CN113201464B - Pichia pastoris engineering bacteria for producing alpha-glucanase and construction method, culture method and application thereof - Google Patents
Pichia pastoris engineering bacteria for producing alpha-glucanase and construction method, culture method and application thereof Download PDFInfo
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Abstract
The invention discloses a pichia pastoris engineering bacterium for producing alpha-glucanase, a construction method, a culture method and application thereof, and belongs to the technical field of genes. The Pichia pastoris engineering bacteria (Pichia pastoris) dip1 for producing the alpha-glucanase is preserved in Guangdong province microorganism strain preservation center in 12 months and 22 days in 2020, and the preservation number is GDMCC No.61378. The strain is subjected to shake flask fermentation culture and fed-batch culture in a fermentation tank in a 250mL triangular flask, the enzyme activity of the alpha-glucanase reaches 220U/mL and 1870U/mL respectively, the enzyme production capacity of the strain is in a leading level relative to the reported alpha-glucanase production strain, and the strain is simple in culture method, strong in enzyme production capacity and stable in genetic characteristic, and is an excellent strain suitable for producing the alpha-glucanase by high-density fermentation.
Description
Technical Field
The invention belongs to the technical field of genes, and relates to a pichia pastoris engineering bacterium for producing alpha-glucanase, a construction method, a culture method and application thereof.
Background
Alpha-glucanases (Dextranase, 1, 6-alpha-D-glucan-6-glucanohydrolase; EC 3.2.1.11), also known as dextranases, are capable of randomly hydrolyzing alpha-1, 6 glycosidic linkages in glucans, with end products including isomaltose, isomaltotriose and glucose and traces of polysaccharides. Alpha-glucanases are divided into two classes, endo-alpha-glucanases (endoglucanases) hydrolyse glycosidic bonds within glucans; exo-alpha-glucanases (exodexanases) cleave from the non-reducing end of the glucan and the end product is typically isomaltose or isomaltotriose, among others. A wide variety of microorganisms produce alpha-glucanases, including Penicillium, aspergillus, mucor, fusarium, sporothrix schenckii, lipomyces starkeyi, bacteroides stomatitis, thermoanaerobacter, streptococcus, and the like.
The alpha-glucanases which are currently commercially available are mainly obtained from cultures of fungi such as Penicillium (Penicillium) and Chaetomium (Chaetomium). Meanwhile, the production of the foreign protein by utilizing the genetically engineered bacteria has great development prospect. In the aspects of gene cloning and heterologous protein expression of alpha-glucanase, both an escherichia coli expression system and a pichia pastoris expression system are reported. Although the escherichia coli expression system has the characteristics of convenient operation and simple culture, the density of fermentation cells is not high, the cells need to be broken for obtaining expressed protein, the protein easily forms inclusion bodies, and the difficulty is brought to purification, separation and denaturation and renaturation. At present, research progress of alpha-glucanase mainly focuses on the production of exogenous alpha-glucanase by methanol-induced pichia pastoris engineering bacteria. The methylotrophic yeast is a yeast which can utilize methanol as a unique carbon source, wherein the gene operation of the Pichia Pastoris is relatively simple, the exogenous protein is expressed in a super high amount, the industrial production is easy, and the post-translational modification processing processes such as proteolytic maturation, glycosylation modification and disulfide bond formation of eukaryotic system proteins can be completed. Pichia pastoris expression vectors are divided into free vectors and integrated vectors, and the current integrated vectors are widely applied, wherein most of the vectors belong to methanol inducible vectors regulated by an alcohol oxidase AOX promoter, and a small part of vectors such as pGAPZ and pGAPZ alpha belong to constitutive vectors regulated by a glyceraldehyde triphosphate dehydrogenase GAP promoter. Chen et al obtained 83.9U/mL enzyme activity in 5L fermentor, and Kang HK et al obtained 134U/mL enzyme activity in 10L fermentor.
Pichia belongs to the ascomycete budding yeast, usually in haploid form. Can be combined to form diploid cell under the condition of nutrient limitation. The exogenous protein expression quantity of the general pichia pastoris engineering bacteria is positively correlated with the gene copy number of the exogenous protein, and in the process of constructing the engineering bacteria, the expression level of the exogenous protein can be improved by integrating a plurality of gene copies into a pichia pastoris haploid. However, after the copy number reaches a certain number, negative correlation effect is often generated, so that the exogenous protein expression yield of the common haploid engineering bacteria is bottleneck and cannot be continuously improved. Therefore, the pichia pastoris engineering strain with the alpha-glucanase production capability at the leading level is provided, and the practical application value is realized.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a pichia pastoris engineering bacterium for producing alpha-glucanase, the diploid engineering bacterium is formed by joining haploid engineering bacteria KM-HZ and KM-HK, and the enzyme activity of the produced alpha-glucanase is higher.
The second purpose of the invention is to provide a construction method of the pichia pastoris engineering bacteria for producing alpha-glucanase.
The third purpose of the invention is to provide the culture method of the pichia pastoris engineering bacteria for producing the alpha-glucanase.
The last purpose of the invention is to provide the application of the pichia pastoris engineering bacteria for producing the alpha-glucanase.
The purpose of the invention is realized by the following technical scheme:
a Pichia pastoris engineering bacterium (Pichia pastoris) dip1 for producing alpha-glucanase is preserved in Guangdong province microorganism strain collection center at 12-22 months 2020, and the preservation number is GDMCC No.61378.
The pichia pastoris engineering bacteria dip1 for producing the alpha-glucanase is applied to the production of the alpha-glucanase.
A construction method of Pichia pastoris engineering bacteria dip1 for producing alpha-glucanase comprises the following steps:
(1) Construction of haploid enzyme-producing engineering bacteria KM-HZ: according to the amino acid sequence (GenBank GI: 37927147) of alpha-glucanase of Penicillium cinnabarinum, alpha-glucanase gene dex is synthesized after codon optimization, the alpha-glucanase gene dex is subjected to enzyme digestion and is connected with plasmid pGAPZ alpha A to construct recombinant expression plasmid pGAPZ alpha A-dex, and the sequence is verified to be correct through sequencing; the expression vector pGAPZ alpha A-dex is subjected to single enzyme digestion by using AvrII to linearize the expression vector, competent pichia pastoris KM71H is transformed by electric shock, positive clone is obtained by screening on a YPD plate containing bleomycin, and engineering bacteria KM-HZ is obtained;
(2) Construction of haploid enzyme-producing engineering bacteria KM-HK: transforming the plasmid pGAPZ alpha A, and replacing the original bleomycin resistance gene and the corresponding regulatory sequence thereof with a kanamycin resistance gene and a corresponding regulatory sequence to obtain a new plasmid pGK;
carrying out enzyme digestion on the synthesized alpha-glucanase gene dex in the step (1), connecting the alpha-glucanase gene dex with a pGK plasmid to construct a recombinant expression plasmid pGK-dex, and carrying out sequencing verification to obtain a correct sequence; the expression vector pGK-dex is subjected to single enzyme digestion by AvrII to be linearized, and then competent Pichia pastoris KM71H is transformed by electric shock, and positive clones are obtained by screening on a YPD plate containing geneticin (geneticin), so that the engineering bacterium KM-HK is obtained;
(3) Respectively streaking engineering bacteria KM-HZ and engineering bacteria KM-HK on a YPD plate, culturing, respectively washing the bacterial colonies on the plate with sterile water, then fully mixing, coating the mixed bacterial liquid on a joint culture medium plate, and culturing; scraping a proper amount of bacterial colonies, putting the bacterial colonies into sterile water, fully mixing the bacterial colonies, coating the bacterial colonies on a screening culture medium, culturing, and carrying out grown positive cloning to obtain diploid engineering bacteria, namely Pichia pastoris (dip 1) for producing alpha-glucanase, wherein the bacterial strains are preserved in the microbial strain preservation center of Guangdong province, guangzhou, michelia Tokyo No. 100 Dazhou No. 59 building 5 with the preservation number of GDMCC No.61378 in 12 months and 22 days in 2020.
The alpha-glucanase gene segment dex in the step (1) is preferably obtained by the following method: according to the amino acid sequence (GenBank GI: 37927147) of alpha-glucanase from Penicillium cinnabarinum, the using preference of pichia pastoris codon, the GC content of the gene and the secondary structure of mRNA are comprehensively considered, and the alpha-glucanase gene dex is synthesized after codon optimization.
The primers used for synthesizing the alpha-glucanase gene dex in the step (1) are preferably:
primer P1: CGCGAATTCCATGGTACTACTACTACTACACAGCTAACACAC;
and (3) primer P2: TGAATGCGGCCCGCAGAAATTTGCCACTCTCC.
The amplification procedure used for the synthesis of the alpha-glucanase gene dex described in step (1) is preferably: denaturation at 95 deg.C for 5min; denaturation at 94 deg.C for 1min; annealing at 53 deg.C for 1min; extension at 72 ℃ for 2min,30 cycles; total extension at 72 ℃ for 10min.
The nucleotide sequence of the alpha-glucanase gene fragment dex in the step (1) is SEQ ID NO.1.
The digestion in step (1) is performed by EcoRI and NotI.
The ligation described in step (1) is preferably performed using T4 DNA ligase.
The method for obtaining the plasmid pGK in the step (2) comprises the following steps: plasmid pGAPZ alpha A is taken as a template, and a primer P3: GTGCGGATCCATGTGAGCAAAAGGCCAAGC and primer P4: GTGCGGATCCGCCAAACGAAGGTCT, performing PCR amplification, purifying, and performing enzyme digestion with BamHI; plasmid pPIC9k was used as template, and primers P5: GTGCGGATCCGTCGGGACAGTGAGTGTAGTAGTCTT and primer P6: performing PCR amplification on GATCAGATCGCTCCCTTATGCGACTCCT, purifying, and performing double enzyme digestion with BamHI and BglII; the two enzyme products are connected by T4 DNA ligase, competent Escherichia coli JM109 is transformed, positive clones are screened on an LB resistant plate (containing 30 mu g/mL kanamycin), and the plasmid pGK is obtained after colony PCR identification and sequencing.
The enzymes used for enzyme digestion of the alpha-glucanase gene fragment dex and the pGK plasmid in the step (2) are EcoRI and NotI.
The streaking culture on the YPD plate in the step (3) is preferably carried out at 30 ℃ for 48 hours.
The components of the joint culture medium plate in the step (3) are 0.2-0.5% of sodium acetate, 0.5-1% of potassium chloride, 0.5-2% of glucose and 1.5-2% of agar.
The conditions for culturing on the joint medium plate described in step (3) are preferably culturing at 25 ℃ for 72 hours.
The screening medium in step (3) is preferably a YPD plate containing bleomycin and geneticin.
The culture in the screening medium in step (3) is preferably carried out at 27 to 30 ℃ for 72 hours.
A shake flask fermentation culture method of Pichia pastoris engineering bacteria dip1 for producing alpha-glucanase comprises the operation of inoculating the Pichia pastoris engineering bacteria dip1 for producing alpha-glucanase into a fermentation culture medium for culture.
The composition of the fermentation medium is preferably: 8-12 g/L of yeast powder, 15-25 g/L of peptone and 2-6% (w/v) of glycerol; more preferably 10g/L yeast powder, 20g/L peptone and 5% (w/v) glycerol.
The culture temperature is preferably 27-30 ℃, the rotating speed is 240-260 r/min, and the culture time is 96-120 h; more preferably, the culture temperature is 30 ℃, the rotation speed is 250r/min, and the culture time is 100h.
A fermentation tank fed-batch culture method of Pichia pastoris engineering bacteria dip1 for producing alpha-glucanase comprises the following steps:
1) Seed culture: after slant culture is carried out on the pichia pastoris engineering bacteria dip1 for producing alpha-glucanase in a YPD culture medium, the culture is transferred into a liquid culture medium for culture;
2) Inoculating the pichia pastoris engineering bacteria dip1 which is cultured in the step 1) and produces the alpha-glucanase into a fermentation tank culture medium for culturing, wherein the culture conditions are as follows: the temperature is 28-30 ℃, the pH value is 5.0-6.0, and the dissolved oxygen is more than 10%;
3) When the content of the glycerol is lower than 0.5 percent, feeding the glycerol in a flowing manner at the beginning, and fermenting by maintaining the dissolved oxygen between 10 percent and 20 percent through feeding the glycerol in a flowing manner;
4) Culturing for 96-130 hours.
The YPD culture medium in the step 1) comprises the following components: contains 100. Mu.g/mL bleomycin, 200. Mu.g/mL geneticin, 2% tryptone, 2% glucose and 2% agar powder.
The conditions for culturing the YPD medium in the step 1) are preferably: culturing at 28-30 deg.c for 2-3 days.
The liquid medium described in step 1) preferably has the following composition: 10g/L of yeast powder, 20g/L of peptone and 5% (w/v) of glycerol.
The culture temperature of the liquid medium described in step 1) is preferably 30 ℃.
The fermentation tank culture medium in the step 2) comprises the following components: adding 12mL/L PTM into BSM culture medium 1 Trace salt solution and 5% (w/v) glycerol; the PMT1 comprises the following components: cuSO 4 ·5H 2 O 6.0g/L、NaI 0.08g/L、MnSO 4 ·H 2 O 3.0g/L、Na 2 MoO 4 ·2H 2 O 0.2g/L、H 3 BO 3 0.02g/L、CoCl 2 0.5g/L、 ZnCl 2 20.0g/L、FeSO 4 ˙7H 2 O65.0 g/L, biotin 0.2g/L, 98% H 2 SO 4 5.0mL/L。
An enzyme preparation, which contains at least one of fermentation culture of pichia pastoris engineering bacteria dip1 which can produce alpha-glucanase and has the collection number of GDMCC No.61378, bacterial strain in the fermentation culture and supernatant in the fermentation culture.
Compared with the prior art, the invention has the following advantages and effects:
1. the strain is subjected to shake flask fermentation culture and fed-batch culture in a fermentation tank in a 250mL triangular flask, the enzyme activity of the alpha-glucanase reaches 220U/mL and 1870U/mL respectively, the enzyme production capacity of the strain is in a leading level relative to the reported alpha-glucanase production strain, and the strain is simple in culture method, strong in enzyme production capacity and stable in genetic characteristic, and is an excellent strain suitable for producing the alpha-glucanase by high-density fermentation.
2. Compared with recombinant pichia pastoris (Huangzeng consortium and the like, and the amplification research on the alpha-glucanase produced by fermenting the recombinant pichia pastoris [ J ]. Sugarcane sugar industry, 2016, 2.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
Methods for defining and measuring enzyme activity in examples:
1. definition of enzyme activity: the amount of enzyme required to catalyze hydrolysis of a substrate to produce 1. Mu. Mol of glucose per minute using the DNS method at 45 ℃ and pH5.5 is 1 enzyme activity unit, expressed as U.
2. The enzyme activity determination method comprises the following steps: centrifuging the fermentation liquor at 12000r/min for 5min, taking supernatant, and diluting by proper times to obtain the enzyme solution to be detected. Taking 900 mu L of 0.02g/mL Dextran T2000 solution as a reaction substrate, placing the solution in a constant-temperature water bath at 45 ℃ for heat preservation for 5min, then adding 100 mu L of enzyme solution for reaction for 10min, adding 2mL of DNS (dinitro sodium salicylate reagent) to stop the reaction, boiling the solution in the water bath for 5min, then rapidly cooling the solution, fixing the volume to 25mL by using distilled water, and measuring the light absorption value at 540 nm. And (4) solving the corresponding glucose amount from a regression equation of the standard curve, and calculating the enzyme activity.
Example 1: construction of Pichia pastoris engineering bacterium dip1 for expressing alpha-glucanase
1. Experimental method
According to an alpha-glucanase amino acid sequence (GenBank GI: 37927147) of Penicillium cinnabarinum (Penicillium miniolutum), the use preference of a pichia pastoris codon, the GC content of a gene and the secondary structure of mRNA are comprehensively considered, and after codon optimization, an alpha-glucanase gene dex (the sequence is shown as SEQ ID NO. 1) is synthesized, wherein the total length is 1722bp, and corresponds to 574aa; the synthesized α -glucanase gene dex was ligated into a pUC57 plasmid (purchased from Koehalo Biotech Co., ltd.) to obtain a cloning vector pUC57-dex. Primer P1 (CGCGAATTCCATGGTACTACTACTACAGCTAACACACAC) and primer P2 (TGAATGCGGCGGCCGCAGAAATTTGCCACTCTCC) were designed. PCR amplification is carried out by taking a cloning vector pUC57-dex as a template and P1 and P2 as primers under the conditions that: denaturation at 95 deg.C for 5min; denaturation at 94 deg.C for 1min; annealing at 53 deg.C for 1min; extension at 72 ℃ for 2min,30 cycles; total extension at 72 ℃ for 10min. And recovering the PCR product by glue to obtain an alpha-glucanase gene fragment dex.
(2) The α -glucanase gene fragment dex obtained in (1) and the plasmid pGAPZ α A (purchased from Invitrogen) were double-digested with EcoRI and NotI, respectively. The enzyme-cleaved products were ligated by T4 DNA ligase, and competent E.coli JM109 (purchased from Beijing Baiolyobo technologies, ltd.) was transformed, and positive clones were selected on LLB resistant plates (containing 25. Mu.g/mL bleomycin). And selecting a plurality of clones to perform colony PCR identification and sequencing identification to obtain the recombinant expression vector pGAPZ alpha A-dex.
(3) Using plasmid pGAPZ alpha A as template, primer P3 (GTGCGGATCCATGTGAGCAAAAGGCCAAGC) and primer P4 (GTGCGGATCCGCCAAAACGAAGGTCT), carrying out PCR amplification, and carrying out digestion with BamHI after purification; carrying out PCR amplification by using a primer P5 (GTGCGGATCCGTCGGACAGTGAGTGTAGTCTT) and a primer P6 (GATCAGATCTCGCTCCCTTATGCGACTCCT) by taking a plasmid pPIC9k (purchased from Invitrogen company) as a template, and carrying out double digestion by using BamHI and BglII after purification; the two enzyme products are connected by T4 DNA ligase, competent Escherichia coli JM109 is transformed, positive clones are screened on an LB resistant plate (containing 30 mu g/mL kanamycin), and the plasmid pGK is obtained through colony PCR identification and sequencing.
The alpha-glucanase gene fragment dex and the plasmid pGK were double digested with EcoRI and NotI, respectively. The enzyme digestion product is connected overnight by T4 DNA ligase, and transformed into competent Escherichia coli JM109, positive clones are screened on an LB resistant plate (containing 30 mu g/mL kanamycin), and the recombinant expression vector pGK-dex is obtained after colony PCR identification and sequencing.
(4) The expression vector pGAPZ alpha A-dex and the expression vector pGK-dex are respectively cut by AvrII enzyme and are kept overnight at 37 ℃, and the cut products are purified by using a DNA fragment purification kit (purchased from Boehringer Bio-engineering Co., ltd.) to obtain linearized vectors. Competent Pichia pastoris KM71H was prepared as described in the Invitrogen Pichia expression Manual. The two obtained linearized vectors were mixed with about 80. Mu.L of competent Pichia pastoris KM71H, and placed in a 1mm electroporation cuvette for 5min in ice bath, followed by electroporation. The electric shock parameters are as follows: 1600V, 25. Mu.F, 200. Omega. After transformation, the yeast cells were plated on 100. Mu.g/mL bleomycin-containing YPD resistant plates (containing 100. Mu.g/mL bleomycin, 1% yeast extract, 2% peptone, and 2% agar) and 200. Mu.g/mL geneticin-containing YPD resistant plates (containing 200. Mu.g/mL geneticin, 1% yeast extract, 2% peptone, and 2% agar), respectively, and cultured at 30 ℃ for 3 to 4 days. And respectively selecting positive clones, and obtaining two haploid engineering bacteria KM-HZ and KM-HK through colony PCR and sequencing verification.
(5) And (3) respectively scribing the two haploid engineering bacteria obtained in the last step on a YPD plate, and culturing for 48h at the temperature of 30 ℃. The colonies on the above plates were washed off with sterile water, respectively, and then mixed well. The mixed bacterial solution was spread on a plate of a conjugate medium (0.5% sodium acetate, 1% potassium chloride, 1% glucose, 2% agar) and cultured at 25 ℃ for 72 hours. Scraping a proper amount of bacterial colony, and fully mixing in sterile water. Spread on YPD plates containing 100. Mu.g/mL bleomycin and 200. Mu.g/mL geneticin, and cultured at 30 ℃ for 72 hours. Selecting positive clones, and performing colony PCR and sequencing verification to obtain enzyme-producing diploid engineering bacteria named as alpha-glucanase-producing Pichia pastoris (Pichia pastoris) dip1, wherein the strains are preserved in Guangdong province microbial strain preservation center at 12-22 months in 2020, and the preservation number is GDMCC No.61378.
Example 2
A method for preparing alpha-glucanase (shake flask fermentation culture) comprises the following steps:
(1) Activating strains: inoculating the Pichia pastoris engineering strain dip1 which is obtained in the example 1 and produces the alpha-glucanase to a YPD slant culture medium (containing 100 mu g/mL bleomycin, 200 mu g/mL geneticin, 1% yeast powder, 2% tryptone, 2% glucose and 2% agar powder), and culturing for about 2-3 days at 30 ℃;
(2) And (3) shake flask culture: transferring the activated strain obtained in the step (1) into a liquid culture medium (culture medium components: 10g/L of yeast powder, 20g/L of peptone and 5% (w/v) of glycerol) for culture under the following culture conditions: the liquid loading amount in a 250mL triangular flask is 40mL, the temperature is 30 ℃, the rotating speed is 250r/min, and the culture is carried out for 120 hours;
(3) After culturing for 120 hours, the enzyme activity of alpha-glucanase in the culture solution is determined to be 220U/mL.
Example 3
A method for preparing alpha-glucanase (fed-batch culture in a fermentation tank) comprises the following steps:
(1) Seed culture: inoculating the Pichia pastoris engineering strain dip1 which is obtained in the example 1 and produces the alpha-glucanase to a YPD culture medium (containing 100 mu g/mL bleomycin, 200 mu g/mL geneticin, 1% yeast powder, 2% tryptone, 2% glucose and 2% agar powder) for slant culture at 30 ℃ for about 2-3 days; the cells were inoculated into a liquid medium (medium composition: yeast powder 10g/L, peptone 20g/L,5% (w/v) glycerol) and cultured under the following conditions: the liquid loading amount of a 250mL triangular flask is 40mL, the temperature is 30 ℃, the rotating speed is 250r/min, and the culture is carried out for 16h; then transferring the mixture into 300mL YPD culture medium, and carrying out shake flask culture at the temperature of 30 ℃ and the rotating speed of 250r/min until OD600 reaches 12-15 to obtain seed liquid;
(2) Inoculation: producing alpha-glucanase cultured in the step (1)1 mL of seed solution of the engineering bacterium Pichia pastoris dip1 mL was inoculated with 2.7L of BSM medium (composition: 85% H) 3 PO 4 26.7ml/L、CaSO 4 ˙2H 2 O 0.93g/L、K 2 SO 4 18.2g/L、MgSO 4 ˙2H 2 14.9g/L of O, 4.13g/L of KOH, 40g/L of 5% (v/v) of glycerol and 1 ml/L of PMT; wherein the PMT1 comprises the following components: cuSO 4 ·5H 2 O 6.0g/L、NaI 0.08g/L、MnSO 4 ·H 2 O 3.0g/L、Na 2 MoO 4 ·2H 2 O 0.2g/L、H 3 BO 3 0.02g/L、CoCl 2 0.5g/L、ZnCl 2 20.0g/L、 FeSO 4 ˙7H 2 O65.0 g/L, biotin 0.2g/L, 98% H 2 SO 4 5.0 mL/L) in a 6.8L fermenter under the following conditions: the temperature is 28-30 ℃, the pH value is 5.0-6.0, and the dissolved oxygen is more than 10%;
(3) When the content of the glycerol is lower than 0.5 percent, feeding the glycerol in a flowing manner, and fermenting by adjusting the feeding amount, the stirring speed and the ventilation quantity of the glycerol and maintaining the dissolved oxygen between 10 percent and 20 percent; after 96 hours of culture, the enzyme activity of the alpha-glucanase in the culture solution is determined to be 1870U/mL.
Example 4
Culture of haploid engineering bacteria KM-HZ
(1) Activating strains: the haploid engineering bacterium KM-HZ obtained in example 1 is inoculated to YPD slant culture medium (containing 1% yeast powder, 2% tryptone, 2% glucose and 2% agar powder), and cultured for about 2-3 days at 30 ℃;
(2) And (3) shake flask culture: inoculating the activated strain into a liquid culture medium (culture medium components: 10g/L of yeast powder, 20g/L of peptone and 5% (w/v) of glycerol) for culture under the following culture conditions: the liquid loading amount in a 250mL triangular flask is 40mL, the temperature is 30 ℃, the rotating speed is 250r/min, and the culture is carried out for 120 hours;
(3) The enzyme activity of the alpha-glucanase in the culture solution is measured to be 150U/mL.
Example 5
Culture of haploid engineering bacteria KM-HK
(1) Activating strains: inoculating the haploid engineering bacterium KM-HK obtained in example 1 to YPD slant culture medium (containing 1% yeast powder, 2% tryptone, 2% glucose and 2% agar powder), and culturing at 30 ℃ for about 2-3 days;
(2) And (3) shake flask culture: inoculating the activated strain into a liquid culture medium (culture medium components: 10g/L of yeast powder, 20g/L of peptone and 5% (w/v) of glycerol) for culture, wherein the culture conditions are as follows: the liquid loading amount in a 250mL triangular flask is 40mL, the temperature is 30 ℃, the rotating speed is 250r/min, and the culture is carried out for 120 hours;
(3) The enzyme activity of alpha-glucanase in the culture solution was determined to be 135U/mL.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such modifications are intended to be included in the scope of the present invention.
Sequence listing
<120> pichia pastoris engineering bacterium for producing alpha-glucanase, construction method, culture method and application thereof
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1722
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> nucleotide sequence of alpha-glucanase gene fragment dex
<400> 1
catggtacta cagctaacac acactgtgga gctgatttct gtacttggtg gcatgactct 60
ggtgaaatta acactcaaac accagttcag cctggaaatg ttagacaatc acataagtac 120
tctgttcagg tttccttggc tggtactaac aacttccacg attctttcgt ttacgagtcc 180
attcctagaa acggaaatgg tagaatctac gctccaacag atccacctaa ctctaatact 240
ttggactctt ccgttgatga cggtatttct atcgaacctt ctattggatt gaacatggct 300
tggtcacaat ttgagtattc tcacgatgtt gacgttaaaa ttttggctac tgatggttca 360
tctttgggat ctccatccga cgttgttatc agacctgttt caatttctta cgctatctcc 420
cagtcagatg acggtggaat tgttatcaga gttcctgctg atgctaacgg tagaaagttc 480
tcagttgaat ttaagacaga tttgtacact ttcttgtctg acggtaacga atacgttaca 540
tccggtggat cagttgttgg agttgagcca actaatgctt tggttatttt cgcttcccca 600
tttttgcctt ctggtatgat cccacatatg actcctgaca acactcaaac aatgactcca 660
ggacctatta acaatggaga ttggggtgct aagtctatct tgtactttcc acctggtgtt 720
tattggatga accaagatca gtctggaaat tccggaaagt tgggatctaa ccacatcaga 780
ttgaactcca acacttactg ggtttatttg gctcctggtg cttacgttaa gggagctatc 840
gaatacttca caaagcaaaa cttctacgct actggacatg gtatcttgtc tggagagaat 900
tacgtttatc aggctaacgc tggagataat tatattgctg ttaagtctga ctccacttca 960
ttgagaatgt ggtggcacaa caatttgggt ggaggtcaaa catggtactg tgttggtcca 1020
actattaacg ctccaccttt caatactatg gactttaacg gtaattcagg aatctcctca 1080
caaatctctg attacaagca ggttggtgct ttctttttcc aaactgacgg accagaaatt 1140
taccctaact ctgttgttca tgatgttttc tggcacgtta acgatgacgc tatcaagatt 1200
tactactctg gtgcttctgt ttccagagct acaatctgga agtgtcataa cgatccaatc 1260
atccaaatgg gttggacttc cagagatatt tcaggagtta caatcgacac tttgaacgtt 1320
atccacacaa gatacatcaa gtctgaaact gttgttccat ccgctattat cggtgcttca 1380
cctttctacg cttcaggaat gtctccagac tccagaaagt caatctctat gacagtttct 1440
aacgttgttt gtgagggttt gtgtccatcc ttgtttagaa tcactccttt gcaaaactac 1500
aagaacttcg ttgttaagaa cgttgctttt cctgatggat tgcagacaaa ttctattgga 1560
actggagagt ccattatccc agctgcttca ggtttgacta tgggattggc tatttccgct 1620
tggacaatcg gaggtcaaaa ggttactatg gaaaacttcc aagctaattc tttgggacag 1680
tttaatattg atggttccta ctggggagag tggcaaattt ct 1722
<210> 2
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer P1
<400> 2
cgcgaattcc atggtactac agctaacaca c 31
<210> 3
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer P2
<400> 3
tgaatgcggc cgcagaaatt tgccactctc c 31
<210> 4
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer P3
<400> 4
gtgcggatcc catgtgagca aaaggccagc 30
<210> 5
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer P4
<400> 5
gtgcggatcc gcacaaacga aggtct 26
<210> 6
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer P5
<400> 6
gtgcggatcc gtcggacagt gagtgtagtc tt 32
<210> 7
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer P6
<400> 7
gatcagatct cgctctccct tatgcgactc ct 32
Claims (8)
1. Pichia pastoris (A) for producing alpha-glucanasePichia pastoris) The engineering bacterium dip1 is deposited in Guangdong province microbial culture collection center in 22 months 12 and 2020, and the deposit number is GDMCC No.61378.
2. The application of the pichia pastoris engineered bacterium dip1 for producing alpha-glucanase of claim 1 in producing alpha-glucanase.
3. A construction method of Pichia pastoris engineering bacteria dip1 for producing alpha-glucanase is characterized by comprising the following steps:
(1) Construction of haploid enzyme-producing engineering bacteria KM-HZ: according to the amino acid sequence GenBank GI 37927147 of the alpha-glucanase of the penicillium cinnabarinum, the alpha-glucanase is synthesized into an alpha-glucanase gene dex after codon optimization, the alpha-glucanase gene dex is subjected to enzyme digestion and is connected with a plasmid pGAPZ alpha A to construct a recombinant expression plasmid pGAPZ alpha A-dex, and the sequence is verified to be correct by sequencing; the expression vector pGAPZ alpha A-dex is subjected to single enzyme digestion by using AvrII to linearize the expression vector, competent pichia pastoris KM71H is transformed by electric shock, positive clone is obtained by screening on a YPD plate containing bleomycin, and engineering bacteria KM-HZ is obtained;
(2) Construction of haploid enzyme-producing engineering bacteria KM-HK: transforming the plasmid pGAPZ alpha A, and replacing the original bleomycin resistance gene with a kanamycin resistance gene to obtain a new plasmid pGK;
carrying out enzyme digestion on the synthesized alpha-glucanase gene dex in the step (1), connecting the enzyme digested alpha-glucanase gene dex with a pGK plasmid to construct a recombinant expression plasmid pGK-dex, and carrying out sequencing verification to obtain a correct sequence; the expression vector pGK-dex is subjected to single enzyme digestion by AvrII to be linearized, competent Pichia pastoris KM71H is transformed by electric shock, and positive clones are obtained by screening on a YPD plate containing geneticin to obtain engineering bacteria KM-HK;
(3) Respectively streaking the engineering bacteria KM-HZ and the engineering bacteria KM-HK on a YPD plate, culturing, respectively washing bacterial colonies on the plate with sterile water, then fully mixing, coating the mixed bacterial liquid on a joint culture medium plate, and culturing; scraping appropriate amount of bacterial colony, mixing in sterile water, spreading on screening culture medium, culturing, and obtaining diploid engineering bacterium named as alpha-glucanase-producing Pichia pastoris (A)Pichia pastoris) The engineering bacteria dip1, which is preserved in Guangdong province microorganism culture collection center in 22 months 12 and 2020, with the preservation number of GDMCC No.61378;
the primers used for synthesizing the alpha-glucanase gene dex in the step (1) are as follows:
primer P1: CGCGAATTCCATGGTACTACTACAGCTAACAC;
and (3) primer P2: TGAATGCGGCCCGCAGAAATTTGCCACTCTCC;
the amplification program used for synthesizing the alpha-glucanase gene dex in the step (1) is as follows: denaturation at 95 deg.C for 5min; denaturation at 94 deg.C for 1min; annealing at 53 deg.C for 1min; extension at 72 ℃ for 2min,30 cycles; total extension at 72 ℃ for 10 min;
the nucleotide sequence of the alpha-glucanase gene dex in the step (1) is SEQ ID NO.1;
the condition of streak culture on YPD plates in the step (3) is culture at 30 ℃ for 48h;
the components of the joint culture medium plate in the step (3) are 0.2-0.5% of sodium acetate, 0.5-1% of potassium chloride, 0.5-2% of glucose and 1.5-2% of agar;
the condition of culturing on the joint medium plate in the step (3) is culturing for 72 hours at 25 ℃;
the screening culture medium in the step (3) is a YPD plate containing bleomycin and geneticin;
the culture condition in the screening culture medium in the step (3) is 27-30 ℃ for 72h.
4. The method for constructing a recombinant plasmid according to claim 3, wherein the α -glucanase gene fragment dex obtained in step (1) is obtained by: according to the amino acid sequence GenBank GI:37927147 of the alpha-glucanase from Penicillium cinnabarinum, the using preference of a pichia pastoris codon, the GC content of the gene and the secondary structure of mRNA are comprehensively considered, and the alpha-glucanase gene dex is synthesized after codon optimization.
5. The construction method according to claim 3,
the method for obtaining the plasmid pGK in the step (2) comprises the following steps: plasmid pGAPZ alpha A is taken as a template, and a primer P3: GTGCGGATCCATGTGAGCAAAAGGCCAAGC and primer P4: GTGCGGATCCGCCAAACGAAGGTCT, performing PCR amplification, purifying, and performing enzyme digestion with BamHI; plasmid pPIC9k was used as template, and primer P5: GTGCGGATCCGTCGGGACAGTGAGTGTAGTAGTCTT and primer P6: performing PCR amplification on GATCAGATCTCGCTCCCTTATGCGACTCCT, purifying, and performing double enzyme digestion with BamHI and BglII; the two enzyme digestion products are connected by T4 DNA ligase, competent Escherichia coli JM109 is transformed, positive clones are screened on an LB resistant plate containing 30 mu g/mL kanamycin, and the plasmid pGK is obtained through colony PCR identification and sequencing.
6. A shake flask fermentation culture method of Pichia pastoris engineering bacteria dip1 for producing alpha-glucanase, which is characterized by comprising inoculating the Pichia pastoris engineering bacteria dip1 for producing alpha-glucanase of claim 1 into a fermentation culture medium for culture;
the fermentation medium comprises the following components: 8-12 g/L of yeast powder, 15-25 g/L of peptone and 2-6% w/v of glycerol;
the culture temperature is 27-30 ℃, the rotating speed is 240-260 r/min, and the culture time is 96-120 h.
7. A fermentation tank fed-batch culture method of Pichia pastoris engineering bacteria dip1 for producing alpha-glucanase comprises the following steps:
1) Seed culture: carrying out slant culture on the pichia pastoris engineering bacteria dip1 for producing alpha-glucanase in a YPD culture medium, and then transferring the pichia pastoris engineering bacteria dip1 into a liquid culture medium for culture;
2) Inoculating the alpha-glucanase-producing pichia pastoris engineering bacteria dip1 cultured in the step 1) into a fermentation tank culture medium for culture, wherein the culture conditions are as follows: the temperature is 28-30 ℃, the pH value is 5.0-6.0, and the dissolved oxygen is more than 10%;
3) When the content of the glycerol is lower than 0.5 percent, feeding the glycerol initially, and fermenting by keeping the dissolved oxygen between 10 and 20 percent through feeding the glycerol;
4) Culturing for 96-130 hours.
8. An enzyme preparation, which comprises the pichia pastoris engineered bacterium dip1 for producing alpha-glucanase according to claim 1.
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