CN103834582B - A kind of improve itaconic acid fermentation output bacterial strain and structure thereof and utilize bacterial strain to produce the method for methylene-succinic acid - Google Patents
A kind of improve itaconic acid fermentation output bacterial strain and structure thereof and utilize bacterial strain to produce the method for methylene-succinic acid Download PDFInfo
- Publication number
- CN103834582B CN103834582B CN201210477312.2A CN201210477312A CN103834582B CN 103834582 B CN103834582 B CN 103834582B CN 201210477312 A CN201210477312 A CN 201210477312A CN 103834582 B CN103834582 B CN 103834582B
- Authority
- CN
- China
- Prior art keywords
- terreus
- bacterial strain
- cis
- methylene
- acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to genetically engineered field, specifically a kind of improve itaconic acid fermentation output bacterial strain and structure thereof and utilize bacterial strain to produce the method for methylene-succinic acid.Described bacterial strain is insert aconitate decarboxylase gene in terreus to obtain terreus of recombinating.By the plasmid of genetic engineering modified structure with cis-aconitic acid decarboxylase gene expression cassette PgpdAt1-cad-TtrpC, be inserted into catalysis cis-aconitic acid in terreus obtain process LAN cis-aconitic acid decarboxylase obtain recombinate terreus.The invention provides the itaconic acid fermentation output of recombinant bacterial strain generally higher than starting strain.
Description
Technical field
The present invention relates to genetically engineered field, specifically a kind of improve itaconic acid fermentation output bacterial strain and structure thereof and utilize bacterial strain to produce the method for methylene-succinic acid.
Technical background
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.The factory that methylene-succinic acid is produced in current domestic and international all submerged fermentations all adopts terreus.As shown in Figure 1, in terreus, the pathways metabolism of methylene-succinic acid is shunted out by tricarboxylic acid cycle, cis-aconitic acid in tricarboxylic acid cycle is at cis-aconitic acid decarboxylase (cis-aconiticaciddecarboxylase, CAD) under katalysis, decarboxylation forms methylene-succinic acid (Kanamasa, S., Dwiarti, L., Okabe, M., ParkE.Y., Cloningandfunctionalcharacterizationofthecis-aconiticaci ddecarboxylase (CAD) genefromAspergillusterreus.Appl.Microbiol.Biotechnol., 2008, 80, 223-229).Li etc. compare analysis to the genetic transcription difference of terreus under high yield and low yield methylene-succinic acid two kinds of conditions, find that the transcriptional level of cad gene under high-yield itaconic acid condition translating into cis-aconitic acid decarboxylase significantly improves (Li, A., vanLuijk, N., Beek, M., Caspers, M., Punt, P., vanderWerf, M., Aclone-basedtranscriptomicsapproachfortheidentificationo fgenesrelevantforitaconicacidproductioninAspergillus.Fun galGenet.Biol., 2011, 48, 602-611), this experimental result shows, cis-aconitic acid decarboxylase is a key enzyme in methylene-succinic acid biosynthetic pathway, the expression level improving cad gene in terreus will contribute to improving the fermentation yield of methylene-succinic acid.
So far two sections of reports are only had about being improved itaconic acid fermentation product quantifier elimination by genetically engineered.Tevz etc. relieve the feedback inhibition for phosphofructokinase by overexpression fructose-1, 6-diphosphate kinase mutants in terreus A156, enhance glycolytic pathway, about 2 times the output increased of methylene-succinic acid, simultaneously fermentation period shorten (Tev, G.,
m.,
m., EnhancingitaconicacidproductionbyAspergillusterreus.Appl .Microbiol.Biotechnol., 2010,87,1657-1664); Lin etc. are by overexpression Vitreoscilla hemoglobin in terreus NRRL1960, improve the tolerance of bacterial strain to hypoxemia, and the fermentation yield of methylene-succinic acid is improve 17%(Lin, Y.H., Li, Y.F., Huang, M.C., Tsai, Y.C., IntracellularexpressionofVitreoscillahemoglobininAspergi llusterreustoalleviateeffectofashortbreakinaerationduirn gculture, Biotechnol.Lett., 2004,26,10671072).But, producing quantifier elimination about improving itaconic acid fermentation by process LAN cis-aconitic acid decarboxylase in terreus, being not reported so far.
Summary of the invention
The object of the invention is that providing a kind of improves bacterial strain and the structure thereof of itaconic acid fermentation output and utilize bacterial strain to produce the method for methylene-succinic acid.
For achieving the above object, the technical solution used in the present invention is:
Improve a bacterial strain for itaconic acid fermentation output, described bacterial strain is by the genetic engineering modified restructuring terreus inserting aconitate decarboxylase expression casette PgpdAt1-cad-TtrpC and obtain in terreus.
Improve the construction process of the bacterial strain of itaconic acid fermentation output, by genetic engineering modified structure cis-aconitic acid decarboxylase gene expression cassette, be inserted in terreus the restructuring terreus obtained containing cis-aconitic acid decarboxylase expression cassette.
The bacterial strain improving itaconic acid fermentation output is utilized to produce the method for methylene-succinic acid, by genetic engineering modified structure cis-aconitic acid decarboxylase gene expression cassette, be inserted in terreus the restructuring terreus obtained containing cis-aconitic acid decarboxylase expression cassette, process LAN cis-aconitic acid decarboxylase strengthens the reaction process that catalysis cis-aconitic acid generates methylene-succinic acid, and then improves methylene-succinic acid output.
Described cis-aconitic acid decarboxylase expression cassette is made up of terreus cis-aconitic acid decarboxylase gene, terreus glyceraldehyde-3-phosphate dehydrogenase promotor and Aspergillus nidulans tryptophan synthetase gene terminator.Be incubated in fermention medium by described restructuring terreus, described fermention medium is: 140gL
-1glucose, 2gL
-1nH
4nO
3, 0.2gL
-1(NH
4)
2hPO
4, 20mgL
-1feSO
4, 0.4gL
-1mgSO
4, 40mgL
-1znSO
4, 40mgL
-1cuSO
4and water, with sulfuric acid adjustment pH to 3.5,115 DEG C of sterilizing 10min.
The advantage that the present invention has: genetic engineering modified terreus provided by the invention is to improve the method for itaconic acid fermentation output, and compared to mutagenesis method such as traditional physics, chemistry, the inventive method is motivated, workable, is convenient to screening.The invention provides the itaconic acid fermentation output of recombinant bacterial strain generally higher than starting strain, the bacterial strain C27 that wherein output is the highest improves about 7g/L(9.5% than starting strain), there is very strong using value.
Accompanying drawing explanation
Fig. 1 is that the terreus mentioned in background technology of the present invention produces methylene-succinic acid pathways metabolism figure.
The carrier pSGF957 plasmid map that Fig. 2 provides for the embodiment of the present invention, hph-casette is hygromycin resistance element; Sgfp is green fluorescence protein gene; TtrpC is Aspergillus nidulans tryptophan synthetase terminator; Pmgpd is monascus glyceraldehyde-3-phosphate dehydrogenase promotor.
The carrier pJJL-2 plasmid map that Fig. 3 provides for the embodiment of the present invention, wherein PgpdAt1 is terreus glyceraldehyde-3-phosphate dehydrogenase promotor.
The carrier pXH-1 plasmid map that Fig. 4 provides for the embodiment of the present invention, PgpdAt1 is terreus glyceraldehyde-3-phosphate dehydrogenase promotor; TtrpC is Aspergillus nidulans tryptophan synthetase terminator.
The carrier pXH-3 plasmid map that Fig. 5 provides for the embodiment of the present invention, PgpdAt1 is terreus glyceraldehyde-3-phosphate dehydrogenase promotor; TtrpC is Aspergillus nidulans tryptophan synthetase terminator, and cad is cis-aconitic acid decarboxylase decarboxylase gene.
The carrier pXH-4 plasmid map that Fig. 6 provides for the embodiment of the present invention, PgpdAt1 is terreus glyceraldehyde-3-phosphate dehydrogenase promotor; TtrpC is Aspergillus nidulans tryptophan synthetase terminator; Hph is hygromycin phosphotransferase gene, this plasmid contain can in terreus process LAN hph gene, thus give Host Strains hygromycin resistance.
The comparative effectiveness figure of the restructuring terreus bacterial strain shake flask fermentation product methylene-succinic acid ability that Fig. 7 provides for the embodiment of the present invention, WT is starting strain CICC40205.
The HPLC that Fig. 8 provides for the embodiment of the present invention analyzes the purity of methylene-succinic acid in bacterial strain fermentation liquor.A: methylene-succinic acid standard substance; B: the fermented liquid of starting strain CICC40205; C: the fermented liquid of recombinant bacterial strain C27; D: the fermented liquid of recombinant bacterial strain C5.
The integration of cad expression casette in the pcr analysis part recombinant bacterial strain that Fig. 9 provides for the embodiment of the present invention.M:200bpDNAmaker; 1: recombinant bacterial strain C5; 2: recombinant bacterial strain C12; 3: recombinant bacterial strain C25; 4: recombinant bacterial strain C27; 5: recombinant bacterial strain C28; 6: recombinant bacterial strain C38; 7:pXH-3(positive control); 8: starting strain CICC40205(negative control).
Embodiment
Carrier (vector) refers to the ring-shaped DNA molecule of a kind of energy self-replacation DNA fragmentation can transferred in recipient cell.
Promotor (promoter) is defined as and a kind ofly and guides polysaccharase to arrive the correct transcription initiation site of encoding sequence thus the DNA sequence dna of initiation transcription in conjunction with RNA polymerase.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.
" 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., Comparisonofbiosequences, Adv.Appl.Math., 1981,2,482-489) local homology algorithm; Needleman and Wunsch(NeedlemanS.B.andWunschC.D., Ageneralmethodapplicabletothesearchforsimilaritiesinthea minoacidsequenceoftwoproteins, J.Mol.Biol., 1970,48,443-453.) homology alignment algorithm; Pearson and Lipman(PearsonW.R., LipmanD.J., Improvedtoolsforbiologicalsequencecomparison, Proc.Natl.Acad.Sci., 1988,85,2444-2452.) search for similarity method; The computerize of these algorithms is implemented (such as, WisconsinGeneticsSoftwarePackage, GeneticsComputerGroup, 575ScienceDr., GAP, BESTFIT, FASTA, BLASTP, BLASTN and TFASTA in Madison, Wis.); Or manual alignment and visual inspection (see, the people such as such as Ausubel, CurrentProtocolsinMolecularBiology(1995 supplementary issue).
Below in conjunction with specific embodiment, technological line of the present invention is described in further details.
In the present invention, plasmid extraction adopts OMEGA company PlasmidMiniKitI test kit (D6942-01), it is adopt OMEGA company Cycle-PureKit test kit (D6492-01) that DNA fragmentation reclaims, and it is adopt OMEGA company GelExtractionKit test kit (D2500-01) that gel reclaims.Terreus CICC40205 bacterial strain is purchased from Chinese industrial Microbiological Culture Collection administrative center.
The structure of embodiment 1 terreus cis-aconitic acid decarboxylase gene cad expression cassette
The structure of 1.1 expression vector pXH-1
With TtrpC-F (5 '-cagacatgtagatctaagcttgcggccgcacttaacgttactgaaatc-3 ') and TtrpC-R (5 '-gtcgtgccagtcgactctaga-3 ') for primer pair, obtained by SeoulNationaluniversity with plasmid pSGF957(, build plasmid map as Fig. 2, Kim, J.G., Choi, Y.D., Chang, Y.J., Kim, S.U., GenetictransformationofMonascuspurpureusDSM1379, BiotechnologyLetters, 2003, 25, 1509 – 1514) carry out pcr amplification TtrpC fragment for template, product is through PciI(Fermentas, and XbaI enzyme cutting purifying CatalogNo.:#ER1871).PgpdAt1 promoter fragment is cloned in (Takara on pMD18-simple carrier by pJJL-2, CatalogNo.:D103A), then obtained (plasmid map is see Fig. 3) by the PciI restriction enzyme site on rite-directed mutagenesis removing pMD18-simple plasmid, its concrete construction process is with reference to Chinese patent: a kind of promotor of filamentous fungus glyceraldehyde-3-phosphate dehydrogenase gene (gpd) and application (application number 201210116163.7) thereof.Carry out enzyme with PciI and XbaI to plasmid pJJL-2 to cut, reclaim carrier segments.The pJJL-2 fragment of recovery and TtrpC fragment are connected through T4 ligase enzyme and obtain plasmid pXH-1(see Fig. 4).
The clone of 1.2 terreus cis-aconitic acid decarboxylase genes
Terreus CICC40205 bacterial strain used in the present invention is purchased from Chinese industrial Microbiological Culture Collection administrative center.The information design primer cad-F1 (5 '-gcattccatgaccaaacaatctgcggacagc-3 ') announced according to terreus NIH2624 genome database and cad-R1(5 '-ggcggatccttataccagtggcgatttcacgg-3 '), with the cDNA of terreus CICC40205 for template amplification terreus cis-aconitic acid decarboxylase gene, product for the purpose of band that length is about 1500bp 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, CatalogNo.:D101A) in, plasmid pXH-2 is obtained after order-checking.The DNA band of sequencing result display clone is terreus cis-aconitic acid decarboxylase gene, and nucleotides sequence is classified as SEQIDNO.1, and corresponding aminoacid sequence is SEQIDNO.2.With complete gene order-checking terreus NIH2624 bacterial strain Gene A TEG_09971 compared with, homology is 96%, the homology of the cad gene (GenBank:AB326105.1) of cloning with people such as Kanamasa is 100%(Kanamasa, S., Dwiarti, L., Okabe, M., ParkE.Y., Cloningandfunctionalcharacterizationofthecis-aconiticaci ddecarboxylase (CAD) genefromAspergillusterreus.Appl.Microbiol.Biotechnol., 2008,80,223-229).
SEQIDNO.1：
atgaccaaacaatctgcggacagcaacgcaaagtcaggagttacgtccgaaatatgtcattgggcatccaacctggccactgacgacatcccttcggacgtattagaaagagcaaaataccttattctcgacggtattgcatgtgcctgggttggtgcaagagtgccttggtcagagaagtatgttcaggcaacgatgagctttgagccgccgggggcctgcagggtgattggatatggacagaaactggggcctgttgcagcagccatgaccaattccgctttcatacaggctacggagcttgacgactaccacagcgaagcccccctacactctgcaagcattgtccttcctgcggtctttgcagcaagtgaggtcttagccgagcagggcaaaacaatttccggtatagatgttattctagccgccattgtggggtttgaatctggcccacggatcggcaaagcaatctacggatcggacctcttgaacaacggctggcattgtggagctgtgtatggcgctccagccggtgcgctggccacaggaaagctcctcggtctaactccagactccatggaagatgctctcggaattgcgtgcacgcaagcctgtggtttaatgtcggcgcaatacggaggcatggtaaagcgtgtgcaacacggattcgcagcgcgtaatggtcttcttgggggactgttggcccatggtgggtacgaggcaatgaaaggtgtcctggagagatcttacggcggtttcctcaagatgttcaccaagggcaacggcagagagcctccctacaaagaggaggaagtggtggctggtctcggttcattctggcatacctttactattcgcatcaagctctatgcctgctgcggacttgtccatggtccagtcgaggctatcgaaaaccttcaggggagataccccgagctcttgaatagagccaacctcagcaacattcgccatgttcatgtacagctttcaacggcctcgaacagtcactgtggatggataccagaggagagacccatcagttcaatcgcagggcagatgagtgtcgcatacattctcgccgtccagctggtcgaccagcaatgtcttttgtcccagttttctgagtttgatgacaacctggagaggccagaagtttgggatctggccaggaaggttacttcatctcaaagcgaagagtttgatcaagacggcaactgtctcagtgcgggtcgcgtgaggattgagttcaacgatggttcttctattacggaaagtgtcgagaagcctcttggtgtcaaagagcccatgccaaacgaacggattctccacaaataccgaacccttgctggtagcgtgacggacgaatcccgggtgaaagagattgaggatcttgtcctcggcctggacaggctcaccgacattagcccattgctggagctgctgaattgccccgtgaaatcgccactggtataa
(a) sequence signature:
● length: 1473bp
● 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: terreus (Aspergillusterreus)
(f) specificity title: cis-aconitic acid decarboxylase gene (cis-aconiticaciddecarboxylasegene, cad)
SEQIDNO.2：
MTKQSADSNAKSGVTSEICHWASNLATDDIPSDVLERAKYLILDGIACAWVGARVPWSEKYVQATMSFEPPGACRVIGYGQKLGPVAAAMTNSAFIQATELDDYHSEAPLHSASIVLPAVFAASEVLAEQGKTISGIDVILAAIVGFESGPRIGKAIYGSDLLNNGWHCGAVYGAPAGALATGKLLGLTPDSMEDALGIACTQACGLMSAQYGGMVKRVQHGFAARNGLLGGLLAHGGYEAMKGVLERSYGGFLKMFTKGNGREPPYKEEEVVAGLGSFWHTFTIRIKLYACCGLVHGPVEAIENLQGRYPELLNRANLSNIRHVHVQLSTASNSHCGWIPEERPISSIAGQMSVAYILAVQLVDQQCLLSQFSEFDDNLERPEVWDLARKVTSSQSEEFDQDGNCLSAGRVRIEFNDGSSITESVEKPLGVKEPMPNERILHKYRTLAGSVTDESRVKEIEDLVLGLDRLTDISPLLELLNCPVKSPLV
(a) sequence signature:
● length: 490aa
● type: aminoacid sequence
(b) molecule type: protein
C () is supposed: no
(d) antisense: no
E () is originated at first: terreus (Aspergillusterreus)
(f) specificity title: cis-aconitic acid decarboxylase (cis-aconiticaciddecarboxylase, CAD)
The structure of 1.3 goal gene cad expression vectors and selection markers hph expression vector
Extract plasmid pXH-2 and plasmid pXH-1 respectively, pXH-2 plasmid restriction enzyme BspHI(Fermentas, and BamHI(Fermentas CatalogNo.:#FD1284), CatalogNo.:#FD0054) carry out enzyme to cut, plasmid pXH-1 restriction enzyme PciI(Fermentas, and BglII(Fermentas CatalogNo.:#ER1871), CatalogNo.:#ER0081) double digestion is carried out, carry out rubber tapping respectively to reclaim, wherein the digestion products of pXH-2 reclaims the cad gene band that size is about 1.5kb, the digestion products recovery size of pXH-1 is about the band of 6.5kb.By the fragment T4 ligase enzyme (Fermentas reclaimed, CatalogNo.:#EL0011) connect, after transforming, picking positive colony is verified, obtains plasmid pXH-3(containing a PgpdAt1-cad-TtrpC expression cassette see Fig. 5 after order-checking is correct).
With primer hph-F (5 '-cattcatgactgaactcaccgcgacgtc-3 ') and hph-R(5 '-gacggatcctattcctttgccctcggacgag-3 ') for primer pair, with plasmid pSGF957 for template, pcr amplification size is about the hygromycin phosphotransferase gene (HygromycinBphosphotransferasegene of 1019bp, hph), PCR fragment BspHI after purifying and BamHI carries out double digestion, hph gene band is reclaimed in rubber tapping, the hph gene of recovery is connected with T4 ligase enzyme with the pXH-2 reclaimed after PciI and BglII enzyme is cut above, after transforming, picking positive colony is verified, plasmid pXH-4(is obtained see Fig. 6) after order-checking is correct.PXH-4 is the carrier that an energy expresses hph gene in filamentous fungus, can be filamentous fungus cotransformation and provides hygromycin resistance selection markers.
Embodiment 2 prepares the restructuring terreus containing PgpdAt1-cad-TtrpC expression cassette
The preparation of 2.1 terreus CICC40205 protoplastiss
1) spore suspension of terreus CICC40205 is seeded in 50mL liquid nutrient medium ME that (substratum ME is 20gL in every premium on currency
-1glucose, 2gL
-1nH
4nO
3, 20mgL
-1(NH
4)
2hPO
4, 20mgL
-1feSO
4, 0.4gL
-1mgSO
4, 4.4mgL
-1znSO
4), the vitriol oil regulates pH to 3.5, makes spore concentration be about 10
7individual/mL, cultivates 12-18h at 200rmp, 32 ° of C.
2) with the mycelia that aseptic individual layer 200 order nylon cloth collecting by filtration grows, and with the 0.6MMgSO 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 ° of C, 60rpm process 1-3h.Enzymolysis solution composition is: 0.8% cellulase (Sigma, CatalogNo.:C1184), 0.8% lyase (Sigma, CatalogNO.:L1412), 0.4% helicase (the raw work in Shanghai, CatalogNo.:SB0870), 0.6MMgSO
4, degerming via the sterile filter of 0.22 μm.
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 ° of C collected by centrifugation protoplastiss, wash once, then (STC are 1.0M sorbyl alcohol, 50mMTrisHCl-pH8.0,50mMCaCl 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.
2.2pXH-3 and pXH-4 cotransformation terreus
1) in above-mentioned protoplast suspension, add about 5 μ g(volumes be no more than 10 μ l) linearizing pXH-3 plasmid and the linearizing pXH-4 plasmid of 1 μ g, (PTC is 40%PEG4000,50mMTris-HClpH8.0,50mMCaCl to reenter 50 μ lPTC
2), mix gently, ice bath 30min.Add 1mLPTC, after mixing, room temperature places 20min; Then (3.9g potato dextrose agar substratum (DifcoTMPotatoDextroseAgar) (BD on the dull and stereotyped PDA-SH of regeneration screening culture medium is poured into after mixing with top-layer agar, and 22g sorbyl alcohol (Sigma LOT:1165825), LOT:071M00271V) be dissolved in 100ml distilled water, sterilizing, Totomycin (Solarbio is added when being cooled to about 55 ° of C, CatalogNo.:M419099) to final concentration be 100 μ g/mL, preparation is dull and stereotyped), under 30 ° of C, dark condition, cultivate 3-4 days.
2) from flat board, transformant is forwarded to screening dull and stereotyped PDA-H on (taking 4g potato dextrose agar substratum is dissolved in 100ml distilled water, sterilizing, adding Totomycin to final concentration when being cooled to about 55 ° of C is 100 μ g/mL, preparation is dull and stereotyped), 3-5 days, i.e. recombinant bacterial strain is cultivated at 30 ° of C.
3) gained recombinant bacterial strain is transferred on PDA-H flat board carries out Secondary Culture, go down to posterity 3 times.And then collection spore uses also physiological saline to carry out suitable gradient dilution respectively, gets 100 μ L and coats on PDA-H flat board, make it grow independently single bacterium colony, be single spore separation.Get spore from single bacterium colony again and again carry out single spore separation, carry out 4 single spore separation Secondary Culture altogether.
Embodiment 3 recombinate terreus fermentation produce methylene-succinic acid
3.1 recombinant bacterial strains produce the shaking flask screening of methylene-succinic acid
1) itaconic acid fermentation substratum IPM:140gL is prepared
-1glucose, 2gL
-1nH
4nO
3, 0.2gL
-1(NH
4)
2hPO
4, 20mgL
-1feSO
4, 0.4gL
-1mgSO
4, 40mgL
-1znSO
4, 40mgL
-1cuSO
4, with sulfuric acid adjustment pH to 3.5,115 DEG C of sterilizing 10min.
2) by above-mentioned gained recombinant bacterial strain, and recombinant bacterial strain go down to posterity after stable restructuring terreus bacterial strain be seeded to terreus respectively and produce spore slant medium (10gL
-1glucose, 2gL
-1naNO
3, 0.2gL
-1, KH
2pO
4, 20mgL
-1feSO
4, 5gL
-1mgSO
4, 0.5gL
-1naCl, 40mgL
-1znSO
4, 40mgL
-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 itaconic acid fermentation substratum is housed) in 500ml triangular flask in itaconic acid fermentation substratum, 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.Except several strain bacterial strain produces the obvious decline of acid, all the other most bacterial strains produce acid and are all better than starting strain (see Fig. 6).
3) according to the sour result of product, choose 22 strains produce acid preferably bacterial strain carry out multiple sieve again, every strain bacterium do three bottles parallel.Measure total acid, as shown in Figure 7, the product acid of 22 strain recombinant bacterial strains is general higher than starting strain CICC40205 (WT), and wherein the highest bacterial strain C27 improves 7g/L(9.5% than starting strain for result).
The purity check of methylene-succinic acid in 3.2 fermented liquids
The fermented supernatant fluid choosing recombinant bacterial strain C5, C27 and starting strain CICC40205 carries out efficient liquid phase chromatographic analysis (HighPerformanceLiquidChromatography, HPLC), with the purity of methylene-succinic acid in com-parison and analysis fermented liquid.Chromatographic column: Bio-radAminexHPX-87-H, 300mmx7.8mm; Moving phase: 4mmol/L sulfuric acid; Flow velocity: 0.8ml/min; Temperature: 30 DEG C; Check temperature: 30 DEG C; UV-detector (210nm).As shown in Figure 8, retention time is methylene-succinic acid at the peak of 13.317min to result, and analytical results shows, recombinant bacterial strain C5 does not have noticeable change, more than 99% with the methylene-succinic acid purity in C27 fermented liquid compared with starting strain CICC40205.
The checking of embodiment 4 high-yield itaconic acid restructuring terreus strain genotype
The higher restructuring terreus bacterial strain of picking product acid is inoculated on PDA-H flat board cultivates spore, then respectively spore inoculating is cultivated in ME liquid nutrient medium, collect mycelia and extract genome, with PgpdAt-seqF(5 '-gctctgtagcttttgccccgtc-3 ') and TtrpC-seqR(5 '-cgagatcctgaacaccatttgtctc-3 ') carry out PCR for primer pair, PgpdAt1-cad-TtrpC element in amplification conversion carrier, agarose gel electrophoresis with 1% analyzes PCR primer, the integration of PgpdAt1-cad-TtrpC element in checking transformant.PgpdAt-seqF primer is positioned in PgpdAt1 promotor, cad gene start codon upstream is about 260bp place, TtrpC-seqR primer is positioned on TtrpC terminator, cad gene end codon downstream is about 120bp place, therefore pcr amplification product 380bp about larger than cad gene, namely about 1850bp(is see Fig. 9).Electrophoresis result shows in the transformant selected the band of the 1850kb that can increase, and using wild-type terreus CICC40205 strain gene group DNA template as negative control, amplification is not to DNA fragmentation.Therefore, all Successful integration Pg are proved in these transformants
pdAt1-cad-Ttr
pc expression cassette.
Claims (5)
1. improve a bacterial strain for itaconic acid fermentation output, it is characterized in that: described bacterial strain is by the genetic engineering modified restructuring terreus inserting aconitate decarboxylase expression casette PgpdAt1-cad-TtrpC and obtain in terreus;
The nucleotide sequence of described aconitate decarboxylase gene is as shown in SEQIDNO.1.
2. the construction process of the bacterial strain of a raising itaconic acid fermentation output according to claim 1, it is characterized in that: by genetic engineering modified structure cis-aconitic acid decarboxylase gene expression cassette, be inserted in terreus the restructuring terreus obtained containing cis-aconitic acid decarboxylase expression cassette.
3. the bacterial strain of a raising itaconic acid fermentation output according to claim 1 produces the method for methylene-succinic acid, it is characterized in that: by genetic engineering modified structure cis-aconitic acid decarboxylase gene expression cassette, be inserted in terreus the restructuring terreus obtained containing cis-aconitic acid decarboxylase expression cassette, process LAN cis-aconitic acid decarboxylase strengthens the reaction process that catalysis cis-aconitic acid generates methylene-succinic acid, and then improves methylene-succinic acid output.
4. produce the method for methylene-succinic acid by the bacterial strain of raising itaconic acid fermentation output according to claim 3, it is characterized in that: described cis-aconitic acid decarboxylase expression cassette is made up of terreus cis-aconitic acid decarboxylase gene, terreus glyceraldehyde-3-phosphate dehydrogenase promotor and Aspergillus nidulans tryptophan synthetase gene terminator.
5. produce the method for methylene-succinic acid by the bacterial strain of raising itaconic acid fermentation output according to claim 3, it is characterized in that: be incubated in fermention medium by described restructuring terreus, described fermention medium is: 140gL
-1glucose, 2gL
-1nH
4nO
3, 0.2gL
-1(NH
4)
2hPO
4, 20mgL
-1feSO
4, 0.4gL
-1mgSO
4, 40mgL
-1znSO
4, 40mgL
-1cuSO
4and water, with sulfuric acid adjustment pH to 3.5,115 DEG C of sterilizing 10min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210477312.2A CN103834582B (en) | 2012-11-22 | 2012-11-22 | A kind of improve itaconic acid fermentation output bacterial strain and structure thereof and utilize bacterial strain to produce the method for methylene-succinic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210477312.2A CN103834582B (en) | 2012-11-22 | 2012-11-22 | A kind of improve itaconic acid fermentation output bacterial strain and structure thereof and utilize bacterial strain to produce the method for methylene-succinic acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103834582A CN103834582A (en) | 2014-06-04 |
| CN103834582B true CN103834582B (en) | 2016-04-27 |
Family
ID=50798480
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210477312.2A Active CN103834582B (en) | 2012-11-22 | 2012-11-22 | A kind of improve itaconic acid fermentation output bacterial strain and structure thereof and utilize bacterial strain to produce the method for methylene-succinic acid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103834582B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11066681B2 (en) * | 2016-08-26 | 2021-07-20 | Lesaffre Et Compagnie | Production of itaconic acid |
| CN107058144A (en) * | 2017-02-15 | 2017-08-18 | 江南大学 | A kind of restructuring yeast strains for producing itaconic acid and its construction method and application |
| CN108384815B (en) * | 2018-05-22 | 2022-03-04 | 中国林业科学研究院林产化学工业研究所 | Method for preparing itaconic acid by using lignocellulose raw material |
| CN110527637B (en) * | 2019-07-18 | 2021-10-15 | 中国科学院青岛生物能源与过程研究所 | Monascus strain for producing aconitic acid and construction method and application thereof |
| CN111944706B (en) * | 2020-08-06 | 2022-06-03 | 中国科学院青岛生物能源与过程研究所 | Recombinant aspergillus terreus strain for producing itaconic acid and construction method and application thereof |
| CN113999807B (en) * | 2021-11-02 | 2023-04-25 | 南京工业大学 | Construction method of recombinant strain and application of recombinant strain in itaconic acid production |
| CN116004677B (en) * | 2022-11-25 | 2024-02-13 | 华东理工大学 | Construction method and application of Trichoderma reesei engineering bacteria for producing itaconic acid |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2017344A1 (en) * | 2007-07-20 | 2009-01-21 | Nederlandse Organisatie voor toegepast- natuurwetenschappelijk onderzoek TNO | Production of itaconic acid |
| CN101886045A (en) * | 2009-05-11 | 2010-11-17 | 财团法人工业技术研究院 | Genetically modified microorganisms for producing itaconic acid with high yields |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100330631A1 (en) * | 2009-06-30 | 2010-12-30 | Industrial Technology Research Institute | Cis-aconitate Decarboxylase Mutants Having Improved Enzymatic Activity |
-
2012
- 2012-11-22 CN CN201210477312.2A patent/CN103834582B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2017344A1 (en) * | 2007-07-20 | 2009-01-21 | Nederlandse Organisatie voor toegepast- natuurwetenschappelijk onderzoek TNO | Production of itaconic acid |
| CN101886045A (en) * | 2009-05-11 | 2010-11-17 | 财团法人工业技术研究院 | Genetically modified microorganisms for producing itaconic acid with high yields |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103834582A (en) | 2014-06-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103834582B (en) | A kind of improve itaconic acid fermentation output bacterial strain and structure thereof and utilize bacterial strain to produce the method for methylene-succinic acid | |
| CN103740629A (en) | Genetic engineering acetic acid bacteria of overexpressing coenzyme PQQ (pyrroloquinoline quinone) synthetic proteins and application of bacteria | |
| CN103614398B (en) | The encoding gene of cis-Epoxysuccinic acid hydratase, the polypeptide of its coding and related application thereof | |
| US10519203B2 (en) | Gene for biosynthesis of core structure of ophiobolin | |
| CN102965324B (en) | Genetic engineering strain for producing (R, R)-2,3-butanediol, as well as construction method and application thereof | |
| CN105255951B (en) | A method of Alcohol Production efficiency is improved by overexpression HAC1 genes | |
| CN111944706B (en) | Recombinant aspergillus terreus strain for producing itaconic acid and construction method and application thereof | |
| CN104232611A (en) | Recombinant beauveria brongniartii proteinase K as well as industrial production and purification method thereof | |
| CN113846023A (en) | Method and strain for reducing by-product succinic acid in L-malic acid fermentation process and application | |
| CN116023449B (en) | Fusarium fumonisin synthesis and pathogenicity related gene FpFUM21 and application thereof | |
| CN110791508A (en) | Codon-optimized pediocin gene, expression vector and recombinant engineering strain | |
| CN103374570B (en) | Promoter of filamentous fungus glyceraldehyde-3-phosphate dehydrogenase gene (gpd) and application of promoter | |
| CN105255952B (en) | A method of Alcohol Production efficiency is improved by overexpression INO2 genes | |
| CN106635847A (en) | Recombinant aspergillus niger capable of improving yield of citric acid and preparation method of recombinant aspergillus niger | |
| CN111378682A (en) | Method for reducing ethyl carbamate content in yellow wine by metabolically modifying saccharomyces cerevisiae | |
| CN112029670A (en) | Recombinant aspergillus terreus for high yield of itaconic acid and construction method and application thereof | |
| CN112011469B (en) | Recombinant aspergillus terreus strain for producing trans-aconitic acid and construction method and application thereof | |
| CN111139208A (en) | High-yield engineering bacterium for producing ivermectin and preparation method and application thereof | |
| CN104419650A (en) | Transformed aspergillus terreus engineering bacteria and application thereof | |
| US20190048353A1 (en) | Marine bacterial gene lfliz and use | |
| CN115960920B (en) | Coroxepin FHb, recombinant bacterium X33-pPICZ alpha A-102C300C-FHb2 and application thereof | |
| CN112481290B (en) | Method for improving citric acid fermentation production level based on morphological gene co-interference | |
| CN115948427B (en) | Coroxepin FHb, recombinant bacterium X33-pPICZ alpha A-102C300C-FHb1 and application thereof | |
| CN113151262B (en) | Yeast promoter with weakened regulation strength and application thereof in metabolic flux regulation | |
| CN112011468B (en) | Recombinant aspergillus terreus for producing trans-aconitic acid and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |