CN103834582A - Itaconic acid fermentation yield improvement bacterial strain, construction method thereof and itaconic acid production method using bacterial strain - Google Patents
Itaconic acid fermentation yield improvement bacterial strain, construction method thereof and itaconic acid production method using bacterial strain Download PDFInfo
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Abstract
本发明涉及基因工程领域，具体的说是一种提高衣康酸发酵产量的菌株及其构建和利用菌株生产衣康酸的方法。所述菌株为土曲霉中插入乌头酸脱羧酶基因得到得到重组土曲霉。通过基因工程改造构建带有顺乌头酸脱羧酶基因表达盒PgpdAt1-cad-TtrpC的质粒，将其插入土曲霉中催化顺乌头酸得到过表达顺乌头酸脱羧酶得到重组土曲霉。本发明提供重组菌株的衣康酸发酵产量普遍高于出发菌株。The invention relates to the field of genetic engineering, in particular to a bacterial strain for improving the fermentation yield of itaconic acid and a method for constructing and utilizing the bacterial strain to produce itaconic acid. The bacterial strain is obtained by inserting the aconitic acid decarboxylase gene into Aspergillus terreus to obtain recombinant Aspergillus terreus. A plasmid carrying the aconitic acid decarboxylase gene expression cassette PgpdAt1-cad-TtrpC was constructed by genetic engineering, and inserted into Aspergillus terreus to catalyze cis-aconitic acid to obtain a recombinant Aspergillus terreus obtained by overexpressing the aconitic acid decarboxylase. The invention provides that the itaconic acid fermentation yield of the recombinant strain is generally higher than that of the starting strain.
Description
Technical field
The present invention relates to genetically engineered field, specifically a kind of method that improves bacterial strain and the structure thereof of itaconic acid fermentation output and utilize bacterial strain production 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 at present 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-aconitic acid decarboxylase, CAD) under katalysis, decarboxylation forms methylene-succinic acid (Kanamasa, S., Dwiarti, L., Okabe, M., Park E.Y., Cloning andfunctional characterization of the cis-aconitic acid decarboxylase (CAD) gene from Aspergillus terreus.Appl.Microbiol.Biotechnol., 2008, 80, 223-229).To terreus, the genetic transcription difference under high yield and two kinds of conditions of low yield methylene-succinic acid compares analysis to Li etc., the transcriptional level of the cad gene that cis-aconitic acid decarboxylase is translated in discovery under high-yield itaconic acid condition significantly improves (Li, A., van Luijk, N., Beek, M., Caspers, M., Punt, P., van der Werf, M., A clone-basedtranscriptomics approach for the identification of genes relevantfor itaconic acid production in Aspergillus.Fungal Genet.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 that improves cad gene in terreus will contribute to improve the fermentation yield of methylene-succinic acid.
So far only there are two pieces of reports about the research that improves itaconic acid fermentation output by genetically engineered.Tevz etc. have removed the feedback inhibition for phosphofructokinase by overexpression fructose-1, 6-diphosphate kinase mutant body in terreus A156, have strengthened glycolytic pathway, and the output of methylene-succinic acid has been improved to about 2 times, fermentation period shortening simultaneously (Tev, G.,
m.,
m., Enhancing itaconic acid production by Aspergillus terreus.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 improved to 17%(Lin, Y.H., Li, Y.F., Huang, M.C., Tsai, Y.C., Intracellular expression of Vitreoscilla hemoglobin inAspergillus terreus to alleviate effect of a short break in aerationduirng culture, Biotechnol.Lett., 2004,26,1,067 1072).But about the research that improves itaconic acid fermentation output by excessively express cis-aconitic acid decarboxylase in terreus, so far there are no reports.
Summary of the invention
The object of the invention is to provide a kind of and 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 in terreus, to insert by genetic engineering modified the restructuring terreus that aconitate decarboxylase expression casette PgpdAt1-cad-TtrpC obtains.
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 into and in terreus, obtain the restructuring terreus that contains cis-aconitic acid decarboxylase expression cassette.
The method of utilizing the bacterial strain that improves itaconic acid fermentation output to produce methylene-succinic acid, by genetic engineering modified structure cis-aconitic acid decarboxylase gene expression cassette, be inserted into and in terreus, obtain the restructuring terreus that contains cis-aconitic acid decarboxylase expression cassette, cross and express cis-aconitic acid decarboxylase and strengthen catalysis cis-aconitic acid and generate the reaction process of methylene-succinic acid, and then improve 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.Described restructuring terreus is incubated in fermention medium, and described fermention medium is: 140g L
-1glucose, 2g L
-1nH
4nO
3, 0.2g L
-1(NH
4)
2hPO
4, 20mg L
-1feSO
4, 0.4g L
-1mgSO
4, 40mgL
-1znSO
4, 40mg L
-1cuSO
4and water, with sulfuric acid adjustment pH to 3.5,115 ℃ of sterilizing 10min.
The present invention has advantages of: genetic engineering modified terreus provided by the invention is to improve the method for itaconic acid fermentation output, and compared to the traditional mutagenesis such as physics, chemistry mode, 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 has improved about 7g/L(9.5% than starting strain), there is very strong using value.
Accompanying drawing explanation
Fig. 1 is that the terreus of mentioning 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, and this plasmid contains can cross expression hph gene in terreus, thereby gives Host Strains hygromycin resistance.
The restructuring terreus bacterial strain shake flask fermentation that Fig. 7 provides for the embodiment of the present invention produces the comparative effectiveness figure of methylene-succinic acid ability, and 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:200bp DNA maker; 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 that DNA fragmentation can be transferred in recipient cell.
Promotor (promoter) thus being defined as a kind of combination RNA polymerase and guides polysaccharase to arrive the initial DNA sequence dna of transcribing of correct transcription initiation site of encoding sequence.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-effect transcriptional regulatory element, as enhanser and can with other nucleotide sequences of transcription factor interaction.
" identity " or " identity per-cent " refers to the sequence identity between two aminoacid sequences or between two nucleotide sequences.For determining the identity percentage ratio of two aminoacid sequences or two nucleic acid, with the best comparison object, sequence is compared.Identity per-cent between two sequences is the function (, the sum of the number/position of identity percentage ratio=same position (for example, lap position) × 100) of the number of the same position that had by these sequences.For example, " identity per-cent " calculates by following manner: in comparison window, compare two sequences through the best comparison, be determined at the number of the position that occurs identical nucleotide base or same amino acid residue in two sequences to produce the number of matched position, by the number of matched position divided by the overall number of position in comparison window (, the size of window), thus and result is multiplied by 100 generation sequence identity per-cents.The best comparison for sequence relatively can be undertaken by following: for example, and Smith and Waterman(Smith, T.F.andWaterman, M.S., Comparison of biosequences, Adv.Appl.Math., 1981,2,482-489) local homology's 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.) sequence analysis algorithm; Pearson and Lipman(Pearson W.R., Lipman D.J., Improved tools for biological sequence comparison, Proc.Natl.Acad.Sci., 1988,85,2444-2452.) similarity searching method; The computerize of these algorithms (is for example implemented, Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wis.); Or manual comparison and visual inspection (referring to, the people such as such as Ausubel, Current Protocols in Molecular Biology(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 Plasmid Mini Kit I test kit (D6942-01), it is to adopt the Cycle-Pure Kit of OMEGA company test kit (D6492-01) that DNA fragmentation reclaims, and it is to adopt OMEGA company Gel Extraction Kit test kit (D2500-01) that gel reclaims.Terreus CICC40205 bacterial strain is purchased from Chinese industrial microbial strains preservation 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
Take TtrpC-F (5 '-cagacatgtagatctaagcttgcggccgcacttaacgttactgaaatc-3 ') and TtrpC-R (5 '-gtcgtgccagtcgactctaga-3 ') as primer pair, obtained by Seoul National university with plasmid pSGF957(, build plasmid map as Fig. 2, Kim, J.G., Choi, Y.D., Chang, Y.J., Kim, S.U., Genetictransformation of Monascus purpureus DSM1379, Biotechnology Letters, 2003, 25, 1509 – 1514) carry out pcr amplification TtrpC fragment for template, product is through Pci I(Fermentas, Catalog No.:#ER1871) and Xba I enzyme is cut and purifying.PgpdAt1 promoter fragment is cloned in (Takara on pMD18-simple carrier by pJJL-2, Catalog No.:D103A), then the Pci I restriction enzyme site of removing on pMD18-simple plasmid by rite-directed mutagenesis obtains (plasmid map is referring to Fig. 3), and its concrete construction process is with reference to Chinese patent: the promotor of a kind of filamentous fungus glyceraldehyde-3-phosphate dehydrogenase gene (gpd) and application thereof (application number 201210116163.7).With Pci I and Xba I, plasmid pJJL-2 is carried out to enzyme and cut, reclaim carrier segments.The pJJL-2 fragment of recovery is connected and obtains plasmid pXH-1(referring to Fig. 4 through T4 ligase enzyme with TtrpC fragment).
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 microbial strains preservation administrative center.The information design primer cad-F1 (5 '-gcattccatgaccaaacaatctgcggacagc-3 ') and the cad-R1(5 '-ggcggatccttataccagtggcgatttcacgg-3 ' that announce according to terreus NIH2624 genome database), take the cDNA of terreus CICC40205 as template amplification terreus cis-aconitic acid decarboxylase gene, the band that is about 1500bp through 1.0% agarose gel electrophoresis detection length is object product, object band product TA after purifying is reclaimed in rubber tapping is cloned into pMD18-T carrier (TaKaRa, Catalog No.:D101A) in, after order-checking, obtain plasmid pXH-2.Sequencing result shows that clone's DNA band is terreus cis-aconitic acid decarboxylase gene, and nucleotides sequence is classified SEQ ID NO.1 as, and corresponding aminoacid sequence is SEQ ID NO.2.Compare with the Gene A TEG_09971 of the terreus NIH2624 bacterial strain that completes gene order-checking, homology is 96%, with the homology of the people such as Kanamasa clone's cad gene (GenBank:AB326105.1) be 100%(Kanamasa, S., Dwiarti, L., Okabe, M., Park E.Y., Cloning andfunctional characterization of the cis-aconitic acid decarboxylase (CAD) gene from Aspergillus terreus.Appl.Microbiol.Biotechnol., 2008,80,223-229).
SEQ?ID?NO.1：
atgaccaaacaatctgcggacagcaacgcaaagtcaggagttacgtccgaaatatgtcattgggcatccaacctggccactgacgacatcccttcggacgtattagaaagagcaaaataccttattctcgacggtattgcatgtgcctgggttggtgcaagagtgccttggtcagagaagtatgttcaggcaacgatgagctttgagccgccgggggcctgcagggtgattggatatggacagaaactggggcctgttgcagcagccatgaccaattccgctttcatacaggctacggagcttgacgactaccacagcgaagcccccctacactctgcaagcattgtccttcctgcggtctttgcagcaagtgaggtcttagccgagcagggcaaaacaatttccggtatagatgttattctagccgccattgtggggtttgaatctggcccacggatcggcaaagcaatctacggatcggacctcttgaacaacggctggcattgtggagctgtgtatggcgctccagccggtgcgctggccacaggaaagctcctcggtctaactccagactccatggaagatgctctcggaattgcgtgcacgcaagcctgtggtttaatgtcggcgcaatacggaggcatggtaaagcgtgtgcaacacggattcgcagcgcgtaatggtcttcttgggggactgttggcccatggtgggtacgaggcaatgaaaggtgtcctggagagatcttacggcggtttcctcaagatgttcaccaagggcaacggcagagagcctccctacaaagaggaggaagtggtggctggtctcggttcattctggcatacctttactattcgcatcaagctctatgcctgctgcggacttgtccatggtccagtcgaggctatcgaaaaccttcaggggagataccccgagctcttgaatagagccaacctcagcaacattcgccatgttcatgtacagctttcaacggcctcgaacagtcactgtggatggataccagaggagagacccatcagttcaatcgcagggcagatgagtgtcgcatacattctcgccgtccagctggtcgaccagcaatgtcttttgtcccagttttctgagtttgatgacaacctggagaggccagaagtttgggatctggccaggaaggttacttcatctcaaagcgaagagtttgatcaagacggcaactgtctcagtgcgggtcgcgtgaggattgagttcaacgatggttcttctattacggaaagtgtcgagaagcctcttggtgtcaaagagcccatgccaaacgaacggattctccacaaataccgaacccttgctggtagcgtgacggacgaatcccgggtgaaagagattgaggatcttgtcctcggcctggacaggctcaccgacattagcccattgctggagctgctgaattgccccgtgaaatcgccactggtataa
(a) sequence signature:
● length: 1473bp
● type: base sequence
● chain: two strands
● topological framework: linearity
(b) molecule type: DNA
(c) suppose: no
(d) antisense: no
(e) originate at first: terreus (Aspergillus terreus)
(f) specificity title: cis-aconitic acid decarboxylase gene (cis-aconitic acid decarboxylasegene, cad)
SEQ?ID?NO.2：
MTKQSADSNAKSGVTSEICHWASNLATDDIPSDVLERAKYLILDGIACAWVGARVPWSEKYVQATMSFEPPGACRVIGYGQKLGPVAAAMTNSAFIQATELDDYHSEAPLHSASIVLPAVFAASEVLAEQGKTISGIDVILAAIVGFESGPRIGKAIYGSDLLNNGWHCGAVYGAPAGALATGKLLGLTPDSMEDALGIACTQACGLMSAQYGGMVKRVQHGFAARNGLLGGLLAHGGYEAMKGVLERSYGGFLKMFTKGNGREPPYKEEEVVAGLGSFWHTFTIRIKLYACCGLVHGPVEAIENLQGRYPELLNRANLSNIRHVHVQLSTASNSHCGWIPEERPISSIAGQMSVAYILAVQLVDQQCLLSQFSEFDDNLERPEVWDLARKVTSSQSEEFDQDGNCLSAGRVRIEFNDGSSITESVEKPLGVKEPMPNERILHKYRTLAGSVTDESRVKEIEDLVLGLDRLTDISPLLELLNCPVKSPLV
(a) sequence signature:
● length: 490aa
● type: aminoacid sequence
(b) molecule type: protein
(c) suppose: no
(d) antisense: no
(e) originate at first: terreus (Aspergillus terreus)
(f) specificity title: cis-aconitic acid decarboxylase (cis-aconitic acid decarboxylase, CAD)
The structure of 1.3 goal gene cad expression vectors and selection markers hph expression vector
Extract respectively plasmid pXH-2 and plasmid pXH-1, restriction enzyme BspH I(Fermentas for pXH-2 plasmid, Catalog No.:#FD1284) and BamH I(Fermentas, CatalogNo.:#FD0054) carrying out enzyme cuts, restriction enzyme Pci I(Fermentas for plasmid pXH-1, Catalog No.:#ER1871) and Bgl II(Fermentas, Catalog No.:#ER0081) carry out double digestion, the recovery of tapping rubber respectively, wherein the enzyme of pXH-2 is cut product and is reclaimed the cad gene band that size is about 1.5kb, the enzyme of pXH-1 is cut product and is reclaimed the band that size is about 6.5kb.By T4 ligase enzyme (Fermentas for the fragment reclaiming, Catalog No.:#EL0011) connect, after transforming, picking positive colony is verified, the plasmid pXH-3(that obtains containing a PgpdAt1-cad-TtrpC expression cassette after order-checking is correct is referring to Fig. 5).
Take primer hph-F (5 '-cattcatgactgaactcaccgcgacgtc-3 ') and hph-R(5 '-gacggatcctattcctttgccctcggacgag-3 ') as primer pair, take plasmid pSGF957 as template, pcr amplification size is about hygromycin phosphotransferase gene (the Hygromycin Bphosphotransferase gene of 1019bp, hph), PCR fragment after purifying is carried out double digestion with BspH I and BamH I, hph gene band is reclaimed in rubber tapping, the hph gene of recovery is connected with the pXH-2 T4 ligase enzyme reclaiming after Pci I and Bgl II enzyme are cut above, after transforming, picking positive colony is verified, after order-checking is correct, obtain plasmid pXH-4(referring to Fig. 6).PXH-4 is the carrier that an energy is expressed hph gene in filamentous fungus, can be filamentous fungus cotransformation hygromycin resistance selection markers is provided.
The preparation of 2.1 terreus CICC40205 protoplastiss
1) spore suspension of terreus CICC40205 is seeded to in 50mL liquid nutrient medium ME, (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, makes spore concentration be about 10
7individual/mL, cultivates 12-18h at 200rmp, 32 ° of C.
2) filter and collect the mycelia growing with aseptic individual layer 200 order nylon cloths, 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, process 1-3h at 30 ° of C, 60rpm.Enzymolysis solution composition is: 0.8% cellulase (Sigma, Catalog No.:C1184), 0.8% lyase (Sigma, Catalog NO.:L1412), 0.4% helicase (the raw work in Shanghai, Catalog No.:SB0870), 0.6M MgSO
4, via the sterilizing filter filtration sterilization of 0.22 μ m.
3) mixed solution after above-mentioned enzymolysis is first filtered with 2 layers of aseptic lens wiping paper, then with 5 layers of lens wiping papers filtration, collect filtrate.4 ° of centrifugal collection protoplastiss of C, wash once with precooling 1.0M Sorbitol Solution USP, then (STC is 1.0M sorbyl alcohol, 50mM TrisHCl-pH 8.0,50mM CaCl to use the STC of precooling
2) washing once, finally protoplastis is resuspended in to the STC of 150 μ l precoolings, and protoplastis concentration is adjusted into 5 × 10 with STC
7individual/mL, obtains protoplastis suspension.
2.2 pXH-3 and pXH-4 cotransformation terreus
1) in above-mentioned protoplastis suspension, add approximately 5 μ g(volumes to be no more than l) linearizing pXH-3 plasmid and the linearizing pXH-4 plasmid of 1 μ g of 10 μ, (PTC is 40%PEG4000,50mM Tris-HCl pH8.0,50mM CaCl to reenter 50 μ l PTC
2), mix gently ice bath 30min.Add 1mL PTC, mix rear room temperature and place 20min; Then after mixing with top-layer agar, be poured into upper (3.9g potato dextrose agar substratum (DifcoTMPotato Dextrose the Agar) (BD of the dull and stereotyped PDA-SH of regeneration screening culture medium, and 22g sorbyl alcohol (Sigma LOT:1165825), LOT:071M00271V) be dissolved in 100ml distilled water, sterilizing, while being cooled to approximately 55 ° of C, add Totomycin (Solarbio, Catalog No.: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) above (taking 4g potato dextrose agar substratum is dissolved in 100ml distilled water transformant to be forwarded to the dull and stereotyped PDA-H of screening from flat board, sterilizing, while being cooled to approximately 55 ° of C, adding Totomycin to final concentration is 100 μ g/mL, preparation is dull and stereotyped), cultivate 3-5 days, i.e. recombinant bacterial strain at 30 ° of C.
3) gained recombinant bacterial strain is transferred to the cultivation of going down to posterity on PDA-H flat board, goes down to posterity 3 times.And then collect respectively spore and use also physiological saline to carry out suitable gradient dilution, get 100 μ L and coat on PDA-H flat board, make it grow independently single bacterium colony, be monospore and separate.Get spore from single bacterium colony again and again carry out monospore separation, carry out altogether 4 monospores and separate the cultivation of going down to posterity.
Methylene-succinic acid is produced in recombinate terreus fermentation of embodiment 3
3.1 recombinant bacterial strains produce the shaking flask screening of methylene-succinic acid
1) preparation itaconic acid fermentation substratum IPM:140g L
-1glucose, 2g L
-1nH
4nO
3, 0.2gL
-1(NH
4)
2hPO
4, 20mg L
-1feSO
4, 0.4g L
-1mgSO
4, 40mg L
-1znSO
4, 40mgL
-1cuSO
4, with sulfuric acid adjustment pH to 3.5,115 ℃ 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 respectively terreus and produce spore slant medium (10g L
-1glucose, 2gL
-1naNO
3, 0.2gL
-1, KH
2pO
4, 20mg L
-1feSO
4, 5g L
-1mgSO
4, 0.5g L
-1naCl, 40mg L
-1znSO
4, 40mgL
-1cuSO
4, 0.5% wheat bran, 1.5% agarose, 115 ℃ of sterilizing 15min, then divide and are filled to test tube, then 115 ℃ of sterilizing 25min, prepare inclined-plane), cultivate for 32 ℃ and within 6 days, obtain ripe spore.Respectively each is cultured to again to ripe spore inoculating and (55ml itaconic acid fermentation substratum is housed) in 500ml triangular flask to itaconic acid fermentation substratum, every strain bacterium do two bottles parallel, 37 ℃, the 220rpm 72h that ferments.Remove by filter the mycelia in fermented liquid, fermented supernatant fluid carries out acid base titration, measures the total acid in fermented liquid.Because the main organic acid in fermented liquid is methylene-succinic acid, therefore conventionally the total acid recording is thought to the amount of methylene-succinic acid, and calculated the content (Liu Jianjun, the research of itaconic acid fermentation, University Of Science and Technology Of Tianjin's doctorate paper, 2003) of methylene-succinic acid with this.Except several strain bacterial strains produce the obvious decline of acid, all the other most bacterial strains produce acid and are all better than starting strain (referring to Fig. 6).
3) according to producing sour result, choose 22 strains produce acid preferably bacterial strain carry out again multiple sieve, 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 generally higher than starting strain CICC40205 (WT) for result, and wherein the highest bacterial strain C27 has improved 7g/L(9.5% than starting strain).
The purity check of methylene-succinic acid in 3.2 fermented liquids
The fermented supernatant fluid of choosing recombinant bacterial strain C5, C27 and starting strain CICC40205 carries out efficient liquid phase chromatographic analysis (High Performance Liquid Chromatography, HPLC), to analyze the purity of methylene-succinic acid in comparison fermented liquid.Chromatographic column: Bio-rad Aminex HPX-87-H, 300mmx7.8mm; Moving phase: 4mmol/L sulfuric acid; Flow velocity: 0.8ml/min; Temperature: 30 ℃; Check temperature: 30 ℃; 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 with the methylene-succinic acid purity in C27 fermented liquid compared with starting strain CICC40205, all more than 99%.
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, take PgpdAt-seqF(5 '-gctctgtagcttttgccccgtc-3 ') and TtrpC-seqR(5 '-cgagatcctgaacaccatttgtctc-3 ') carry out PCR as primer pair, PgpdAt1-cad-TtrpC element in amplification conversion carrier, agarose gel electrophoresis with 1% is analyzed PCR product, the integration of PgpdAt1-cad-TtrpC element in checking transformant.PgpdAt-seqF primer is positioned in PgpdAt1 promotor, about 260bp place, cad gene start codon upstream, TtrpC-seqR primer is positioned on TtrpC terminator, about 120bp place, cad gene terminator codon downstream, therefore pcr amplification product is than the approximately large 380bp of cad gene, and about 1850bp(is referring to Fig. 9).Electrophoresis result shows the band of the 1850kb that can increase in the transformant of selecting, and using wild-type terreus CICC40205 strain gene group DNA template as negative control, amplification is not to DNA fragmentation.Therefore, prove all successfully to have integrated in these transformants Pg
pdAt1-cad-Ttr
pc expression cassette.
Claims (5)
1. a bacterial strain that improves itaconic acid fermentation output, is characterized in that: described bacterial strain is in terreus, to insert by genetic engineering modified the restructuring terreus that aconitate decarboxylase expression casette PgpdAt1-cad-TtrpC obtains.
2. the construction process of the bacterial strain of a raising itaconic acid fermentation output claimed in claim 1, it is characterized in that: by genetic engineering modified structure cis-aconitic acid decarboxylase gene expression cassette, be inserted into and in terreus, obtain the restructuring terreus that contains cis-aconitic acid decarboxylase expression cassette.
3. the method that the bacterial strain of a utilization raising itaconic acid fermentation output claimed in claim 1 is produced methylene-succinic acid, it is characterized in that: by genetic engineering modified structure cis-aconitic acid decarboxylase gene expression cassette, be inserted into and in terreus, obtain the restructuring terreus that contains cis-aconitic acid decarboxylase expression cassette, cross and express cis-aconitic acid decarboxylase and strengthen catalysis cis-aconitic acid and generate the reaction process of methylene-succinic acid, and then improve methylene-succinic acid output.
4. the method that improves the bacterial strain production methylene-succinic acid of itaconic acid fermentation output by utilization claimed in claim 3, 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. the method that improves the bacterial strain production methylene-succinic acid of itaconic acid fermentation output by utilization claimed in claim 3, is characterized in that: described restructuring terreus is incubated in fermention medium, and described fermention medium is: 140gL
-1glucose, 2g L
-1nH
4nO
3, 0.2g L
-1(NH
4)
2hPO
4, 20mg L
-1feSO
4, 0.4g L
-1mgSO
4, 40mg L
-1znSO
4, 40mg L
-1cuSO
4and water, with sulfuric acid adjustment pH to 3.5,115 ℃ of sterilizing 10min.
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| 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 |
| CN108384815A (en) * | 2018-05-22 | 2018-08-10 | 中国林业科学研究院林产化学工业研究所 | A method of preparing itaconic acid using lignocellulosic material |
| CN110036114A (en) * | 2016-08-26 | 2019-07-19 | 勒萨弗尔公司 | The itaconic acid of raising produces |
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| CN110036114A (en) * | 2016-08-26 | 2019-07-19 | 勒萨弗尔公司 | The itaconic acid of raising produces |
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| CN110527637A (en) * | 2019-07-18 | 2019-12-03 | 中国科学院青岛生物能源与过程研究所 | A kind of Aspergillus terreus bacterial strain producing aconitic acid and its construction method and application |
| CN111944706A (en) * | 2020-08-06 | 2020-11-17 | 中国科学院青岛生物能源与过程研究所 | A recombinant Aspergillus terreus strain producing itaconic acid and its construction method and application |
| CN113999807A (en) * | 2021-11-02 | 2022-02-01 | 南京工业大学 | Construction method of a kind of recombinant strain and its application in itaconic acid production |
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