CN110863005A - Method for improving α -galactosidase constitutive expression quantity in pichia pastoris engineering bacteria through co-expression of hemoglobin - Google Patents
Method for improving α -galactosidase constitutive expression quantity in pichia pastoris engineering bacteria through co-expression of hemoglobin Download PDFInfo
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Abstract
The invention relates to a method for improving pichia pastoris engineering bacteria by coexpression of hemoglobinαThe method for improving constitutive expression quantity of galactosidase can effectively solve the problem of improving pichia pastoris engineering bacteriaαThe constitutive expression quantity of galactosidase and the application thereof, by constructing expression plasmid pGAPZαA‑GalA(ii) a Construction of constitutive expressionαStarting strain of Pichia engineering bacteria of galactosidase, DNA restriction endonucleaseBamH I enzyme cuts gene fragment PVT and pichia pastoris expression vector pPIC9K, then T4 DNA ligase is used for connecting the PVT into the vector pPIC9K, and a co-expression plasmid pPIC9K-PVT is formed; preparing an original strain into a competent cell, electrically transforming a co-expression plasmid pPIC9K-PVT into the competent cell, coating the competent cell on an MD (MD) plate, and culturing and screening positive clones to obtain a co-expression pichia pastoris engineering strain; the preparation method is simple, novel, unique, easy to operate and good in expression effect.
Description
Technical Field
The invention relates to chemical engineering, in particular to a method for improving pichia pastoris engineering bacteria by coexpression of hemoglobinα-constitutive expression of galactosidase.
Background
α-galactoseThe glycosidase (EC 3.2.1.12) can pass α -galactooligosaccharide, galactomannan side chain, galactolipid and glycoproteinαThe galactoside residues of the galactoside-linked non-reducing end are hydrolyzed off one by one. The enzyme has wide application in food, feed and other industries. In the sugar industry, it is known that,αgalactosidase can decompose raffinose in beet pulp to improve sucrose recovery. In the feed industry, it is known that,αthe galactosidase can decompose the raffinose family oligosaccharide anti-nutritional substances in the feed, reduce the problems of abdominal distension and diarrhea caused by the feed for monogastric animals, and improve the utilization rate of the feed. In the paper industry, it is known that,αgalactosidase can also be used to increase the bleaching effect of mannanase on pulp. Therefore, it is not only easy to useαThe galactosidase has wide market prospect. In the aspect of production, the construction of high-efficiency expression strains by utilizing genetic engineering means is nowαThe main way of producing galactosidase, methanol-induced pichia pastoris engineering bacteria expression is a common expression means, but the methanol use process has the risks of flammability and explosiveness, residues in the product can bring adverse effects to downstream use, the use of methanol is avoided, and the improvement of pichia pastoris engineering bacteriaαExpression level of galactosidase by constitutive secretionαMethod for co-expressing vitreoscilla hemoglobin in pichia pastoris engineering strain of galactosidase to improve content of pichia pastoris engineering strain fermentation liquorαThe expression level of galactosidase has a significant improvement effect, and the co-expression of vitreoscilla hemoglobin will be compared to the starting strainαThe expression level of galactosidase is increased, but it has not been reported in a public way.
Disclosure of Invention
In view of the above situation, the present invention aims to overcome the defects of the prior art by providing a method for increasing the content of pichia pastoris engineering bacteria by co-expression of hemoglobinαThe method for improving constitutive expression quantity of galactosidase can effectively solve the problem of improving pichia pastoris engineering bacteriaαConstitutive expression of galactosidase and its application.
The technical scheme of the invention is that the method and the device share the tableIncreasing content of Pichia pastoris by hemoglobinα-a method for constitutive expression of galactosidase comprising the steps of:
(1) construction of expression plasmid pGAPZαA-GalA: using DNA restriction endonucleasesEcoRⅠ/NotI pairα-galactosidase geneGalAAnd Pichia constitutive expression vector pGAPZαA is subjected to double enzyme digestion simultaneously, and α -galactosidase gene is connected into a vector pGAPZ by using T4 DNA ligaseαIn A, the expression plasmid pGAPZ is formedαA-GalA；
Saidα-galactosidase geneGalAVibrio fischeri optimized for codon, mRNA secondary structure and GC content: (fecheri) One of the sourcesα-a galactosidase gene;
(2) construction of constitutive expressionαStarting strain of pichia pastoris engineering bacteria of galactosidase: plasmid pGAPZ was transformed according to the yeast transformation method of InvitrogenαA-GalATransforming into Pichia pastoris GS115 competent cells, coating on YPDS plates containing 100 mug/mL bleomycin to screen positive clones as starting strains;
the plasmid pGAPZαA-GalAIs a DNA restriction endonucleasePmeI, enzyme-digested plasmid;
(3) construction of the co-expression plasmid pPIC 9K-PVT: using DNA restriction endonucleasesBamH I enzyme cuts gene fragment PVT and pichia pastoris expression vector pPIC9K, then T4 DNA ligase is used for connecting the PVT into the vector pPIC9K, and a co-expression plasmid pPIC9K-PVT is formed;
the gene segment PVT is Vitreoscilla hemoglobin genevgbPichia stipitis hypoxia-inducible promoter placed in tree trunkPsADH2And a DNA fragment formed between the terminators;
(4) constructing a co-expression pichia pastoris engineering strain: according to a yeast transformation method of Invitrogen company, the initial strain obtained in the step (2) is made into a competent cell, a co-expression plasmid pPIC9K-PVT is electrically transformed into the competent cell, and the competent cell is coated on an MD plate and cultured for 4 days at 30 ℃ to screen positive clones, so that a co-expression Pichia pastoris engineering strain is obtained;
the co-expression plasmid pPIC9K-PVT is DNA restriction endonucleasePmeI plasmid after enzyme digestion.
The pichia pastoris engineering strain is applied to raffinose hydrolysis.
The preparation method is simple, novel, unique, easy to operate and good in expression effect, and can effectively solve the problem of improving the content of the engineering bacteria of pichia pastoris by coexpression of hemoglobinα-galactosidase methanol induced expression quantity and realizes the pichia pastoris engineering bacteriaαThe application of the galactosidase methanol induction expression quantity has obvious economic and social benefits.
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FIG. 1 is a graph showing that the enzyme activities of fermentation broth screened by shake flask expression high-activity strains are all significantly improved.
Detailed Description
The following examples and specific examples will explain the present invention in detail.
In the specific implementation of the invention, the improvement of the content of the engineering bacteria of pichia pastoris by coexpression of hemoglobinα-a method for constitutive expression of galactosidase comprising the steps of:
(1) construction of expression plasmid pGAPZαA-GalA
The expression gene is Vibrio fischeri: (fecheri) One of the sourcesα-galactosidase geneGalAOptimizing the pichia pastoris according to the codon preference, reasonably optimizing the mRNA secondary structure, GC content and the like of the pichia pastoris, and finally determining the optimal sequence as shown in SEQ 1; after gene synthesis, DNA restriction endonuclease was usedEcoRⅠ/NotI double digestion of α -galactosidase gene and Pichia constitutive expression vector pGAPZ α A, followed by ligation of α -galactosidase gene into vector pGAPZ with T4 DNA ligaseαIn A, the expression plasmid pGAPZ is formedαA-GalAUsing DNA restriction endonucleasesEcoRⅠ/NotI, carrying out enzyme digestion detection on the plasmid to ensure that the plasmid is successfully constructed;
(2) construction of constitutive expressionαPichia of galactosidaseParent engineering bacterium starting strain
Transformation method according to the yeast transformation method of Invitrogen corporation, DNA restriction enzyme was usedPmeI pairs plasmid pGAPZαA-GalAAfter linearization, electrotransformation is carried out to Pichia pastoris GS115 competent cells, the cells are spread on YPDS plates containing 100 mu g/mL bleomycin to screen positive clones, the monoclone is selected and inoculated to 50mL (250 mL conical flask) YPD culture medium for 3 days, and the determination is carried outαGalactosidase enzyme activity, mixing 100 muL fermentation liquor and 100 muL pNPG solution with the concentration of 10 mmol/L, reacting for 10min at 60 ℃, and adding 800 muL 0.5 mo/L Na2CO3Stopping the reaction of the solution, adding 200 mu L of the solution into a 96-well plate, measuring an absorption value at 405nm by using an enzyme-linked immunosorbent assay (ELIASA), and substituting the absorption value into a standard curve which is measured by using p-nitrobenzene (pNP) in advance to obtainαThe activity value of galactosidase, selecting the clone with the highest preservation activity as the starting strain of pichia pastoris engineering bacteria;
saidαGalactosidase enzyme activity is defined as the amount of enzyme required to hydrolyze p-nitrophenyl- α -D-galactoside (pNPG) to produce 1 μmol of p-nitrophenol (pNP) per minute, defined as 1 α -galactosidase enzyme activity unit;
the YPDS plate is prepared by adding 10g of yeast powder, 20g of peptone, 20g of glucose, 182g of sorbitol and 15g of agar powder into 1000mL of water, and sterilizing at 108 ℃ for 20 minutes;
the YPD culture medium comprises 10g of yeast powder, 20g of peptone and 20g of glucose, water is added to the mixture to reach 1000mL, and the mixture is sterilized at 108 ℃ for 20 minutes;
(3) construction of the Co-expression plasmid pPIC9K-PVT
In order to improve the content of pichia pastoris engineering bacteriaαThe expression quantity of galactosidase co-expresses a vitreoscilla hemoglobin gene in engineering bacteria so as to improve the utilization rate of oxygen by pichia pastoris, improve the tolerance of pichia pastoris to low oxygen environment during high-density fermentation and improveα-the amount of expression of a galactosidase gene; optimizing pichia pastoris codon preference, reasonably optimizing mRNA secondary structure and GC content, and synthesizing vitreoscilla hemoglobin genevgbThe sequence of which is underlined in SEQ2, by splicing PCRMethod for placing pichia stipitis under low-oxygen induction promoterPsADH2And a terminator, a DNA fragment PVT is formed, the sequence is SEQ2, and DNA restriction endonuclease is usedBamThe PVT and the Pichia expression vector pPIC9K are digested by HI, then the PVT is connected into the vector pPIC9K by using T4 DNA ligase to form a co-expression plasmid pPIC9K-PVT, and DNA restriction endonuclease is usedBamH I, carrying out enzyme digestion detection on the plasmid to ensure that the plasmid is successfully constructed;
(4) construction of co-expression Pichia pastoris engineering strain
According to the yeast transformation method of Invitrogen, the starting strain obtained in step (2) was made into competent cells, and DNA restriction enzyme was usedPmeI linearized co-expression plasmid pPIC9K-PVT was electroporated into it, plated on MD plates at 30 ℃ for 4 days to select positive clones, picked up and inoculated into 50mL (250 mL Erlenmeyer flask) YPD medium for 3 days, and assayed in the same manner as in step 2αAnd (3) galactosidase enzyme activity, wherein α -galactosidase enzyme activity in the fermentation broth is the highest, so that construction of the co-expression pichia pastoris engineering strain is realized.
Accessories:
SEQ1：
TTGGTTAGACCAGGTAACGTTGGTAAGTTGCCAGCTTTGGGTTGGAACTCTTGGAACGCTTTCGGTTGTGATATTGATGCCACCAAGATTATGACTGCCGCTAACGAAGTTGTTAACTTGGGTTTGAAGAACTTGGGTTACGAGTACATCAACATTGATGATTGCTGGTCCGTTAAGTCTGGTAGAGATGCTTCTACTCAAAGAATGGTTCCAGACCCAGAAAAGTTTCCAGATGGCATTTCTGGTTTGGCTGACCAAATTCACGATTTGGGTTTGAAGGTTGGTATTTACTCTTCTGCTGGTTTGACTACTTGTGCTGGTTACCCAGCTTCTTTGGGTTACGAAGATATTGACGCCCAAACTTTTGCTGAATGGGGTATCGATTACTTGAAGTACGACAACTGTGGTGTTCCATCTAACTGGACTGATGCTTACTCTTACTGTGTTCCAGATCCAGGTTCTAAGGCTACTAACGGTACTTGTCCAGATAACAAGAACCCAGCTCCAGCTGGTTACGATTGGAGAACTTCTTTGACTGCTGAGAGATACAGAAGAATGAGAGATGCTTTGGTTTCTGTCGACAGAACCATTTTGTACTCTTTGTGCAACTGGGGTCAAGCTGATGTTAACAACTGGGGTAACGAAACTGGTAACTCTTGGAGAACTACCGGTGATATTACTCCATCTTGGCCAAGAATTGCTGCTATTGCTAACGAAAACTCCTTCTTGATGAACTACGTCGACTTTTGGGGTTACCCAGATCCAGATATGTTGGAAGTTGGTAACGGTAACTTGACTTTGGCTGAAAACCGTGCTCATTTTGCTTTGTGGGCTGCTATGAAGTCTCCATTGATTATTGGTACTGCCTTGGATTCCATTTCCCAAGACCATTTGGCTATTTTGTCCAACAAAATCTTGTTGAAGTTCCACCAAGATCCAGTTGTTGGTAGACCAGCTCATCCATACAAGTGGGGTTACAACCCAGATTGGACTTTTGATCCAGCTCATCCAGCTGAATACTGGTCTGGTGTTTCTTCTGCTTTGGGTGGTACTTTGGTTTTGATGTTGAACTCTGAAGACACCAAGCAAAGAAGAACTGCTGTCTGGAAGGAAATTCCAGAGTTGAAGGATGTTTTGGGTAGACAAGGTAAAAGACGTACTGGTTTTAGAGTTACCGATGTTTGGACTGGTAAGGATTTGGGTTGTGTCAGAGATCATTACTCTGTCGAATTGGAGTCTCATGATGTTGCTGCTTTGGTTGTTGGTAGAGCTTGT
SEQ2：
GCGGAAGCACAGTCTAATGCTGATTCTTGATAGTGCTCATCGCCGACAGCCAGATTCGAAGAAAGGGGGGACGAGATCCGGGTTCATCTGCAAGAGACACAGAAAATAAAAAACATACGATCCTTTTCAGCTACCAAGCGCTTAACCAGGAAATCCACTGCTGGAGTGGCCAGCATGTCACGAGGTGGCAGATTCCGATAATGTGTGATTGGAGTGTAGCATTGGCGCAAGTCGAATTTCGGTCATATTCCGTGTCTGGATATTATTCCACTATTTTTTAATTTTTCAGGTTGGATGCGATTGTTCCCTTTATGTCTGGACGATGCCTGAAGCCCCAGGTCTATATAAGGGGCTCGAAAGTCCTTTGACCAGCTGGTTGATTTGATTTTGTTTGTTCCTTTCTTTCTTTCATCTACCATCACTCAATTGCATTCGCAATTACCCATTAATACATATTTCACTTGCTCCACATATTGCACCCATCTGCATAAGTGCTGCGATCCATCCAAATTATCATGTTGGACCAACAAACCAT CAACATCATCAAGGCTACTGTTCCAGTTTTGAAGGAACATGGTGTCACTATCACTACTACCTTCTACAAGAACTTGT TTGCCAAGCATCCAGAAGTTAGACCATTGTTTGACATGGGTAGACAGGAATCTTTGGAACAACCAAAGGCTTTGGCT ATGACTGTTTTGGCTGCTGCTCAAAACATTGAAAACTTGCCCGCTATTTTGCCAGCTGTTAAGAAGATTGCCGTCAA GCATTGTCAAGCTGGTGTTGCTGCTGCTCATTACCCAATTGTTGGCCAAGAATTGTTGGGTGCCATTAAGGAAGTTT TGGGTGATGCTGCTACTGATGATATTTTGGATGCTTGGGGTAAGGCTTACGGTGTTATTGCTGACGTCTTCATTCAA GTTGAAGCCGATTTGTACGCTCAAGCTGTTGAATAAACAAGCCGTGCTAGATAGTGTTTTGTTCTATTCCACCTCGTGTTTTATTTACCTTTTAACTAGTGCTAATTTACGAATAATGTACGTTAGAAAAATTGACCTATTTATATATGGGTGCAAAAAAGTTGCGTCCTGTCCGAAGGTTGCATTTCCCCTCAAGCAATTTCTTTGCCCCATAACAACCAAAAGGAGTTAGGATCTGGGAACTATTTAACGCCGCTGGGGCAACTATTGAGATGTACCTAGATGCCACGCCCCACCTGACCAATCTAATTGCTGTAATTCATATCCTTAAGATTAGTTCTCGCCGTGAACCTAGAGATGTGACCCTTAGCTTAGAAGTTTTCTAAGAAAAATCGTAACTTTAAGAAACTATTAATTAGGCGGCTTTTTCAGAACTTTTTCATTAGCATCATTGAGCTTTTCGGCTTCTACCTGAGCTCCGC
the co-expression pichia pastoris engineering strain can be effectively used for hydrolyzing raffinose, and the application of the co-expression pichia pastoris engineering strain in the hydrolysis of raffinose is realized.
The method is simple and easy to operate, and the co-expression of the hemoglobin is used for improving the content of the engineering bacteria of the pichia pastorisαThe co-expression pichia pastoris engineering strain prepared by the method has good hydrolysis effect on raffinose and can hydrolyze the raffinoseαThe complete hydrolysis of the galactoside bond can be used for producing actual needs, and the technical effect which is very satisfactory is achieved by field application and test, and the related information is as follows:
experiment one: construction of expression plasmid pGAPZαA-GalA
The expression gene is Vibrio fischeri: (fecheri) One of the sourcesα-galactosidase geneGalAOptimizing the pichia pastoris according to the codon preference, reasonably optimizing the mRNA secondary structure, GC content and the like of the pichia pastoris, and finally determining the optimal sequence as shown in SEQ 1; after gene synthesis, DNA restriction endonuclease was usedEcoRⅠ/NotI double digestion of α -galactosidase gene and Pichia constitutive expression vector pGAPZ α A, followed by ligation of α -galactosidase gene into vector pGAPZ with T4 DNA ligaseαIn A, the expression plasmid pGAPZ is formedαA-GalAUsing DNA restriction endonucleasesEcoRⅠ/NotI, carrying out enzyme digestion detection on the plasmid to ensure the successful construction of the plasmid, wherein the result of the enzyme digestion detection of the plasmid is visible in agarose gel electrophoresis,EcoRⅠ/Noti restriction enzyme plasmid pGAPZαA-GalAOne vector strip and the corresponding gene strip can be cut out, and the success of vector construction is proved.
Experiment two: construction of constitutive expressionαPichia pastoris engineering bacteria starting strain of galactosidase
The transformation method was referred to as yeast transformation method by Invitrogen. Using DNA restriction endonucleasesPmeI pairs plasmid pGAPZαA-GalAAfter linearization, the cells are electrically transformed into pichia pastoris GS115 competent cells, the competent cells are spread on a YPDS plate containing 100 mu g/mL bleomycin to screen positive clones, and the single clones are selected and inoculated into 50mL (250 mL conical flask) YPD culture medium to be cultured for 3 daysMeasurement ofαPerforming galactosidase enzyme activity, mixing 100 muL of fermentation liquor properly diluted with 100 muL of pNPG solution (10 mmol/L), reacting for 10min at 60 ℃, adding 800 muL of 0.5 mo/L of Na2CO3 solution to terminate the reaction, adding 200 muL into a 96-well plate, measuring an absorption value at 405nm by using an enzyme-labeling instrument, substituting into a standard curve measured by p-nitrobenzene (pNP) in advance to obtain the productαThe activity value of galactosidase, selecting the clone with the highest preservation activity as the starting strain of pichia pastoris engineering bacteria;
experiment three: construction of the Co-expression plasmid pPIC9K-PVT
In order to improve the content of pichia pastoris engineering bacteriaαThe expression quantity of galactosidase needs to co-express a vitreoscilla hemoglobin gene in engineering bacteria, so that the utilization rate of oxygen by pichia pastoris can be improved, the tolerance of pichia pastoris to a low-oxygen environment during high-density fermentation can be improved, and finally, the improvement of the tolerance of pichia pastoris to a low-oxygen environment is realizedα-expression level of galactosidase gene. Optimizing pichia pastoris codon preference, reasonably optimizing mRNA secondary structure, GC content and the like, and synthesizing vitreoscilla hemoglobin genevgbThe sequence is shown in the underlined part of SEQ2, and the Pichia stipitis is placed in a pichia stipitis hypoxia-inducible promoter by a splicing PCR methodPsADH2And a terminator to form a DNA fragment PVT with the sequence shown in SEQ2BamThe PVT and the Pichia expression vector pPIC9K are digested by HI, then the PVT is connected into the vector pPIC9K by using T4 DNA ligase to form a co-expression plasmid pPIC9K-PVT, and DNA restriction endonuclease is usedBamH I carries out enzyme digestion detection on the plasmid to ensure that the plasmid construction is successful.
Experiment four: construction of Co-expression Pichia engineering Strain
The starting strain obtained in step (2) (experiment two) was prepared as a competent cell by the yeast transformation method of Invitrogen corporation, and DNA restriction enzyme was usedPmeI linearized co-expression plasmid pPIC9K-PVT was electroporated into it, plated on MD plates at 30 ℃ for 4 days to select positive clones, picked up and inoculated into 50mL (250 mL Erlenmeyer flask) YPD medium for 3 days, and assayed in the same manner as in step 2 (experiment two)α-halfThe lactase enzyme activity and the shake flask expression high activity strain screening show that the fermentation liquor enzyme activity of the total No. 1-3 expression strains is obviously improved relative to the original strain, the fermentation liquor enzyme activity of the No. 2 strain is obviously improved to the most obvious degree and reaches 126.68 percent of the original strain, the α -galactosidase enzyme activity in the fermentation liquor of the No. 2 strain is the highest, and relative to the original strain, the fermentation liquor of the No. 2 strain has the highest α -galactosidase enzyme activityαThe enzyme activity of the galactosidase is improved by 26.68 percent, and the constitutive secretory expression can be seenαThe method for co-expressing vitreoscilla hemoglobin in pichia pastoris engineering strains of galactosidase can obviously improve the content of fermentation liquor of the pichia pastoris engineering strainsαThe expression level of galactosidase is also avoided, at the same time as the adverse effects of the use of methanol are avoided.
Experiment five: application of co-expression pichia pastoris engineering strain
Selecting co-expression Pichia pastoris engineering strain, inoculating to 2 mL YPD liquid culture medium, shake culturing at 30 deg.C for 1 day, transferring to 250 mL YPD liquid culture medium (500 mL conical flask), shake culturing at 30 deg.C to OD600Between 2 and 6, the cells were transferred to 2.5L of 4% glycerol in mineral salts medium, NH4H2PO438.66 g/L、CaSO4∙2H2O 1.17 g/L、KSO418.2 g/L、MgSO4∙7H2O14.9 g/L, KOH 4.13.13 g/L and phosphoric acid with the mass content of 85 percent, 3.76 ml/L, water and constant volume; adjusting pH to 5.5 with ammonia water, introducing air in an amount of 3L/min, stirring at 800 rpm, culturing at 30 deg.C for 20 hr, and measuring dissolved oxygen value to about 100%; then, the stirring speed was increased to 1200rpm, and 50% glycerol (12 mL of PTM per liter) was started to be fed1Inorganic salt nutrient solution), the feeding speed is 54 mL per hour, and the total time is 3-4 hours; stopping glycerol supplementation when the wet weight of the cells reaches about 200 g/L, and beginning to feed inorganic salt culture medium (12 mL PTM per liter) containing 4% glucose at the speed of 10-100mL per hour1Inorganic salt nutrient solution), maintaining the dissolved oxygen amount of more than 20%, fermenting for 288 hours in total, sampling in the fermentation process (0, 24, 48, 64, 72, 96, 120, 144, 168, 192, 216, 240, 264 and 288 hours), and performing SDS-PAGE electrophoresis, wherein the structure is as follows: fermentation liquorSDS-PAGE shows a clear band around 50-70KD, andαthe molecular weight of galactosidase GalA is consistent with 55KD, which indicates that the coexpression strain can actually secrete and express during fermentationαGalactosidase GalA, which is expressed in the fermentation liquor in a large amount and secreted in the late fermentation stageα-a galactosidase; measurement in fermentation broth at 60 ℃ and pH4.5αThe activity of galactosidase can reach 1100U/mL (α-galactosidase enzyme activity is defined as: hydrolysis of p-nitrobenzene per minute under certain conditionsαThe amount of enzyme required for the production of 1. mu. mol of p-nitrophenol (pNP) by D-galactoside (pNPG) was defined as 1α-galactosidase enzyme activity unit). The fermentation liquor is applied to hydrolysis of natural raffinose to prepare a reaction system containing raffinose, wherein the reaction system contains 4 mg/mL sugar and 10 enzyme activity unitsαGalactosidase and 50mmol/L buffer pH4.5 (sodium acetate buffer), reacted at 60 ℃, sampled at various time points, and the samples taken immediately after boiling for inactivation. A sample (chromatography is carried out twice) is spread on a Silica Gel chromatography plate (Merck Silica Gel 60F 254, Germany) by using a chromatography liquid (propanol: acetic acid: water =1:1.5: 0.1), after a color development liquid (methanol: concentrated sulfuric acid =95: 5) is sprayed, the chromatography plate is placed in an oven at 105 ℃ for heating and color development, and after a visible reaction is carried out for 60 minutes, raffinose is completely hydrolyzed into cane sugar and galactose, so that the co-expression pichia pastoris engineering strain prepared by the method is shown to be produced by fermentationαThe galactosidase has good hydrolysis effect on raffinose, and can hydrolyze raffinoseαComplete hydrolysis of the galactoside linkage, useful for the production of the actual need.
Experiments show that the invention expresses the protein through constitutive secretionαMethod for co-expressing vitreoscilla hemoglobin in pichia pastoris engineering strain of galactosidase to improve content of pichia pastoris engineering strain fermentation liquorαThe expression level of galactosidase has a significant improvement effect, and the co-expression of vitreoscilla hemoglobin will be compared to the starting strainαThe expression quantity of the galactosidase is improved by 26.68 percent, and the galactosidase can be effectively used for the fermentation production of the co-expression pichia pastoris engineering strainαThe effect of galactosidase on the hydrolysis of raffinose, which can be found in raffinoseαThe galactose glycosidic bond is completely hydrolyzed into sucrose and galactose, which can meet the actual requirement of production and has obvious economic and social benefits.
Sequence listing
<110> institute of biological sciences, Inc. of Henan province
<120> method for improving α -galactosidase constitutive expression quantity in pichia pastoris engineering bacteria by coexpression of hemoglobin
<160>2
<170>SIPOSequenceListing 1.0
<210>1
<211>1275
<212>DNA
<213> Artificial sequence ()
<400>1
ttggttagac caggtaacgt tggtaagttg ccagctttgg gttggaactc ttggaacgct 60
ttcggttgtg atattgatgc caccaagatt atgactgccg ctaacgaagt tgttaacttg 120
ggtttgaaga acttgggtta cgagtacatc aacattgatg attgctggtc cgttaagtct 180
ggtagagatg cttctactca aagaatggtt ccagacccag aaaagtttcc agatggcatt 240
tctggtttgg ctgaccaaat tcacgatttg ggtttgaagg ttggtattta ctcttctgct 300
ggtttgacta cttgtgctgg ttacccagct tctttgggtt acgaagatat tgacgcccaa 360
acttttgctg aatggggtat cgattacttg aagtacgaca actgtggtgt tccatctaac 420
tggactgatg cttactctta ctgtgttcca gatccaggtt ctaaggctac taacggtact 480
tgtccagata acaagaaccc agctccagct ggttacgatt ggagaacttc tttgactgct 540
gagagataca gaagaatgag agatgctttg gtttctgtcg acagaaccat tttgtactct 600
ttgtgcaact ggggtcaagc tgatgttaac aactggggta acgaaactgg taactcttgg 660
agaactaccg gtgatattac tccatcttgg ccaagaattg ctgctattgc taacgaaaac 720
tccttcttga tgaactacgt cgacttttgg ggttacccag atccagatat gttggaagtt 780
ggtaacggta acttgacttt ggctgaaaac cgtgctcatt ttgctttgtg ggctgctatg840
aagtctccat tgattattgg tactgccttg gattccattt cccaagacca tttggctatt 900
ttgtccaaca aaatcttgtt gaagttccac caagatccag ttgttggtag accagctcat 960
ccatacaagt ggggttacaa cccagattgg acttttgatc cagctcatcc agctgaatac 1020
tggtctggtg tttcttctgc tttgggtggt actttggttt tgatgttgaa ctctgaagac 1080
accaagcaaa gaagaactgc tgtctggaag gaaattccag agttgaagga tgttttgggt 1140
agacaaggta aaagacgtac tggttttaga gttaccgatg tttggactgg taaggatttg 1200
ggttgtgtca gagatcatta ctctgtcgaa ttggagtctc atgatgttgc tgctttggtt 1260
gttggtagag cttgt 1275
<210>2
<211>1402
<212>DNA
<213> Artificial sequence ()
<400>2
gcggaagcac agtctaatgc tgattcttga tagtgctcat cgccgacagc cagattcgaa 60
gaaagggggg acgagatccg ggttcatctg caagagacac agaaaataaa aaacatacga 120
tccttttcag ctaccaagcg cttaaccagg aaatccactg ctggagtggc cagcatgtca 180
cgaggtggca gattccgata atgtgtgatt ggagtgtagc attggcgcaa gtcgaatttc 240
ggtcatattc cgtgtctgga tattattcca ctatttttta atttttcagg ttggatgcga 300
ttgttccctt tatgtctgga cgatgcctga agccccaggt ctatataagg ggctcgaaag 360
tcctttgacc agctggttga tttgattttg tttgttcctt tctttctttc atctaccatc 420
actcaattgc attcgcaatt acccattaat acatatttca cttgctccac atattgcacc 480
catctgcata agtgctgcga tccatccaaa ttatcatgtt ggaccaacaa accatcaaca 540
tcatcaaggc tactgttcca gttttgaagg aacatggtgt cactatcact actaccttct 600
acaagaactt gtttgccaag catccagaag ttagaccatt gtttgacatg ggtagacagg 660
aatctttgga acaaccaaag gctttggcta tgactgtttt ggctgctgct caaaacattg 720
aaaacttgcc cgctattttg ccagctgtta agaagattgc cgtcaagcat tgtcaagctg 780
gtgttgctgc tgctcattac ccaattgttg gccaagaatt gttgggtgcc attaaggaag 840
ttttgggtga tgctgctact gatgatattt tggatgcttg gggtaaggct tacggtgtta 900
ttgctgacgt cttcattcaa gttgaagccg atttgtacgc tcaagctgtt gaataaacaa 960
gccgtgctag atagtgtttt gttctattcc acctcgtgtt ttatttacct tttaactagt 1020
gctaatttac gaataatgta cgttagaaaa attgacctat ttatatatgg gtgcaaaaaa 1080
gttgcgtcct gtccgaaggt tgcatttccc ctcaagcaat ttctttgccc cataacaacc 1140
aaaaggagtt aggatctggg aactatttaa cgccgctggg gcaactattg agatgtacct 1200
agatgccacg ccccacctga ccaatctaat tgctgtaatt catatcctta agattagttc 1260
tcgccgtgaa cctagagatg tgacccttag cttagaagtt ttctaagaaa aatcgtaact 1320
ttaagaaact attaattagg cggctttttc agaacttttt cattagcatc attgagcttt 1380
tcggcttcta cctgagctcc gc 1402
Claims (3)
1. Improving content of pichia pastoris engineering bacteria by coexpression of hemoglobinα-a method for constitutive expression of galactosidase, comprising the steps of:
(1) construction of expression plasmid pGAPZαA-GalA: using DNA restriction endonucleasesEcoRⅠ/NotI pairα-galactosidase geneGalAAnd Pichia constitutive expression vector pGAPZαA is subjected to double enzyme digestion simultaneously, and α -galactosidase gene is connected into a vector pGAPZ by using T4 DNA ligaseαIn A, the expression plasmid pGAPZ is formedαA-GalA；
Saidα-galactosidase geneGalAVibrio fischeri optimized for codon, mRNA secondary structure and GC content: (fecheri) One of the sourcesα-a galactosidase gene;
(2) construction of constitutive expressionαStarting strain of pichia pastoris engineering bacteria of galactosidase: plasmid pGAPZ was transformed according to the yeast transformation method of InvitrogenαA-GalATransforming into Pichia pastoris GS115 competent cells, coating on YPDS plates containing 100 mug/mL bleomycin to screen positive clones as starting strains;
the plasmid pGAPZαA-GalAIs a DNA restriction endonucleasePmeI, enzyme-digested plasmid;
(3) construction of the co-expression plasmid pPIC 9K-PVT: using DNA restriction endonucleasesBamH I enzyme cuts gene fragment PVT and pichia pastoris expression vector pPIC9K, then T4 DNA ligase is used for connecting the PVT into the vector pPIC9K, and a co-expression plasmid pPIC9K-PVT is formed;
the gene segment PVT is Vitreoscilla hemoglobin genevgbPichia stipitis hypoxia-inducible promoter placed in tree trunkPsADH2And a DNA fragment formed between the terminators;
(4) constructing a co-expression pichia pastoris engineering strain: according to a yeast transformation method of Invitrogen company, the initial strain obtained in the step (2) is made into a competent cell, a co-expression plasmid pPIC9K-PVT is electrically transformed into the competent cell, and the competent cell is coated on an MD plate and cultured for 4 days at 30 ℃ to screen positive clones, so that a co-expression Pichia pastoris engineering strain is obtained;
the co-expression plasmid pPIC9K-PVT is DNA restriction endonucleasePmeI plasmid after enzyme digestion.
2. The method for improving the content of the engineering bacteria of Pichia pastoris by co-expression of hemoglobin according to claim 1α-a method for constitutive expression of galactosidase, comprising the steps of:
(1) construction of expression plasmid pGAPZαA-GalA
The expression gene is Vibrio fischeri: (fecheri) One of the sourcesα-galactosidase geneGalAOptimizing the secondary structure and GC content of mRNA according to the codon preference of pichia pastoris, and determining the optimal sequence as SEQ 1; after gene synthesis, DNA restriction endonuclease was usedEcoRⅠ/NotI double digestion of α -galactosidase gene and Pichia constitutive expression vector pGAPZ α A, followed by ligation of α -galactosidase gene into vector pGAPZ with T4 DNA ligaseαIn A, the expression plasmid pGAPZ is formedαA-GalAUsing DNA restriction endonucleasesEcoRⅠ/NotI, carrying out enzyme digestion detection on the plasmid to ensure that the plasmid is successfully constructed;
(2) construction of constitutive expressionαPichia pastoris engineering bacteria starting strain of galactosidase
Transformation method according to the yeast transformation method of Invitrogen corporation, DNA restriction enzyme was usedPmeI pairs plasmid pGAPZαA-GalAAfter linearization, electrotransformation is carried out to Pichia pastoris GS115 competent cells, the cells are spread on a YPDS plate containing 100 mu g/mL bleomycin to screen positive clones, the monoclone is selected and inoculated to a conical flask filled with 50mL YPD culture medium to be cultured for 3 days, and the determination is carried outαGalactosidase enzyme activity, mixing 100 muL fermentation liquor and 100 muL pNPG solution with the concentration of 10 mmol/L, reacting for 10min at 60 ℃, and adding 800 muL 0.5 mo/L Na2CO3Stopping the reaction of the solution, adding 200 mu L of the solution into a 96-well plate, measuring an absorption value at 405nm by using an enzyme-linked immunosorbent assay, and substituting the absorption value into a standard curve which is measured by using p-nitrobenzene in advance to obtainαThe activity value of galactosidase, selecting the clone with the highest preservation activity as the starting strain of pichia pastoris engineering bacteria;
saidαGalactosidase enzyme activity is defined as the amount of enzyme required to hydrolyze p-nitrophenyl- α -D-galactoside per minute to produce 1 μmol of p-nitrophenolMeaning 1 α -galactosidase enzyme activity unit;
the YPDS plate is prepared by adding 10g of yeast powder, 20g of peptone, 20g of glucose, 182g of sorbitol and 15g of agar powder into 1000mL of water, and sterilizing at 108 ℃ for 20 minutes;
the YPD culture medium comprises 10g of yeast powder, 20g of peptone and 20g of glucose, water is added to the mixture to reach 1000mL, and the mixture is sterilized at 108 ℃ for 20 minutes;
(3) construction of the Co-expression plasmid pPIC9K-PVT
In order to improve the content of pichia pastoris engineering bacteriaαThe expression quantity of galactosidase co-expresses a vitreoscilla hemoglobin gene in engineering bacteria, improves the utilization rate of oxygen by pichia pastoris, improves the tolerance of pichia pastoris to low oxygen environment during high-density fermentation, and improvesα-the amount of expression of a galactosidase gene; optimizing mRNA secondary structure and GC content according to pichia pastoris codon preference to synthesize vitreoscilla hemoglobin genevgbThe sequence is underlined in SEQ2, and is placed in a Pichia stipitis hypoxia-inducible promoter by a splicing PCR methodPsADH2And a terminator, a DNA fragment PVT is formed, the sequence is SEQ2, and DNA restriction endonuclease is usedBamThe PVT and the Pichia expression vector pPIC9K are digested by HI, then the PVT is connected into the vector pPIC9K by using T4 DNA ligase to form a co-expression plasmid pPIC9K-PVT, and DNA restriction endonuclease is usedBamH I, carrying out enzyme digestion detection on the plasmid to ensure that the plasmid is successfully constructed;
(4) construction of co-expression Pichia pastoris engineering strain
According to the yeast transformation method of Invitrogen, the starting strain obtained in step (2) was made into competent cells, and DNA restriction enzyme was usedPmeI linearized co-expression plasmid pPIC9K-PVT was electroporated into it, plated on MD plates and cultured at 30 ℃ for 4 days to select positive clones, and single clones were selected and inoculated into Erlenmeyer flasks containing 50mL of YPD medium for 3 days, and assayed in the same manner as in step (2)αAnd (3) galactosidase enzyme activity, wherein α -galactosidase enzyme activity in the fermentation broth is the highest, and the construction of the co-expression pichia pastoris engineering strain is realized.
3. The application of the co-expression pichia pastoris engineering strain constructed by the method of claim 1 or 2 in raffinose hydrolysis.
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