CN107043757A - The recombinant yeast pichia pastoris of one plant of heterologous high efficient expression rhizomucor miehei lipase and its application - Google Patents
The recombinant yeast pichia pastoris of one plant of heterologous high efficient expression rhizomucor miehei lipase and its application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
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Abstract
The invention discloses the recombinant yeast pichia pastoris of one plant of heterologous high efficient expression lipase and its application.The plasmid SEC31 pPIC3.5K that recombinant yeast pichia pastoris is overexpressed SEC31 genes by being converted into the Recombinant Pichia pastoris X 33 that can express Pro RML containing 4 copy number pro rml genes are obtained.The present invention has been effectively promoted the expression of rhizomucor miehei lipase, improves the secernment efficiency of the enzyme, during shake flask fermentation 144h, and enzyme activity reaches 996U/mL, and bacterial strain enzymatic activities secernment efficiency reaches 38U/OD600, enzymatic activities secernment efficiency (26U/OD of the secernment efficiency higher than the Pichia pastoris recombinant bacterial strain of 2 rhizomucor miehei lipase copy numbers involved by patent CN103361327A600)。
Description
Technical field
The invention belongs to biological technical field, be related to one plant of heterologous high efficient expression lipase recombinant yeast pichia pastoris and its should
With.
Background technology
Lipase (EC 3.1.1.3) is can to hydrolyze the class of enzymes of triacylglycerol.A variety of chemical reactions can be catalyzed such as：Water
Solution, esterification, transesterification, ammonolysis etc..Because lipase-catalyzed reaction has a variety of advantages compared with chemical method：Reaction condition is gentle,
Accessory substance is few, pollution-free etc., is widely used in the industries such as food, medicine, cosmetics, oil, and be referred to as the third-largest
Industrial enzymes.
At present, the main strategy using heterologous protein expression of production of lipase.But influence during heterologous protein expression because
It is plain a lot, it is broadly divided into two major classes：External cause and internal cause.External cause includes：Culture medium, condition of culture etc.；Internal cause has gene codon
Preferences, gene copy number, the performance of carrier, the ability to bear of host's oneself protein synthesis secretion approach etc..Due to limitation
Factor is more, the expression quantity of most enzymes is not reached the requirement of industrialized production still.How host secretion heterogenous lipase is improved
Ability is the problem of lipase industrial applications need to be solved.
Heterologous protein synthesis secretion in host cell is related to multiple steps, target gene transcription, translates into nascent peptide and enters
Enter endoplasmic reticulum, golgiosome is entered by vesica after carrying out folding modification in endoplasmic reticulum, carried out in golgiosome further
Fold after modification, be secreted into by vesica extracellular.During heterologous protein expression, the step of being related to vesicle transport.Especially
It is during heterologous protein overexpression, and the quantity of vesica may be insufficient for the transhipment of so many nascent peptide.Therefore it is logical
Overexpression vesica constitutive protein in host is crossed, such as SEC31 may help the timely transhipment of nascent peptide.
Rhizomucor miehei lipase is a kind of lipase of 1,3 position specifics, can be used for many of biodiesel preparation
Individual aspect.The CN103361327A of applicant's early stage application discloses a kind of heterologous efficient secretory expression rhizomucor miehei lipase
Recombinant yeast pichia pastoris, above-mentioned recombinant yeast pichia pastoris is that by using Protocols in Molecular Biology, the codings rice for introducing 2 copies is black
Root miehei lipase gene (band own leader peptide), builds what can be obtained in the carrier of pichia pastoris phaff expression.This is heavy
Although group Pichia pastoris realizes the heterologous secretion of rhizomucor miehei lipase, and the recombinase enzyme activity of secretion is higher, but it is secreted
Amount is to influence the weakness of its popularization and application all the time.
The content of the invention
It is red that first purpose of the present invention is that the restructuring for providing the rhizomucor miehei lipase of a plant height secernment efficiency is finished
Yeast strain.
Second object of the present invention is to provide the method for improving above-mentioned lipase secernment efficiency.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of efficient heterologous secretion expresses the genetic engineering bacterium of rhizomucor miehei lipase, and described genetic engineering bacterium passes through
Convert and be overexpressed into the Recombinant Pichia pastoris X-33 that can express Pro-RML containing 4 copy number pro-rml genes
The plasmid SEC31-pPIC3.5K of SEC31 genes is obtained.
The Recombinant Pichia pastoris X-33 of the described pro-rml genes containing 4 copy numbers is by that will contain leader peptide
Lipase gene pro-rml be inserted on pichia pastoris phaff expression vector pPICZ α A build obtain containing 2 copy pro-
The expression vector pPICZ α A-2prorml of rml genes, its plasmid construction process is as shown in Figure 2.By pPICZ α A-
2prorml be transformed into pichia pastoris phaff X-33 by qPCR methods screen obtain containing 4 copy number pro-rml genes can
Express Pro-RML Recombinant Pichia pastoris X-33.
The present invention is cloned into the 1,3- positions comprising leader peptide from rhizomucor miehei (Rhizomucor miehei) bacterial strain
Lipase (Pro-RML) gene pro-rml of selectivity, its nucleotide sequence is as shown in SEQ ID No.1, and the full length gene is
(1017) bp, analysis shows, G/C content is (48.9) %, the albumen of the individual amino acid composition of coding (339).
Present invention discover that being produced in the genetic engineering bacterium that can express Pro-RML of 4 copy number target gene unfolded
Or the albumen of false folding is accumulated in endoplasmic, endoplasmic reticulum is caused to produce pressure.Vesica is constituted into GFP SEC31 to connect
(Fig. 5) is connected on pPIC3.5K carriers, is transformed into m α -4pRML-X33, the 4 copy number pro- for being overexpressed SEC31 are obtained
Rml genes can express Pro-RML engineering strain m α -4pRML-X33S.
Application of the genetic engineering bacterium of the present invention in heterologous secretion expresses rhizomucor miehei lipase.
A kind of method that efficient heterologous secretion expresses rhizomucor miehei lipase, it is by cultivating above-mentioned overexpression
The genetic engineering bacterium of SEC31 4 copy number pro-rml genes, lipase Pro-RML is obtained through induced expression.The culture bar
Part is 28 DEG C, and initial pH is that 7,200rpm is cultivated 144 hours.The inductive condition is inducer first of every 24 hours supplements
Alcohol, this method is used to methanol final concentration of 1.0% (v/v), Shaking culture 144 hours, and using olive oil as substrate, enzyme activity is
996U/mL.And find that the enzymatic activities secernment efficiency of the bacterial strain is improved significantly and reaches 38U/OD600, higher than patent
(enzymatic activities secernment efficiency is 26U/OD to 2 copy bacterial strains involved by CN103361327A600)。
Beneficial technique effect：
This research optimizes its gene copy number by the way that the lipase is expressed in Pichia pastoris, obtains one plant and contains 4
The Pichia pastoris recombinant bacterial strain of rhizomucor miehei lipase copy number, during fermentation 144h, enzymatic activities are 704U/mL, enzyme activity point
Efficiency is secreted for 29U/OD600.Find to generate UPR reactions, substantial amounts of unfolded or false folding egg inside the bacterial strain in addition
Accumulated in vain in endoplasmic.This experiment has been effectively promoted rice by being overexpressed vesica constitutive protein SEC31 in the bacterial strain
The secretion of Rhizomocur miehei lipase, improves the enzymatic activities secernment efficiency of the enzyme, during shake flask fermentation 144h, enzyme activity reaches
996U/mL, bacterial strain enzyme activity secernment efficiency reaches 38U/OD600, the secernment efficiency is higher than 2 involved by patent CN103361327A
(enzymatic activities secernment efficiency is 26U/OD to the Pichia pastoris recombinant bacterial strain of individual rhizomucor miehei lipase copy number600)。
Brief description of the drawings
Fig. 1 target gene DNA enzymatics cut back to close electrophoretogram, and swimming lane 1 is DNA standard molecular weights (kb):4.5,3.0,2.0,
1.2,0.8,0.5 0.2；Swimming lane 2:Target gene DNA enzymatic cuts back to close 1017bp fragments (arrow is signified)；
The copy expression plasmids of Fig. 22 build flow chart；
Fig. 32 copies expression plasmid double digestion electrophoretogram, wherein, swimming lane 1 is DNA standard molecular weights (kb):15,10,
7.5,5,2.5,1,0.25；Swimming lane 2：2 copy expression plasmid pPICZ α+M α -2 prorml are through Bgl II and BamH I double digestions
Result afterwards, arrow meaning is expression cassette.
The standard curve that Fig. 4 copy numbers bacterial strain is determined
Fig. 5 is overexpressed the building process of SEC31 carriers
Fig. 6 is overexpressed SEC31 bacterial strain shake flask fermentation results contrast
A：The cell growth of each bacterial strain in fermentation process；B：The enzymatic activities of each bacterial strain in fermentation process；C：Fermentation process
In each bacterial strain enzyme activity secernment efficiency；
Embodiment：
Following examples further illustrate present disclosure, but should not be construed as limiting the invention.Without departing substantially from
In the case of spirit and essence of the invention, the modifications or substitutions made to the inventive method, step or condition belong to the present invention
Scope.
Unless otherwise specified, the conventional meanses that technological means used in embodiment is well known to those skilled in the art.
The percentage sign " % " being related in the present invention, if not specified, refers to mass percent；But the percentage of solution
Than unless otherwise specified, referring to contain some grams of solute in solution 100ml；Percentage between liquid, refers to hold at 20 DEG C
The ratio of amount.The normal experiment operating procedures such as involved digestion, connection, recovery, conversion, PCR amplifications are referred to《Molecular cloning
(third edition)》.Primer is synthesized and is sequenced and completed by English fine horse (Invitrogen) biotech firm.
The rhizomucor miehei cDNA of embodiment 1 preparation
The extraction of 1.1 rhizomucor miehei total serum IgEs
(1) appropriate rhizomucor miehei mycelia is taken, filter paper suck dry moisture, liquid nitrogen grinding adds 1ml Trizol reagents
(Invitrogen), oscillator vibration 5min, is stored at room temperature 1min；
(2) 0.2ml chloroforms are added, 15s is vibrated, 2min is stood；
(3) 4 DEG C, 12000rpm, 15min；
(4) supernatant is drawn, isometric isopropanol, -20 DEG C of precipitation 30min is added；
(5) 4 DEG C, 12000rpm, 15min；
(6) supernatant is outwelled, precipitation, 7500rpm, 4 DEG C, 5min are washed with the ethanol of 1ml 75%；
(7) (6) step is repeated once；
(8) supernatant is outwelled, 10min is dried；
(9) appropriate DEPC water dissolving is added, total serum IgE is obtained；
The preparation of 1.2 the first chains of rhizomucor miehei cDNA
Reverse transcription uses reverse transcriptase (MMLV) concrete operations produced by Promega companies as follows：
Following component is added in the centrifuge tube of a nuclease free by 25 μ l reaction systems：
95 DEG C of heating 5min terminating reactions, freezen protective.
The structure for the pPICZ α A-2prorml plasmids that embodiment 22 is copied
2.1 design of primers
According to the sequence (GenBank accession number is A02536.1) of rml genes in GenBank, design has been synthesized with next
To primer：
FW(P1)：5’—CGGAATTCGTGCCAATCAAGAG—3’(SEQ ID NO.2)
REV(P2)：5’—TAGTCTAGAGTACAGAGGCCTGTG—3’(SEQ ID NO.3)
P1, P2 two ends, which are respectively designed with EcoR I and Not I restriction enzyme sites, (see italic in above-mentioned sequence and has underscore
Part)
The 2.2 rhizomucor miehei lipase pro-rml containing leader peptide PCR amplifications
Using P1, P2 primer, with rhizomucor miehei (Boel E, Huge-Jensen B, Christensen M, Thim L,
Fiil N:Rhizomucor miehei triglyceride lipase is synthesized as a
precursor.Lipids1988,23(7):701-706.) (preservation of this room) cDNA is template, and PCR reaction systems are：
Reaction condition is：95 DEG C of 5min, 5 DEG C of 40s, 60 DEG C of 40s, 72 DEG C of 1min, are circulated 30 times, 72 DEG C of 10min, 4 DEG C
2min。
2.3 reclaim purpose fragment from PCR reaction products
Purifying reclaims target gene fragment using the method that glue crosses post is cut from PCR primer, and PCR reaction products pass through agar
After sugared gel electrophoresis, target gene DNA is cut under the irradiation of uviol lamp, target gene length is 1017bp (Fig. 1), according to
The method of DNA QIAquick Gel Extraction Kits specification (being purchased from Tiangeng company, production code member is DP209-02) is reclaimed.
2.4 TA are cloned
PCR recovery products are connected on carrier pMD18-T-Simple and (are purchased from TaKaRa companies), coupled reaction according to
Kit (Code No.D104A) specification operation that TaKaRa companies are provided.
2.5 target gene are connected to pichia pastoris phaff expression vector pPICZ α A.
2.5.1 2 copy expression plasmids are built
Double digestion is carried out to pMD18-T-prorml and pPICZ α A respectively with restriction enzyme EcoR I and Not I,
Then purpose fragment is reclaimed, connected with T4 ligases, connection product is converted into bacillus coli DH 5 alpha (is purchased from Shanghai
Sheng Gong bioengineering Co., Ltd).Obtain single copy expression plasmid pPICZ α A-prorml.Will be with single copy pPICZ α A-
Prorml carries out BamH I and the double digestions of Bgl II, obtains the expression cassette containing pro-rml fragments；PPICZ α A-prorml are entered
The single endonuclease digestions of row BamH I obtain the pPICZ α A carrier segments containing single copy pro-rml；Above-mentioned expression cassette and carrier segments are connected
Connect the pPICZ α A-2prorml carriers for obtaining 2 copies.PPICZ α A-2prorml are transferred in bacillus coli DH 5 alpha and carried out
Expand and PCR detections, send invitrogn companies to be sequenced.Build flow chart and see accompanying drawing 2, double digestion detection figure is shown in accompanying drawing 3.
The Pichia pastoris recombinant bacterial strain screening of the copy pro-rml genes of embodiment 34
The preparation of 3.1 pichia pastoris phaff X-33 (purchase of Invitrogen companies) Electroporation-competent cells and its electricity
Hit conversion
(1) the fresh single bacterium of picking is fallen within 5ml YPD fluid nutrient mediums, and in 30 DEG C, 250rpm is cultivated 12-14 hours；
(2) it is inoculated into 0.1% inoculum concentration in the 2L triangular flasks of the culture mediums of YPD containing 500ml, in 30 DEG C, 250rpm trainings
Support 12-14 hours, make its OD600=1.3-1.5；
(3) 1500rpm is centrifuged 5 minutes at 4 DEG C, collects cell；
(4) the sterile water washing cell of 500-250ml ice precoolings is used twice；
(5) cell is washed once with the 1M sorbitol solutions of 20ml ice precoolings；
(6) cell is resuspended with the 1M sorbitol solutions of 1ml ice precoolings, is 1.5ml or so to final volume, is sub-packed in 80 μ l
Small centrifuge tube；
3.2 pichia pastoris phaff yeast cells it is electroporated
(1) by the μ l of expression plasmid about 10 of ready about 100 μ g/ μ l 2 copy target gene of non-linearization, with 80
μ l competent yeast cells are mixed, and are placed on ice about 5 minutes；
(2) competent cell for being mixed with DNA is transferred to the 0.2cm of ice precooling electric revolving cup；
Converted under 1.5 kilovolts of voltage；
(3) the 1M sorbitol solutions of 1ml ice precoolings and then are at once added in inverted cell, cell is mixed, turned
Enter the small centrifuge tubes of 1.5ml, 30 DEG C of static 1-2h.
(4) 50-200ul is taken to be coated on containing 100ug/ml YPDS flat boards (yeast extract 1%, peptone
2%, dextrose 2%, Sorbitol 1M, agar 2%), in 30 DEG C of cultures, 2 to 3 days observation results.
3.3. the screening of the recombinant bacterial strain of the pro-rml genes containing 4 copy numbers
3.3.1 the transformant that the identification of yeast colony PCR method is correctly integrated
Positive bacterium colony is chosen on flat board, using 5 ' AOX1,3 ' AOX1 as primer, one is entered using the method for yeast colony PCR
The transformant that step card is correctly integrated.
Primer sequence is：5’AOX1：5′-GACTGGTTCCAATTGACAAGC-3′(SEQ ID NO.4)
3’AOX1：The processing of 5 '-GCAAATGGCATTCTGACATCC-3 ' (SEQ ID NO.5) template
Method：
(1) it is a little with sterile suction nozzle picking colony, it is dissolved into 50 μ l D2-Buffer (1L：Guanidinium isothiocyanate
472.64g, 1mol/L pH8.0TrisHCl buffer solutions 50ml, beta -mercaptoethanol 7ml) middle mixing；
(2) by mixed liquor in 100 DEG C of boiling water bath 5min；
(3) 12000rpm, centrifuges 30s, abandons supernatant；
(4) precipitated 2 times with sterile water washing；
(5) ddH for being dissolved in 20 μ l will be precipitated2O, 95 DEG C of effect 5min；
(6) supernatant as template is obtained after centrifuging.
PCR reaction systems：
Reaction condition：95℃5min；95 DEG C of 40s, 60 DEG C of 40s, 72 DEG C of 1min 30s, 30cycles；72℃10min.
3.3.2 4 copy bacterial strains are determined using qPCR methods
With TIANGEN pastoris genomic dnas extracts kit (TIANamp Yeast DNA Kit, article No.：DP307-02)
The genome for the Pichia pastoris recon that screening is obtained is extracted, with the gene for the recon for comprising only a copy pro-rml
Group is template, and it is reference gene to choose glyceraldehyde-3-phosphate dehydrogenase gene (gap), and template is carried out to the dilution of various concentrations,
Q-PCR is detected, respectively obtains the standard between the log values and Ct values of target gene pro-rml and reference gene gap template amount
Curve (Fig. 4).Table 1 is the qPCR data of positive strain, by the Ct (pro-rml) and reference gene of the target gene of unknown sample
Ct (gap) substitutes into the template amount pro-rml (copy quantity) and gap (copy that formula obtains target gene and reference gene
quantity).The bacterial strain m α -4pRML-X33 of 4 copies are obtained according to following copy number calculation formula：
It the results are shown in Table 1：
Table 1 determines the copy number of recombinant bacterial strain
SEC31 genes are overexpressed by embodiment 4 in the above-mentioned bacterium of 4 copies
The acquisition of 4.1 SEC31 genetic fragments
With PSEC31-F(CGGGATCCACCATGGTGGTACCCCTAAGTCAGTT (SEQ ID NO.6)) and PSEC31-R
(ATAAGAATGCGGCCGCCTAGTTGATGATGTAATTCAGACTGC (SEQ ID NO.7)) it is primer, pichia pastoris X-33
Genome be template (TIANGEN pastoris genomic dnas extracts kit (TIANamp Yeast DNA Kit, article No.：
DP307-02) extract), the fragment of SEC31 genes is obtained with following PCR system and amplification condition.
PCR reaction systems：
Reaction condition：95℃5min；95 DEG C of 40s, 60 DEG C of 40s, 72 DEG C of 2min, 30cycles；72℃10min.
4.2 are overexpressed plasmid SEC31-pPIC3.5K structure
The PCR recovery products of SEC31 genes are connected on carrier pMD18-T-Simple and (are purchased from TaKaRa companies), even
Reversed kit (Code No.D104A) specification operation that should be provided according to TaKaRa companies.Obtain correct through sequencing
SEC31-pMD18T plasmids.With restriction enzyme BamH I and Not I double digestion SEC31-pMD18T and pPIC3.5K, difference
SEC31 fragments and pPIC3.5K carriers are reclaimed in rubber tapping.Connected with T4 ligases, connection product is converted into bacillus coli DH 5
α.Obtain through plasmid extraction, the checking screening of PCR, BamHI and Not I double digestions and be overexpressed plasmid SEC31-pPIC3.5K.Build
Process is shown in Fig. 5.
4.3 over-express vector SEC31-pPIC3.5K electricity are transformed into Pichia pastoris and sun containing 4 copy lipase genes
Property bacterial strain screening.
SEC31-pPIC3.5K is extracted, linearisation and electric method for transformation are with 3.2, the screening process same 3.3 of positive strain, sieve
Choosing primer used is D-SEC31-F (CGTGTTCCTCAATCAGCCA (SEQ ID NO.8)) and D-SEC31-R
(GCAAATGGCATTCTGACATCC(SEQ ID NO.9)).PCR reaction systems and amplification condition are with 3.3.It is overexpressed
SEC31 bacterial strain m α -4pRML-X33S.
The expression of purpose lipase in 4.4 pichia pastoris phaffs
NaOH titration measuring lipase activities
(1) 0.05M NaOH preparation：First with without CO2Water configures 5M NaOH liquid storages；Again after 50 times of accurate dilutions, weigh
100 DEG C of Potassium Hydrogen Phthalate 0.38g for drying to constant weight, are dissolved in 80ml without CO2In water, its actual concentrations is calibrated, then calculate
Go out stock concentrations；0.05M NaOH solution is with without CO2Water liquid storage matching while using；
(2) preparation of PVA- olives oil emulsion substrate：By olive oil 100ml, 300ml 2%PVA1750 (polyethylene
Alcohol) mixing, heating is dissolved, with ultrasonic emulsification, power 300W, ultrasonic 3s, interval 4s, 99 times, circulation 2 times；
(3) 5ml emulsion substrates are taken, 4ml 0.1M pH6.0 disodium hydrogen phosphate-citrate buffer solution is added to 150ml
In triangular flask, 35 DEG C of water bath with thermostatic control shaking table is placed in, 150rpm incubates 5min；
(4) enzyme liquid for taking 1ml suitably to dilute is added in substrate and buffer solution, is added after 35 DEG C of 150rpm reactions 10min
15ml absolute ethyl alcohol terminating reactions；
(5) 4 are added dropwise and drip phenolphthalein indicator, the aliphatic acid for digesting and producing is titrated with 0.05M NaOH, powder is become to reaction solution
Red stops；
Blank operation is consistent with the above, will simply be added to after zymotic fluid and absolute ethyl alcohol hybrid reaction 10min substrate and
In buffer solution.
Enzyme activity is defined as under the condition determination, and it is an enzyme-activity unit that 1min, which discharges the enzyme amount of 1 μm of ol aliphatic acid,.
Picking is overexpressed SEC31 4 copy pro-rml genes bacterial strains (m α -4pRML-X33S), is not overexpressed the 4 of SEC31
Copy pro-rml genes bacterial strain (m α -4pRML-X33) and patent CN103361327A involved by 2 copy pro-rml gene bacterium
The single bacterium colony of strain (m α -2pRML-X33), is inoculated in 25ml BMGY culture mediums (1% dusty yeast, 2% peptone, 1% glycerine),
In 28 DEG C, 200rpm shaking table cultures to OD600For 4.0-8.0 or so, be transferred to equipped with 50ml BMMY culture mediums (1% dusty yeast,
2% peptone, 1.0% methanol, 100mmol/l phosphate buffers, pH 7.0) 500ml triangular flasks in, same culture conditions after
Continuous culture, adds 100% methanol for every 24 hours in culture medium to final concentration of 1.0% (v/v), induced expression 6 days.Every certain
Time sampling, measure its cell density (OD600) and extracellular rhizomucor miehei lipase enzyme activity, and calculate its ectoenzyme
Secernment efficiency living, testing result is shown in Fig. 6.
(Fig. 6 A) is grown from each strain cell, shake flask fermentation 144h, m α -2pRML-X33 (OD600=39) cell life
Long situation is better than m α -4pRML-X33S (OD600=25.9) and m α -4pRML-X33 (OD600=24.25).Illustrate to be overexpressed
The recombinant yeast pichia pastoris cell growth condition of SEC31 4 copy pro-rml genes is slightly below the restructuring of 2 copy pro-rml genes
Pichia pastoris, but change little compared with the recombinant yeast pichia pastoris of 4 copy pro-rml genes.
Each bacterial strain enzymatic activities secretion situation see Fig. 6 B, when fermenting between be 144h when, m α -4pRML-X33S and m α -
2pRML-X33 enzyme activity is close, and enzyme activity highest is respectively 996U/ml and 1023U/ml, is above m α -4pRML-X33 704U/
mL.Compared with the recombinant yeast pichia pastoris of 4 copy pro-rml genes for not being overexpressed SEC31, rice can be promoted by being overexpressed SEC31
The secretion of Rhizomocur miehei lipase.
The secernment efficiency of recombinant bacterial strain is calculated according to enzyme activity and cell growth condition, Fig. 6 C are as a result seen.mα-4pRML-X33S
Enzymatic activities secernment efficiency be higher than m α -2pRML-X33 and m α -4pRML-X33, fermentation 144h when, m α -4pRML-X33H, m α -
2pRML-X33 and m α -4pRML-X33 enzymatic activities secernment efficiency is respectively 38U/OD600、26U/OD600And 29U/OD600.Should
As a result explanation is overexpressed the enzymatic activities secernment efficiency that SEC31 improves the recombinant yeast pichia pastoris of 4 copy pro-rml genes.
The result of complex chart 6, under conditions of BMMY culture mediums (initial pH is 7, and cultivation temperature is 28 DEG C) Shaking culture
144 hours, the pichia pastoris phaff recon enzymatic activities containing 4 copy pro-rml genes for being overexpressed SEC31 reached most
High 996U/ml, enzymatic activities secernment efficiency is up to 38U/OD600Higher than 2 copy bacterial strains involved by patent CN103361327A
26U/OD600。
<110>Jiangsu Normal University
<120>The recombinant yeast pichia pastoris of one plant of heterologous high efficient expression lipase and its application
<160> 9
<210> 1
<211> 1017
<212>
<213>Rhizomucor miehei（Rhizomucor miehei）
<220>
<223>Pro-rml genes
<400> 1
gtgccaatca agagacaatc aaacagcacg gtggatagtc tgccacccct catcccctct 60
cgaacctcgg caccttcatc atcaccaagc acaaccgacc ctgaagctcc agccatgagt 120
cgcaatggac cgctgccctc ggatgtagag actaaatatg gcatggcttt gaatgctact 180
tcctatccgg attctgtggt ccaagcaatg agcattgatg gtggtatccg cgctgcgacc 240
tcgcaagaaa tcaatgaatt gacttattac actacactat ctgccaactc gtactgccgc 300
actgtcattc ctggagctac ctgggactgt atccactgtg atgcaacgga ggatctcaag 360
attatcaaga cttggagcac gctcatctat gatacaaatg caatggttgc acgtggtgac 420
agcgaaaaaa ctatctatat cgttttccga ggttcgagct ctatccgcaa ctggattgct 480
gatctcacct ttgtgccagt ttcatatcct ccggtcagtg gtacaaaagt acacaaggga 540
ttcctggaca gttacgggga agttcaaaac gagcttgttg ctactgttct tgatcaattc 600
aagcaatatc caagctacaa ggttgctgtt acaggtcact cactcggtgg tgctactgcg 660
ttgctttgcg ccctgggtct ctatcaacga gaagaaggac tctcatccag caacttgttc 720
ctttacactc aaggtcaacc acgggtaggc gaccctgcct ttgccaacta cgttgttagc 780
accggcattc cttacaggcg cacggtcaat gaacgagata tcgttcctca tcttccacct 840
gctgcttttg gttttctcca cgctggcgag gagtattgga ttactgacaa tagcccagag 900
actgttcagg tctgcacaag cgatctggaa acctctgatt gctctaacag cattgttccc 960
ttcacaagtg ttcttgacca tctctcgtac tttggtatca acacaggcct ctgtact 1017
<210> 2
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<223>Primer P1
<400> 2
cggaattcgt gccaatcaag ag 22
<210> 3
<211> 24
<212> DNA
<213>Artificial sequence
<220>
<223>Primer P2
<400> 3
tagtctagag tacagaggcc tgtg 24
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>The AOX1 of primer 5 '
<400> 4
gactggttcc aattgacaag c 21
<210> 5
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>The AOX1 of primer 3 '
<400> 5
gcaaatggca ttctgacatc c 21
<210> 6
<211> 34
<212> DNA
<213>Artificial sequence
<220>
<223>Primer PSEC31-F
<400> 6
cgggatccac catggtggta cccctaagtc agtt 34
<210> 7
<211> 34
<212> DNA
<213>Artificial sequence
<220>
<223>Primer PSEC31-R
<400> 7
ataagaatgc ggccgcctag ttgatgatgt aattcagact gc 42
<210> 8
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<223>Primer D-SEC31-F
<400> 8
cgtgttcctc aatcagcca 19
<210> 9
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>Primer D-SEC31-R
<400> 9
gcaaatggca ttctgacatc c 21
<210> 10
<211> 1116
<212> DNA
<213>Pichia pastoris X-33
<220>
<223>SEC31 genes
<400> 10
atggtggtac ccctaagtca gttcccttcg ccgcccaact caagtggcga cggctgcgag 60
aactcaagta cagtcttttc caaggactgt gtcaagagga ctgcatcgcc attagaccct 120
caatctcaat ttagagtctc cccaaactcg tacactacat ccacaaacta ttctaccccg 180
gtttcttccc acgaaacttt gtcctcctca acatccggga tctcagtgac aggcgttctg 240
ctaccatcaa tagattcatt agcacgctca atctcctata caggacgaga cgctgttcct 300
ttcaacttgt ccacttggac tgacaagtca acacctgtta ctaattcttt tgtcatctct 360
cagtatgaag acacgcgtgt tcctcaatca gccaccagtt ctacacatcc aaacatacct 420
aaacacgcca aagagtatcc gttagcaaat gggccacctg ggtggtgttg gaattcccat 480
tccagtatgt cgacagacca accaatatat ccaggacacc aatatccacc accgcttcag 540
cagcactacc actttgcttc acccaggcaa ctatcaaact ctagctctgg gacgtcatcc 600
gttcctttcc aaccaccccc tgctggtcaa ttacaaccac aaggtaattc tatgttcata 660
cacatgccat tttcgctaaa tggcccacca gctgctggac agcaattgat accaccccaa 720
ggactagcct caatacctgt cggccccggc aacaacagtt ccctattggt tagccaaggt 780
gcacctggcg gctattcttt agcttcacca gcgttgtcac cggtagatgc gaccttcgaa 840
gatcccgtca agagactgcc caaaaagcgg acaaaaactg gatgtctcac ttgccgtaag 900
agacgaatca aatgtgacga acgcaagccg ttctgtttca actgtgaaaa aagcaaaaag 960
gtgtgtactg gttttacgca tctattcaaa gatcccccta gcaaatccta ccctcccagt 1020
tcagatggtg cctcccctgt tgccaatgac caccctgtcc ccccaaggca aaactttggt 1080
gaattgaggg gcagtctgaa ttacatcatc aactag 1116
Claims (7)
1. a kind of efficient heterologous secretion expresses the genetic engineering bacterium of rhizomucor miehei lipase, it is characterised in that described gene work
Journey bacterium into the Recombinant Pichia pastoris X-33 that can express Pro-RML containing 4 copy number pro-rml genes by converting
The plasmid SEC31-pPIC3.5K for being overexpressed SEC31 genes is obtained.
2. genetic engineering bacterium according to claim 1, it is characterised in that the described pro-rml genes containing 4 copy numbers
Recombinant Pichia pastoris X-33 is by the way that the lipase gene pro-rml containing leader peptide is inserted into pichia pastoris phaff
Built on expression vector pPICZ α A and obtain the expression vector pPICZ α A-2prorml containing 2 copy pro-rml genes；By pPICZ α
A-2prorml, which is transformed into screen by qPCR methods in pichia pastoris phaff X-33, obtains the energy containing 4 copy number pro-rml genes
Enough express Pro-RML Recombinant Pichia pastoris X-33；Wherein, described pro-rml gene nucleotide series such as SEQ
Shown in ID NO.1.
3. genetic engineering bacterium according to claim 1, it is characterised in that described overexpression plasmid SEC31-pPIC3.5K
It is by gained between SEC31 genes insertion pPIC3.5K carrier B amH I and Not I restriction enzyme sites.
4. genetic engineering bacterium the answering in heterologous secretion expresses rhizomucor miehei lipase any one of claim 1-3
With.
5. a kind of method that efficient heterologous secretion expresses rhizomucor miehei lipase, it is characterised in that by cultivating claim 1-
Genetic engineering bacterium any one of 3, lipase Pro-RML is obtained through induced expression.
6. method according to claim 5, it is characterised in that the condition of the culture is 28 DEG C, initial pH is 7,200rpm
Culture 144 hours.
7. method according to claim 5, it is characterised in that the condition of the induction is that every 24 hours supplements are once induced
Thing methanol, to methanol final concentration of 1.0% (v/v).
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| CN110923216A (en) * | 2018-09-19 | 2020-03-27 | 江苏师范大学 | Method for producing rhizomucor miehei lipase pRML enzyme powder |
| CN110923260A (en) * | 2018-09-19 | 2020-03-27 | 江苏师范大学 | Engineering strain for producing rhizomucor miehei lipase powder and application thereof |
| CN111763665A (en) * | 2019-04-01 | 2020-10-13 | 江苏师范大学 | Preparation method of high-activity sweet potato chitinase and application of high-activity sweet potato chitinase in preparation of plant pathogenic fungus antibacterial agent |
| CN114574617A (en) * | 2022-03-08 | 2022-06-03 | 江南大学 | Reference gene of pichia pastoris under different carbon sources and primer and application thereof |
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| CN103361327A (en) * | 2013-07-19 | 2013-10-23 | 中国农业大学 | Recombinant pichia pastoris for heterogenous high level expression of lipase |
| CN103981197A (en) * | 2014-04-21 | 2014-08-13 | 中国科学院广州能源研究所 | Novel leader peptide-containing rhizomucor mieheilipase gene and expression of rhizomucor mieheilipase gene in pichia pastoris |
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| CN103361327A (en) * | 2013-07-19 | 2013-10-23 | 中国农业大学 | Recombinant pichia pastoris for heterogenous high level expression of lipase |
| CN103981197A (en) * | 2014-04-21 | 2014-08-13 | 中国科学院广州能源研究所 | Novel leader peptide-containing rhizomucor mieheilipase gene and expression of rhizomucor mieheilipase gene in pichia pastoris |
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| CN110923216A (en) * | 2018-09-19 | 2020-03-27 | 江苏师范大学 | Method for producing rhizomucor miehei lipase pRML enzyme powder |
| CN110923260A (en) * | 2018-09-19 | 2020-03-27 | 江苏师范大学 | Engineering strain for producing rhizomucor miehei lipase powder and application thereof |
| CN110923216B (en) * | 2018-09-19 | 2021-08-06 | 江苏师范大学 | Method for producing rhizomucor miehei lipase pRML enzyme powder |
| CN110923260B (en) * | 2018-09-19 | 2022-06-03 | 江苏师范大学 | Engineering strain for producing rhizomucor miehei lipase powder and application thereof |
| CN111763665A (en) * | 2019-04-01 | 2020-10-13 | 江苏师范大学 | Preparation method of high-activity sweet potato chitinase and application of high-activity sweet potato chitinase in preparation of plant pathogenic fungus antibacterial agent |
| CN111763665B (en) * | 2019-04-01 | 2022-08-12 | 江苏师范大学 | Preparation method of high-activity sweet potato chitinase and application of high-activity sweet potato chitinase in preparation of plant pathogenic fungus antibacterial agent |
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| CN114574617B (en) * | 2022-03-08 | 2022-12-27 | 江南大学 | Reference gene of pichia pastoris under different carbon sources, and primer and application thereof |
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