CN104894048B - Method for improving ferulic acid stress resistance of clostridium beijerinckii - Google Patents
Method for improving ferulic acid stress resistance of clostridium beijerinckii Download PDFInfo
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- CN104894048B CN104894048B CN201510323508.XA CN201510323508A CN104894048B CN 104894048 B CN104894048 B CN 104894048B CN 201510323508 A CN201510323508 A CN 201510323508A CN 104894048 B CN104894048 B CN 104894048B
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- 241000193454 Clostridium beijerinckii Species 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 20
- 230000006518 acidic stress Effects 0.000 title abstract description 3
- KSEBMYQBYZTDHS-HWKANZROSA-M (E)-Ferulic acid Natural products COC1=CC(\C=C\C([O-])=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-M 0.000 title abstract 2
- KSEBMYQBYZTDHS-HWKANZROSA-N ferulic acid Chemical compound COC1=CC(\C=C\C(O)=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-N 0.000 title abstract 2
- 229940114124 ferulic acid Drugs 0.000 title abstract 2
- KSEBMYQBYZTDHS-UHFFFAOYSA-N ferulic acid Natural products COC1=CC(C=CC(O)=O)=CC=C1O KSEBMYQBYZTDHS-UHFFFAOYSA-N 0.000 title abstract 2
- 235000001785 ferulic acid Nutrition 0.000 title abstract 2
- QURCVMIEKCOAJU-UHFFFAOYSA-N trans-isoferulic acid Natural products COC1=CC=C(C=CC(O)=O)C=C1O QURCVMIEKCOAJU-UHFFFAOYSA-N 0.000 title abstract 2
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 22
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims abstract description 15
- 108010014531 FMN Reductase Proteins 0.000 claims abstract description 13
- 239000002773 nucleotide Substances 0.000 claims abstract description 9
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 9
- ACFIXJIJDZMPPO-NNYOXOHSSA-N NADPH Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](OP(O)(O)=O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 ACFIXJIJDZMPPO-NNYOXOHSSA-N 0.000 claims abstract 8
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 32
- 239000002253 acid Substances 0.000 claims description 26
- 239000013612 plasmid Substances 0.000 claims description 22
- 241000894006 Bacteria Species 0.000 claims description 21
- 238000000855 fermentation Methods 0.000 claims description 21
- 230000004151 fermentation Effects 0.000 claims description 21
- 238000003209 gene knockout Methods 0.000 claims description 10
- 230000001419 dependent effect Effects 0.000 claims description 9
- 230000002779 inactivation Effects 0.000 claims description 8
- 238000003780 insertion Methods 0.000 claims description 7
- 230000037431 insertion Effects 0.000 claims description 7
- 230000001580 bacterial effect Effects 0.000 claims description 6
- 241001110912 Clostridium beijerinckii NCIMB 8052 Species 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 2
- 241000193403 Clostridium Species 0.000 claims 1
- 238000010353 genetic engineering Methods 0.000 abstract description 2
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- 239000002609 medium Substances 0.000 description 18
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- 239000005695 Ammonium acetate Substances 0.000 description 13
- 229940043376 ammonium acetate Drugs 0.000 description 13
- 235000019257 ammonium acetate Nutrition 0.000 description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 12
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 9
- 239000008103 glucose Substances 0.000 description 9
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 9
- 235000019341 magnesium sulphate Nutrition 0.000 description 9
- 235000019796 monopotassium phosphate Nutrition 0.000 description 9
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 9
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- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 7
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- 238000006243 chemical reaction Methods 0.000 description 3
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- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 3
- 159000000003 magnesium salts Chemical class 0.000 description 3
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 description 3
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- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical group NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 description 2
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- HYBBIBNJHNGZAN-UHFFFAOYSA-N furfural Chemical compound O=CC1=CC=CO1 HYBBIBNJHNGZAN-UHFFFAOYSA-N 0.000 description 2
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a method for improving ferulic acid stress resistance of clostridium beijerinckii, which belongs to the technical field of genetic engineering, wherein a coding FMN reductase gene depending on NADPH in clostridium beijerinckii is inactivated, so that the gene can not be normally expressed in clostridium beijerinckii, the clostridium beijerinckii is NCIMB 8052, the coding FMN reductase gene depending on NADPH is Cbei _4693, and the nucleotide sequence is shown as SEQ ID NO: 1.
Description
Technical field
The invention belongs to gene engineering technology field, and in particular to a kind of side for improving Clostridium beijerinckii forulic acid resistance
Method.
Background technology
With the exhaustion of petroleum-based energy, biological butanol turns into study hotspot as second generation power source.Traditional acetone-fourth
Alcohol-alcohol fermentation (ABE fermentations) substrate raw material is directly high using glucose, xylose etc., consuming cost.Using it is cheap it is discarded can
The lignocellulosic material (corncob, bagasse, stalk etc.) of regeneration, research is turned into by Production by Microorganism Fermentation butanol
One of emphasis.But lignocellulosic material pretreatment turn into microorganism it is available sugared while also degrade out largely and have
The mortifiers such as machine acid, furfural, 5 hydroxymethyl furfural, phenolic compound, but in the technique for removing the mortifier, cost consumption
Height, therefore, the resistance for improving microorganism itself are even more important.
At present, the degeneration-resistant Journal of Sex Research of biological butanol is concentrated mainly on Clostridium beijerinckii and utilizes ligno-cellulose hydrolysate aspect,
The toxic action of its phenolic compound is especially pronounced.High concentration aldehydes matter can change cell membrane hydrophobic, destroy cell membrane,
Cause thalline dead.Thaddeus Ezeji etc. (Biotechnol.Bioeng.2007,97,1460-1469.) report Bai Shi shuttles
Bacterium mutant strain BA101 does not grow completely under 2g/L asafoetide acid stress；In addition, (the Bioresource such as Thaddeus Ezeji
Technol.2008,99:BA101 5915-5922) is utilized, with the corncob dilute sulfuric acid hydrolysis of XAD-4 resin detoxifications and enzymolysis liquid
For fermentation substrate, it is 9.3g/L to obtain total solvent yield；However, BA101 hardly produces fourth in the dilute acid hydrolysis liquid of non-detoxification
Alcohol.Guo Ting etc. (J and Microbiol Biotechnol., 2012,39 (3), 401-407) researchs find, when corncob and
When 0.5g/L is arrived in the concentration lifting of phenolic compound in bagasse hydrolyzate, Clostridium beijerinckii (Clostridium
Beijerinckii) 8052 do not grow substantially.Richmond etc. has found that syringaldehyde suppresses CoAT expression, has blocked acetic acid and fourth
The conversion of acid, and cause the reduction of quantity of solvent.(Appl Environ Microbiol, 2003,69 (8) such as Tomas:4951-
4965) encoding Heat Shock Protein groESL genes are overexpressed in clostridium acetobutylicum, make inhibitory action of the butanol to somatic cells
85% is reduced, and finally production concentration is improved 33%.The specific mechanism of suppression of the pattern phenolic compound to thalline is not yet
Clearly, but by genetic engineering means the recombinant bacterium obtained produces the resistance of solvent bacterium to improve, and improving butanol yield problem will
Paid attention to.
As can be seen here, Clostridium beijerinckii is improved to toxin such as phenolic compounds in cheap lignocellulosic material hydrolyzate
Resistance, will be one of key issue that lignocellulosic material production butanol industrialization must solve.
The content of the invention
The technical problem to be solved in the invention is to provide a kind of method for improving Clostridium beijerinckii forulic acid resistance.
In order to solve the above technical problems, the present invention adopts the following technical scheme that：
A kind of method for improving Clostridium beijerinckii forulic acid resistance, this method are to depend on coding in Clostridium beijerinckii
NADPH FMN reductase genes inactivation, makes the gene be unable to normal expression in Clostridium beijerinckii.
Wherein, described Clostridium beijerinckii is Clostridium beijerinckii NCIMB 8052, and described coding depends on NADPH FMN also
Nitroreductase gene is Cbei_4693, its nucleotide sequence such as SEQ ID NO：Shown in 1.
Wherein, it is described to inactivate FMN reductase gene of the coding in Clostridium beijerinckii dependent on NADPH, it is by two types
Coding is set to be inactivated dependent on NADPH FMN reductase genes containing sub- gene Knockout.
Wherein, it is described to make FMN reductase gene of the coding dependent on NADPH by two type introne gene Knockouts
Inactivation, comprises the following steps：
(1) software analysis SEQ ID No are utilized：Sequence shown in 1, obtain the nucleotide sequence of introne and inserting for gene
Angle of striking；
(2) obtained intron sequences are building up in two type introne gene knockout plasmids, obtain recombinant plasmid；
(3) the recombinant plasmid transformed Clostridium beijerinckii for obtaining step (2), screening obtain coding and depend on NADPH FMN also
The bacterial strain of nitroreductase gene inactivation.
In step (1), the nucleotide sequence such as SEQ ID No of the introne：Shown in 2, the insertion point of the gene
For SEQ ID No：Between 335bp and 336bp in 1.
In step (2), the two types introne gene knockout plasmid is pWJ plasmids, the nucleotide sequence such as SEQ of the plasmid
ID No：Shown in 5.
The Clostridium beijerinckii that the method for above-mentioned raising Clostridium beijerinckii forulic acid resistance builds to obtain is in protection model of the invention
Within enclosing.
Application of the above-mentioned Clostridium beijerinckii in fermentation prepares butanol is also within protection scope of the present invention.：
The present invention high degeneration-resistant Clostridium beijerinckii (Clostridium beijerinckii) recombinant bacterium, to forulic acid with compared with
Strong resistance, can in the fermentation medium containing the phenolic compound fermenting and producing butanol.(forulic acid is aldehydes matter ratio
A kind of more representational compound,
Wherein, the method for described fermenting and producing butanol comprises the following steps：
1) flat board culture：The high anti-forulic acid restructuring Clostridium beijerinckii of the present invention is seeded to plating medium Anaerobic culturel,
37 DEG C of cultivation temperature, 12~18h of incubation time；
2) seed culture：The high anti-forulic acid restructuring Clostridium beijerinckii of flat board culture is inoculated into seed culture medium, 100mL
The bottled liquid measure 50mL of anaerobism, fills N23min, 37 DEG C of cultivation temperature, 12~18h of incubation time；
3) fermentation production butanol：Seed culture fluid is inoculated into fermentation medium, inoculum concentration 10 (v/v) %, fills N23min,
37 DEG C of fermentation temperature, fermented incubation time are 60~72h.
Described plating medium includes the component of following mass percent：Carbon source 0.3%~1%, nitrogen source 0.5%~
1%th, inorganic salts 0.5%~0.8%, agar 1.5%~2%, remaining be water；Wherein, the carbon source is starch or glucose；Institute
It is organic or inorganic nitrogen-containing compound to state nitrogen source, and wherein inorganic nitrogen-containing compound is the one or more in ammonium acetate, ammonium chloride,
Nitrogen-containing organic compound is the one or more in peptone, dusty yeast and corn steep liquor；Inorganic salts are sodium salt, sylvite, magnesium salts, phosphorus
One or more in hydrochlorate, ferrous salt, agar is added in solid medium.Plating medium is preferably：Dusty yeast 3g/L, egg
White peptone 5g/L, soluble starch 10g/L, ammonium acetate 2g/L, sodium chloride 3g/L, bitter salt 3g/L, potassium dihydrogen phosphate 1g/
L, dipotassium hydrogen phosphate 1g/L, green vitriol 0.1g/L, agar 15g/L, remaining is water, pH 6.
Described seed culture medium includes the component of following mass percent：Carbon source 0.5%~1%, nitrogen source 0.5%~
1%th, inorganic salts 0.5%~0.8%, remaining be water；Described carbon source is starch or glucose；The nitrogen source is organic or inorganic
Nitrogen-containing compound, wherein inorganic nitrogen-containing compound are the mixing of one or both of ammonium acetate and ammonium chloride, organic nitrogen-containing
Compound is one or more of mixing in peptone, dusty yeast and corn steep liquor；Described inorganic salts be sylvite, magnesium salts, calcium salt,
One or more in phosphate, ferrous salt.Seed culture medium is preferably：Dusty yeast 3g/L, peptone 5g/L, soluble starch
10g/L, ammonium acetate 2g/L, sodium chloride 3g/L, bitter salt 3g/L, potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 1g/L, seven
Ferrous sulfate hydrate 0.1g/L, remaining is water, pH 6.
Described fermentation medium includes the component of following mass percent：Carbon source 3%~6%, nitrogen source 0.1%~
0.3%th, inorganic salts 0.1%~0.2%, growth factor 0.05%~0.1%, forulic acid 0.05%, remaining be water；Described carbon
Source is glucose；Described nitrogen source is the one or more in ammonium acetate, ammonium chloride and dusty yeast；Described inorganic salts are sodium
One or more in salt, sylvite, magnesium salts, calcium salt, phosphate, ferrous salt；The growth factor is p-aminobenzoic acid, dimension life
One or more of mixing in plain B1, biotin and corn steep liquor.Fermentation medium is preferably：Glucose 30g/L, ammonium acetate
2.2g/L, potassium dihydrogen phosphate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, sodium chloride 0.01g/L, bitter salt 0.2g/L, seven
Ferrous sulfate hydrate 0.01g/L, Manganous sulfate monohydrate 0.01g/L, corn steep liquor 0.1 (v/v) %, forulic acid 0.5g/L, remaining is
Water, pH 6.6.
Beneficial effect：
(1) present invention is inserted coding in Clostridium beijerinckii dependent on NADPH FMN reductase genes Cbei_4693
After inactivation so that this gene is unable to normal expression, obtains the recombinant bacterial strain of Cbei_4693 gene disruptions.
(2) the invention provides a kind of simple, efficient method for improving Clostridium beijerinckii resistance, obtained by the method
Recombinant bacterial strain have forulic acid compared with high resistance to cold and diseases, when using glucose as carbon source, containing forulic acid in 100ml anaerobism bottles
During fermentation medium culture, butanol yield reaches 5.6g/L, and the starting strain of equal conditions culture does not produce alcohol substantially.
(3) it is high using the recombinant bacterial strain of the inventive method structure, its forulic acid strong stress resistance, butanol yield.
Brief description of the drawings
Fig. 1 is two type introne gene knockout plasmid pWJ of present invention plasmid map.
Fig. 2 is the mechanism figure that the present invention is inactivated using two type Intron insertions.
Fig. 3 is conversion daughter colony PCR electrophoretograms.
Fig. 4 is that recombinant bacterium and starting strain are investigated to the resistance of forulic acid.
Embodiment
According to following embodiments, the present invention may be better understood.It is however, as it will be easily appreciated by one skilled in the art that real
Apply the content described by example and be merely to illustrate the present invention, without should be also without limitation on sheet described in detail in claims
Invention.
Embodiment 1：The construction method of Clostridium beijerinckii Cbei_4693 gene Inactivating mutations strains.
1. design introne：
Cbei_4693 gene orders (such as SEQ ID No for the Clostridium beijerinckii included according to ncbi database：Shown in 1), borrow
Help Software for Design suitably insert gene loci (http://www.clostron.com), selection is inserted in the 335-336 alkali
Between base, and intron sequences are generated, synthesis its sequence of intron sequences S-335 such as SEQ ID NO：Shown in 2.
2.Cbei_4693 inserts the structure of inactivating vectors：
Distinguishing double digestion carrier pWJ with Xho I and BsrG I, (Shanghai Sheng Ke institutes teacher Yang Sheng provides, its sequence such as SEQ ID
NO：Shown in 5) and DNA fragmentation S-335.The purified kit of digestion products (Takara) after purification, passes through T4 ligases
(Takara) connection overnight.By the recombinant plasmid transformed of connection to E. coli DH5a (this laboratory), it is applied to and contains
There are 50ug/ml ammonia benzyl chloramphenicol resistance LB flat boards, 37 DEG C of culture 12-16h, picking transformant, be connected to liquid and contain 50ug/ml ammonia benzyls
In mycin LB culture mediums, 37 DEG C, 200rpm culture 12h, extraction recombinant plasmid (AXYGEN plasmid extraction kits), sequencing is tested
Card, obtain two type introne gene knockout carrier pWJ-335 of Cbei_4693 genes.
3. carrier pWJ-335 methylates：
The Competent in E.coli Top 10/pAN2 (this laboratory) is prepared, successful Cbei_4693 will be sequenced and insert
Enter inactivating vectors pWJ-335 and be transformed into E. coli Top 10, because pAN2 plasmids have tetracyclin resistance, therefore apply
Cloth is to containing 50ug/ml ammonia benzyl mycins and the Double LB flat boards of 10ug/ml tetracyclines, 37 DEG C of culture 12-16h, picking transformant,
It is connected to liquid to contain in 50ug/ml ammonia benzyl mycins and 10ug/ml tetracycline LB culture mediums, 37 DEG C, 200r culture 12h, extracts first
Base deleted carrier pWJ-335 (pAN2 plasmids contain a bacillus subtilis phage gene, can encode transmethylase,
Exogenous plasmid methylating in Escherichia coli can be realized).
4. the electricity knockout plasmid that methylates of conversion is to Clostridium beijerinckii (Clostridium beijerinckii NCIMB
8052)：
1) Clostridium beijerinckii NCIMB 8052 (this laboratory) are seeded to YPS seed culture mediums
(dusty yeast 3g/L, peptone 5g/L, soluble starch 10g/L, ammonium acetate 2g/L, NaCl 3g/L, MgSO4·7H2O3g/L,
KH2PO41g/L, K2HPO41g/L, FeSO4·7H2O 0.1g/L) 37 DEG C be incubated overnight, next day is inoculated into YPS with 5% ratio and trained
Base is supported, 37 DEG C of culture 6-8h, 2 × YTG culture mediums (dusty yeast 16g/L, peptone 10g/L, glucose 5g/ are inoculated into 10%
L, sodium chloride 5g/L) 37 DEG C of culture 3h, OD600=1.
2) 50ml bacterium solutions are taken, 5000rpm, 4 DEG C of centrifugation 10min, abandon supernatant.Again with ETM buffer solutions (270mM sucrose,
0.6mM Na2HPO4, 4.4mM Na2HPO4, 10mM MgCl2·7H2O) it is resuspended；Ibid centrifuge, remove supernatant, buffered again with ETM
Liquid is resuspended, and ibid centrifuges, thoroughly takes supernatant.
3) the pWJ-335 plasmids for taking 1ug to methylate are added to the electric revolving cup of 0.2cm precoolings；1mL ET buffer solutions are used again
(270mM sucrose, 0.6mM Na2HPO4, 4.4mM Na2HPO4) step 2) bacterium mud is resuspended, take 200ul to be added to electric revolving cup, gently
Mix.
4) turned using MicroPulserTM electroporations electricity, condition be voltage 1.8kV, resistance 200 Ω, electric capacity 2.5uF, electric
1mL 2 × YTG culture mediums are added immediately after hitting, are transferred to recovery 2-3h in sterile centrifugation tube.
5) the above-mentioned bacterium solutions of 200ul are taken, are applied to the YPS solid mediums containing 10ug/ml erythromycin, are cultivated 2-3 days.
5. screen Cbei_4693 insertion Inactivating mutations strains：
Picking transformant, use primer Cbei-4693-T-S (its sequence such as SEQ ID NO：Shown in 3) and Cbei-4693-
T-A (its sequence such as SEQ ID NO：Shown in 4), bacterium colony PCR checkings are carried out to transformant, filter out Intron insertion genome
Mutant strain (after insertion, PCR is amplified on gene band electrophoretogram than wild type about 1Kbp).The mutant strain being correctly inserted into is passed
In generation, three times, while is coated on containing Erythromycinresistant and without on the YPS solid mediums of Erythromycinresistant, filters out knockout matter
The mutant strain (mutant strain that can not be grown on Erythromycinresistant flat board) that grain is lost.
Embodiment 2：The mitotic stability of the recombinant bacterium of Cbei_4693 genes inactivation.
Culture medium prescription described in the present embodiment：
Plating medium：Dusty yeast 3g/L, peptone 5g/L, soluble starch 10g/L, ammonium acetate 2g/L, sodium chloride 3g/
L, bitter salt 3g/L, potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 1g/L, green vitriol 0.1g/L, agar
15g/L, remaining is water, pH 6.
Seed culture medium：Dusty yeast 3g/L, peptone 5g/L, soluble starch 10g/L, ammonium acetate 2g/L, sodium chloride 3g/
L, bitter salt 3g/L, potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 1g/L, green vitriol 0.1g/L, remaining is
Water, pH 6.
Fermentation medium：Glucose 30g/L, ammonium acetate 2.2g/L, potassium dihydrogen phosphate 0.5g/L, dipotassium hydrogen phosphate 0.5g/
L, sodium chloride 0.01g/L, bitter salt 0.2g/L, green vitriol 0.01g/L, Manganous sulfate monohydrate 0.01g/L,
Corn steep liquor 0.1% (v/v), remaining is water, pH 6.6.
The Clostridium beijerinckii recombinant bacterium of the Cbei_4693 gene disruptions built in embodiment 1 is seeded to flat board culture
Base Anaerobic culturel, 37 DEG C of cultivation temperature, incubation time 12h.The recombinant bacterium of flat board culture is inoculated into seed culture medium, cultivated
37 DEG C of temperature, the bottled liquid measure 15mL of 25mL Xiao Te anaerobism, fills N23min, 37 DEG C of cultivation temperature, incubation time 12h；Seed is connect
Kind inoculum concentration 10% (v/v), 37 DEG C of fermentation temperature, the bottled liquid measure 50mL of 100mL Xiao Te anaerobism, is filled into fermentation medium
N23min, fermented and cultured 72h.
The Clostridium beijerinckii recombinant bacterium of the Cbei_4693 genes inactivation built in embodiment 1 continuously turns on solid medium
Connect 7 times, it is found that obtained strains and starting strain are identical in morphological feature and cultural characteristic, growth conditions are good.With grape
For sugar in the fermentation medium of carbon source, to detect the mitotic stability of recombinant bacterium, recombinant bacterium passes on the fermentation test result such as institute of table 1
Show.
Table 1
It was found from experimental result, by 7 continuous passages, the total solvent yield and butanol yield of recombinant bacterium are relatively stable, tool
There is preferable mitotic stability, can be as the production bacterial strain further researched and developed.
Embodiment 3：Recombinant bacterium and starting strain are investigated to forulic acid resistance.
Plating medium：Dusty yeast 3g/L, peptone 5g/L, soluble starch 10g/L, ammonium acetate 2g/L, sodium chloride 3g/
L, bitter salt 3g/L, potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 1g/L, green vitriol 0.1g/L, agar
15g/L, remaining is water, pH 6.
Seed culture medium：Dusty yeast 3g/L, peptone 5g/L, soluble starch 10g/L, ammonium acetate 2g/L, sodium chloride 3g/
L, bitter salt 3g/L, potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 1g/L, green vitriol 0.1g/L, remaining is
Water, pH 6.
Fermentation medium：Glucose 30g/L, ammonium acetate 2.2g/L, potassium dihydrogen phosphate 0.5g/L, dipotassium hydrogen phosphate 0.5g/
L, sodium chloride 0.01g/L, bitter salt 0.2g/L, green vitriol 0.01g/L, Manganous sulfate monohydrate 0.01g/L,
Corn steep liquor 0.1 (v/v) %, forulic acid 0.5g/L, remaining is water, pH 6.6.
By the Clostridium beijerinckii recombinant bacterium and starting strain 8052 of the Cbei_4693 gene disruptions built in embodiment 1
It is seeded to plating medium Anaerobic culturel, 37 DEG C of cultivation temperature, incubation time 12h.The recombinant bacterium of flat board culture is inoculated into kind
In sub- culture medium, 37 DEG C of cultivation temperature, the bottled liquid measure 15mL of 25mL Xiao Te anaerobism, N is filled23min, 37 DEG C of cultivation temperature, during culture
Between 12h；Seed is inoculated into fermentation medium, inoculum concentration 10 (v/v) %, 37 DEG C of fermentation temperature, 100mL Xiao Te anaerobism is bottled
Liquid measure 50mL, fills N23min, fermented and cultured 72h.Vapor detection is carried out, final result is shown in Fig. 4, hence it is evident that highlights restructuring of the present invention
Bacterium is to forulic acid high resistance to cold and diseases, and butanol yield reaches 5.6g/L, and the butanol yield of starting strain 8052 (is hardly produced for 0.7g/L
Alcohol).
Claims (7)
- A kind of 1. method for improving Clostridium beijerinckii forulic acid resistance, it is characterised in that this method is will to be encoded in Clostridium beijerinckii FMN reductase genes dependent on NADPH inactivate, and the gene is unable to normal expression in Clostridium beijerinckii；Described Clostridium beijerinckii is Clostridium beijerinckii NCIMB 8052, and described coding is dependent on NADPH FMN reductase genes Cbei_4693, its nucleotide sequence such as SEQ ID NO：Shown in 1.
- 2. the method according to claim 1 for improving Clostridium beijerinckii forulic acid resistance, it is characterised in that described by Bai Shi In clostridium coding dependent on NADPH FMN reductase genes inactivate, be by two type introne gene Knockouts make coding according to Rely the FMN reductase genes inactivation in NADPH.
- 3. the method according to claim 2 for improving Clostridium beijerinckii forulic acid resistance, it is characterised in that described passes through Two type introne gene Knockouts make coding be inactivated dependent on NADPH FMN reductase genes, comprise the following steps：(1) software analysis SEQ ID No are utilized：Sequence shown in 1, obtain the nucleotide sequence of introne and the insertion position of gene Point；(2) obtained intron sequences are building up in two type introne gene knockout plasmids, obtain recombinant plasmid；(3) the recombinant plasmid transformed Clostridium beijerinckii for obtaining step (2), screening obtain FMN reductase of the coding dependent on NADPH The bacterial strain of gene inactivation.
- 4. the method according to claim 3 for improving Clostridium beijerinckii forulic acid resistance, it is characterised in that in step (1), The nucleotide sequence of the introne such as SEQ ID No：Shown in 2, the insertion point of the gene is SEQ ID No：In 1 Between 335bp and 336bp.
- 5. the method according to claim 3 for improving Clostridium beijerinckii forulic acid resistance, it is characterised in that in step (2), The two types introne gene knockout plasmid is pWJ plasmids, the nucleotide sequence such as SEQ ID No of the plasmid：Shown in 5.
- 6. the method for the raising Clostridium beijerinckii forulic acid resistance described in any one of Claims 1 to 5 builds obtained Bai Shi shuttles Bacterium.
- 7. application of the Clostridium beijerinckii in fermentation prepares butanol described in claim 6.
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| WO2008144060A2 (en) * | 2007-05-17 | 2008-11-27 | Tetravitae Bioscience, Inc. | Methods and compositions for producing solvents |
| CN103820367A (en) * | 2014-02-27 | 2014-05-28 | 南京工业大学 | Genetic engineering strain for high yield of butanol and application thereof |
| CN103911334A (en) * | 2014-04-16 | 2014-07-09 | 广州甘蔗糖业研究所 | Clostridium beijerinckii with high stress resistance and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008144060A2 (en) * | 2007-05-17 | 2008-11-27 | Tetravitae Bioscience, Inc. | Methods and compositions for producing solvents |
| CN103820367A (en) * | 2014-02-27 | 2014-05-28 | 南京工业大学 | Genetic engineering strain for high yield of butanol and application thereof |
| CN103911334A (en) * | 2014-04-16 | 2014-07-09 | 广州甘蔗糖业研究所 | Clostridium beijerinckii with high stress resistance and application thereof |
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