CN102604878A - Genetically-engineered bacteria for producing 3-hydroxy butanone as well as construction method and application thereof - Google Patents
Genetically-engineered bacteria for producing 3-hydroxy butanone as well as construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a genetically-engineered bacteria for producing 3-hydroxy butanone. The genetically-engineered bacteria is obtained by transforming klebsiella pneumoniae CICC10011 by using a Nox gene through an expression carrier pDK7. The invention further discloses a preparation method and application of the genetically-engineered bacteria. The genetically-engineered bacteria can reduce the intracellular NADH/NAD<+> ratio significantly, improve the accumulation amount of the 3-hydroxy butanone, increase the conversion rate of the glucose, reduce the content of the byproduct which is 2,3-butanediol and shorten the fermentation time. The genetically-engineered bacteria has important industrial application value to the preparation of the 3-hydroxy butanone through the biological method.
Description
Technical field
The invention belongs to gene engineering technology field, be specifically related to a kind of genetic engineering bacterium and construction process and application of the 3-of producing oxobutanol.
Background technology
The 3-oxobutanol has another name called acetoin, methyl acetylcarbinol; Natural being present in dairy products and some fruit is a kind of widely used flavouring agent, and it has pleasant cream fragrance; It is edible that China GB2760-86 stipulates that it allows, and U.S. food is 2008 with extracting association (FEMA) security number.The 3-oxobutanol is extremely extensive as the fragrance applications scope, and consumption is also very big.In addition, the 3-oxobutanol can also be widely used in various fields such as daily use chemicals food, pharmacy, coating, liquid crystal material as a kind of hardware and software platform compound.2004, USDOE classified it as one of hardware and software platform compound of 30 kinds of preferential developments utilizations, and in recent years, Along with people's is to the continuous growth of 3-oxobutanol demand, and the working method and the applied research of relevant 3-oxobutanol have caused people's extensive concern.
Main two kinds of the compound method of present 3-oxobutanol: the one, chemical synthesis, the 2nd, biological synthesis process.It is low that chemical synthesis exists product yield; Shortcomings such as environmental pollution is more serious; And its quality product is difficult to reach the maximum consumer field of present 3-oxobutanol---the requirement of flavouring agent; The raw material of chemical synthesis that even more serious is mostly is to utilize non-renewable fossil resource---oil, and raw material sources also are restricted.Biological synthesis process comprises enzyme process and microbe fermentation method again; Enzyme process is to be that raw material generates the 3-oxobutanol under the effect of specific enzymes with dimethyl diketone or butyleneglycol; This method transformation efficiency can reach 100%; And product has specific rotation, but the same raw material sources with chemical method are restricted, and obtains very difficulty of a large amount of specific enzymes.The bacterial strain that is used for production of 3-oxobutanol and research at present mainly contains bacillus (Bacillus), Klebsiella (Klebisella), enterobacter (Enterobacter), serratia (Serratia) and lactococcus genus (Lactococcus) etc., and also having the investigator to find also can little accumulation 3-oxobutanol in some yeast.But in most of bacterial strain metabolic processes, the accumulation volume of 3-oxobutanol is all very low, mostly as 2, and the metabolic by-prods of 3-butyleneglycol and producing.
Summary of the invention
Technical problem to be solved by this invention provides a kind of genetic engineering bacterium of high yield 3-oxobutanol.
The technical problem that the present invention also will solve provides the construction process of said gene engineering bacteria.
The technical problem that the present invention will solve at last provides the application of said gene engineering bacteria.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is following:
The genetic engineering bacterium of 3-oxobutanol is produced in one strain, and it is that the Nox gene is transformed the genetic engineering bacterium that Klebsiella pneumonia (Klebsiella pneumoniae) CICC10011 obtains through expression vector pDK7.
The construction process of said gene engineering bacteria; Utilize round pcr from plasmid vector pTEF-Sup35C, to amplify the Nox gene; The Nox gene is connected in expression vector pDK7 MCS place, obtains recombinant plasmid pDK7-Nox, again recombinant plasmid pDK7-Nox is transformed Klebsiella pneumonia (Klebsiella pneumoniae) CICC10011; The Klebsiella pneumonia that obtains recombinating promptly produces the genetic engineering bacterium Klebsiella pneumoniae CICC10011-pDK7-Nox of 3-oxobutanol.
Expression vector pDK7 of the present invention derives from Mike J.Merrick professor (AFRC-IPSR Nitrogen Fixation Laboratory; University of Sussex; Brighton BNI 9RQ, UK), its construction process reference Construction of Multicopy Expression Vectors for Regulated Overproduction of Proteins in Klebsiella pneumoniae and Other Enteric Bacteria [J] .DIETMELM KLEINER; WYATT PAUL AND MIKE J.MERRICK.Journal of General Microbiology; 1988,134,1779-1784.
Above-mentioned construction process specifically may further comprise the steps:
(1) clone of gene Nox:
According to the middle Nox gene (Gen Bank:AF014458.2) of streptococcus pneumoniae (S.pneumoniae) that Genebank announces, sequences Design is closed primer:
P1：5’-CGG
GGTACC(Kpn?I)TAAGGAGGATATACATATGA?AAGTCACAGTT-3’；
P2：5’-CGG
CTGCAG(Pst?I)TTAAGCGTTAACTGATT-3’；
The primer two ends are introduced restriction enzyme site Kpn I and Pst I respectively, are that template is accomplished the PCR reaction with pTEF-Sup35C;
The 8 tube reaction systems of PCR are: Buffer 22.5uL, ddH
2O 141.3uL, MgCl
213.5uL, dNTP-mix 18uL, primer P14.5uL, primer P24.5uL, Taq enzyme 2.7uL; After mixing, be divided into 8 pipes, add the 2uL template DNA to every pipe again; PCR reaction conditions: 94 ℃ of preparatory sex change 3.5min; Carry out 30 circulating reactions: 94 ℃ of sex change 45s, 52.6 ℃ of annealing 45s, 72 ℃ are extended 90s; Last 72 ℃ of insulation 10min, reaction finishes back 4 ℃ of preservations, behind the reaction terminating; 0.8% agarose gel electrophoresis detects, and behind PCR purification kit purifying, is connected with the pUC18 carrier; Carry out sequencing, Kpn I and Pst I enzyme are cut reorganization pUC18 carrier, reclaim the 1.35Kb fragment; KpnI and Pst I enzyme are cut the pDK7 carrier, reclaim big fragment product, the double digestion product are connected obtain recombinant plasmid pDK7-Nox under the effect of T4DNA ligase enzyme;
(2) acquisition of recombination engineering bacteria:
Recombinant plasmid pDK7-Nox is transformed Klebsiella pneumonia (Klebsiella pneumoniae) CICC10011; Coating contains the paraxin flat board of 40 μ g/mL; The picking positive transformant; And carry out bacterium colony PCR and identify, obtain the Klebsiella pneumonia of recombinating, called after Klebsiella pneumoniae CICC10011-pDK7-Nox.
The application of said gene engineering bacteria in producing the 3-oxobutanol.
Utilizing genetic engineering bacterium, is that the concrete technology of fermenting substrate production 3-oxobutanol is following with glucose:
(1) starting strain: Klebsiella pneumoniae CICC10011-pDK7-Nox;
(2) seed culture:
Seed culture medium: glucose 50～80g/L, peptone 5～10g/L, yeast extract paste 3～7g/L, NaCl 5～10g/L, paraxin 20～40 μ g/mL, solvent is a water;
Culture condition: the 250mL triangular flask, liquid amount 50mL, 31～37 ℃ of culture temperature, shaking speed 120～180r/min cultivates 12～24h;
(3) fermentation culture:
Substratum is formed: glucose 80～200g/L, and peptone 5～10g/L, yeast extract paste 3～7g/L, NaCl 5～10g/L, paraxin 20～40 μ g/mL, solvent is a water;
Culture condition: inoculum size 10% (v/v), 31～37 ℃ of leavening temperatures add sec.-propyl-β-D-sulfo-galactopyranoside (IPTG) and induce when OD reaches 0.6～0.8, the final concentration 1.0mmol/L of IPTG, shaking speed 180～250r/min, fermentation 72～96h.
Through Klebsiella pneumonia metabolism network analysis is found: during the fermentation; Klebsiella pneumonia Klebsiella pneumoniae CICC10011 often mainly accumulates a large amount of 2; The 3-butyleneglycol; Can prevent to accumulate excessive sour pair cell like this and cause murder by poisoning, can also can also regulate the interior NADH/NAD of born of the same parents as the effect such as utilizations grade of can degrading again when the needs of deposit carbon source
+Thereby, regulating cell metabolism flow path direction., cell is partial to NADH/NAD in the born of the same parents when being in one than the oxidation state environment
+The 3-oxobutanol accumulation of just having a meeting when low.So when in the cell during overexpression NADH oxidase, NADH is oxidized to NAD
+, NADH/NAD in the born of the same parents
+Significantly reduce, because the amount of available NADH reduces in the born of the same parents, the redox constant needs more to select to accumulate oxidized form meta-bolites 3-oxobutanol cell in the born of the same parents in order to keep.Bacterial strain of the present invention can significantly reduce NADH/NAD in the born of the same parents
+Ratio, the accumulation volume of raising 3-oxobutanol, the transformation efficiency of increase glucose effectively reduces by product 2 simultaneously, 3-butyleneglycol content, shortening fermentation time.
Beneficial effect:
Genetic engineering bacterium of the present invention utilizes glucose fermentation to produce the 3-oxobutanol under aerobic condition, has broken through the limitation of the inefficient accumulation of original strain fermentation 3-oxobutanol.Fed-batch fermentation finishes: 200g/L glucose capable of using, obtain 3-oxobutanol 74g/L, and transformation efficiency 37%, theoretical peak rate of conversion are 50%; Under the same conditions; Original strain can only utilize 160g/L glucose, obtains the 3-oxobutanol of 25g/L, compares with original klebsiella; The ability that recombinant bacterial strain is produced the 3-oxobutanol significantly improves, and has improved substrate glucose utilized ability and transformation efficiency.Produce 3-oxobutanol output with present bibliographical information Klebsiella pneumonia and compare, the product final concentration also higher (Production of (and 2S, 3S)-2; 3-butanediol and (3S)-acetoin from glucose using resting cells of Klebsiella pneumonia and Bacillus subtilis [J] .Zhen Liu, Jiayang Qin, Chao Gao; Et al.BIORESOURCE TECHNOLOGY; 2011,102 (22): the Klebsiella pneumonia that utilizes that 10741-10744.) present document is reported is produced 3-oxobutanol output about 56.7g/L; Add micro-paraxin in addition during the fermentation; Reach the purpose that suppresses the assorted bacterium of part, significantly reduced more incident microbiological contamination phenomenon in course of industrialization, improve the quality of products; Fermentation time also shortens to some extent, for microbe fermentation method suitability for industrialized production 3-oxobutanol is had laid a good foundation.
Description of drawings
Fig. 1 is the NOX gene fragment that amplifies.Wherein, M:DNA Marker DL5000; 1,2:Nox;
Fig. 2 is that NOX is connected the result with pDK7.Wherein, M:DNA Marker DL5000; 1,2:pDK7-Nox; 3,4:pDK7.
Embodiment
According to following embodiment, can understand the present invention better.Yet, those skilled in the art will readily understand that the described content of embodiment only is used to explain the present invention, and the present invention that should also can not limit in claims to be described in detail.
Embodiment 1: the clone of gene Nox.
According to the middle Nox gene (GenBank:AF014458.2) of streptococcus pneumoniae (S.pneumoniae) that Genebank announces, sequences Design is closed primer:
P1：5’-CGG
GGTACC(Kpn?I)TAAGGAGGATATACATATGAAAGTCACAGTT-3’；
P2：5’-CGG
CTGCAG(Pst?I)TTAAGCGTTAACTGATT-3’；
The primer two ends are introduced restriction enzyme site KPn I and Pst I (underscore part) respectively, are that template is accomplished the PCR reaction with pTEF-Sup35C; The 8 tube reaction systems of PCR are Buffer 22.5uL, ddH
2O 141.3uL, MgCl
213.5uL, dNTP-mix 18uL, primer P14.5uL, primer P24.5uL, Taq enzyme 2.7uL.After mixing, be divided into 8 pipes, add the 2uL template DNA to every pipe again.PCR reaction conditions: 94 ℃ of preparatory sex change 3.5min; Carry out 30 circulating reactions: 94 ℃ of sex change 45s, 52.6 ℃ of annealing 45s, 72 ℃ are extended 90s; Last 72 ℃ of insulation 10min, reaction finishes back 4 ℃ of preservations, behind the reaction terminating; 0.8% agarose gel electrophoresis detects, and behind PCR purification kit purifying, is connected with the pUC18 carrier; Carry out sequencing, Kpn I and Pst I enzyme are cut reorganization pUC18 carrier, reclaim the 1.35Kb fragment; Kpn I and Pst I enzyme are cut the pDK7 carrier, reclaim big fragment product, the double digestion product are connected obtain recombinant plasmid pDK7-Nox under the effect of T4DNA ligase enzyme;
Embodiment 2: recombinant plasmid pDK7-Nox transforms Klebsiella pneumonia Klebsiella pneumoniae CICC10011.
Utilize electroporation apparatus that recombinant plasmid pDK7-Nox is transformed Klebsiella pneumonia Klebsiella pneumoniae CICC10011, electricity transforms parameter and is: voltage 2.5kv, 200 Ω, burst length 4.5msec.Electric shock transforms and finishes, and will coat on the LB substratum that contains 40 μ g/L paraxin through the Klebsiella pneumoniae CICC10011 that electric shock was handled, and obtains positive colony Klebsiella pneumoniae CICC10011-pDK7-Nox.
Embodiment 3: the plasmid stability of engineering bacteria Klebsiella pneumoniae CICC10011-pDK7-Nox is investigated.
Investigation method: do not contain to 3mL the LB substratum of paraxin from the dull and stereotyped upward picking list colony inoculation of fresh conversion, cultivate 12h down for 37 ℃ and make seed, change kind of a cultivation 24h; Be 20 generations of propagating, change kind of cultivation 5 times, be for 100 generations; Above-mentioned bacterium liquid coated do not contain on the antibiotic LB flat board; Therefrom 100 single bacterium colonies of picking are put respectively and are being added and do not adding on the flat board of paraxin, cultivate 12～16h down for 37 ℃, and relatively the colony count on the flat board that adds and do not add paraxin is its stability.
Investigate the result: its plasmid stability is 99% after 100 generations of propagating.
Embodiment 4: NADH oxidase enzyme activity determination among the engineering bacteria Klebsiella pneumoniae CICC 10011-pDK7-Nox.
(1) starting strain: Klebsiella pneumoniae CICC 10011-pDK7-Nox
(2) used substratum: yeast extract paste 5g/L, peptone 10g/L, NaCl 10g/L, IPTG 1mmol/L, paraxin 40 μ g/L
(3) enzyme activity determination method: reaction TV 1mL comprises 50mM 0.1mol/L potassium phosphate buffer (pH 7.0); 0.3 mM EDTA, 10 μ M FAD, 0.29mM β-NADH; Add the reaction of 10 μ L crude enzyme liquids then after 10 minutes, 340nm surveys the variation of solution light absorption value.1 enzyme unit definition alive is that catalysis 1 μ molNADH oxidation forms NAD in 25 ℃ of following per minutes
+Required enzyme amount.The preparation of enzyme extract is following: reorganization bacterium Klebsiella pneumoniae CICC10011-pDK7-Nox induces 8h under under the condition that adds IPTG 32 ℃; The centrifugal supernatant of abandoning; With the resuspended thalline of 0.1mol/L potassiumphosphate (pH 7.0); The centrifugal again supernatant that goes, thalline is resuspended with the 0.1mol/L potassiumphosphate (pH 7.0) of 10% volume; The ultrasonication cell, centrifugal, supernatant is as enzyme extract.Protein determination is measured with the Bradford method.
Investigate the result: exist IPTG to induce under the situation, NADH oxidase enzyme is lived and is 2.72U/mg among the reorganization bacterium Klebsiella pneumoniae CICC10011-pDK7-Nox.
Comparative Examples 1: NADH oxidase enzyme activity determination among the original strain Klebsiella pneumoniae CICC10011.
(1) starting strain: Klebsiella pneumoniae CICC10011
(2) used substratum: yeast extract paste 5g/L, peptone 10g/L, NaCl 10g/L
(3) enzyme activity determination method: reaction TV 1mL comprises 50mM 0.1mol/L potassium phosphate buffer (pH7.0); 0.3mM EDTA, 10 μ M FAD, 0.29mM β-NADH; Add the reaction of 10 μ L crude enzyme liquids then after 10 minutes, 340nm surveys the variation of solution light absorption value.1 enzyme unit definition alive is that catalysis 1 μ molNADH oxidation forms NAD in 25 ℃ of following per minutes
+Required enzyme amount.The preparation of enzyme extract is following: Klebsiella pneumoniae CICC10011 cultivates 8h down at 32 ℃; The centrifugal supernatant of abandoning; With the resuspended thalline of 0.1mol/L potassiumphosphate (pH 7.0), the centrifugal again supernatant that goes, thalline is resuspended with the 0.1mol/L potassiumphosphate (pH 7.0) of 10% volume; The ultrasonication cell, centrifugal, supernatant is as enzyme extract.Protein determination is measured with the Bradford method.
Investigate the result: NADH oxidase enzyme is lived and is 0.01U/mg among the original bacterium Klebsiella pneumoniae CICC10011.
Embodiment 5: NADH/NAD among the engineering bacteria Klebsiella pneumoniae CICC10011-pDK7-Nox
+Measure.
(1) starting strain: Klebsiella pneumoniae CICC 10011-pDK7-Nox;
(2) used substratum: yeast extract paste 5g/L, peptone 10g/L, NaCl 10g/L, IPTG 1mmol/L, paraxin 40 μ g/L;
(3) NADH extracting process: get 1mL bacterium liquid, 12000r/min, centrifugal 5min removes supernatant, adds 300 μ LNaOH (0.2mol/L) extraction NADH, destroys NAD
+, 50 ℃ of water-bath 10min are placed on rapidly then and are cooled to 0 ℃ in the ice, then drop by drop interpolation 300 μ LHCl (0.1mol/L) neutralize, 12000r/min, centrifugal 10min, supernatant is moved in another pipe for use ,-20 ℃ of preservations.
NAD
+Extracting process: get 1mL bacterium liquid, 12000r/min, centrifugal 5min removes supernatant, adds 300 μ LHCl (0.2mol/L) extraction NAD
+, destroy NADH, 50 ℃ of water-bath 10min are placed on rapidly then and are cooled to 0 ℃ in the ice, then drop by drop interpolation 300 μ LNaOH (0.1mol/L) neutralize, 12000r/min, centrifugal 10min, supernatant is moved in another pipe for use ,-20 ℃ of preservations.
Mixed reaction solution is formed: the 16.6mmol/L PES of isopyknic 1.0mol/L Bicine buffer (pH8.0), straight alcohol, 40mmol/LEDTA (pH8.0), 4.2mmol/L MTT and double volume is mixed 30 ℃ of water-bath 10min.
NADH, NAD
+Measuring method: during reaction; Add the pure water of 50 μ L neutral cell extracts, 0.3 mL, the mixed reaction solution of 0.6mL and the ethanol dehydrogenase (500U/mL) of 50 μ L successively; Behind the mixing, placing wavelength is the ultraviolet-visible pectrophotometer measurement down of 570nm rapidly.
Investigate the result: NADH/NAD among the reorganization bacterium Klebsiella pneumoniae CICC10011-pDK7-Nox
+Be 0.45 ± 0.02.
Comparative Examples 2: NADH/NAD among the original strain Klebsiella pneumoniae CICC 10011
+Measure.
(1) starting strain: Klebsiella pneumoniae CICC10011;
(2) used substratum: yeast extract paste 5g/L, peptone 10g/L, NaCl 10g/L, IPTG 1mmol/L, paraxin 40 μ g/L;
(3) NADH extracting process: get 1mL bacterium liquid, 12000r/min, centrifugal 5min removes supernatant, adds 300 μ LNaOH (0.2mol/L) extraction NADH, destroys NAD
+, 50 ℃ of water-bath 10min are placed on rapidly then and are cooled to 0 ℃ in the ice, then drop by drop interpolation 300 μ LHCl (0.1mol/L) neutralize, 12000r/min, centrifugal 10min, supernatant is moved in another pipe for use ,-20 ℃ of preservations.
NAD
+Extracting process: get 1mL bacterium liquid, 12000r/min, centrifugal 5min removes supernatant, adds 300 μ LHCl (0.2mol/L) extraction NAD
+, destroy NADH, 50 ℃ of water-bath 10min are placed on rapidly then and are cooled to 0 ℃ in the ice, then drop by drop interpolation 300 μ LNaOH (0.1mol/L) neutralize, 12000r/min, centrifugal 10min, supernatant is moved in another pipe for use ,-20 ℃ of preservations.
Mixed reaction solution is formed: the 16.6mmol/L PES of isopyknic 1.0mol/L Bicine buffer (pH8.0), straight alcohol, 40mmol/LEDTA (pH8.0), 4.2mmol/L MTT and double volume is mixed 30 ℃ of water-bath 10min.
NADH, NAD
+Measuring method: during reaction; Add the pure water of 50 μ L neutral cell extracts, 0.3 mL, the mixed reaction solution of 0.6mL and the ethanol dehydrogenase (500U/mL) of 50 μ L successively; Behind the mixing, placing wavelength is the ultraviolet-visible pectrophotometer measurement down of 570nm rapidly.
Investigate the result: NADH/NAD among the reorganization bacterium Klebsiella pneumoniae CICC10011
+Be 0.67 ± 0.02.
Embodiment 6: engineering bacteria Klebsiella pneumoniae CICC10011-pDK7-Nox utilizes the glucose shake flask fermentation to produce the 3-oxobutanol.
(1) starting strain: Klebsiella pneumoniae CICC 10011-pDK7-Nox
(2) seed culture:
Seed culture medium: glucose 50g/L, yeast extract paste 5g/L, peptone 10g/L, NaCl 10g/L; Paraxin 40ug/mL;
Culture condition: the 250mL triangular flask, liquid amount 50mL, 37 ℃ of culture temperature, shaking speed 180r/min cultivates 12h.
(3) fermentation culture:
Fermention medium: glucose 200g/L, yeast extract paste 5g/L, peptone 10g/L, NaCl 10g/L; Paraxin 40ug/mL;
Culture condition: the 250mL triangular flask, liquid amount 50mL, inoculum size 10% (v/v), 37 ℃ of leavening temperatures are induced when OD adds IPTG 0.6～0.8 the time, the final concentration 1.0mmol/L of IPTG, shaking speed 180r/min is about fermentation 72h.
The fermentation result: 200g/L glucose obtains 56g/L 3-oxobutanol through conversion.
Comparative Examples 3: original bacterium Klebsiella pneumoniae CICC10011 utilizes glucose in shaking bottle, to ferment and produces the 3-oxobutanol.
(1) starting strain: Klebsiella pneumoniae CICC10011
(2) seed culture:
Seed culture medium: glucose 50g/L, yeast extract paste 5g/L, peptone 10g/L, NaCl 10g/L;
Culture condition: 250mL triangular flask, liquid amount are 50mL, 37 ℃ of culture temperature, and shaking speed 180r/min cultivates 12h.
(3) fermentation culture:
Fermention medium: glucose 200g/L, yeast extract paste 5g/L, peptone 10g/L, NaCl 10g/L;
Culture condition: the 250mL triangular flask, liquid amount 50mL, inoculum size 10% (v/v), 37 ℃ of leavening temperatures, shaking speed 180r/min is about fermentation 72h.
The fermentation result: 200g/L glucose obtains 15g/L 3-oxobutanol through conversion.
Embodiment 7: engineering bacteria Klebsiella pneumoniae CICC10011-pDK7-Nox utilizes glucose fed-batch fermentation in the 3L fermentor tank to produce the 3-oxobutanol.
(1) starting strain: Klebsiella pneumoniae CICC10011-pDK7-Nox
(2) seed culture
Seed culture medium: glucose 50g/L, yeast extract paste 5g/L, peptone 10g/L, NaCl 10g/L; Paraxin 40ug/mL;
Culture condition: the 250mL triangular flask, liquid amount 50mL, culture temperature is 37 ℃, shaking speed 180r/min cultivates 12h.
(3) fermentation culture:
Fermention medium: glucose 200g/L, yeast extract paste 5g/L, peptone 10g/L, NaCl 10g/L; Paraxin 40ug/mL;
Culture condition: the 3L fermentor tank (New Brunswick Scientific, NJ), inoculum size 10% (v/v); 37 ℃ of leavening temperatures; When adding IPTG 0.6～0.8 the time, OD induces the final concentration 1.0mmol/L of IPTG, mixing speed 220rpm; About fermentation 72h, batch feeding stream adds glucose and keeps glucose concn about 30g/L.
Fermentation result: substrate glucose consumption 200g/L, product 3-oxobutanol concentration 74g/L.
Comparative Examples 4: original bacterium Klebsiella pneumoniae CICC10011 utilizes glucose fed-batch fermentation in the 3L fermentor tank to produce the 3-oxobutanol.
(1) starting strain: Klebsiella pneumoniae CICC10011
(2) seed culture
Seed culture medium: glucose 50g/L, yeast extract paste 5g/L, peptone 10g/L, NaCl 10g/L;
Culture condition: the 250mL triangular flask, liquid amount 50mL, culture temperature is 37 ℃, shaking speed 180r/min cultivates 12h.
(3) fermentation culture:
Fermention medium: glucose 200g/L, yeast extract paste 5g/L, peptone 10g/L, NaCl 10g/L;
Culture condition: the 3L fermentor tank (New Brunswick Scientific, NJ), inoculum size 10% (v/v), 37 ℃ of leavening temperatures, mixing speed 220rpm, about fermentation 72h, batch feeding stream adds glucose and keeps glucose concn about 30g/L.
Fermentation result: substrate glucose consumption 160g/L, product 3-oxobutanol concentration 25g/L.
Embodiment 8:
With the method for embodiment 7, different is:
(2) seed culture:
Seed culture medium: glucose 80g/L, peptone 5g/L, yeast extract paste 7g/L, NaCl 5g/L, paraxin 20 μ g/mL, solvent is a water;
Culture condition: the 250mL triangular flask, liquid amount 50mL, 31 ℃ of culture temperature, shaking speed 120r/min cultivates 24h;
(3) fermentation culture:
Substratum is formed: glucose 100g/L, and peptone 5g/L, yeast extract paste 3g/L, NaCl 5g/L, paraxin 20 μ g/mL, solvent is a water;
Culture condition: inoculum size 10% (v/v), 31 ℃ of leavening temperatures add IPTG and induce when OD reaches 0.6～0.8, the final concentration 1.0mmol/L of IPTG, shaking speed 180r/min, fermentation 96h.
Claims (5)
1. the genetic engineering bacterium of 3-oxobutanol is produced in a strain, it is characterized in that, it is that the Nox gene is transformed the genetic engineering bacterium that Klebsiella pneumonia (Klebsiella pneumoniae) CICC10011 obtains through expression vector pDK7.
2. the construction process of the described genetic engineering bacterium of claim 1; It is characterized in that; Utilize round pcr from plasmid vector pTEF-Sup35C, to amplify the Nox gene; The Nox gene is connected in expression vector pDK7 MCS place, obtains recombinant plasmid pDK7-Nox, again recombinant plasmid pDK7-Nox is transformed Klebsiella pneumonia (Klebsiella pneumoniae) CICC10011; The Klebsiella pneumonia that obtains recombinating promptly produces the genetic engineering bacterium Klebsiella pneumoniae CICC 10011-pDK7-Nox of 3-oxobutanol.
3. construction process according to claim 2 is characterized in that it may further comprise the steps:
(1) clone of gene Nox:
Primer is closed in the middle Nox gene order design of streptococcus pneumoniae (S.pneumoniae) according to Genebank announces:
P1：5’-CGG
GGTACCTAAGGAGGATATACATATGAAAGTCACAGTT-3’；
P2：5’-CGG
CTGCAGTTAAGCGTTAACTGATT-3’；
The primer two ends are introduced restriction enzyme site Kpn I and Pst I respectively, are that template is accomplished the PCR reaction with pTEF-Sup35C;
The 8 tube reaction systems of PCR are: Buffer 22.5uL, ddH
2O 141.3uL, MgCl
213.5uL, dNTP-mix18uL, primer P14.5uL, primer P24.5uL, Taq enzyme 2.7uL; After mixing, be divided into 8 pipes, add the 2uL template DNA to every pipe again; PCR reaction conditions: 94 ℃ of preparatory sex change 3.5min; Carry out 30 circulating reactions: 94 ℃ of sex change 45s, 52.6 ℃ of annealing 45s, 72 ℃ are extended 90s; Last 72 ℃ of insulation 10min, reaction finishes back 4 ℃ of preservations, behind the reaction terminating; 0.8% agarose gel electrophoresis detects, and behind PCR purification kit purifying, is connected with the pUC18 carrier; Carry out sequencing, Kpn I and Pst I enzyme are cut reorganization pUC18 carrier, reclaim the 1.35Kb fragment; KpnI and Pst I enzyme are cut the pDK7 carrier, reclaim big fragment product, the double digestion product are connected obtain recombinant plasmid pDK7-Nox under the effect of T4DNA ligase enzyme;
(2) acquisition of recombination engineering bacteria:
Recombinant plasmid pDK7-Nox is transformed Klebsiella pneumonia (Klebsiella pneumoniae) CICC10011; Coating contains the paraxin flat board of 40 μ g/mL; The picking positive transformant; And carry out bacterium colony PCR and identify, obtain the Klebsiella pneumonia of recombinating, called after Klebsiella pneumoniae CICC 10011-pDK7-Nox.
4. the application of the described genetic engineering bacterium of claim 1 in producing the 3-oxobutanol.
5. application according to claim 4 is characterized in that, utilizes genetic engineering bacterium, is that the technology of fermenting substrate production 3-oxobutanol is following with glucose:
(1) starting strain: Klebsiella pneumoniae CICC10011-pDK7-Nox;
(2) seed culture:
Seed culture medium: glucose 50～80g/L, peptone 5～10g/L, yeast extract paste 3～7g/L, NaCl 5～10g/L, paraxin 20～40 μ g/mL, solvent is a water;
Culture condition: the 250mL triangular flask, liquid amount 50mL, 31～37 ℃ of culture temperature, shaking speed 120～180r/min cultivates 12～24h;
(3) fermentation culture:
Substratum is formed: glucose 80～200g/L, and peptone 5～10g/L, yeast extract paste 3～7g/L, NaCl 5～10g/L, paraxin 20～40 μ g/mL, solvent is a water;
Culture condition: inoculum size 10% (v/v); 31～37 ℃ of leavening temperatures add sec.-propyl-β-D-sulfo-galactopyranoside and induce, the final concentration 1.0mmol/L of sec.-propyl-β-D-sulfo-galactopyranoside when OD reaches 0.6～0.8; Shaking speed 180～250r/min, fermentation 72～96h.
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| CN101008019A (en) * | 2007-01-29 | 2007-08-01 | 山东省食品发酵工业研究设计院 | Use of Bacillus subtilis (Ehrenberg)Cohn in preparing 3-hydroxy butanone |
| EP1820848A1 (en) * | 2004-11-19 | 2007-08-22 | Shanghai Apple Flavor&Fragrance Co. Ltd. | A acetoin high yield bacillus pumilus strain |
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| CN101008019A (en) * | 2007-01-29 | 2007-08-01 | 山东省食品发酵工业研究设计院 | Use of Bacillus subtilis (Ehrenberg)Cohn in preparing 3-hydroxy butanone |
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