitosan using bioenzyme technology is the main way to prepare chitosan oligosaccharides. However, the current process of producing chitosan oligosaccharides by chitosanase still has problems such as lack of high-activity specific enzymes, high cost of enzyme preparations, low conversion efficiency, and difficult refining process. The use of whole-cell efficient catalytic strategy to produce chitosan oligosaccharides not only simplifies the production process, but also does not require enzyme extraction and purification, reduces enzyme activity loss, and is expected to significantly reduce production costs. However, when using this method, it is still faced with low chitosanase activity and the antibacterial property of the product, which makes the whole-cell catalytic efficiency low. Therefore, establishing a stable and tolerant whole-cell catalytic synthesis of chitosan oligosaccharide production process has great application value and industrialization potential. Summary of the invention

[0006] In view of the problems existing in the prior art, the technical problem to be solved by the present invention is to provide a strain of Kluyveromyces marxianus genetically engineered bacteria that can tolerate high concentrations of chitosan oligosaccharides to meet the use requirements for preparing chitosan oligosaccharides. Another technical problem to be solved by the present application is to provide a method for preparing the above-mentioned Kluyveromyces marxianus genetically engineered bacteria. Another technical problem to be solved by the present application is to provide an application of the Kluyveromyces marxianus genetically engineered bacteria.

[0007] In order to solve the above technical problems, the technical solution adopted in this application is:

[0008] A strain of Kluyveromyces marxianus genetic engineering bacteria capable of tolerating high concentration of chitosan oligosaccharide is disclosed. A chitosanase gene ChiA is cloned in the Kluyveromyces marxianus. The nucleotide sequence of the chitosanase gene is shown in SEQ ID NO.1.

[0009] The signal peptide of the inulinase gene is fused to the N-terminus of the chitosanase gene ChiA; the signal peptide sequence of the inulinase gene is shown in SEQ ID NO.2.

[0010] The Kluyveromyces marxianus can tolerate no less than 50 g / L chitosan oligosaccharide.

[0011] The method for constructing the Kluyveromyces marxianus genetically engineered bacteria capable of tolerating high concentrations of chitosan oligosaccharides comprises the following steps:

[0012] 1) Using 4%-6% chitosan oligosaccharide as the screening pressure, the Kluyveromyces marxianus strain was continuously cultured and subcultured to obtain a dominant strain that can tolerate high concentrations of chitosan oligosaccharide;

[0013] 2) The chitosanase gene SPinu-ChiA fused with the inulinase gene signal peptide was connected to the Kluyveromyces marxianus expression plasmid pRSG to obtain the recombinant plasmid pRSG-SPinu-ChiA; the recombinant plasmid was transformed into Kluyveromyces marxianus Km35 using the PEG-lithium acetate transformation method to obtain the recombinant Kluyveromyces marxianus.

[0014] In step 1), during screening culture, chitosan oligosaccharide concentrations are 40 g / L, 50 g / L and 60 g / L, respectively, and three stages of domestication culture are performed. The first stage of domestication is completed on the 20th day, and the last batch of culture populations harvested are diluted and spread for clone selection and phenotypic testing; 10 colonies are randomly selected from the plate, and the dominant strain with the fastest growth rate is selected for the next round of domestication; after three rounds of domestication, a dominant strain with tolerance to high concentrations of chitosan oligosaccharides is finally obtained.

[0015] In step 2), the specific construction process is as follows:

[0016] (1) Using the Kluyveromyces marxianus Km35 genome as a template, primers SPinu-F and SPinu-R were used to amplify the nucleotide sequence of the inulinase gene signal peptide shown in SEQ ID NO.2; using the pUC57-ChiA plasmid as a template, primers ChiA-F and ChiA-R were used to amplify the nucleotide sequence of the chitosanase gene without the original signal peptide shown in SEQ ID NO.1; the sequences of each primer are as follows:

[0017] SPinu-F: 5'-aaagatacaaaaatggtcgacATGAAGTTAGCATACTCCCTCTTGC-3';

[0018] SPinu-R: 5'-cttttggtctttatttagcccggcagcCTTGTAATTGATCACTGAAGCA-3';

[0019] ChiA-F: 5'-tgcttcagtgatcaattacaagGCTTGCCGGGCTAAATAAAGACCAAAAG-3';

[0020] ChiA-R: 5'-taactaattacatgacccgggTTACTTAATTACAAAGTTACCATATTCGTTAC-3';

[0021] The PCR amplification system was as follows: template plasmid pUC57-ChiA or Kluyveromyces marxianus Km35 genome: 2 μL DNA, upstream primer and downstream primer: 2 μL each, PrimeSTAR high-fidelity enzyme: 12.5 μL, ddH 2 O: 6.5 μL;

[0022] The PCR reaction program was as follows: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, and 30 cycles;

[0023] The PCR amplification products SPinu and ChiA gene fragments were recovered separately and fused into fragment SPinu-ChiA by overlapping PCR. The overlapping PCR amplification system was as follows: SPinu and ChiA gene fragments: 2 μL each, upstream primers and downstream primers: 2 μL each, PrimeSTAR high-fidelity enzyme: 12.5 μL, ddH 2 O: 4.5 μL; overlapping PCR reaction program: 94℃ pre-denaturation for 10 min, 94℃ denaturation for 30 s; then 55℃ annealing for 1 min, 72℃ extension for 2 min, 35 cycles;

[0024] The fused DNA fragment SPinu-ChiA was connected with the plasmid pRGS after double digestion with restriction endonucleases Sal I and Sma I using the one-step cloning method under the action of Exnase II, and the transformation products were transformed into Escherichia coli DH5α respectively; the transformants were picked from the ampicillin resistance plate and transferred to 5 mL of LB liquid culture medium containing 25 μg / mL ampicillin, and cultured overnight at 37°C and 200 rpm. After plasmid extraction, the plasmid was double digested with restriction endonucleases Sal I and Sma I to verify that it was a positive clone strain;

[0025] (2) The successfully verified recombinant plasmid pRGS-SPinu-ChiA was transformed into Kluyveromyces marxianus Km35, spread on a solid culture medium containing 50 μg / mL G418 resistance, and cultured at 37°C for 12-16 hours to obtain preliminary positive clones; after the yeast transformants were screened by the resistance culture medium and transferred to liquid culture, the plasmid double enzyme digestion verification was performed using a yeast plasmid extraction kit. According to the electrophoresis results, the strain corresponding to the plasmid containing the 800 bp fragment was the recombinant Kluyveromyces marxianus containing the chitosanase gene.

[0026] The invention discloses an application of the Kluyveromyces marxianus genetic engineering bacteria capable of tolerating high concentration of chitosan oligosaccharides in the preparation of chitosan oligosaccharides.

[0027] The application described, the steps are as follows:

[0028] 1) Activate the engineered Kluyveromyces marxianus at 30-37°C, inoculate the strain into YPD medium, and culture at 30-37°C and 100-250 rpm for 10-16 h until OD600 is greater than 4.0;

[0029] 2) inoculating the cultured seed liquid into a fermentation medium containing a chitosan substrate at an inoculum size of 2-10%, and aerobically culturing for 60 hours at 30-37° C. and 100-250 rpm; wherein the fermentation medium comprises the following components: 1-50 g / L glucose, 1-5 g / L yeast powder, 1-10 g / L peptone, 1-10 g / L KH2PO4, 0.31-10 g / L MgSO4, 0.1-0.5% Tween-80, and the rest water, pH 5.0;

[0030] 3) The fermentation was scaled up using a 7.5 L fermentation tank, and catalytic fermentation was carried out using 50 g / L chitosan oligosaccharide as a substrate. After the fermentation was completed, the chitosanase activity, chitosan oligosaccharide content and degree of polymerization in the fermentation broth of the engineered strain were determined.

[0031] The application uses 50 g / L chitosan oligosaccharide as a substrate for catalytic fermentation to obtain 37 g / L chitosan oligosaccharide, and the molecular weight of the chitosan oligosaccharide is lower than 2000 Da.

[0032] In the application, the chitosanase of the engineered strain is secreted and expressed extracellularly, and the volume enzyme activity reaches 2300U / mL.

[0033] Beneficial effects: Compared with the prior art, this application has the following advantages:

[0034] 1) Using the chitosan oligosaccharide-tolerant Kluyveromyces marxianus genetically engineered bacteria provided in the present application, chitosan oligosaccharides are directly prepared by a fermentation method. The method is simple and feasible, and avoids the enzyme activity loss and cost waste in the process of chitosanase separation and purification;

[0035] 2) The chitosan oligosaccharide-tolerant Kluyveromyces marxianus genetically engineered bacteria provided in the present application are used to prepare chitosan oligosaccharides, which can synthesize chitosan oligosaccharides with a specific degree of polymerization and have a high conversion efficiency, thereby providing a new method and effective approach for the efficient preparation of chitosan oligosaccharides.

[0036] 3) The present application uses food-grade Kluyveromyces marxianus as the production strain, which is not only safe and reliable, but also provides an effective reference and reference for the industrial green production of chitosan oligosaccharides.

[0037] 4) The enzyme activity assay results of the present application show that the chitosanase of the engineered strain has achieved extracellular secretion expression, and the volume enzyme activity has reached 2300U / mL. This process can be used to realize a new fermentation process for extracellular synthesis of chitosan oligosaccharides with a specific degree of polymerization. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 : is a diagram showing the results of strain performance testing before and after chitosan oligosaccharide tolerant yeast domestication; in the figure, A is a laboratory domestication flow chart, B is a schematic diagram of the morphology of Kluyveromyces marxianus Km35 obtained by domestication, and C is a diagram showing the domestication process and the comparison of the fermentation performance of the strain before and after domestication;

[0039] Figure 2 It is a schematic diagram of the construction of the recombinant plasmid pRGS-SPinu-ChiAO and the verification result diagram; Note: Lane 1: DL15000Marker; Lane 2: pRGS-SPinu-ChiA double-digested with Sal I and Sma I (target band 800bp);

[0040] Figure 3 This is the SDS-PAGE protein gel image verification result of the supernatant of the recombinant yeast fermentation broth;

[0041] Figure 4 This is a graph showing the conversion rate of chitosan oligosaccharides during the fermentation process of recombinant yeast. DETAILED DESCRIPTION

[0042] The present application is further described below in conjunction with specific embodiments.

[0043] The main materials and kits used in the following examples are: E. coli DH5α competent state (purchased from Novazon), primer synthesis and DNA sequencing (Nanjing GenScript Biotechnology Co., Ltd.), PrimeSTAR high-fidelity enzyme (Takara), restriction endonucleases Sal I and Sma I (Takara), DNA marker (Takara), plasmid extraction kit (Nanjing Novazon Biotechnology Co., Ltd.), gel recovery kit (Nanjing Novazon Biotechnology Co., Ltd.), one-step cloning and ligation Exnase II kit (Nanjing Novazon Biotechnology Co., Ltd.), pRSG plasmid is a self-constructed Kluyveromyces marxianus expression plasmid (the construction process is as follows Figure 2 ).

[0044] Chitosan oligosaccharide detection method in fermentation broth: The product was determined using the LC-1260 system, and the separation column was Alltech chromPrevail Carbohydrate ES5μ column (4.6mm×250mm), (Santa Clara, CA). The mobile phase consisted of acetonitrile and water, and the gradient elution conditions were: 0min, 75% acetonitrile; 7min, 75% acetonitrile; 8min, 65% acetonitrile; 15min, 65% acetonitrile; 16min, 75% acetonitrile; 22min, 75% acetonitrile. The column temperature was 40℃, and the flow rate was 1mL / min. The qualitative analysis of the product was based on the standard sample; the quantitative analysis was based on the peak area of ​​different concentrations of chitosan oligosaccharide (DP2~6) standard samples.

[0045] Strain: The strain used in this application is Kluyveromyces marxianus, which is a strain of Kluyveromyces marxianus isolated and screened by the applicant. It has been identified to be the same as other Kluyveromyces marxianus in the field. Kluyveromyces marxianus KM35 was domesticated by the method of Example 1. Those skilled in the art can use other Kluyveromyces marxianus to conduct experiments using the method of this application to obtain the target effect.

[0046] Example 1 Construction of genetically engineered Kluyveromyces marxianus

[0047] The method for constructing an engineered strain of Kluyveromyces marxianus comprises the following steps:

[0048] 1) Due to the antibacterial properties of chitosan oligosaccharides, in order to obtain a chassis that can tolerate chitosan oligosaccharide products, the Kmarxianus strain was continuously cultured and subcultured with 4%-6% chitosan oligosaccharide as the screening pressure using laboratory acclimation technology. Figure 1 As shown, it shows that in the medium rich in high concentration of chitosan oligosaccharide (40g / L, 50g / L and 60g / L), the bacterial biomass (OD 600 ) changes with the increase of chitosan oligosaccharide concentration. The process went through three stages and took a total of 58 days. The first stage of domestication was completed on the 20th day, and the last batch of culture populations harvested were diluted and spread for clone selection and phenotypic testing. Ten colonies were randomly selected from the plate, and the dominant strain with the fastest growth rate was selected for the next round of domestication ( Figure 1 ). After three rounds of domestication, a dominant strain with tolerance to high concentrations of chitosan oligosaccharides was obtained and named K. marxianus Km35.

[0049] 2) Connecting the chitosanase gene SPinu-ChiA fused with the inulinase gene signal peptide to the Kluyveromyces marxianus expression plasmid pRSG to obtain the recombinant plasmid pRSG-SPinu-ChiA; transforming the recombinant plasmid into Kluyveromyces marxianus Km35 by PEG-lithium acetate transformation method to obtain the recombinant Kluyveromyces marxianus ( Figure 2 ). The specific construction method is as follows:

[0050] (1) Using the Kluyveromyces marxianus Km35 genome as a template, primers SPinu-F and SPinu-R were used to amplify the nucleotide sequence of the inulinase gene signal peptide shown in SEQ ID NO.2; using the pUC57-ChiA plasmid as a template, primers ChiA-F and ChiA-R were used to amplify the nucleotide sequence of the chitosanase gene without the original signal peptide shown in SEQ ID NO.1; the sequences of each primer are as follows:

[0051] SPinu-F: 5'-aaagatacaaaaatggtcgacATGAAGTTAGCATACTCCCTCTTGC-3';

[0052] SPinu-R: 5'-cttttggtctttatttagcccggcagcCTTGTAATTGATCACTGAAGCA-3';

[0053] ChiA-F: 5'-tgcttcagtgatcaattacaagGCTTGCCGGGCTAAATAAAGACCAAAAG-3';

[0054] ChiA-R: 5'-taactaattacatgacccgggTTACTTAATTACAAAGTTACCATATTCGTTAC-3';

[0055] The PCR amplification system was as follows: template plasmid pUC57-ChiA or Kluyveromyces marxianus Km35 genome: 2 μL DNA, primer SPinu-F and primer SPinu-R (primer ChiA-F or primer ChiA-R): 2 μL each, PrimeSTAR high-fidelity enzyme: 12.5 μL, ddH 2 O: 6.5 μL.

[0056] The PCR reaction program was as follows: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, and 30 cycles.

[0057] The PCR amplification products SPinu and ChiA gene fragments were recovered separately and fused into fragment SPinu-ChiA by overlapping PCR. The overlapping PCR amplification system was as follows: SPinu and ChiA gene fragments: 2 μL each, primers SPinu-F and ChiA-R: 2 μL each, PrimeSTAR high-fidelity enzyme: 12.5 μL, ddH 2 O: 4.5 μL; overlapping PCR reaction program: 94 ° C pre-denaturation for 10 min, 94 ° C denaturation for 30 s; then 55 ° C annealing for 1 min, 72 ° C extension for 2 min, and 35 cycles.

[0058] The fused DNA fragment SPinu-ChiA and the plasmid pRGS after double digestion with restriction endonucleases Sal I and Sma I were connected using the one-step cloning method under the action of Exnase II, and the transformation products were transformed into Escherichia coli DH5α. Transformants were picked from the ampicillin-resistant plate and transferred to 5 mL of LB liquid culture medium containing 25 μg / mL ampicillin, and cultured overnight at 37°C and 200 rpm. After plasmid extraction, the plasmid was double digested with restriction endonucleases Sal I and Sma I. The recombinant plasmid pRGS-SPinu-ChiA was successfully constructed as shown in the following figure. Figure 2 The positive clone strains are shown.

[0059] (2) The successfully verified recombinant plasmid pRGS-SPinu-ChiA was transformed into Kluyveromyces marxianus Km35, spread on a solid culture medium containing 50 μg / mL G418 resistance, and cultured at 37°C for 12-16 hours to obtain preliminary positive clones; after the yeast transformants were screened by the resistance culture medium and transferred to liquid culture, the plasmid double enzyme digestion verification was performed using a yeast plasmid extraction kit. According to the electrophoresis results, the strain corresponding to the plasmid containing the 800 bp fragment was the recombinant Kluyveromyces marxianus Km35 containing the chitosanase gene.

[0060] Example 2 Whole cell catalytic synthesis of chitosan oligosaccharides

[0061] The steps of catalytic synthesis of chitosan oligosaccharides using the whole cell of the genetically engineered strain prepared in Example 1 are as follows:

[0062] 1) The successfully constructed recombinant Kluyveromyces marxianus Km35 was activated at 30-37°C, and the strain was inoculated into YPD medium and cultured at 30-37°C and 100-250 rpm for 10-16 h until the OD 600 Greater than 4.0.

[0063] 2) Inoculate the cultured seed liquid into a fermentation medium containing chitosan substrate at an inoculum of 2-10%, and culture aerobically at 30-37°C and 100-250rpm for 60h. The fermentation medium contains the following components: 1-50g / L glucose, 1-5g / L yeast powder, 1-10g / L peptone, KH 2 PO 4 1~10g / L, MgSO 4 0.31~10g / L, Tween-800.1~0.5%, the rest is water, pH 5.0.

[0064] 3) The fermentation was carried out in a 7.5L fermentation tank, and catalytic fermentation was carried out using 50g / L chitosan oligosaccharide as substrate. After the fermentation was completed, the chitosanase activity, chitosan oligosaccharide content and polymerization degree in the fermentation broth of the recombinant strain were determined. The results were as follows: Figure 3 and Figure 4 As shown, 37g / L chitosan oligosaccharide was finally obtained, the molecular weight of chitosan oligosaccharide was lower than 2000Da, the main degree of polymerization of the product was DP2-DP5, the yields accounted for 4%, 16%, 30%, 50% respectively, the chitosan oligosaccharide conversion rate reached 74%, and the enzyme activity assay results showed that the chitosanase of the recombinant strain achieved extracellular secretion expression, and the volume enzyme activity reached 2300U / mL. This process can be used to realize a new fermentation process for extracellular synthesis of chitosan oligosaccharide with a specific degree of polymerization. Sequence Listing <110> Yangzhou Rixing Biotechnology Co., Ltd. <120> Genetically engineered Kluyveromyces marxianus bacteria capable of tolerating high concentration of chitosan oligosaccharides, preparation method and application thereof <130> 100 <160> 7 <170> SIPOSequenceListing 1.0 <210> 1 <211> 265 <212> PRT <213> Chitosanase protein sequence (Artificial Sequence) <400> 1 Met Lys Leu Ala Tyr Ser Leu Leu Leu Pro Leu Ala Gly Val Ser Ala 1 5 10 15 Ser Val Ile Asn Tyr Lys Ala Ala Gly Leu Asn Lys Asp Gln Lys Arg 20 25 30 Arg Ala Glu Gln Leu Thr Ser Ile Phe Glu Asn Gly Thr Thr Glu Ile 35 40 45 Gln Tyr Gly Tyr Val Glu Arg Leu Asp Asp Gly Arg Gly Tyr Thr Cys 50 55 60 Gly Arg Ala Gly Phe Thr Thr Ala Thr Gly Asp Ala Leu Glu Val Val 65 70 75 80 Glu Val Tyr Thr Lys Ala Val Pro Asn Asn Lys Leu Lys Lys Tyr Leu 85 90 95 Pro Glu Leu Arg Arg Leu Ala Lys Glu Glu Ser Asp Asp Thr Ser Asn 100 105 110 Leu Lys Gly Phe Ala Ser Ala Trp Lys Ser Leu Ala Asn Asp Lys Glu 115 120 125 Phe Arg Ala Ala Gln Asp Lys Val Asn Asp His Leu Tyr Tyr Gln Pro 130 135 140 Ala Met Lys Arg Ser Asp Asn Ala Gly Leu Lys Thr Ala Leu Ala Arg 145 150 155 160 Ala Val Met Tyr Asp Thr Val Ile Gln His Gly Asp Gly Asp Asp Pro 165 170 175 Asp Ser Phe Tyr Ala Leu Ile Lys Arg Thr Asn Lys Lys Ala Gly Gly 180 185 190 Ser Pro Lys Asp Gly Ile Asp Glu Lys Lys Trp Leu Asn Lys Phe Leu 195 200 205 Asp Val Arg Tyr Asp Asp Leu Met Asn Pro Ala Asn His Asp Thr Arg 210 215 220 Asp Glu Trp Arg Glu Ser Val Ala Arg Val Asp Val Leu Arg Ser Ile 225 230 235 240 Ala Lys Glu Asn Asn Tyr Asn Leu Asn Gly Pro Ile His Val Arg Ser 245 250 255 Asn Glu Tyr Gly Asn Phe Val Ile Lys 260 265 <210> 2 <211> 732 <212> DNA <213> Artificial Sequence <400> 2 gctgccgggc taaataaga ccaaaagcgt agagcggaac aattaacttc tattttcgaa 60 aacggtacaa ctgagataca gtatggatat gttgaaagat tagatgacgg tagaggatat 120 acgtgcggtc gtgccggttt tacaaccgct acgggggatg ccttggaagt tgttgaagtt 180 tatacaaagg cagtccccaa caataacta aaaaaatatc taccggaatt acgtagattg 240 gccaaagagg aatctgatga cacttcaaat ttgaaaggtt ttgctagcgc ctggaaatct 300 ctagctaatg ataaagaatt tagggcagct caagataagg taaatgatca cctatattac 360 caacccgcca tgaagaggtc tgataacgca ggcctgaaaa ccgccttagc tagagccgta 420 atgtatgaca cagtaattca acatggtgat ggagacgacc ctgacagctt ttatgctttg 480 atcaaaagaa caaataagaa ggctggtggt tctccgaaag atggtatcga tgaaaagaag 540 tggcttaata aattccttga tgttcgttat gacgatctaa tgaatcctgc aaaccatgat 600 accagagatg aatggaggga atctgtcgct cgtgttgatg tcttgaggtc aattgcaaaa 660 gaaaacaatt acaatctgaa tggtcccatc cacgtacgta gtaacgaata tggtaacttt 720 gtaattaagt aa 732 <210> 3 <211> 66 <212> DNA <213> Signal peptide sequence (Artificial Sequence) <400> 3 atgaagttag catactccct cttgcttcca ttggcaggag tcagtgcttc agtgatcaat 60 tacaag 66 <210> 4 <211> 46 <212> DNA <213> Primer SPinu-F sequence (Artificial Sequence) <400> 4 aaagatacaa aaatggtcga catgaagtta gcatactccc tcttgc 46 <210> 5 <211> 49 <212> DNA <213> Primer SPinu-R sequence (Artificial Sequence) <400> 5 cttttggtct ttattagcc cggcagcctt gtaattgatc actgaagca 49 <210> 6 <211> 49 <212> DNA <213> Primer ChiA-F sequence (Artificial Sequence) <400> 6 tgcttcagtg atcaattaca aggctgccgg gctaaataaa gaccaaaag 49 <210> 7 <211> 53 <212> DNA <213> Primer ChiA-R sequence (Artificial Sequence) <400> 7 taactaatta catgacccgg gttacttaat tacaaagtta ccatattcgt tac 53

Claims

1. A method for constructing a genetically engineered strain of Kluyveromyces marxianus that can tolerate high concentrations of chitosan oligosaccharides, characterized in that: The steps include: 1) Using 4%-6% chitosan oligosaccharide as the screening pressure, the Kluyveromyces marxianus strain was continuously cultured and subcultured to obtain the dominant strain KM35 that can tolerate high concentrations of chitosan oligosaccharides; the specific process is as follows: During the screening culture, chitosan oligosaccharide concentrations were 40 g / L, 50 g / L and 60 g / L in three stages of acclimatization. The first stage of acclimatization was completed on the 20th day, and the last batch of culture populations harvested were diluted and spread for clone selection and phenotypic testing. Ten colonies were randomly selected from the plate, and the dominant strain with the fastest growth rate was selected for the next round of acclimatization. After three rounds of acclimatization, the dominant strain KM35 with tolerance to high concentrations of chitosan oligosaccharides was finally obtained. 2) The chitosanase gene SPinu-ChiA fused with the inulinase gene signal peptide was connected to the Kluyveromyces marxianus expression plasmid pRSG to obtain the recombinant plasmid pRSG-SPinu-ChiA; the recombinant plasmid was transformed into Kluyveromyces marxianus KM35 using the PEG-lithium acetate transformation method to obtain the recombinant Kluyveromyces marxianus; the specific construction process is as follows: (1) The genome of Kluyveromyces marxianus Km35 was used as a template, and primers SPinu-F and SPinu-R were used to amplify the nucleotide sequence of the inulinase gene signal peptide shown in SEQ ID NO.2; the nucleotide sequence of the chitosanase gene without the original signal peptide shown in SEQ ID NO.1 was amplified using primers ChiA-F and ChiA-R using the pUC57-ChiA plasmid as a template; the sequences of the primers are as follows: SPinu-F: 5'-aaagatacaaaaatggtcgacATGAAGTTAGCATACTCCCTCTTGC-3'; SPinu-R: 5'-cttttggtctttatttagcccggcagcCTTGTAATTGATCACTGAAGCA-3'; ChiA-F: 5'-tgcttcagtgatcaattacaagGCTTGCCGGGCTAAATAAAGACCAAAAG-3'; ChiA-R: 5'-taactaattacatgacccgggTTACTTAATTACAAAGTTACCATATTCGTTAC-3'; The PCR amplification system was as follows: template plasmid pUC57-ChiA or Kluyveromyces marxianus Km35 genome: DNA 2 μL, upstream primer and downstream primer: 2 μL each, PrimeSTAR high-fidelity enzyme: 12.5 μL, ddH2O: 6.5 μL; The PCR reaction program was as follows: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, and 30 cycles; The PCR amplification products SPinu and ChiA gene fragments were recovered separately and fused into the fragment SPinu-ChiA by overlapping PCR. The overlapping PCR amplification system was as follows: SPinu and ChiA gene fragments: 2 μL each, upstream primer and downstream primer: 2 μL each, PrimeSTAR high-fidelity enzyme: 12.5 μL, ddH2O: 4.5 μL; the overlapping PCR reaction program was as follows: pre-denaturation at 94°C for 10 min, denaturation at 94°C for 30 s; then annealing at 55°C for 1 min, extension at 72°C for 2 min, and 35 cycles; The fused DNA fragment SPinu-ChiA was connected with the plasmid pRGS after double digestion with restriction endonucleases Sal I and Sma I using the one-step cloning method under the action of Exnase II, and the transformation products were transformed into Escherichia coli DH5α respectively; the transformants were picked from the ampicillin-resistant plate and transferred to 5 mL LB liquid culture medium containing 25 μg / mL ampicillin, and cultured overnight at 37°C and 200 rpm. After plasmid extraction, the plasmid was double digested with restriction endonucleases Sal I and Sma I to verify that it was a positive clone strain; (2) The successfully verified recombinant plasmid pRGS-SPinu-ChiA was transformed into Kluyveromyces marxianus Km35, spread on a solid culture medium containing 50 µg / mL G418 resistance, and cultured at 37°C for 12-16 hours to obtain preliminary positive clones; after the yeast transformants were screened through the resistance culture medium and transferred to liquid culture, the plasmid double enzyme digestion verification was performed using a yeast plasmid extraction kit. According to the electrophoresis results, the strain corresponding to the plasmid containing the 800 bp fragment was the recombinant Kluyveromyces marxianus containing the chitosanase gene.

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