CN112608935A - Method for producing acetone by modifying metabolic pathway of clostridium acetobutylicum - Google Patents
Method for producing acetone by modifying metabolic pathway of clostridium acetobutylicum Download PDFInfo
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- CN112608935A CN112608935A CN202011498111.1A CN202011498111A CN112608935A CN 112608935 A CN112608935 A CN 112608935A CN 202011498111 A CN202011498111 A CN 202011498111A CN 112608935 A CN112608935 A CN 112608935A
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Abstract
A method for modifying a clostridium acetobutylicum metabolic pathway to produce acetone belongs to the field of genetic engineering. According to the invention, the recombinant plasmid is constructed and introduced into the clostridium acetobutylicum, and the transformed clostridium acetobutylicum is fermented to produce acetone. The key point is to construct a recombinant plasmid containing three genes of ThlA, ctfB and adc, and enable the recombinant plasmid to be expressed in clostridium acetobutylicum to generate a target product of acetone. The invention constructs a recombinant plasmid PIMP1-ThlA-ctfB-adc, and successfully introduces the recombinant plasmid PIMP1-ThlA-ctfB-adc into clostridium acetobutylicum to express and produce acetone.
Description
Technical Field
A method for producing acetone by fermenting modified clostridium acetobutylicum by constructing recombinant plasmids and introducing the recombinant plasmids into the clostridium acetobutylicum belongs to the field of genetic engineering.
Background
The production of chemicals by means of microbial fermentation is a hotspot of current research, and the production meets the requirements of constructing clean, low-carbon, safe and efficient energy systems proposed by nineteen parties because of the advantages of energy conservation, environmental protection, high efficiency and the like. With the development of scientific technology and the modernization of the world, the dependence of human beings on energy is increasing. The mass exploitation and utilization of fossil energy in the last century leads the fossil energy and raw material to be nearly exhausted, the emission of waste gas and fertilizer can bring huge pollution to the environment in the process of processing and using fossil fuel, greenhouse gas generated in the process of fuel combustion also brings huge influence to the earth climate, and thinking about how to produce renewable energy and how to reduce greenhouse gas becomes a subject of more and more attention.
Acetone is an important raw material in chemical production and is mainly used as a solvent and a precursor of methyl methacrylate and bisphenol a required for synthesizing polycarbonate plastics. The global production of acetone is based on fossil resources and is combined with phenol production and cumene production processes. Information processing services reports show that global acetone demand is increasing every year as the world population increases. While recognizing the consumable nature of petroleum as a resource and the continuing pressure on the environment from carbon dioxide pollution, research and industry are also striving to find more environmentally sustainable processes for producing large volumes of chemicals and fuels.
Clostridium acetobacter is a strictly anaerobic, acetic acid-producing bacterium, which can either be H2+CO2The fertilizer can grow in an autotrophic mode by using an energy source and a carbon source, and can grow in a heterotrophic mode by using fructose, pyruvic acid and fumaric acid as carbon sources. Acetoicum can transform H by Wood-Ljungdahl pathway2+CO2Conversion to acetyl-coa, which in turn can convert fructose to acetyl-coa via the glycolytic pathway and further metabolize to acetate. Acetyl coenzyme A can be further converted into acetone by exogenously introducing three genes of ThlA (thiolase), ctfB (butyrate coenzyme A transferase) and adc (acetoacetate decarboxylase), so that the production process of acetone is realized.
Disclosure of Invention
The invention utilizes the gene engineering technology to transform the clostridium acetobutylicum, so that the clostridium acetobutylicum can produce acetone. The key point is to construct a recombinant plasmid containing three genes of ThlA, ctfB and adc, and enable the recombinant plasmid to be expressed in clostridium acetobutylicum to generate a target product of acetone.
The construction process of the recombinant plasmid PIMP1-ThlA-ctfB-adc comprises the following specific steps:
1. inoculating the preserved strain Clostridium propionicum into fresh CGM culture medium, and culturing at 30 deg.C in a constant temperature box overnight to recover the strain.
The formula of the CGM culture medium is as follows: (NH)4)2SO4 2g、K2HPO4 1g、KH2PO4 0.5g、MgSO4·7H20.1g of O, 4g of peptone, 2g of yeast powder, 20g of glucose and FeSO4·7H2O 0.015g、CaCl2·2H2O 0.015g、MnSO4·H2O 0.01g、CoCl2 0.02g、ZnSO40.002g, made up to 1L with deionized water.
2. Inoculating the recovered clostridium butyricum into a fresh CGM culture medium, culturing for 12-16 hours in a constant temperature box at 30 ℃, and centrifugally collecting thalli. The whole genome of the clostridium propionicum is extracted by using a bacterial whole genome extraction kit and is used as a PCR amplification DNA template.
3. Primers were designed according to thlA, CtfB, adc, with the forward primer being F and the reverse primer being R.
ThlA-F cgggatcccgatgaaagaagttgtaatagc
ThlA-R cgggatcccgctagcacttttctagcaata
CtfB-F cggaattccgatgattaatgataaaaacct
CtfB-R cggaattccgctaaacagccatgggtcta
Adc-F cgatcgatcgatgttaaaggatgaagta
Adc-R cgatcgatcgttacttaagataatcatat
Respectively amplifying target fragments ThlA, CtfB and adc by PCR, wherein the single enzyme cutting sites are respectively selected as follows: BamHI, EcoRI, ClaI. The length of the ThlA gene fragment is 1179bp, the length of the CtfB gene fragment is 666bp, and the length of the adc gene fragment is 735 bp. The method specifically comprises the following steps:
step (1): preparing a reaction system according to the following proportion:
step (2): the PCR program was set as: 1. pre-denaturation at 98 ℃ for 3 min; 2. denaturation at 98 ℃ for 10 s; 3. renaturation is carried out for 30s at ThlA 57 ℃, CtfB 53 ℃ and adc 51 ℃; 4. extension at 72 ℃, thlA 72s, CtfB 40s, adc 45 s; 5. extending for 10min at 72 ℃; 6. storing at constant temperature of 4 ℃. Wherein 2-4 cycles are set by steps for 30 times.
And (3): and detecting an amplification result by agarose electrophoresis, wherein whether a target band appears between 1000bp and 2000bp of ThlA and between 500bp and 750bp of CtfB and adc is seen.
4. Connecting the target fragment with a vector PIMP, wherein the system is as follows: vector PIMP 2.5. mu.L, target fragment 6. mu.L, 10 XBuffer 1. mu.L, T4 ligase 0.5. mu.L, total 10. mu.L. The system was left to react overnight at 4 ℃.
5. The ligation product was transformed into E.coli trans10 competent cells for preservation
The LB liquid culture medium for culturing the escherichia coli comprises the following components: NaCl, 5 g; 5g of yeast extract powder; tryptone, 10 g. Adding deionized water to a constant volume of 1L, and sterilizing at 121 deg.C. 1.8g of agar powder was added per 100mL of LB solid medium.
Secondly, the recombinant plasmid is introduced into a Clostridium acetobacter (Clostridium acetoicum) strain, and the specific operation is as follows:
(1) taking out the C.aceticium glycerin tube from a refrigerator at the temperature of-80 ℃, completely pouring the C.aceticium glycerin tube into the C.aceticium culture medium, immediately placing the culture medium inoculated with the clostridium acetate spore suspension into a water bath at the temperature of 70 ℃, thermally shocking for 10 minutes, then cooling the culture medium to the room temperature, and standing, culturing and activating the culture medium at the temperature of 37 ℃. Inoculating the clostridium acetobacter aceti in a C.aceticum culture medium by using the spore suspension according to the inoculation amount of 1 percent of volume fraction, and performing static culture at 37 ℃.
Formula of aceticum culture medium: resazurin 0.5mg, NH4Cl 1g，K2HPO4 0.45g，KH2PO4 0.33g，MgSO4·7H20.1g of O, 20mL of vitamin solution, 20mL of trace element solution, 2g of yeast powder and NaHCO310g of L-cysteine-HCl 0.5g and 10g of fructose, adding deionized water to a constant volume of 1L, adjusting the pH value to 7.4-7.8, subpackaging into 10 250ml infusion bottles, and filling N2Removing oxygen and recharging CO2And sterilizing at 121 deg.C for 30 min. Wherein 1L of the vitamin solution comprises: biotin 2mg, folic acid 2mg, pyridoxic acid HCl 10mg, thiamine HCl 2H2O5 mg, riboflavin 5mg, nicotinic acid 5mg, D-Ca-pantothenate 5mg, vitamin B120.1mg, 5mg of p-aminobenzoic acid and 5mg of lipoic acid. 1L of the trace element solution contained: nitrilotriacetic acid 1.5g, MgSO4·7H2O 3g、MnSO4·H2O 0.5g、NaCl 1g、FeSO4·7H2O 0.1g、CoSO4·7H2O 0.18g、CaCl2·2H2O 0.1g、ZnSO4·7H2O 0.18g、CuSO4·5H2O 0.01g、KAl(SO4)2·12H2O 0.02g、H3BO3 0.01g、Na2MoO4·2H2O 0.01g、NiCl2·6H2O 0.01g、Na2SeO3·5H2O 0.3mg、NaWO4·2H2O 0.4mg。
(2) When the OD value of clostridium acetobacter reaches 0.6-0.8, preparing a clostridium acetobacter competence, taking 190 mu L of the clostridium acetobacter competence, taking 10 mu L of recombinant plasmid PIMP1-ThlA-CtfB-adc, adding the recombinant plasmid into an ice-precooled electric transfer cup, introducing the recombinant plasmid into the clostridium acetobacter competence in an electric transfer mode, immediately adding a prepared 400 mu LC. Adding 2g of agar powder into 100mL of C.aceticum culture medium to obtain a solid culture medium, adding 100 mu L of erythromycin with the mass concentration of 10mg/mL into 100mL of C.aceticum solid culture medium during plate pouring, taking 100 mu L of erythromycin-coated resistant plates from the bacterial liquid cultured for 4-5 hours, and culturing at 37 ℃. A plurality of single clones are picked for colony PCR verification. And (4) selecting bacteria from the bacterial colonies which are verified to be correct, culturing overnight, and preserving the strains.
(3) And (3) inoculating the recombinant bacteria verified to be correct in the step (2) into a C.aceticum culture medium again, fermenting again in three parallel, and sampling at intervals of 8 hours. Measuring acetone content in C.aceticum fermentation liquor by using high performance liquid chromatography area external standard method, and taking 5mmol/L H2SO4As a mobile phase, a 300X 7.8mm organic acid column from BIO-RAD company was used as a separation column, a differential refractometer was used, the separation temperature was controlled at 65 ℃ and the separation flow rate was 0.6mL/min, and an acetone sample was measured. Then preparing the acetone standard substance into concentration gradients of 5mg/mL, 4mg/mL, 3mg/mL, 2mg/mL and 1mg/mL, making a standard working curve by using the peak area to the mass concentration, and then calculating the actual concentration of the sample in the sample according to the peak areas of the components in the sample.
Drawings
FIG. 1 is a diagram of the construction of expression vector PIMP 1-ThlA-ctfB-adc.
Detailed Description
First, acquisition of target gene and construction of recombinant plasmid
1. Inoculating the preserved strain Clostridium propionicum into fresh CGM culture medium, and culturing at 30 deg.C in a constant temperature box overnight to recover the strain.
2. Inoculating the recovered clostridium propionicum into a fresh CGM culture medium, culturing in a constant temperature box at 30 ℃ for 12-16 hours, and centrifugally collecting thalli. The whole genome of the clostridium propionicum is extracted by using a bacterial whole genome extraction kit and is used as a PCR amplification DNA template.
3. Designing a swimming primer according to the ThlA, CtfB and adc. Respectively amplifying target fragments ThlA, CtfB and adc by PCR, wherein the single enzyme cutting sites are respectively selected as follows: BamHI, EcoRI, ClaI. The length of the ThlA gene fragment is 1179bp, the length of the CtfB gene fragment is 666bp, and the length of the adc gene fragment is 735 bp. The method specifically comprises the following steps:
step (1): preparing a reaction system according to the following proportion:
wherein, the concentration of the DNA template is 100-200 ng/muL, and the concentrations of the upstream primer and the downstream primer are 10 mmol/muL;
step (2): the PCR program was set as: 1. pre-denaturation at 98 ℃ for 3 min; 2. denaturation at 98 ℃ for 10 s; 3. renaturation is carried out for 30s at ThlA 57 ℃, CtfB 53 ℃ and adc 51 ℃; 4. extension at 72 ℃, thlA 72s, CtfB 40s, adc 45 s; 5. extending for 10min at 72 ℃; 6. storing at constant temperature of 4 ℃. Wherein 2-4 cycles are set in a floating manner, and the times are 30 times.
And (3): and detecting an amplification result by agarose electrophoresis, wherein whether a target band appears between 1000bp and 2000bp of ThlA and between 500bp and 750bp of CtfB and adc is seen.
4. Connecting the obtained target fragment with a vector PIMP1, wherein the system is as follows: vector PIMP 12.5. mu.L, target fragment 6. mu.L, 10 XBuffer 1. mu.L, T4 ligase 0.5. mu.L, total 10. mu.L. The system was left to react overnight at 4 ℃.
5. The ligation product is transformed into escherichia coli trans10 competent cells for preservation, and the method specifically comprises the following steps:
step (1): adding 10 μ L of the ligation product into the trans10 competent cells of Escherichia coli, and transforming the product into trans10 competent cells by ice-water bath at 0 deg.C for 30min, heat shock at 42 deg.C for 45s, and ice-water bath at 0 deg.C for 3-5 min;
step (2): adding 500 mu L LB liquid into the system for culturing, and culturing for 45-60 min at 37 ℃ and 200rmp by shaking table to express resistance genes on plasmids and recover thalli;
and (3): centrifuging the system for 30s under the condition of 4000-6000 rmp, pouring out part of supernatant, uniformly mixing the rest, taking 100 mu L of the mixture, coating the mixture on an LB solid culture medium added with ampicillin, and pouring the mixture into a 37 ℃ incubator for standing and culturing overnight. Wherein 100. mu.L of 0.1g/ml ampicillin was added to 100ml of LB solid culture rack.
And (4): selecting a single colony on the plate for colony PCR verification, and setting a PCR program as follows: 1. pre-denaturation at 94 ℃ for 3 min; 2. denaturation at 94 ℃ for 30 s; 3. renaturation is carried out for 30s at ThlA 57 ℃, CtfB 53 ℃ and adc 51 ℃; 4. extension at 72 ℃, thlA 72s, CtfB 40s, adc 45 s; 5. extending for 10min at 72 ℃; 6. storing at constant temperature of 4 ℃. Wherein 2-4 cycles are set by steps for 30 times. Colonies with a bright band between 1000bp to 2000bp and between 500bp to 750bp of CtfB and adc were picked up by ThlA, and inoculated into 4. mu.L of 0.1g/mL ampicillin 4mL of LB liquid medium for 12 hours.
And (5): extracting plasmids for sequencing, and storing strains with correct sequencing for later use.
Second, clostridium acetobutylicum electrocution conversion
1, the recombinant plasmid PIMP1-ThlA-ctfB-adc was extracted and transformed into Clostridium acetobacter by electroporation in an anaerobic chamber. The specific steps of the clostridium acetobutylicum conversion are as follows:
(1) picking single colony of clostridium acetobacter on a plate, inoculating into a C.aceticum culture medium, performing static culture at 37 ℃, and taking out when the OD value of the clostridium acetobacter reaches 0.6-0.8.
(2) Precooling 100mL of the bacterial liquid obtained in the step (1) and a 50mL centrifuge tube on ice, subpackaging the bacterial liquid into the 50mL centrifuge tube, placing on ice for 5-10min, then centrifuging at 4500rpm for 10min at 4 ℃, and discarding the supernatant.
(3) The pellet was broken up with 20-30mL of ETM buffer, centrifuged at 4500rpm at 4 ℃ for 10min, and the supernatant was discarded. And finally, 2.5mL of ET buffer solution is added to break the bacteria and mix uniformly, so that the C.aceticum competence is completed.
(4) And (3) adding 10 mu L of the recombinant plasmid PIMP1-ThlA-ctfB-adc into 190 mu L of the C.aceticum competent cells obtained in the step (3), precooling on ice for 10-15min, then carrying out an electric transformation process, and discharging for 4-5 ms by using a 1mm electric shock cup and 2.5 kV.
(5) Immediately adding 400 mul of C.aceticum culture solution prepared in advance after the electric conversion is finished, uniformly mixing, completely taking out, adding another 400 mul of C.aceticum culture solution, and culturing for 4-5 hours. Adding 2g of agar powder into 100mL of C.aceticum culture medium to obtain a solid culture medium, adding 100 mu L of erythromycin with the mass concentration of 10mg/mL into 100mL of C.aceticum solid culture medium during plate turnover, taking 100 mu L of erythromycin-coated resistant plates from a bacterial solution cultured for 4-5 hours, culturing at 37 ℃, and selecting a plurality of monoclones for colony PCR verification. And (4) selecting bacteria from the bacterial colonies which are verified to be correct, culturing overnight, and preserving the strains.
The preparation method of the ET buffer solution and the ETM buffer solution comprises the following steps: firstly, 46.2105g of cane sugar and Na are added2HPO4 0.0426g、NaH2PO40.2637g of the mixture is dissolved by purified water to be 500mL, 50mL of the mixture is taken out to be ET buffer solution, and MgCl is added20.4725g of the buffer solution is dissolved in 450mL of purified water to obtain an ETM buffer solution, and the ETM buffer solution is subpackaged into 5 bottles of 90mL of ETM buffer solution. ET buffer solution, ETM buffer solution all dispose in the anaerobic phase, and the pure water is boiled in advance and is put in the anaerobism case and oxygen removal.
2. The expression process of the target gene is as follows:
(1) and (3) inoculating the positive clone bacteria obtained in the step (1) into a C.aceticum culture medium, and standing overnight at 37 ℃ for culture to recover the strains.
(3) And inoculating the obtained recovered recombinant bacteria into a C.aceticum culture medium again, wherein 100 mg/ml of erythromycin is added into every 100ml of the C.aceticum culture medium, performing three-parallel secondary fermentation at 37 ℃, and sampling 1ml every eight hours. Measuring by high performance liquid chromatography area external standard methodDetermining acetone content in C.aceticum fermentation liquor by 5mmol/L H2SO4As a mobile phase, an organic acid column of 300X 7.8mm manufactured by BIO-RAD company is used as a separation column, a differential refraction detector is selected, the separation temperature is controlled to be 65 ℃, the separation flow rate is 0.6mL/min, and an acetone sample is measured. Then preparing the acetone standard substance into concentration gradients of 5mg/mL, 4mg/mL, 3mg/mL, 2mg/mL and 1mg/mL, and making a standard working curve according to peak area to mass concentration to obtain a standard curve y which is 144679x-11756R2At fermentation time 56 hours, 0.9998, a maximum yield of 1.5mg/ml to 2.5mg/ml was achieved.
The innovation point of the patent is that the recombinant plasmid PIMP1-ThlA-ctfB-adc is constructed and successfully introduced into clostridium acetobutylicum to be expressed to produce acetone.
The parameters such as temperature and time in the above embodiments of the present invention are preferably selected, and experiments prove that the purpose of the present invention can be achieved beyond the scope of the above embodiments, and therefore, all the values cannot be exhausted, and are not described herein again.
The above embodiments are only exemplary embodiments of the present invention, and are not intended to limit the present invention, and the scope of the present invention is defined by the claims. Various modifications and equivalents may be made by those skilled in the art within the spirit and scope of the present invention, and such modifications and equivalents should also be considered as falling within the scope of the present invention.
Claims (3)
1. A method for modifying a metabolic pathway of clostridium acetobutylicum to produce acetone is characterized in that the construction process of a recombinant plasmid PIMP1-ThlA-ctfB-adc specifically comprises the following operations:
1) inoculating the preserved clostridium propionicum into a fresh CGM culture medium, and culturing in a 30 ℃ incubator overnight to recover the strain;
the formula of the CGM culture medium is as follows: (NH)4)2SO4 2g、K2HPO4 1g、KH2PO4 0.5g、MgSO4·7H20.1g of O, 4g of peptone, 2g of yeast powder, 20g of glucose and FeSO4·7H2O 0.015g、CaCl2·2H2O 0.015g、MnSO4·H2O 0.01g、CoCl20.02g、ZnSO40.002g, adding deionized water to 1L;
2) inoculating the recovered clostridium butyricum into a fresh CGM culture medium, placing the culture medium in a constant temperature box at 30 ℃ for culturing for 12-16 hours, and centrifugally collecting thalli; extracting the whole genome of the clostridium propionicum as a PCR amplification DNA template by using a bacterial whole genome extraction kit;
3) designing primers according to the ThlA, CtfB and adc, wherein an upstream primer is F, and a downstream primer is R, which is shown in the specification;
ThlA-F cgggatcccgatgaaagaagttgtaatagc
ThlA-R cgggatcccgctagcacttttctagcaata
CtfB-F cggaattccgatgattaatgataaaaacct
CtfB-R cggaattccgctaaacagccatgggtcta
Adc-F cgatcgatcgatgttaaaggatgaagta
Adc-R cgatcgatcgttacttaagataatcatat
respectively amplifying target fragments ThlA, CtfB and adc by PCR, wherein the single enzyme cutting sites are respectively selected as follows: BamH1, EcoR1, Cla 1; the length of the ThlA gene fragment is 1179bp, the length of the CtfB gene fragment is 666bp, and the length of the adc gene fragment is 735 bp;
the method specifically comprises the following steps:
step (1): preparing a reaction system according to the following proportion:
step (2): the PCR program was set as: 1. pre-denaturation at 98 ℃ for 3 min; 2. denaturation at 98 ℃ for 10 s; 3. ThlA 57 ℃, CtfB 53 ℃, adc 51 ℃ renaturation 30s 4, 72 ℃ extension, ThlA 72s, CtfB 40s, adc 45 s; 5. extending for 10min at 72 ℃; 6. preserving at constant temperature of 4 ℃; wherein 2-4 steps are set for circulation, and the times are 30 times;
and (3): detecting an amplification result by agarose electrophoresis, and judging whether a target band appears when ThlA is 1000bp-2000bp and CtfB and adc are 500bp-750 bp;
step (4), connecting the obtained target fragment with a vector PIMP1, wherein the system is as follows: vector PIMP 12.5 μ L, target fragment 6 μ L, 10 XBuffer 1 μ L, T4 ligase 0.5 μ L, total 10 μ L; the system is placed at 4 ℃ for reaction overnight;
and (5) transforming the ligation product into an escherichia coli trans10 competent cell for preservation.
2. The method of claim 1, wherein step (5) comprises the steps of:
step (1): adding 10 μ L of the ligation product into the trans10 competent cells of Escherichia coli, and transforming the product into trans10 competent cells by ice-water bath at 0 deg.C for 30min, heat shock at 42 deg.C for 45s, and ice-water bath at 0 deg.C for 3-5 min;
step (2): adding 500 mu L LB liquid into the system for culturing, and culturing for 45-60 min at 37 ℃ and 200rmp by shaking table to express resistance genes on plasmids and recover thalli;
and (3): centrifuging the system for 30s under the condition of 4000-6000 rmp, pouring off part of supernatant, uniformly mixing the rest 100-200 mu L of supernatant and precipitate, coating the mixture on an LB solid culture medium added with ampicillin, and pouring the mixture in a 37 ℃ incubator for standing culture overnight; wherein 100 mul of 0.1g/ml ampicillin is added into 100ml LB solid culture frame;
and (4): selecting a single colony on the plate for colony PCR verification, and setting a PCR program as follows: 1. pre-denaturation at 94 ℃ for 3 min; 2. denaturation at 94 ℃ for 30 s; 3. renaturation is carried out for 30s at ThlA 57 ℃, CtfB 53 ℃ and adc 51 ℃; 4. extension at 72 ℃, thlA 72s, CtfB 40s, adc 45 s; 5. extending for 10min at 72 ℃; 6. preserving at constant temperature of 4 ℃; wherein 2-4 steps are set for circulation, and the times are 30 times; selecting a colony with a brighter band between 1000bp to 2000bp of ThlA and between 500bp to 750bp of CtfB and adc, and inoculating 4 mu L of LB liquid culture medium with mass concentration of 0.1g/mL ampicillin for culturing for 12 hours;
and (5): extracting plasmids for sequencing, and storing strains with correct sequencing for later use.
3. The method of claim 1, wherein step (5) further comprises the steps of:
extracting the recombinant plasmid PIMP1-ThlA-CtfB-adc, and transforming the recombinant plasmid into clostridium acetobutylicum in an anaerobic box through electric shock; the specific operation of the clostridium acetobutylicum conversion is as follows:
(1) taking out the C.aceticum glycerin tube from a refrigerator at minus 80 ℃, completely pouring the C.aceticum glycerin tube into the C.aceticum culture medium, immediately placing the C.aceticum glycerin tube into a water bath at 70 ℃, thermally shocking the C.aceticum culture medium for 10 minutes, then cooling the C.aceticum glycerin tube to room temperature, and standing, culturing and activating the C.aceticum culture medium at 37 ℃; inoculating clostridium acetobacter in a C.aceticum culture medium by using a spore suspension with the inoculation amount of 1 percent of volume fraction, and performing standing culture at 37 ℃;
formula of aceticum culture medium: resazurin 0.5mg, NH4Cl 1g，K2HPO4 0.45g，KH2PO4 0.33g，MgSO4·7H20.1g of O, 20mL of vitamin solution, 20mL of trace element solution, 2g of yeast powder and NaHCO310g of L-cysteine-HCl 0.5g and 10g of fructose, adding deionized water to a constant volume of 1L, adjusting the pH value to 7.4-7.8, subpackaging into 10 250ml infusion bottles, and filling N2Removing oxygen and recharging CO2Sterilizing at 121 deg.C for 30 min; wherein 1L of the vitamin solution comprises: biotin 2mg, folic acid 2mg, pyridoxic acid HCl 10mg, thiamine HCl 2H2O5 mg, riboflavin 5mg, nicotinic acid 5mg, D-Ca-pantothenate 5mg, vitamin B120.1mg, 5mg of p-aminobenzoic acid and 5mg of lipoic acid; 1L of the trace element solution contained: nitrilotriacetic acid 1.5g, MgSO4·7H2O 3g、MnSO4·H2O 0.5g、NaCl 1g、FeSO4·7H2O 0.1g、CoSO4·7H2O 0.18g、CaCl2·2H2O 0.1g、ZnSO4·7H2O 0.18g、CuSO4·5H2O 0.01g、KAl(SO4)2·12H2O 0.02g、H3BO30.01g、Na2MoO4·2H2O 0.01g、NiCl2·6H2O 0.01g、Na2SeO3·5H2O 0.3mg、NaWO4·2H2O 0.4mg；
Picking single clostridium acetobacter colonies on a flat plate, inoculating the single clostridium acetobacter culture medium, performing static culture at 37 ℃, and taking out the single clostridium acetobacter culture medium when the OD value of the single clostridium acetobacter reaches 0.6-0.8;
(2) precooling 100mL of bacterial liquid with OD value of 0.6-0.8 obtained in the step (1) and 50mL of centrifuge tube on ice, subpackaging the bacterial liquid into 50mL of centrifuge tube, placing on ice for 5-10min, centrifuging at 4500rpm at 4 ℃ for 10min, and discarding supernatant;
(3) dispersing the precipitate with 20-30mL ETM buffer solution, centrifuging at 4500rpm at 4 deg.C for 10min, and discarding the supernatant; finally, 2.5mL of ET buffer solution is added to break up the bacteria and mix uniformly, so as to prepare the C.aceticum competence;
(4) adding 10 mu L of recombinant plasmid PIMP1-ThlA-ctfB-adc into 190 mu L of C.aceticum competent cells obtained in the step (3), precooling on ice for 10-15min, then carrying out an electric transformation process, discharging at 2.5kV by using a 1mm electric shock cup, and discharging for 4-5 ms;
(5) immediately adding 400 mul of C.aceticum culture solution prepared in advance after the electrotransformation is finished, uniformly mixing, completely taking out, adding another 400 mul of C.aceticum culture solution, and culturing for 4-5 hours; adding 2g of agar powder into 100mL of C.aceticum culture medium to obtain a solid culture medium, adding 100 mu L of erythromycin with the mass concentration of 10mg/mL into 100mL of C.aceticum solid culture medium during plate pouring, taking 100 mu L of erythromycin-coated resistant plates from a bacterial solution cultured for 4-5 hours, and culturing at 37 ℃; selecting a plurality of monoclonals for colony PCR verification; selecting bacteria from the bacterial colony which is verified to be correct, culturing overnight, and preserving the strains;
the preparation method of the ET buffer solution and the ETM buffer solution comprises the following steps: firstly, 46.2105g of cane sugar and Na are added2HPO4 0.0426g、NaH2PO40.2637g of the mixture is dissolved by purified water to be 500mL, 50mL of the mixture is taken out to be ET buffer solution, and MgCl is added20.4725g of the buffer solution is dissolved in 450mL of purified water and is uniformly dissolved to obtain an ETM buffer solution; ET buffer solution, ETM buffer solution all dispose in the anaerobic phase, and the pure water is boiled in advance and is put in the anaerobism case and oxygen removal.
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| CN101423850A (en) * | 2007-11-02 | 2009-05-06 | 赢创德固赛有限责任公司 | Fermentative production of acetone from renewable resources by means of novel metabolic pathway |
| CN102414314A (en) * | 2009-04-23 | 2012-04-11 | 赢创德固赛有限责任公司 | Cells and method for producing acetone |
| CN102796692A (en) * | 2011-05-25 | 2012-11-28 | 中国科学院上海生命科学研究院 | Method for improving sugar utilization rate of clostridium acetobutylicum in fermentation of mixed sugar |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101423850A (en) * | 2007-11-02 | 2009-05-06 | 赢创德固赛有限责任公司 | Fermentative production of acetone from renewable resources by means of novel metabolic pathway |
| CN102414314A (en) * | 2009-04-23 | 2012-04-11 | 赢创德固赛有限责任公司 | Cells and method for producing acetone |
| US20120101304A1 (en) * | 2009-04-23 | 2012-04-26 | Evonik Degussa Gmbh | Cells and method for producing acetone |
| CN102796692A (en) * | 2011-05-25 | 2012-11-28 | 中国科学院上海生命科学研究院 | Method for improving sugar utilization rate of clostridium acetobutylicum in fermentation of mixed sugar |
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