CN103865869A - Genetically engineered bacterium for producing alpha-ketobutyric acid and application of genetically engineered bacterium - Google Patents
Genetically engineered bacterium for producing alpha-ketobutyric acid and application of genetically engineered bacterium Download PDFInfo
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Abstract
The invention relates to a genetically engineered bacterium for producing alpha-ketobutyric acid as well as a construction method and application of the genetically engineered bacterium, belonging to the technical field of biology. The strain is relatively high in production efficiency when used for producing alpha-ketobutyric acid through fermentation. The genetically engineered bacterium is obtained through carrying out genetic engineering modification on Escherichia coli MG1655; the genetic engineering modification is realized by knocking out a large-subunit encoding gene ilvB of acetohydroxyacid synthase I, a large-subunit encoding gene ilvI of acetohydroxyacid synthase III and a threonine operon leading peptide encoding gene thrL in an over-expressive threonine dehydratase encoding gene ilvA. When the bacterium is used for producing alpha-ketobutyric acid by using a fermentation method, defects such as complex reaction conditions, high energy consumption, serious pollution or high production cost, serious pollution and the like existing in a chemical synthesis method, an enzyme method or a microbial conversion method can be overcome; after the bacterium is fermented for 20-24h, the yield of alpha-ketobutyric acid is up to 8.5-15.7g/L; aerobic fermentation is adopted in a fermentation process; the bacterium is rapid in growth, short in fermentation period and high in acid production rate; any reports for producing alpha-ketobutyric acid by using a direct fermentation method are not found at present.
Description
Technical field:
The present invention relates to a kind of genetic engineering bacterium and construction process and application of the α of production-one base butyric acid, utilize this strain fermentation to produce α-one base butyric acid and there is higher production efficiency, belong to biological technical field.
Background technology:
α-one base butyric acid (α-ketobutyric acid), claim again 2-Oxobutyric acid (2-Oxobutyric acid), 2-Oxobutyric acid (2-Ketobutyric acid) etc., be the intermediate of synthetic multiple important chemical substance, be widely used in the fields such as medicine, food, chemistry.Can be used for the materials such as synthetic ILE, food spice, 1-propyl alcohol, furanone, paulomycin, L-butyrine and D-alpha-hydroxybutyric acid as α-one base butyric acid.Wherein L-butyrine is the precursor of the antiepileptic drugs such as synthetic Levetiracetam, and D-alpha-hydroxybutyric acid is for preparing azinothricin family cancer therapy drug.
The production method of α-one base butyric acid comprises chemical synthesis and biological synthesis process at present.The former can obtain α-one base butyric acid by ethyl propionate and oxalic acid diethyl ester hydrolysis.And the synthetic α-one base butyric acid of the biological process of hitherto reported mainly comprises enzyme process and microbe transformation method.Nakahara etc. (1994) are carbon source and substrate with 1,2-butyleneglycol, adopt Rhodococcus equi (Rhodococcus equi) IF03730 to be translated into α-one base butyric acid, and cultivating 32h transformation efficiency is 68.2%.Research subsequently finds that pseudomonas putida (Pseudomonas sputita) and Pseudomonas stutzeri (Pseudomonas stutzeri) SDM can be converted into α-one base butyric acid by β-crotonic acid and DL-2-hydroxybutyric acid respectively, and its output is 4.8g/L.Zu Er Balbriggan (2005, CN159278) adopts the Ilv-3 mutant of a strain neurospora crassa to produce α-one base butyric acid take L-threonine as fermenting substrate, and under optimal conditions, output reaches 8g/L.Ma Cuiqing etc. (2011) have reported that the Pseudomonas stutzeri SDM cell that contains L-threonine dehydratase take L-threonine as substrate utilization prepares the method for α-one base butyric acid as biological catalyst, and reaction 20h transformation efficiency is 49.9%.
Although at present industrial production α-one base butyric acid mainly adopts chemical synthesis, the method exist reaction conditions complexity, energy consumption large, the deficiency such as pollute heavily.And enzyme process or microbe transformation method exist the high pollution of substrate production cost large, be difficult to realize the deficiencies such as scale operation.If can adopt microorganism fermentation direct production α-one base butyric acid, can address the above problem and deficiency.
L-threonine is the precursor substance of synthetic α-one base butyric acid, and therefore obtaining the bacterial strain that can accumulate L-threonine is the prerequisite of fermentative Production α-one base butyric acid.In the present invention by knocking out threonine operon leading peptide encoding gene thrL, the attenuation of the key enzyme encoding gene in L-threonine route of synthesis (thrA, thrB, thrC) being transcribed to remove ThrL, reaches and increases the synthetic object of L-threonine.
α-one base butyric acid can be generated 2-ethanoyl-2-hydroxybutyric acid by the further metabolism of Acetohydroxyacid synthase; and in intestinal bacteria, contain altogether 3 Acetohydroxyacid synthases, be respectively Acetohydroxyacid synthase I (by ilvBN genes encoding), Acetohydroxyacid synthase II (by ilvGM genes encoding) and Acetohydroxyacid synthase III (by ilvIH genes encoding).In this patent, the large subunit coding gene ilvG of Acetohydroxyacid synthase II of starting strain intestinal bacteria used (Escherichia coli) MG1655 does not express because of phase shift mutation, therefore only need knock out the further metabolism that Acetohydroxyacid synthase I and Acetohydroxyacid synthase III encoding gene can stop α-one base butyric acid.
In sum, this patent flows by reinforcing alpha-one base butyric acid anabolism and blocks its further pathways metabolism and realize fermentative Production α-one base butyric acid, the object that not only can reach pollution abatement, reduces costs, can also alleviate the pressure of L-threonine production capacity surplus.But the method have not been reported at present.
Summary of the invention:
The problem to be solved in the present invention be overcome reaction conditions complexity, energy consumption that chemical synthesis, enzyme process or microbe transformation method exist large, pollute the deficiencies such as heavy or production cost high pollution is large, a kind of genetic engineering bacterium of the α of producing-one base butyric acid is provided and adopts the method for this bacterium fermentative production α-one base butyric acid.
The present invention solves the problems of the technologies described above one of technical scheme of employing: a kind of Recombinant organism of the α of production-one base butyric acid is provided, and described Recombinant organism is in intestinal bacteria (Escherichia coli) MG1655 (ATCC47076), to cross to express from the threonine dehydra(ta)se encoding gene ilvA of this bacterium, knock out the large subunit coding gene ilvB of Acetohydroxyacid synthase I, the large subunit coding gene ilvI of Acetohydroxyacid synthase III and and the mutant strain that obtains of threonine operon leading peptide encoding gene thrL.
The nucleotides sequence of described encoding gene ilvA is classified the SEQ ID NO:1 in sequence table as;
The nucleotides sequence of described encoding gene ilvB is classified the SEQ ID NO:2 in sequence table as;
The nucleotides sequence of described encoding gene ilvI is classified the SEQ ID NO:3 in sequence table as;
The nucleotides sequence of described encoding gene thrL is classified the SEQ ID NO:4 in sequence table as;
Two of the technical scheme that the present invention solves the problems of the technologies described above is: a kind of method that builds said gene engineering bacteria is provided, comprises the steps:
(1) knocking out of ilvB gene
1, adopt round pcr take intestinal bacteria MG1655 as template, according to (GeneID:948181) gene 5 ' of ilvB in MG1655 and 3 ' end 400bp sequences Design homology arm primer, the upstream and downstream homology arm of ilvB gene is obtained in amplification.
2, adopt round pcr take pKD3 plasmid as template, design primer, amplification chloramphenicol resistance gene box fragment.
3, obtain ilvB gene knockout fragment take 1,2 amplified fragments that obtain as template by overlapping PCR, described gene knockout fragment is made up of upstream and downstream homology arm gene fragment and the chloramphenicol resistance gene box fragment of the large subunit coding gene ilvB of Acetohydroxyacid synthase I.
4, said gene is knocked out to fragment importing and obtain positive transformant containing in the intestinal bacteria MG1655 competent cell of pKD46 plasmid, after the chloramphenicol resistance gene in elimination positive transformant, obtain the degerming of ilvB clpp gene.
(2) knocking out of ilvI gene
1, with 1-3 in step () identical method build ilvI (GeneID:947267) gene knockout fragment.
2, the gene knockout fragment in (two)-1 is imported in the ilvB gene knockout bacterium competence cell that contains pKD46 plasmid and obtains positive transformant, after the chloramphenicol resistance gene in elimination positive transformant, obtain ilvB, the degerming of ilvI clpp gene.
(3) knocking out of thrL gene
1, with 1-3 in step () identical method build thrL (GeneID:948283) base and knock out fragment.
2, the gene knockout fragment in (three)-1 is imported in the ilvB that contains pKD46 plasmid, ilvI gene knockout bacterium competence cell and obtain positive transformant, obtain ilvB, ilvI, thrL clpp gene degerming THRZ after eliminating the chloramphenicol resistance gene in positive transformant.
(4) crossing of ilvA gene expressed
1, after being carried out to double digestion with Xba I with BamH I, ilvA gene and expression vector pWSK29 thereof be connected, connect product and be converted in the degerming of above-mentioned THRZ clpp gene, identify that by bacterium colony PCR the positive transformant obtaining is Recombinant organism of the present invention.
The present invention addresses the above problem three of adopted technical scheme: a kind of method of fermentative production α-one base butyric acid is provided, comprises the following steps:
1. seed culture: will be seeded to the 5L fermentor tank that 1L seed culture medium is housed after activated Recombinant organism of the present invention, the ammoniacal liquor that stream adds 25% (W/V) regulates fermented liquid pH to 6.8-7.2, dissolved oxygen maintains 30-50%, ventilation 3-5m
3/ h, mixing speed 200-600rpm, cultivates 6-8h for 32-37 ℃.
2. ferment tank: with 5%-10% inoculum size, step inoculum is 1. connected to the 10L fermentor tank that 6L fermention medium is housed and carries out fermentation culture, leavening temperature 32-37 ℃, ventilation 3-5m
3/ h, mixing speed 300-1000rpm, dissolved oxygen maintains 30-60%, stream adds the glucose solution that concentration is 60-80% (W/V), maintaining remaining sugar concentration is 0.1-0.5% (W/V), the ammoniacal liquor that stream adds 25% (W/V) regulates fermented liquid pH to 6.8-7.2, fermentation period 20-24h.
3. the detection of α-one base butyric acid in fermented liquid: fermented liquid is got supernatant liquor and with after 5 times of deionized water dilutions, adopted UltiMate3000 (Thermo Scientific) high performance liquid chromatograph to measure the content of α-one base butyric acid after the centrifugal 10min of 8000 × g.Testing conditions is: chromatographic column REzex RoA-organic Acid H+, moving phase 5mmol/L H
2sO
4, flow velocity 0.5mL/min, 30 ℃ of column temperatures, detect wavelength 215nm, and sample size is 20 μ L.
Described seed culture based component is: sucrose 15-25g/L, corn steep liquor 15-25mL/L, yeast powder 1-4g/L, (NH
4)
2sO
41-4g/L, KH
2pO
40.5-1.5g/L, MgSO
40.1-0.5g/L, FeSO
47H
2o0.01-0.05g/L, MnSO
4h
2o0.010.05g/L, pH7.0,0.075MPa high pressure steam sterilization 15min.
Described fermentation culture based component is: glucose 15-45g/L, yeast powder 1-2g/L, soya-bean cake hydrolyzed solution 5-15mL/L, corn steep liquor 5-15mL/L, KH
2pO
41-5g/L, citric acid 0.5-2g/L, MgSO
40.5-1g/L, FeSO
47H
2o0.1-0.5g/L, MnSO
4h
2o0.1-0.5g/L, pH7.0,0.075MPa high pressure steam sterilization 15min.
Beneficial effect:
(1) the present invention is take E.coli MG1655 (ATCC47076) as starting strain, utilize metabolic engineering means, built the Recombinant organism of production α-one base butyric acid by strengthening and extend the approach of the synthetic α-one base butyric acid of L-threonine and the approach of the further metabolism of cut-out the latter.After fermentation 20-24h, α-one base butyric acid output reaches 8.5-15.7g/L.This fermenting process is aerobic fermentation, and thalli growth is fast, and fermentation period is short, and rate of producing acid is high, has no at present the report of direct fermentation production α-one base butyric acid.
(2) zymotechnique that the method adopts is simple, is easy to control, and production cost is low, is conducive to the promotion and application of suitability for industrialized production.
Accompanying drawing explanation:
Fig. 1 ilvB knocks out strain and builds schematic diagram;
Fig. 2 ilvB gene knockout PCR identifies collection of illustrative plates;
Wherein M is Marker; 1 is ilvB upstream region of gene 400bp fragment; 2 is ilvB gene downstream 400bp fragment; 3 is ilvBK;
4 is the fragment take E.coli MG1655 genome as template, CH1 and CH2 as primer amplification;
5 for ilvBK is incorporated into E.coli MG1655 postgenome, the fragment take CH1 and CH2 as primer amplification;
6 is the fragment take ilvB gene knockout pnca gene group as template, ID1 and ID2 as primer amplification;
7 is the fragment take E.coli MG1655 genome as template, ID1 and ID2 as primer amplification;
Fig. 3 ilvI knocks out strain and builds schematic diagram;
Fig. 4 ilvI gene knockout PCR identifies collection of illustrative plates;
Wherein M is Marker; 1 is ilvI upstream region of gene 400bp fragment; 2 is ilvI gene downstream 400bp fragment; 3 is ilvIK;
4 is the fragment take E.coli MG1655ilvB gene knockout pnca gene group as template, CH1 and CH2 as primer amplification;
5 for ilvIK is incorporated into after E.coli MG1655ilvB gene knockout pnca gene group, the fragment take CH1 and CH2 as primer amplification;
6 is take the dual-gene fragment of pnca gene group as template, ID3 and ID4 as primer amplification that knock out of ilvB, ilvI;
7 is take E.coli MG1655ilvB gene knockout strain and the fragment as template, ID3 and ID4 as primer amplification;
Fig. 5 thrL knocks out strain and builds schematic diagram;
Fig. 6 thrL gene knockout PCR identifies collection of illustrative plates;
Wherein M is Marker; 1 is thrL upstream region of gene 400bp fragment; 2 is thrL gene downstream 400bp fragment; 3 is thrL K
4 is the fragment take E.coli MG1655ilvB gene knockout pnca gene group as template, CH1 and CH2 as primer amplification;
5 for thrLK is incorporated into after E.coli MG1655ilvB gene knockout pnca gene group, the fragment take CH5 and CH6 as primer amplification;
6 for take THRZ genome as template, ID5 and ID6 be the fragment of primer amplification;
7 is take E.coli MG1655ilvB gene knockout strain and the fragment as template, ID5 and ID6 as primer amplification;
Fig. 7 recombinant plasmid pW-ilvA and engineering strain KBA build schematic diagram;
The restriction enzyme digestion and electrophoresis collection of illustrative plates of Fig. 8 recombinant plasmid pW-ilvA;
Wherein M-Marker1-pW-ilvA cuts through Xba I and BamH I enzyme;
Fig. 9 genetic engineering bacterium KBA stability test figure.
Embodiment:
The embodiment 1:E.coli MG1655 acyl alcohol acid large subunit coding gene ilvB of synthetic enzyme I knocks out
According to ilvB (GeneID:948181) gene 5 ' in intestinal bacteria in ncbi database (Escherichia coli) MG1655 and 3 ' end 400bp sequences Design upstream and downstream homology arm amplimer ilvB1-F, ilvB1-R, ilvB3-F and ilvB3-R, and take this bacterium genomic dna as template amplification homology arm fragment.
PCR condition is 94 ℃ 5min1 circulation, 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ 40s30 circulation, and 72 ℃ 10min1 circulation, reaction system is 100 μ L.
PCR product is cut after 1.5% agarose gel electrophoresis to glue and reclaimed, by the fragment obtaining called after ilvB1, ilvB3 respectively.
According to chloramphenicol resistance gene box sequences Design amplimer ilvB2-F, ilvB2-R in plasmid pKD3, and take this plasmid as template amplification chloramphenicol resistance gene box fragment.
PCR condition is 94 ℃ 5min1 circulation, 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ 70s30 circulation, and 72 ℃ 10min1 circulation, reaction system is 100 μ L.
PCR product is cut after 1.5% agarose gel electrophoresis to glue and reclaimed, by the fragment called after ilvB2 obtaining.
Take ilvB1 and ilvB2 fragment as template, utilize primer ilvB1-F and ilvB2R to carry out overlapping PCR.
PCR condition is 94 ℃ 5min1 circulation, 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ 100s30 circulation, and 72 ℃ 10min1 circulation, reaction system is 100 μ L.
PCR product is cut after 1.5% agarose gel electrophoresis to glue and reclaimed, called after ilvB1-2.Take ilvB1-2 and ilvB3 fragment as template, utilize primer ilvB1-F and ilvB3-R to carry out overlapping PCR, PCR condition is 94 ℃ 5min1 circulation, 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ 120s30 circulation, 72 ℃ 10min1 circulation, reaction system is 100 μ L.PCR product is cut after 1.5% agarose gel electrophoresis to glue and reclaimed, obtain ilvB and knock out fragment, called after ilvBK.
IlvBK electricity is converted into (electricity transforms voltage and the time is respectively 2500V and 5ms) in the E.coli MG1655 competent cell containing pKD46 plasmid.In 1mL SOC substratum, after 37 ℃, 150rpm recovery 1h, coat containing on paraxin (25 μ g/mL) LB solid medium flat board rapidly.After being inverted cultivation 24h, adopt primers designed CH1 and CH2 to identify positive transformant by the method for bacterium colony PCR, chloramphenicol resistance gene box is successfully incorporated into the about 1054bp of its amplified fragments of genomic bacterium colony.PCP20 plasmid is converted into above-mentioned positive transformant to eliminate chloramphenicol resistance gene, after 42 ℃ of incubated overnight, screening can be grown and contained single bacterium colony of not growing on paraxin flat board and adopting primers designed ID1 and ID2 to verify on non-resistant flat board, the about 234bp of bacterial strain amplified fragments that ilvB is successfully knocked out, as shown in Figure 1, Fig. 2 is PCR checking to building process.
Primer sequence is in table 1.
Wherein: LB solid medium (g/L): tryptone 10, yeast extract 5, NaCl10, agar powder 15, is settled to 1000mL, 121 ℃ of high pressure steam sterilization 20min with distilled water.
SOC substratum (g/L): tryptone 20, yeast extract 5, NaCl0.5, KCl0.2, MgCl
20.95, glucose 3.6.
Embodiment 2:E.coli MG1655ilvB knocks out knocking out of the large subunit coding gene ilvI of strain Acetohydroxyacid synthase III
According to intestinal bacteria in ncbi database (Escherichia coli) MG1655ilvI (GeneID:947267) gene 5 ' and 3 ' end 400bp sequences Design homology arm amplimer ilvI1-F, ilvI1-R, ilvI3-F and ilvI3-R, and take this bacterium genomic dna as template amplification homology arm fragment.
PCR condition is 94 ℃ 5min1 circulation, 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ 40s30 circulation, and 72 ℃ 10min1 circulation, reaction system is 100 μ L.
According to chloramphenicol resistance gene box sequences Design amplimer ilvI2-F, ilvI2-R in plasmid pKD3, and take this plasmid as template amplification chloramphenicol resistance gene box fragment, PCR condition is 94 ℃ 5min1 circulation, 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ 70s30 circulation, 72 ℃ 10min1 circulation, reaction system is 100 μ L.PCR product is cut after 1.5% agarose gel electrophoresis to glue and reclaimed, by the fragment obtaining called after ilvI1, ilvI3 and ilvI2 respectively.
Take ilvI1 and ilvI2 fragment as template, utilize primer ilvI1-F and ilvI2R to carry out overlapping PCR, PCR condition is 94 ℃ 5min1 circulation, 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ 100s30 circulation, 72 ℃ 10min1 circulation, reaction system is 100 μ L.PCR product is cut after 1.5% agarose gel electrophoresis to glue and reclaimed, called after ilvI1-2.Take ilvI1-2 and ilvI3 fragment as template, utilize primer ilvI1-F and ilvI3-R to carry out overlapping PCR, PCR condition is 94 ℃ 5min1 circulation, 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ 120s30 circulation, 72 ℃ 10min1 circulation, reaction system is 100 μ L.PCR product is cut after 1.5% agarose gel electrophoresis to glue and reclaimed, obtain ilvI and knock out fragment, called after ilvIK.
IlvIK electricity is converted into (electricity transforms voltage and the time is respectively 2500V and 5ms) in the E.coli MG1655ilvB gene knockout strain competent cell containing pKD46 plasmid.In 1mL SOC substratum, after 37 ℃, 150rpm recovery 1h, coat containing on paraxin (25 μ g/mL) LB solid medium flat board rapidly.After being inverted cultivation 24h, adopt primers designed CH1 and CH2 to identify positive transformant by the method for bacterium colony PCR, chloramphenicol resistance gene box is successfully incorporated into the about 1054bp of its amplified fragments of genomic bacterium colony.PCP20 plasmid is converted into above-mentioned positive transformant to eliminate chloramphenicol resistance gene, after 42 ℃ of incubated overnight, screening can be grown and contained single bacterium colony of not growing on paraxin flat board and adopting primers designed ID3 and ID4 to verify on non-resistant flat board, the about 233bp of bacterial strain amplified fragments that ilvI is successfully knocked out.
As shown in Figure 3, Fig. 4 is PCR checking to building process.
Primer sequence is in table 1.
Wherein: LB solid medium (g/L): peptone 10, yeast extract 5, NaCl10, agar powder 15, is settled to 1000mL, 121 ℃ of high pressure steam sterilization 20min with distilled water.
The structure of embodiment 3THRZ bacterial strain
According to thrL (GeneID:948283) gene 5 ' in intestinal bacteria E.coli MG1655 in ncbi database and 3 ' end 400bp sequences Design homology arm amplimer thrL1-F, thrL1-R, thrL3-F and thrL3-R, and take this bacterium genomic dna as template amplification homology arm fragment.
PCR condition is 94 ℃ 5min1 circulation, 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ 40s30 circulation, and 72 ℃ 10min1 circulation, reaction system is 100 μ L.
PCR product is cut after 1.5% agarose gel electrophoresis to glue and reclaimed, by the fragment obtaining called after thrL1, thrL3 respectively.
According to chloramphenicol resistance gene box sequences Design amplimer thrL2-F, thrL2-R in plasmid pKD3, and take this plasmid as template amplification chloramphenicol resistance gene box fragment.
PCR condition is 94 ℃ 5min1 circulation, 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ 70s30 circulation, and 72 ℃ 10min1 circulation, reaction system is 100 μ L.
PCR product is cut after 1.5% agarose gel electrophoresis to glue and reclaimed, by the fragment called after thrL2 obtaining.
Take thrL1 and thrL2 fragment as template, utilize primer thrL1-F and thrL2R to carry out overlapping PCR.
PCR condition is 94 ℃ 5min1 circulation, 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ 100s30 circulation, and 72 ℃ 10min1 circulation, reaction system is 100 μ L.
PCR product is cut after 1.5% agarose gel electrophoresis to glue and reclaimed, called after thrL1-2.Take thrL1-2 and thrL3 fragment as template, utilize primer thrL1-F and thrL3-R to carry out overlapping PCR, PCR condition is 94 ℃ 5min1 circulation, 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ 120s30 circulation, 72 ℃ 10min1 circulation, reaction system is 100 μ L.PCR product is cut after 1.5% agarose gel electrophoresis to glue and reclaimed, obtain thrL and knock out fragment, called after thrLK.
ThrLK electricity is converted into (electricity transforms voltage and the time is respectively 2500V and 5ms) in the ilvB that obtains containing the embodiment 2 of pKD46 plasmid, ilvI gene knockout strain competent cell.In 1mL SOC substratum, after 37 ℃, 150rpm recovery 1h, coat containing on paraxin (25 μ g/mL) LB solid medium flat board rapidly.After being inverted cultivation 24h, adopt primers designed CH1 and CH2 to identify positive transformant by the method for bacterium colony PCR, chloramphenicol resistance gene box is successfully incorporated into the about 1054bp of its amplified fragments of genomic bacterium colony.PCP20 plasmid is converted into above-mentioned positive transformant to eliminate chloramphenicol resistance gene, after 42 ℃ of incubated overnight, screening can be grown and contained single bacterium colony of not growing on paraxin flat board and adopting primers designed ID5 and ID6 to verify on non-resistant flat board, the about 246bp of bacterial strain amplified fragments that thrL is successfully knocked out.
As shown in Figure 5, Fig. 6 is PCR checking to building process.By the engineering strain called after THRZ successfully constructing.
Crossing of embodiment 4:ilvA gene expressed
According to ilvA gene (GeneID:948287) primers ILA-F and ILA-R in intestinal bacteria in ncbi database (Escherichia coli) MG1655, take this bacterium genomic dna as template amplification ilvA gene.
PCR condition is 94 ℃ 5min1 circulation, 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ 130s30 circulation, and 72 ℃ 10min1 circulation, reaction system is 100 μ L.
Get the band that 10 μ L PCR products obtain 1.5-1.6kb after 1.5% agarose gel electrophoresis, consistent with gene actual size (1573bp).After residue 90 μ L amplified fragments are reclaimed, adopt Xba I and BamH I to carry out double digestion, after 1.5% agarose gel electrophoresis, cut glue and reclaim and get the recovery fragment employing T that adapts to volume
4ligase enzyme is connected to the expression vector pWSK29 cutting through same enzyme, adopts CaCl
2method is converted in intestinal bacteria DH10 β competent cell.Picking can and utilize primer P29F and P29R carries out bacterium colony PCR evaluation at the bacterium colony containing growing in the LB solid culture of ammonia benzyl mycin (100 μ g/mL).Extract through being accredited as positive transformant plasmid and entrust Beijing Liuhe Huada Genomics Technology Co., Ltd to check order, sequencing result is consistent with expection, shows plasmid construction success, by its called after pW-ilvA.Building process is as Fig. 7, and checking is as Fig. 8.
Adopt CaCl
2recombinant plasmid pW-ilvA is converted into ilvB, ilvI, the degerming of thrL clpp gene that embodiment 3 obtains by method.Until adopting primer P29F and P29R to carry out bacterium colony PCR evaluation, will be genetic engineering bacterium of the present invention, called after KBA through being accredited as positive transformant after growing bacterium colony in the LB solid culture of ammonia benzyl mycin (100 μ g/mL).
Primer sequence is in table 1.
Table 1 knocks out fragment for pcr amplification and crosses the primer sequence of expressing gene
Embodiment 5: the 10L ferment tank of genetic engineering bacterium KBA
(1) seed culture: 3-5 is propped up through the intestinal bacteria KBA of fresh slant activation and is all seeded to the 5L fermentor tank that 1L seed culture medium is housed with transfering loop, the ammoniacal liquor that stream adds 25% (W/V) regulates fermented liquid pH to 6.8-7.2, dissolved oxygen maintains 30-50%, ventilation 3-5m
3/ h, mixing speed 200-600rpm, cultivates 6-8h for 32-37 ℃.
(2) ferment tank: with 5%-10% inoculum size, the inoculum of step (1) is connected to the 10L fermentor tank that 6L fermention medium is housed and sends out tank and cultivate, leavening temperature 32-37 ℃, ventilation 3-5m
3/ h, mixing speed 300-1000rpm, dissolved oxygen maintains 30-60%, stream adds the glucose solution that concentration is 60-80% (W/V), maintaining remaining sugar concentration is 0.1-0.5% (W/V), the ammoniacal liquor that stream adds 25% (W/V) regulates fermented liquid pH to 6.8-7.2, fermentation period 20-24h.
(3) detection of α-one base butyric acid in fermented liquid: fermented liquid is got supernatant liquor and with after 5 times of deionized water dilutions, adopted UltiMate3000 (Thermo Scientific) high performance liquid chromatograph to measure the content of α-one base butyric acid after the centrifugal 10min of 8000 × g.Testing conditions is: chromatographic column REzex RoA-organic Acid H+, moving phase 5mmol/L H
2sO
4, flow velocity 0.5mL/min, 30 ℃ of column temperatures, detect wavelength 215nm, and sample size is 20 μ L.Detected result shows, the appearance time of α-one base butyric acid is about 12.5min, and output is 8.5-15.7g/L.
Wherein: seed culture based component is:
Sucrose 15-25g/L, corn steep liquor 15-25mL/L, yeast powder 1-4g/L, (NH
4)
2sO
41-4g/L, KH
2pO
40.5-1.5g/L, MgSO
40.1-0.5g/L, FeSO
47H
2o0.01-0.05g/L, MnSO
4h
2o0.010.05g/L, pH7.0,0.075MPa high pressure steam sterilization 15min.
Fermentation culture based component is:
Glucose 15-45g/L, yeast powder 1-2g/L, soya-bean cake hydrolyzed solution 5-15mL/L, corn steep liquor 5-15mL/L, KH
2pO
41-5g/L, citric acid 0.5-2g/L, MgSO
40.5-1g/L, FeSO
47H
2o0.1-0.5g/L, MnSO
4h
2o0.1-0.5g/L, pH7.0,0.075MPa high pressure steam sterilization 15min.
Embodiment 6: the 10L ferment tank of genetic engineering bacterium KBA
(1) seed culture: 3 intestinal bacteria KBA through fresh slant activation are all seeded to the 5L fermentor tank that 1L seed culture medium is housed with transfering loop, the ammoniacal liquor that stream adds 25% (W/V) regulates fermented liquid pH to 7.0, dissolved oxygen maintains 35%, ventilation 3m
3/ h, mixing speed 200-600rpm, cultivates 6h for 37 ℃.
(2) ferment tank: with 8% inoculum size, the inoculum of step (1) is connected to the 10L fermentor tank that 6L fermention medium is housed and sends out tank and cultivate, 37 ℃ of leavening temperatures, ventilation 4m
3/ h, mixing speed 300-1000rpm, dissolved oxygen maintains 40%, stream adds the glucose solution that concentration is 80% (W/V), maintaining remaining sugar concentration is 0.15% (W/V), and the ammoniacal liquor that stream adds 25% (W/V) regulates fermented liquid pH to 7.0, fermentation period 20h.
(3) detection of α-one base butyric acid in fermented liquid: fermented liquid is got supernatant liquor and with after 5 times of deionized water dilutions, adopted UltiMate3000 (Thermo Scientific) high performance liquid chromatograph to measure the content of α-one base butyric acid after the centrifugal 10min of 8000 × g.Testing conditions is: chromatographic column REzex RoA-organic Acid H+, moving phase 5mmol/L H
2sO
4, flow velocity 0.5mL/min, 30 ℃ of column temperatures, detect wavelength 215nm, and sample size is 20 μ L.Detected result shows, the appearance time of α-one base butyric acid is about 12.5min, and output is 11.6g/L.
Wherein: seed culture based component is:
Sucrose 15.5g/L, corn steep liquor 15mL/L, yeast powder 2.5g/L, (NH
4)
2sO
42.0g/L, KH
2pO
40.7g/L, MgSO
40.2g/L, FeSO
47H
2o0.02g/L, MnSO
4h
2o0.01g/L, pH7.0,0.075MPa high pressure steam sterilization 15min.
Fermentation culture based component is:
Glucose 20g/L, yeast powder 1.5g/L, soya-bean cake hydrolyzed solution 5mL/L, corn steep liquor 8mL/L, KH
2pO
41.5g/L, citric acid 0.7g/L, MgSO
40.5g/L, FeSO
47H
2o0.3g/L, MnSO
4h
2o0.2g/L, pH7.0,0.075MPa high pressure steam sterilization 15min.
Embodiment 7: the 10L ferment tank of genetic engineering bacterium KBA
(1) seed culture: 4 intestinal bacteria KBA through fresh slant activation are all seeded to the 5L fermentor tank that 1L seed culture medium is housed with transfering loop, the ammoniacal liquor that stream adds 25% (W/V) regulates fermented liquid pH to 7.0, dissolved oxygen maintains 35%, ventilation 4m
3/ h, mixing speed 200-600rpm, cultivates 6h for 37 ℃.
(2) ferment tank: with 10% inoculum size, the inoculum of step (1) is connected to the 10L fermentor tank that 6L fermention medium is housed and sends out tank and cultivate, 37 ℃ of leavening temperatures, ventilation 4m
3/ h, mixing speed 300-1000rpm, dissolved oxygen maintains 40%, stream adds the glucose solution that concentration is 80% (W/V), maintaining remaining sugar concentration is 0.15% (W/V), and the ammoniacal liquor that stream adds 25% (W/V) regulates fermented liquid pH to 7.0, fermentation period 24h.
(3) detection of α-one base butyric acid in fermented liquid: fermented liquid is got supernatant liquor and with after 5 times of deionized water dilutions, adopted UltiMate3000 (Thermo Scientific) high performance liquid chromatograph to measure the content of α-one base butyric acid after the centrifugal 10min of 8000 × g.Testing conditions is: chromatographic column REzex RoA-organic Acid H+, moving phase 5mmol/L H
2sO
4, flow velocity 0.5mL/min, 30 ℃ of column temperatures, detect wavelength 215nm, and sample size is 20 μ L.Detected result shows, the appearance time of α-one base butyric acid is about 12.5min, and output is 15.7g/L.
Wherein: seed culture based component is:
Sucrose 15.5g/L, corn steep liquor 20mL/L, yeast powder 2.5g/L, (NH
4)
2sO
42.0g/L, KH
2pO
40.7g/L, MgSO
40.2g/L, FeSO
47H
2o0.02g/L, MnSO
4h
2o0.01g/L, pH7.0,0.075MPa high pressure steam sterilization 15min.
Fermentation culture based component is:
Glucose 20g/L, yeast powder 1.5g/L, soya-bean cake hydrolyzed solution 6mL/L, corn steep liquor 10mL/L, KH
2pO
42.5g/L, citric acid 0.7g/L, MgSO
40.5g/L, FeSO
47H
2o0.1g/L, MnSO
4h
2o0.25g/L, pH7.0,0.075MPa high pressure steam sterilization 15min.
Embodiment 8: genetic engineering bacterium KBA plasmid stability
Adopt the dull and stereotyped dilution method of counting, frozen genetic engineering bacterium KBA in-80 ℃ of glycerine pipes is being rule containing on the LB solid plate of ammonia benzyl mycin (100 μ g/mL), be inverted for 37 ℃ and cultivate after 48h, with transfering loop picking list colony inoculation in 5ml containing in the LB liquid nutrient medium of ammonia benzyl mycin (100 μ g/mL), after 37 ℃ of shaking table shaking culture 24h, proceed in 5ml LB liquid nutrient medium in 37 ℃ of shaking table shaking culture with 1% inoculum size.Once, inoculum size is 1% to every 24h transferred species, and continuous passage 50 times is carried out the dull and stereotyped disappearance degree that detects plasmid of diluting every 5 sub-samplings.Detecting step is, get bacterium liquid lmL sterilized water stepwise dilution, get three suitable gradients, be coated on respectively on the LB solid plate containing ammonia benzyl mycin (100 μ g/mL) and antibiotic-free, then in 37 ℃ of incubators, be inverted and cultivate 24h, calculate the colony number in each ware, and be calculated as follows plasmid loss rate, as shown in Figure 7, after 50 times go down to posterity, plasmid loss rate is only 7.66% to result.
Claims (9)
1. a genetic engineering bacterium is that starting strain intestinal bacteria (Escherichia coli) MG1655 is carried out to the bacterium of genetic engineering modified acquisition; Described genetic engineering modified be expression threonine dehydra(ta)se encoding gene ilvA, and knock out the large subunit coding gene ilvB of Acetohydroxyacid synthase I, the large subunit coding gene ilvI of Acetohydroxyacid synthase III and and threonine operon leading peptide encoding gene thrL.
2. genetic engineering bacterium according to claim 1, is characterized in that: described threonine dehydra(ta)se encoding gene ilvA is from intestinal bacteria E.coli MG1655, GeneID:948287, and nucleotide sequence is as the SEQ ID NO:1 in sequence table.
3. genetic engineering bacterium according to claim 1, it is characterized in that: the nucleotide sequence of the large subunit coding gene ilvB of described Acetohydroxyacid synthase I is as SEQ ID NO:2 in sequence table, the nucleotide sequence of the large subunit coding gene ilvI of described Acetohydroxyacid synthase III is as SEQ ID NO:3 in sequence table, and the nucleotide sequence of described threonine operon leading peptide encoding gene thrL is as SEQ ID NO:4 in sequence table.
4. a method that builds genetic engineering bacterium claimed in claim 1, comprises the steps:
(1) knocking out of ilvB gene
(1) adopt round pcr take intestinal bacteria MG1655 genome as template, according to (GeneID:948181) gene 5 ' of ilvB in MG1655 and 3 ' end 400bp sequences Design homology arm primer, the upstream and downstream homology arm of ilvB gene is obtained in amplification;
(2) adopt round pcr take pKD3 plasmid as template, design primer, amplification chloramphenicol resistance gene box fragment;
(3) amplified fragments obtaining take step (1), (2) is as template is by overlapping PCR acquisition ilvB gene knockout fragment, and described gene knockout fragment is made up of upstream and downstream homology arm gene fragment and the chloramphenicol resistance gene box fragment of the large subunit coding gene ilvB of Acetohydroxyacid synthase I;
(4) said gene is knocked out to fragment and import in the starting strain competent cell that contains pKD46 plasmid, obtain positive transformant, after the chloramphenicol resistance gene in elimination positive transformant, obtain the degerming of ilvB clpp gene;
(2) knocking out of ilvI gene
(1) with (1)-(3) in step () identical method build ilvI (GeneID:947267) gene knockout fragment, described ilvI gene knockout fragment is made up of upstream and downstream homology arm gene fragment and the chloramphenicol resistance gene box fragment of ilvI;
(2) the gene knockout fragment in (two)-(1) is imported in the ilvB gene knockout bacterium competence cell that contains pKD46 plasmid and obtains positive transformant, after the chloramphenicol resistance gene in elimination positive transformant, obtain ilvB, the degerming of ilvI clpp gene;
(3) knocking out of thrL gene
(1) with 1-3 in step () identical method build thrL (GeneID:948283) base and knock out fragment;
(2) the gene knockout fragment in (three)-(1) is imported in the ilvB that contains pKD46 plasmid, ilvI gene knockout bacterium competence cell and obtain positive transformant, obtain ilvB, ilvI, the degerming of thrL clpp gene after eliminating the chloramphenicol resistance gene in positive transformant;
(4) crossing of ilvA gene expressed
After being carried out to double digestion with Xba I with BamH I, ilvA gene and expression vector pWSK29 thereof be connected, connect product and be converted into above-mentioned ilvB, ilvI, the degerming of thrL clpp gene, identify that by bacterium colony PCR the positive transformant obtaining is Recombinant organism of the present invention.
5. a kind of genetic engineering bacterium described in claim 1, it is characterized in that, starting strain is from Escherichia (Escherichia), Corynebacterium (Corynebacterium), genus arthrobacter (Arthrobacter), Rhodopseudomonas (Pseudomounas) or bacillus (Bacillus).
6. the application of genetic engineering bacterium claimed in claim 1 in fermentative Production α-one base butyric acid.
7. the method for utilizing the genetic engineering bacterium fermentative production α-one base butyric acid described in claim 1, step is as follows:
(1) seed culture: by after genetic engineering bacterium activation of the present invention, be seeded to the 5L fermentor tank that 1L seed culture medium is housed, the ammoniacal liquor that stream adds 25%W/V regulates fermented liquid pH to 6.8-7.2, and dissolved oxygen maintains 30-50%, ventilation 3-5m
3/ h, mixing speed 200-600rpm, cultivates 6-8h for 32-37 ℃.
(2) ferment tank: with 5%-10% inoculum size, the inoculum of step (1) is connected to the 10L fermentor tank that 6L fermention medium is housed and carries out fermentation culture, leavening temperature 32-37 ℃, ventilation 3-5m
3/ h, mixing speed 300-1000rpm, dissolved oxygen maintains 30-60%, and stream adds the glucose solution that concentration is 60-80%W/V, and maintaining remaining sugar concentration is 0.1-0.5%W/V, and the ammoniacal liquor that stream adds 25%W/V regulates fermented liquid pH to 6.8-7.2, fermentation period 20-24h.
8. the method for genetic engineering bacterium fermentative production α-one base butyric acid according to claim 6, it is characterized in that: in fermented liquid, the detection method of α-one base butyric acid is as follows: after fermented liquid is got supernatant liquor and diluted 5 times with deionized water after the centrifugal 10min of 8000 × g, adopt high performance liquid chromatograph to measure the content of α-one base butyric acid, testing conditions is: chromatographic column REzex RoA-organic Acid H+, moving phase 5mmol/L H
2sO
4, flow velocity 0.5mL/min, 30 ℃ of column temperatures, detect wavelength 215nm, and sample size is 20 μ L.
9. the method for genetic engineering bacterium fermentative production α-one base butyric acid according to claim 6, is characterized in that:
Described seed culture based component is: sucrose 15-25g/L, corn steep liquor 15-25mL/L, yeast powder 1-4g/L, (NH
4)
2sO
41-4, KH
2pO
40.5-1.5g/L, MgSO
40.1-0.5g/L, FeSO
47H
2o0.01-0.05g/L, MnSO
4h
2o0.010.05g/L, pH7.0,0.075MPa high pressure steam sterilization 15min;
Described fermentation culture based component is: glucose 15-45g/L, yeast powder 1-2g/L, soya-bean cake hydrolyzed solution 5-15mL/L, corn steep liquor 5-15mL/L, KH
2pO
41-5g/L, citric acid 0.5-2g/L, MgSO
40.5-1g/L, FeSO
47H
2o0.1-0.5g/L, MnSO
4h
2o0.1-0.5g/L, pH7.0,0.075MPa high pressure steam sterilization 15min.
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| CN105695519A (en) * | 2016-04-22 | 2016-06-22 | 中国科学院天津工业生物技术研究所 | Novel biological preparation method of optically pure 2-hydroxy butyric acid |
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| CN116376995A (en) * | 2022-04-02 | 2023-07-04 | 元素驱动(杭州)生物科技有限公司 | Method for preparing glycine, acetyl-CoA and acetyl-CoA derivatives by using threonine |
| CN116376995B (en) * | 2022-04-02 | 2024-01-26 | 元素驱动(杭州)生物科技有限公司 | Method for preparing glycine, acetyl-CoA and acetyl-CoA derivatives by using threonine |
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