CN104419650A - Transformed aspergillus terreus engineering bacteria and application thereof - Google Patents
Transformed aspergillus terreus engineering bacteria and application thereof Download PDFInfo
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- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01003—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
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Abstract
The invention relates to the field of gene engineering and particularly relates to transformed aspergillus terreus engineering bacteria and an application thereof. A bacterial strain is recombinant aspergillus terreus which is obtained by a saccharifying enzyme expression frame which is inserted into the aspergillus terreus through genetic engineering transformation. The genetic engineering transformation is carried out aiming at the aspergillus terreus strain; and the ability of the aspergillus terreus for expressing secreted accharifying enzyme is improved from the molecular level, and thus the ability of the aspergillus terreus for producing itaconic acid by directly utilizing liquefied starch is improved, and the production process of producing the itaconic acid by aspergillus terreus fermentation is simplified.
Description
Technical field
The present invention relates to genetically engineered field, particularly relate to a kind of terreus engineering bacteria and and application thereof of transformation.
Background technology
Methylene-succinic acid is a kind of important Chemicals, is thought one of 12 important high valuable chemicals by USDOE, is mainly used in the fields such as acrylic fibers chemical fibre, resin, rubber, coating, papermaking, medicine, agricultural chemicals, light industry, food, silk.Occurring in nature has multiple filamentous fungus to convert monose to methylene-succinic acid, and wherein terreus is produced bacterial strain as methylene-succinic acid and is widely used in industrial production.At present, methylene-succinic acid carbon source mainly glucose used is produced in terreus fermentation, and glucose in industrial production mainly starch obtain through two steps hydrolysis, first starch obtains liquefied starch after α-amylaseliquefied, and then finally obtain saccharified liquid through the saccharification of saccharifying enzyme, namely Glucose Liquid (Gao Shanli. the application of Novel saccharification enzyme in starch glucose. Anhui chemical industry, 2012,38(4): 24-26.).Aspergillus niger amylatic enzyme system is more flourishing, can directly by utilizing starch to grow as carbon source, and the ability of its efficient secretion saccharifying enzyme is particularly outstanding, by industrial applications in production saccharifying enzyme.Therefore produce at some fermentation of Aspergillus niger in the technique of citric acid and directly adopt liquefied starch to ferment as raw material, eliminate and add the process that saccharifying enzyme carries out saccharification.Produce methylene-succinic acid technique to simplify terreus fermentation thus reduce production cost, bacterial strain being improved, develops the bacterial strain tool with more good characteristics and be of great significance.
Summary of the invention
The object of the invention is the terreus engineering bacteria and and the application thereof that provide a kind of transformation.
For achieving the above object, the present invention adopts technical scheme to be:
A terreus engineering bacteria for transformation, described bacterial strain is by the genetic engineering modified restructuring terreus inserting glucoamylase expression frame and obtain in terreus.
Described glucoamylase expression frame is made up of filamentous fungus promoter, the coding nucleotide sequence of saccharifying enzyme and terminator; Wherein, promotor is the promotor of the citric acid synthesized enzyme gene of terreus self; The nucleotides sequence of coding saccharifying enzyme is classified as the glucoamylase gene of aspergillus niger; Terminator is the TtrpC terminator of Aspergillus nidulans.
The construction process of the terreus engineering bacteria of transformation, by genetic engineering modified structure glucoamylase expression frame, is inserted in terreus the restructuring terreus obtained containing glucoamylase expression frame.
The application of the terreus engineering bacteria of transformation, utilizes the terreus engineering bacterium fermentation of transformation to produce methylene-succinic acid.
Adopt the terreus engineering bacteria of transformation as fermentation strain in using liquefied starch or starch saccharificating liquid as fermentation production of itaconic acid in the fermention medium of carbon source.
Genetic engineering modified terreus engineering strain provided by the invention, includes a glucoamylase expression frame (expression cassette), and this expression cassette is made up of a filamentous fungus promoter, the coding nucleotide sequence of saccharifying enzyme and terminator.The filamentous fungus promoter adopted is the promotor of the citric acid synthesized enzyme gene deriving from terreus self, by 613 based compositions.The nucleotide sequence of code used saccharifying enzyme is the glucoamylase gene coming from aspergillus niger, and size is for 1923bp is as shown in sequence.The terminator adopted is the TtrpC terminator deriving from Aspergillus nidulans.
The terreus bacterial strain preparing this genetic modification comprises the following steps:
1) the plasmid pXH43-gla1 carrying the expression cassette Pcs3-gla1-TtrpC of glucoamylase gene is built;
2) utilize plasmid pXH43-gla1 vertisol aspergillus CICC40205, obtain the terreus engineering strain containing glucoamylase gene expression cassette Pcs3-gla1-TtrpC.
Utilize the method for above-mentioned terreus engineering strain fermentation production of itaconic acid, the carbon source that fermention medium uses in the method can be the liquefied starch without saccharification, also can be the product (shortening saccharification time) after liquefied starch partially saccharifying.This terreus is adopted to produce methylene-succinic acid as fermentation strain, the Mashing process or shortening saccharification time that utilize Glucoamylase hydrolysis can be saved, and the oligosaccharides such as maltose residual in the Glucoamylase of Aspergillus niger energy hydrolysis sugar liquid of this self secreting, expressing of terreus engineering strain, the final utilization ratio that can improve residual sugar, reduces the production cost of methylene-succinic acid.
The advantage that the present invention has: utilize the present invention to build terreus fermentation and produce methylene-succinic acid, make to omit Mashing process in sugar refining technology, or shortening saccharification time, through the method transformation terreus engineering strain during the fermentation can efficiency utilization liquefied starch as raw material production methylene-succinic acid.
Terreus engineering strain provided by the invention is adopted to be that methylene-succinic acid is produced in raw material direct fermentation with liquefied starch, in the shake flask fermentation stage, terminal methylene-succinic acid output is 55.6g/L, and under the same conditions, the terminal methylene-succinic acid output without the starting strain terreus CICC40205 of the present invention's transformation is 17.1g/L.
The present invention is directed to terreus bacterial strain implements genetic engineering modified, the ability of terreus expression-secretion saccharifying enzyme is improved from molecular level, thus improve the ability that terreus directly utilizes liquefied starch fermentation product methylene-succinic acid, simplify the production technique that methylene-succinic acid is produced in terreus fermentation.
Accompanying drawing explanation
The carrier pJL-8 plasmid map that Fig. 1 provides for the embodiment of the present invention.Wherein hph-casette is hygromycin resistance element; Sgfp is green fluorescence protein gene; TtrpC is Aspergillus nidulans tryptophan synthetase terminator; Pcs3 is terreus citric acid synthesized enzyme gene promotor.
The carrier pXH43 plasmid map that Fig. 2 provides for the embodiment of the present invention.Wherein hph-casette is hygromycin resistance element; TtrpC is Aspergillus nidulans tryptophan synthetase terminator; Pcs3 is terreus citric acid synthesized enzyme gene promotor.
The carrier pXH43-gla1 plasmid map that Fig. 3 provides for the embodiment of the present invention.Wherein hph-casette is hygromycin resistance element; Gla1 is Glucoamylase Gene from Aspergillus niger 1; TtrpC is Aspergillus nidulans tryptophan synthetase terminator; Pcs3 is terreus citric acid synthesized enzyme gene promotor.
The integration figure of gla1 expression casette in the pcr analysis genetically engineered terreus bacterial strain that Fig. 4 provides for the embodiment of the present invention.Wherein M:DNA maker; 1: recombinant bacterial strain G1-1; 2: recombinant bacterial strain G1-2; 3: recombinant bacterial strain G1-3; 4: recombinant bacterial strain G1-4; 5: recombinant bacterial strain G1-5; 6: recombinant bacterial strain G1-6; 7: recombinant bacterial strain G1-7; 8: recombinant bacterial strain G1-8; 9: starting strain CICC40205(negative control).
Utilize liquefied starch to ferment in the restructuring terreus bacterial strain shaking flask of the structure that Fig. 5 provides for the embodiment of the present invention and produce the comparison diagram of methylene-succinic acid ability, wherein, CICC40205 is starting strain, and all the other bacterial strains are the terreus engineering strain of process LAN Glucoamylase Gene from Aspergillus niger gla1.
The HPLC that Fig. 6 provides for the embodiment of the present invention analyzes the purification effect figure of methylene-succinic acid in bacterial strain fermentation liquor.Wherein, a: the fermented liquid of starting strain CICC40205; B: the fermented liquid of recombinant bacterial strain G1-5.
Embodiment
In embodiments of the invention, filamentous fungus promoter is defined as and a kind ofly polysaccharase can be and guided to arrive the correct downstream transcription initiation site of the nucleotide sequence of encoding human material thus the DNA sequence dna of transcriptional start in conjunction with RNA polymerase in filamentous fungus.The assembling of the information RNA of the effective catalysis of RNA polymerase energy and coding region suitable DNA chain complementation.Promotor it is also understood that for comprise for after transcript mRNA for translating 5 ' non-coding region (between promotor and transcription initiation site), cis-acting transcription controlling element, as enhanser and can with other nucleotide sequences of transcription factor interaction.
Carrier (vector) refers to the ring-shaped DNA molecule of a kind of energy self-replacation DNA fragmentation can transferred in recipient cell.
Liquefied starch described herein means the product that starch obtains after amylorrhexis process.Generally adopt amylase-saccharifying enzyme double-enzyme method that Starch Conversion is become glucose in industrial production.Namely first obtain liquefied starch (being mainly glucose, maltose, limit dextrin etc.) with amylorrhexis process starch, then add saccharifying enzyme and be hydrolyzed further and obtain saccharified liquid (main component is glucose).
Saccharifying enzyme (Glucoamylase used herein, EC.3.2.1.3.) or gla mean abiogenous saccharifying enzyme (such as, be described in " Boel E.; Hansen M.T.; Hjort I.; Hoegh I., Fiil N.P., Two different types of intervening sequences in theglucoamylase gene from Aspergillus niger.EMBO J.3:1581-1585 (1984) "
Boel E.,Hjort I.,Svensson B.,Norris F.,Norris K.E.,FiilN.P."Glucoamylases G1and G2from Aspergillus niger are synthesizedfrom two different but closely related mRNAs."EMBO J.3:1097-1102(1984)
Sorimachi K., Jacks A.J., le Gal-Coeffet M.-F., Williamson G., Archer D.B., Williamson M.P. " Solution structure of the granularstarch binding domain of glucoamylase from Aspergillus niger bynuclear magnetic resonance spectroscopy. " J.Mol.Biol.259:970-987 (1996) and its function equivalent (functional equivalent).
What be provided as demonstration in the present invention is Glucoamylase of Aspergillus niger.The example of other saccharifying enzyme arranges in table 1 below:
The GenBank accession number of the saccharifying enzyme that table 1. is demonstrated
As used herein, the function equivalent of reference enzyme (i.e. Glucoamylase of Aspergillus niger) is polypeptide, and its identity is 60%(such as 85%, 90%, 95% or 99% at least) aminoacid sequence of same reference enzyme, and have the enzymic activity identical with reference enzyme.
" identity " or " identity per-cent " refers to the sequence iden between two aminoacid sequences or between two nucleotide sequences.For determining the identity percentage ratio of two aminoacid sequences or two nucleic acid, comparing object with the best and sequence is compared.Identity per-cent between two sequences is the function (that is, the sum (such as, lap position) × 100 of the number/position of identity percentage ratio=same position) of the number of the same position had by these sequences.Such as, " identity per-cent " is calculated by following manner: in comparison window, compare two sequences through best comparison, measure and occur that the number of the position of identical nucleotide base or same amino acid residue is to produce the number of matched position in the two sequences, by the number of matched position divided by position in comparison window overall number (namely, the size of window), and result is multiplied by 100 thus produces Percentage of sequence identity.Best comparison for the sequence compared can be undertaken by following: such as, Smith and Waterman(Smith, T.F.andWaterman, M.S., Comparison of biosequences, Adv.Appl.Math., 1981,2,482-489) local homology algorithm; Needleman and Wunsch(Needleman S.B.andWunsch C.D., A general method applicable to the search forsimilarities in the amino acid sequence of two proteins, J.Mol.Biol., 1970,48,443-453.) homology alignment algorithm; Pearson and Lipman(Pearson W.R., Lipman D.J., Improved tools for biological sequence comparison, Proc.Natl.Acad.Sci., 1988,85,2444-2452.) search for similarity method; The computerize of these algorithms is implemented (such as, Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Madison, Wis.); Or manual alignment and visual inspection (see, the people such as such as Ausubel, Current Protocols in Molecular Biology(1995 supplementary issue).
In the present invention, plasmid extraction adopts OMEGA company Plasmid Mini Kit I test kit (D6942-01), it is adopt OMEGA company Cycle-Pure Kit test kit (D6492-01) that DNA fragmentation reclaims, and it is adopt OMEGA company Gel Extraction Kit test kit (D2500-01) that gel reclaims.
Below in conjunction with specific embodiment, concrete route of the present invention is further elaborated
The structure of embodiment 1 Glucoamylase Gene from Aspergillus niger expression cassette Pcs3-gla1-TtrpC
Aspergillus niger Co827 is purchased from Shanghai City Industry Wei Biological Research Institute; Terreus CICC40205 is purchased from Chinese industrial Culture Collection.Aspergillus niger spore substratum is solid potato culture medium, and culture temperature is 30 DEG C; Aspergillus niger mycelial growth adopts potato liquid nutrient medium, and culture temperature is 30 DEG C, and rotating speed is 200rpm.
The preparation of potato liquid nutrient medium, 20g peeled potatoes, is cut into block and adds water 100ml, boil 30min, by filtered through gauze, add 20g glucose, supply water to 100ml.
The preparation of solid potato culture medium be add in above-mentioned solid medium 2% agar powder, autoclaving.
1) structure of terreus expression vector pXH-43: with TtrpC-F (5 '-CAGTTCGAAATGCTCGAGCGAAGCTTAGATCTACTTAACGTTACTGAAATC-3 ') and TtrpC-R (5 '-GTAATCGATACGAAGATCACTGGGAACAACTGGC-3 ') for primer pair, with plasmid pJL-8(plasmid map see Fig. 1, its concrete construction process is with reference to Chinese patent: a kind of filamentous fungus promoter and application thereof, application number: 201210116460.1) for template carries out pcr amplification TtrpC fragment, product is through Bsp119I(Fermentas, Catalog No.:#FD0124) and Cla I(Fermentas, Catalog No.:#FD0143) enzyme cuts and purifying.Similarly carry out enzyme with Bsp119I to plasmid pJL-8 to cut, reclaim the carrier segments that size is about 6.3kb.By the pJL-8 carrier segments of recovery and TtrpC fragment transformation of E. coli DH5 α after T4 ligase enzyme is connected, use primer Pcs-F3(5 '-GATCGTCGACGGTACCAACCAAGGACCGCGATGACC-3 ') and TtrpC-R PCR screening is carried out to transformant, needed for the transformant that the product size that can increase is 1.5kb is, namely sequence verification correctly obtains plasmid pXH43(see Fig. 2).
2) clone of the glucoamylase gene gla1 of aspergillus niger Co827
With the cDNA of aspergillus niger Co827 for template, with gla1-F1(5 '-GACT TTCGAAATGTCGTTCCGATCTCTACTCGC-3 ') and gla1-R1(5 '-CATA AGATCTCTACCGCCAGGTGTCAGTCAC-3 ') be the glucoamylase gene gla1 of primer amplification aspergillus niger, product for the purpose of band that length is about 1.9kb is detected through 1.0% agarose gel electrophoresis, object band product reclaims TA after purifying through rubber tapping and is cloned into pMD18-T carrier (Takara, Catalog No.:D101A) in, plasmid pMD18-gla1 is obtained after order-checking, this gene order is SEQ ID NO.1.The DNA band of sequencing result display clone is Glucoamylase Gene from Aspergillus niger, the Glucoamylase of Aspergillus niger sequence GenBank:HQ537427.1 announced with GenBank is completely the same, completely the same with the aspergillus niger Gene A n03g06550 of the aspergillus niger CBS513.88 bacterial strain completing gene order-checking.
Glucoamylase Gene from Aspergillus niger
SEQ ID NO.1：
atgtcgttccgatctctactcgccctgagcggcctcgtctgcacagggttggcaaatgtgatttccaagcgcgcgaccttggattcatggttgagcaacgaagcgaccgtggctcgtactgccatcctgaataacatcggggcggacggtgcttgggtgtcgggcgcggactctggcattgtcgttgctagtcccagcacggataacccggactacttctacacctggactcgcgactctggtctcgtcctcaagaccctcgtcgatctcttccgaaatggagataccagtctcctctccaccattgagaactacatctccgcccaggcaattgtccagggtatcagtaacccctctggtgatctgtccagcggcgctggtctcggtgaacccaagttcaatgtcgatgagactgcctacactggttcttggggacggccgcagcgagatggtccggctctgagagcaactgctatgatcggcttcgggcagtggctgcttgacaatggctacaccagcaccgcaacggacattgtttggcccctcgttaggaacgacctgtcgtatgtggctcaatactggaaccagacaggatatgatctctgggaagaagtcaatggctcgtctttctttacgattgctgtgcaacaccgcgcccttgtcgaaggtagtgccttcgcgacggccgtcggctcgtcctgctcctggtgtgattctcaggcacccgaaattctctgctacctgcagtccttctggaccggcagcttcattctggccaacttcgatagcagccgttccggcaaggacgcaaacaccctcctgggaagcatccacacctttgatcctgaggccgcatgcgacgactccaccttccagccctgctccccgcgcgcgctcgccaaccacaaggaggttgtagactctttccgctcaatctataccctcaacgatggtctcagtgacagcgaggctgttgcggtgggtcggtaccctgaggacacgtactacaacggcaacccgtggttcctgtgcaccttggctgccgcagagcagttgtacgatgctctataccagtgggacaagcaggggtcgttggaggtcacagatgtgtcgctggacttcttcaaggcactgtacagcgatgctgctactggcacctactcttcgtccagttcgacttatagtagcattgtagatgccgtgaagactttcgccgatggcttcgtctctattgtggaaactcacgccgcaagcaacggctccatgtccgagcaatacgacaagtctgatggcgagcagctttccgctcgcgacctgacctggtcttatgctgctctgctgaccgccaacaaccgtcgtaactccgtcgtgcctgcttcttggggcgagacctctgccagcagcgtgcccggcacctgtgcggccacatctgccattggtacctacagcagtgtgactgtcacctcgtggccgagtatcgtggctactggcggcaccactacgacggctacccccactggatccggcagcgtgacctcgaccagcaagaccaccgcgactgctagcaagaccagcaccagtacgtcatcaacctcctgtaccactcccaccgccgtggctgtgactttcgatctgacagctaccaccacctacggcgagaacatctacctggtcggatcgatctctcagctgggtgactgggaaaccagcgacggcatagctctgagtgctgacaagtacacttccagcgacccgctctggtatgtcactgtgactctgccggctggtgagtcgtttgagtacaagtttatccgcattgagagcgatgactccgtggagtgggagagtgatcccaaccgagaatacaccgttcctcaggcgtgcggaacgtcgaccgcgacggtgactgacacctggcggtag
(a) sequence signature:
● length: 1923bp
● type: base sequence
● chain: double-strand
● topological framework: linear
(b) molecule type: DNA
C () is supposed: no
(d) antisense: no
E () is originated at first: aspergillus niger (Aspergillus niger)
(f) specificity title: glucoamylase gene (glucoamylase gene, gla1)
SEQ ID NO.2：
MSFRSLLALSGLVCTGLANVISKRATLDSWLSNEATVARTAILNNIGADGAWVSGADSGIVVASPSTDNPDYFYTWTRDSGLVLKTLVDLFRNGDTSLLSTIENYISAQAIVQGISNPSGDLSSGAGLGEPKFNVDETAYTGSWGRPQRDGPALRATAMIGFGQWLLDNGYTSTATDIVWPLVRNDLSYVAQYWNQTGYDLWEEVNGSSFFTIAVQHRALVEGSAFATAVGSSCSWCDSQAPEILCYLQSFWTGSFILANFDSSRSGKDANTLLGSIHTFDPEAACDDSTFQPCSPRALANHKEVVDSFRSIYTLNDGLSDSEAVAVGRYPEDTYYNGNPWFLCTLAAAEQLYDALYQWDKQGSLEVTDVSLDFFKALYSDAATGTYSSSSSTYSSIVDAVKTFADGFVSIVETHAASNGSMSEQYDKSDGEQLSARDLTWSYAALLTANNRRNSVVPASWGETSASSVPGTCAATSAIGTYSSVTVTSWPSIVATGGTTTTATPTGSGSVTSTSKTTATASKTSTSTSSTSCTTPTAVAVTFDLTATTTYGENIYLVGSISQLGDWETSDGIALSADKYTSSDPLWYVTVTLPAGESFEYKFIRIESDDSVEWESDPNREYTVPQACGTSTATVTDTWR
(a) sequence signature:
● length: 640aa
● type: aminoacid sequence
(b) molecule type: protein
C () is supposed: no
(d) antisense: no
E () is originated at first: aspergillus niger (Aspergillus niger)
(f) specificity title: saccharifying enzyme (glucoamylase)
3) structure of glucoamylase expression cassette Pcs3-gla1-TtrpC
Extract plasmid pMD18-gla1 and plasmid pXH43 respectively, restriction enzyme Bsp119I and Bgl II is all used to carry out double digestion to it, carry out rubber tapping respectively to reclaim, wherein the digestion products of pMD18-gla1 reclaims the gla1 band that size is about 1.9kb, and the digestion products recovery size of pXH43 is about the carrier ribbon of 7.3kb.Connected by the fragment of recovery T4 ligase enzyme, after transforming, picking positive colony is verified, obtains plasmid pXH43-gla1(see Fig. 3 after order-checking is correct).
Embodiment 2 prepares the restructuring terreus containing Pcs3-gla-TtrpC expression cassette
1) preparation of terreus CICC40205 protoplastis
1.1) spore suspension of terreus CICC40205 is seeded in 50mL liquid nutrient medium ME that (substratum ME is 20g L in every premium on currency
-1glucose, 2g L
-1nH
4nO
3, 20mg L
-1(NH
4)
2hPO
4, 20mg L
-1feSO
4, 0.4g L
-1mgSO
4, 4.4mg L
-1znSO
4), the vitriol oil regulates pH to 3.5, spore concentration is reached and is about 10
7individual/mL, at 200rmp, 32 DEG C of cultivation 12-18h.
1.2) with the mycelia that aseptic individual layer 200 order nylon cloth collecting by filtration grows, and with the 0.6M MgSO of sterilizing
4solution rinses three times, press dry and is placed in aseptic 50ml triangular flask; By taking 1g mycelia, add 10ml enzymolysis solution, at 30 DEG C, 60rpm process 1-3h.Enzymolysis solution composition is: 0.8% cellulase (Sigma, Catalog No.:C1184), 0.8% lyase (Sigma, CatalogNO.:L1412), 0.4% helicase (the raw work in Shanghai, Catalog No.:SB0870), 0.6M MgSO
4, degerming via the sterile filter of 0.22 μm.
1.3) mixed solution after above-mentioned enzymolysis is first filtered with 2 layers of aseptic lens wiping paper, then filter with 5 layers of lens wiping paper, collection filtrate.4 DEG C of collected by centrifugation protoplastiss, wash once, then (STC are 1.0M sorbyl alcohol, 50mM TrisHCl-pH8.0,50mM CaCl to use the STC of precooling with precooling 1.0M Sorbitol Solution USP
2) wash once, finally protoplastis is resuspended in the STC of 150 μ l precoolings, and with STC, protoplast concentration is adjusted to 5 × 10
7individual/mL, obtains protoplast suspension.
2) conversion of terreus and screening
2.1) in above-mentioned protoplast suspension, adding about 2 μ g(volumes be no more than 10 μ l) (PTC is 40%PEG4000,50mMTris-HCl-pH8.0,50mM CaCl to linearizing above-mentioned plasmid pXH43-gla1 and 50 μ l PTC
2), mix gently, ice bath 30min.Add 1mL PTC again after ice bath, after mixing, room temperature places 20min; Get 200 μ l mixed solutions to coat on the dull and stereotyped PDA-SH of regeneration screening culture medium and (take 4g Difco
tMpotato dextrose agar (BD, and 22g sorbyl alcohol (Sigma LOT:1165825), LOT:071M00271V), be dissolved in 100ml with distilled water, sterilizing, when being cooled to about 55 DEG C, add Totomycin (Solarbio, Catalog No.:M419099) to final concentration be 100 μ g/mL, preparation is dull and stereotyped), 30 DEG C, cultivate 3-4 days under dark condition, obtain recombinant bacterial strain.
2.2) gained recombinant bacterial strain is transferred on PDA-H flat board (takes 4g Difco
tMpotato dextrose agar is dissolved in 100ml distilled water, sterilizing, and adding Totomycin to final concentration when being cooled to about 55 DEG C is 100 μ g/mL, and preparation is dull and stereotyped) carry out Secondary Culture, go down to posterity 3 times.And then collect respectively spore with and physiological saline carries out suitable gradient dilution, be about 50-200/mL to spore concentration, get 100 μ L and coat on PDA-H flat board and make it grow independently single bacterium colony, be single spore separation.Get spore from above-mentioned independently single bacterium colony again and again carry out single spore separation in the manner described above, carry out 4 single spore separation Secondary Culture altogether.
2.3) transformant on picking screening flat board is cultivated in ME liquid nutrient medium, collect mycelia and extract genome, with M13-47(5 '-CGCCAGGGTTTTCCCAGTCACGAC-3 ') and gla1-R1 be that primer pair carries out PCR, amplified production is the Pcs3-gla1 element in conversion carrier, agarose gel electrophoresis with 1% analyzes PCR primer, checking transformant.M13-47 primer is positioned at Pcs3 promotor upstream and is about 90bp place, therefore pcr amplification product 90bp about larger than Pcs3-gla1 fragment, and namely 2.6kb(is see Fig. 4).Electrophoresis result shows transformant G1-1, G1-2, G1-3, G1-4, G1-5, G1-6, G1-7, G1-8 of selecting size that can increase and is about the band of 2.6kb, using wild-type terreus CICC40205 strain gene group DNA template as negative control, amplification is not to DNA fragmentation.This to illustrate in these transformants all Successful integration Pcs3-gla1-TtrpC expression cassettes.
Embodiment 3 terreus of recombinating utilizes liquefied starch to produce the analysis of methylene-succinic acid ability
1) preparation of itaconic acid fermentation substratum
Dissolving calcium chloride is added by the water being heated to 60 DEG C, final concentration is made to be 0.05%, obtain lysate, then in 1:1(V/W) ratio in above-mentioned lysate, drop into starch, adjust about pH to 6.0, then add cumulative volume 0.05% amylase, fully steam ejection liquefaction after mixing, rear 96.5 DEG C maintain about 1h to iodine examine into brick-red after, what obtain is liquefied starch.
Above-mentioned liquefied starch is continued adjustment pH to 4.0, when temperature is down to 60 DEG C, the saccharifying enzyme adding 0.04% of cumulative volume is incubated about 20h, is hydrolyzed into wine inspection qualified (1-2 drips in saccharified liquid to 10ml alcohol and produces without precipitation), namely makes starch saccharificating liquid.
Then utilize above-mentioned gained liquefied starch and starch saccharificating liquid to prepare fermention medium A and fermention medium B, be specially:
Fermention medium A:300ml L
-1starch saccharificating liquid, 2g L
-1nH
4nO
3, 0.2g L
-1(NH
4)
2hPO
4, 20mg L
-1feSO
4, 0.4g L
-1mgSO
4, 10mg L
-1znSO
4, 10mg L
-1cuSO
4, Dried Corn Steep Liquor Powder 0.1%.
Fermention medium B:300ml L
-1liquefied starch, 2g L
-1nH
4nO
3, 0.2g L
-1(NH
4)
2hPO
4, 20mg L
-1feSO
4, 0.4g L
-1mgSO
4, 10mg L
-1znSO
4, 10mg L
-1cuSO
4, Dried Corn Steep Liquor Powder 0.1%.
2) recombinant bacterial strain produces the shaking flask screening of methylene-succinic acid
By set out terreus bacterial strain CICC40205 and after going down to posterity stable restructuring terreus bacterial strain G1-1, G1-2, G1-3, G1-4, G1-5, G1-6, G1-7, G1-8 be seeded to terreus respectively and produce spore slant medium (10g L
-1glucose, 2g L
-1naNO
3, 0.2g L
-1, KH
2pO
4, 20mg L
-1feSO
4, 5g L
-1mgSO
4, 0.5g L
-1naCl, 40mg L
-1znSO
4, 40mg L
-1cuSO
4, 0.5% wheat bran, 1.5% agarose, 115 DEG C of sterilizing 15min, then divide and are filled to test tube, then 115 DEG C of sterilizing 25min, prepare inclined-plane), cultivate for 32 DEG C and obtain ripe spore in 6 days.Respectively each is cultured to ripe spore inoculating again and (55ml fermention medium B is housed) in 500ml triangular flask in fermention medium B, every strain bacterium do two bottles parallel, 37 DEG C, 220rpm ferments 72h.Cross the mycelia filtered in fermented liquid, fermented supernatant fluid then carries out acid base titration, measures the total acid in fermented liquid.Because the main organic acid in fermented liquid is methylene-succinic acid, the therefore usual amount total acid recorded being thought methylene-succinic acid, and the content (Liu Jianjun, the research of itaconic acid fermentation, University Of Science and Technology Of Tianjin Ph.D. Dissertation, 2003) calculating methylene-succinic acid with this.Result display starting strain terreus CICC40205 output is 17.1g/L, major part engineering strain produces acid and is all better than starting strain, the output of wherein the highest bacterial strain G1-5 is 55.6g/L, improves 38.5g/L than starting strain, namely improves about 2.25 times (see Fig. 5).The restructuring terreus bacterial strain of process LAN Glucoamylase Gene from Aspergillus niger gla1 utilizes the ability of liquefied starch fermentation production of itaconic acid to be significantly improved as can be seen here.
3) purity check of methylene-succinic acid in fermented liquid
The fermented supernatant fluid choosing engineering strain G1-5 and starting strain CICC40205 carries out efficient liquid phase chromatographic analysis (High Performance Liquid Chromatography, HPLC), with the purity of methylene-succinic acid in com-parison and analysis fermented liquid.Chromatographic column: Bio-rad Aminex HPX-87-H, 300mmx7.8mm; Moving phase: 4mmol/L sulfuric acid; Flow velocity: 0.6ml/min; Column temperature: 35 DEG C; Check temperature: 35 DEG C; UV-detector (210nm).As shown in Figure 6, retention time is methylene-succinic acid at the peak of 13.30min to result, and analytical results shows, the methylene-succinic acid purity in recombinant bacterial strain G1-5 fermented liquid does not have noticeable change, all more than 99% compared with starting strain CICC40205.
Claims (5)
1. a terreus engineering bacteria for transformation, is characterized in that: described bacterial strain is by the genetic engineering modified restructuring terreus inserting glucoamylase expression frame and obtain in terreus.
2. by the terreus engineering bacteria of transformation according to claim 1, it is characterized in that: described glucoamylase expression frame is made up of the nucleotide sequence of filamentous fungus promoter, coding saccharifying enzyme and terminator; Wherein, promotor is the promotor of the citric acid synthesized enzyme gene of terreus self; The nucleotides sequence of coding saccharifying enzyme is classified as the glucoamylase gene of aspergillus niger; Terminator is the TtrpC terminator of Aspergillus nidulans.
3. a construction process for the terreus engineering bacteria of transformation according to claim 1, is characterized in that: by genetic engineering modified structure glucoamylase expression frame, be inserted in terreus the restructuring terreus obtained containing glucoamylase expression frame.
4. an application for the terreus engineering bacteria of transformation according to claim 1, is characterized in that: utilize the terreus engineering bacterium fermentation of transformation to produce methylene-succinic acid.
5. by the application of terreus engineering bacteria of transformation according to claim 4, it is characterized in that: adopt the terreus engineering bacteria of transformation as fermentation strain in using liquefied starch or starch saccharificating liquid as fermentation production of itaconic acid in the fermention medium of carbon source.
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