CN103865869B - 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 producing α-one base butanoic acid and construction method thereof and application, utilize this bacterial strain
Fermenting and producing α-one base butanoic acid has higher production efficiency, belongs to biological technical field.
Background technology:
α-one base butanoic acid (α-ketobutyric acid), also known as 2-Oxobutyric acid (2-Oxobutyric acid), 2-ketone
Base butanoic acid (2-Ketobutyric acid) etc., are the intermediate synthesizing multiple important chemical substance, are widely used in medicine, food
The field such as product, chemistry.Such as α-one base butanoic acid, to can be used for synthesizing ILE, food flavor, 1-propanol, furanone, guarantor Lip river mould
The materials such as element, L-butyrine and D-alpha-hydroxybutyric acid.Wherein L-butyrine is to synthesize levetiracetam etc. to resist insane
The precursor of epilepsy agent, D-alpha-hydroxybutyric acid is used to prepare azinothricin family cancer therapy drug.
The production method of α-one base butanoic acid includes chemical synthesis and biological synthesis process at present.The former can by ethyl propionate and
Ethyl oxalate hydrolysis obtains α-one base butanoic acid.And the bioanalysis synthesis α-one base butanoic acid of hitherto reported mainly includes enzyme process and micro-
Biotransformation method.Nakahara etc. (1994) are with 1, and 2-butanediol is carbon source and substrate, use Rhodococcus equi (Rhodococcus
Equi) IF03730 is translated into α-one base butanoic acid, and cultivating 32h conversion ratio is 68.2%.Research subsequently finds Pseudomonas putida
Bacterium (Pseudomonas sputita) and Pseudomonas stutzeri (Pseudomonas stutzeri) SDM respectively can be by .beta.-methylacrylic acids
Being converted into α-one base butanoic acid with DL-2-hydroxybutyric acid, its yield is 4.8g/L.Zu Er Balbriggan (2005, CN159278) uses
The Ilv-3 mutant of one strain neurospora crassa produces α-one base butanoic acid with L-threonine for fermenting substrate, produces with optimal conditions
Amount reaches 8g/L.Ma Cuiqing etc. (2011) report the Amur vacation unit cell containing L-threonine dehydratase with L-threonine for substrate utilization
Bacterium SDM cell is the method that biocatalyzer prepares α-one base butanoic acid, and reaction 20h conversion ratio is 49.9%.
Although commercial production α-one base butanoic acid mainly uses chemical synthesis at present, but there is reaction condition again in the method
Miscellaneous, energy consumption is big, pollute the deficiency such as heavily.And enzyme process or microbe transformation method also exist, and substrate production cost high pollution is big, be difficult to reality
The deficiencies such as existing large-scale production.If fermentable can be used to be directly produced α-one base butanoic acid, then can solve the problems referred to above and
Not enough.
L-threonine is the precursor substance of synthesis α-one base butanoic acid, and therefore acquisition can accumulate the bacterial strain of L-threonine is to send out
Ferment method produces the prerequisite of α-one base butanoic acid.By knocking out threonine operon leader peptide encoding gene thrL in the present invention,
To release the attenuation that the key enzyme encoding gene (thrA, thrB, thrC) in L-threonine route of synthesis is transcribed by ThrL,
Reach to increase the purpose of L-threonine synthesis.
α-one base butanoic acid can be generated 2-acetyl group-2-hydroxybutyric acid by the further metabolism of Acetohydroxyacid synthase, and big
Altogether containing 3 Acetohydroxyacid synthases, respectively Acetohydroxyacid synthase I (by ilvBN gene code), acetyl in enterobacteria
Hydroxy acid synthetase II (by ilvGM gene code) and Acetohydroxyacid synthase III (by ilvIH gene code).This patent
The Acetohydroxyacid synthase II large subunit coding of starting strain escherichia coli (Escherichia coli) MG1655 used by
Gene ilvG does not expresses because of frameshift mutation, therefore only need to knock out Acetohydroxyacid synthase I and Acetohydroxyacid synthase III volume
Code gene can stop the further metabolism of α-one base butanoic acid.
In sum, this patent by reinforcing alpha-one base butanoic acid anabolism stream and blocks the realization of its further metabolic pathway
Fermentative Production α-one base butanoic acid, is not only able to reach pollution abatement, the purpose of reduction cost, moreover it is possible to alleviate L-threonine production capacity
Superfluous pressure.But, current the method have not been reported.
Summary of the invention:
The problem to be solved in the present invention is that the reaction condition overcoming chemical synthesis, enzyme process or microbe transformation method to exist is multiple
The deficiencies such as miscellaneous, energy consumption is big, pollute weight or production cost high pollution is big, it is provided that the genetic engineering bacterium of a kind of α of producing-one base butanoic acid and adopting
Method with this bacterium fermenting and producing α-one base butanoic acid.
The present invention solves one of technical scheme of above-mentioned technical problem employing: provides a kind of and produces the big of α-one base butanoic acid
Enterobacteria genetic engineering bacterium, described Recombinant organism is at escherichia coli (Escherichia coli) MG1655
(ATCC47076) in process LAN from this bacterium threonine dehydratase encoding gene ilvA, to knock out Acetohydroxyacid synthase I big
Subunit coding gene ilvB, Acetohydroxyacid synthase III large subunit encoding gene ilvI and and threonine operon leader peptide
The mutant that encoding gene thrL is obtained.
The SEQ ID NO:1 that the nucleotides sequence of described encoding gene ilvA is classified as in sequence table；
The SEQ ID NO:2 that the nucleotides sequence of described encoding gene ilvB is classified as in sequence table；
The SEQ ID NO:3 that the nucleotides sequence of described encoding gene ilvI is classified as in sequence table；
The SEQ ID NO:4 that the nucleotides sequence of described encoding gene thrL is classified as in sequence table；
The present invention solves the two of the technical scheme of above-mentioned technical problem: provide a kind of side building said gene engineering bacteria
Method, comprises the steps:
(1) the knocking out of ilvB gene
1, employing round pcr is with escherichia coli MG1655 as template, according to ilvB in MG1655 (GeneID:948181) base
Because of 5 ' and 3 ' end 400bp sequential design homology arm primers, amplification obtains the upstream and downstream homology arm of ilvB gene.
2, use round pcr with pKD3 plasmid as template, design primer, expand chloramphenicol resistance gene box fragment.
3, ilvB gene knockout fragment, described clpp gene are obtained for template by over-lap PCR with 1,2 amplified fragments obtained
Except fragment is by the upstream and downstream homology arm genetic fragment of Acetohydroxyacid synthase I large subunit encoding gene ilvB and chloromycetin
Resistant gene box fragment forms.
4, said gene is knocked out fragment and imports acquisition sun in the escherichia coli MG1655 competent cell containing pKD46 plasmid
Property transformant, obtain ilvB clpp gene after eliminating the chloramphenicol resistance gene in positive transformant degerming.
(2) the 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 the ilvB gene knockout bacterium competence cell containing pKD46 plasmid
Middle acquisition positive transformant, obtains ilvB, ilvI clpp gene after eliminating the chloramphenicol resistance gene in positive transformant degerming.
(3) the knocking out of thrL gene
1, with 1-3 in step () identical method build thrL (GeneID:948283) base knock out fragment.
2, the gene knockout fragment in (three)-1 is imported the degerming impression of ilvB, ilvI clpp gene containing pKD46 plasmid
State cell obtains positive transformant, after eliminating the chloramphenicol resistance gene in positive transformant, obtains ilvB, ilvI, thrL base
Because knocking out bacterium THRZ.
(4) process LAN of ilvA gene
1, connect after ilvA gene and expression vector pWSK29 Xba I and BamH I thereof are carried out double digestion, connect and produce
Thing convert to above-mentioned THRZ clpp gene degerming in, by bacterium colony PCR identify obtain positive transformant be of the present invention
Recombinant organism.
The present invention solves the three of the technical scheme that the problems referred to above are used: provide a kind of fermenting and producing α-one base butanoic acid
Method, comprises the following steps:
1. seed culture: be seeded to train equipped with 1L seed after activated for Recombinant organism of the present invention
Support base 5L fermentation tank, stream add 25% (W/V) ammonia regulation fermentation liquid pH to 6.8-7.2, dissolved oxygen maintains 30-50%, ventilate
Amount 3-5m3/ h, speed of agitator 200-600rpm, cultivate 6-8h for 32-37 DEG C.
2. ferment tank: step inoculum 1. is connected to equipped with 6L fermentation medium with 5%-10% inoculum concentration
10L fermentation tank carries out fermentation culture, fermentation temperature 32-37 DEG C, ventilation 3-5m3/ h, speed of agitator 300-1000rpm, dissolved oxygen
Maintaining 30-60%, stream adds the glucose solution that concentration is 60-80% (W/V), and maintenance remaining sugar concentration is 0.1-0.5% (W/V), stream
Add 25% (W/V) ammonia regulation fermentation liquid pH to 6.8-7.2, fermentation period 20-24h.
3. the detection of α-one base butanoic acid in fermentation liquid: fermentation liquor 8000 × g take after being centrifuged 10min supernatant and spend from
After sub-water dilutes 5 times, UltiMate3000 (Thermo Scientific) high performance liquid chromatograph is used to measure α-one base butanoic acid
Content.Testing conditions is: chromatographic column REzex RoA-organic Acid H+, and flow phase 5mmol/L H2SO4, flow velocity
0.5mL/min, column temperature 30 DEG C, detect wavelength 215nm, sample size is 20 μ L.
Described seed culture based component is: sucrose 15-25g/L, Semen Maydis pulp 15-25mL/L, yeast powder 1-4g/L, (NH4)2SO41-4g/L, KH2PO40.5-1.5g/L, MgSO40.1-0.5g/L, FeSO4·7H2O0.01-0.05g/L, MnSO4·
H2O0.010.05g/L, pH7.0,0.075MPa high pressure steam sterilization 15min.
Described fermentation medium components is: glucose 15-45g/L, yeast powder 1-2g/L, soybean cake hydrolyzed solution 5-15mL/L,
Semen Maydis pulp 5-15mL/L, KH2PO41-5g/L, citric acid 0.5-2g/L, MgSO40.5-1g/L, FeSO4·7H2O0.1-0.5g/L,
MnSO4·H2O0.1-0.5g/L, pH7.0,0.075MPa high pressure steam sterilization 15min.
Beneficial effect:
(1) present invention is with E.coli MG1655 (ATCC47076) as starting strain, utilizes metabolic engineering means, by by force
Change and extend L-threonine synthesis α-one base butanoic acid approach and cut off the further metabolism of the latter approach construct production α-
The Recombinant organism of alpha-ketobutyric acid.After fermentation 20-24h, α-one base butyric acid yield reaches 8.5-15.7g/L.This
Ferment process is aerobic fermentation, and thalli growth is fast, and fermentation period is short, and rate of producing acid is high, has no that direct fermentation produces α-one at present
The report of base butanoic acid.
(2) fermentation technology that the method uses is simple, it is easy to controlling, production cost is low, beneficially the pushing away of industrialized production
Wide and application.
Accompanying drawing illustrates:
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 downstream of gene 400bp fragment；3 are
ilvBK；
4 be with E.coli MG1655 genome as template, CH1 and the CH2 fragment as primer amplification;
5 are incorporated into E.coli MG1655 postgenome, the fragment with CH1 and CH2 as primer amplification for ilvBK;
6 be with ilvB gene knockout pnca gene group as template, ID1 and the ID2 fragment as primer amplification；
7 be with E.coli MG1655 genome as template, ID1 and the ID2 fragment 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 downstream of gene 400bp fragment;3 are
ilvIK；
4 be with E.coli MG1655ilvB gene knockout pnca gene group as template, CH1 and the CH2 sheet as primer amplification
Section;
After 5 are incorporated into E.coli MG1655ilvB gene knockout pnca gene group for ilvIK, expand with CH1 and CH2 for primer
The fragment increased;
6 for knock out, so that ilvB, ilvI are dual-gene, the fragment that pnca gene group is as template, ID3 and ID4 is as primer amplification；
7 be with E.coli MG1655ilvB gene knockout strain and as template, ID3 and the ID4 fragment 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 downstream of gene 400bp fragment;3 are
thrL K
4 be with E.coli MG1655ilvB gene knockout pnca gene group as template, CH1 and the CH2 sheet as primer amplification
Section;
After 5 are incorporated into E.coli MG1655ilvB gene knockout pnca gene group for thrLK, expand with CH5 and CH6 for primer
The fragment increased;
6 be with THRZ genome as template, ID5 and the ID6 fragment as primer amplification；
7 be with E.coli MG1655ilvB gene knockout strain and as template, ID5 and the ID6 fragment as primer amplification；
Fig. 7 recombiant plasmid pW-ilvA and engineered strain KBA builds schematic diagram；
The restriction enzyme digestion and electrophoresis collection of illustrative plates of Fig. 8 recombiant plasmid pW-ilvA；
Wherein M-Marker1-pW-ilvA is through Xba I and BamH I enzyme action；
Fig. 9 genetic engineering bacterium KBA stability test figure.
Embodiment:
Embodiment 1:E.coli MG1655 acyl hydroxy acid synzyme I large subunit encoding gene ilvB knocks out
According to ilvB (GeneID:948181) in escherichia coli in ncbi database (Escherichia coli) MG1655
Gene 5 ' and 3 ' end 400bp sequential design upstream and downstream homology arm amplimer ilvB1-F, ilvB1-R, ilvB3-F and ilvB3-
R, and with this bacterium genomic DNA for template amplification homology arm fragment.
PCR condition is 94 DEG C 5min1 circulation, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C 40s30 circulations, 72 DEG C 10min1
Circulation, reaction system is 100 μ L.
PCR primer is reclaimed through 1.5% agarose gel electrophoresis rear cutout glue, the fragment of acquisition is respectively designated as ilvB1,
ilvB3。
According to chloramphenicol resistance gene box sequential design amplimer ilvB2-F, ilvB2-R in plasmid pKD3, and with this
Plasmid is template amplification chloramphenicol resistance gene box fragment.
PCR condition is 94 DEG C 5min1 circulation, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C 70s30 circulations, 72 DEG C 10min1
Circulation, reaction system is 100 μ L.
PCR primer is reclaimed through 1.5% agarose gel electrophoresis rear cutout glue, the named ilvB2 of fragment that will obtain.
With ilvB1 and ilvB2 fragment as template, primer ilvB1-F and ilvB2R is utilized to carry out over-lap PCR.
PCR condition is 94 DEG C 5min1 circulation, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C 100s30 circulations, 72 DEG C of 10min1
Individual circulation, reaction system is 100 μ L.
PCR primer is reclaimed through 1.5% agarose gel electrophoresis rear cutout glue, named ilvB1-2.With ilvB1-2 and
IlvB3 fragment is template, utilizes primer ilvB1-F and ilvB3-R to carry out over-lap PCR, and PCR condition is 94 DEG C 5min1 circulation,
94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C 120s30 circulations, 72 DEG C 10min1 circulation, reaction system is 100 μ L.By PCR primer warp
1.5% agarose gel electrophoresis rear cutout glue reclaims, it is thus achieved that ilvB knocks out fragment, named ilvBK.
IlvBK electricity is converted to E.coli MG1655 competent cell containing pKD46 plasmid (electricity convert voltage and time
Between be respectively 2500V and 5ms).Rapidly in 1mL SOC culture medium 37 DEG C, coat containing chloromycetin (25 after 150rpm recovery 1h
μ g/mL) on LB solid medium flat board.It is inverted after cultivating 24h to use and identifies that primer CH1 and CH2 is reflected by the method for bacterium colony PCR
Determining positive transformant, chloramphenicol resistance gene box is successfully incorporated into bacterium colony its amplified fragments about 1054bp of genome.By pCP20
Plastid transformation is to above-mentioned positive transformant to eliminate chloramphenicol resistance gene, and after 42 DEG C of incubated overnight, screening can be in nonreactive
Property flat board on growth and containing the single bacterium colony not grown on chloromycetin flat board and use qualification primer I D1 and ID2 verify,
IlvB is by the bacterial strain amplified fragments about 234bp of successful knockout, and building process is as it is shown in figure 1, Fig. 2 is PCR checking.
Primer sequence is shown in Table 1.
Wherein: LB solid medium (g/L): tryptone 10, yeast extract 5, NaCl10, agar powder 15, with steaming
Distilled water is settled to 1000mL, 121 DEG C of high pressure steam sterilization 20min.
SOC culture medium (g/L): tryptone 20, yeast extract 5, NaCl0.5, KCl0.2, MgCl20.95, Fructus Vitis viniferae
Sugar 3.6.
Embodiment 2:E.coli MG1655ilvB knocks out strain Acetohydroxyacid synthase III large subunit encoding gene ilvI's
Knock out
According to escherichia coli in ncbi database (Escherichia coli) MG1655ilvI (GeneID:947267) base
Because of 5 ' and 3 ' end 400bp sequential design homology arm amplimer ilvI1-F, ilvI1-R, ilvI3-F and ilvI3-R, and with this
Bacterium genomic DNA is template amplification homology arm fragment.
PCR condition is 94 DEG C 5min1 circulation, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C 40s30 circulations, 72 DEG C 10min1
Circulation, reaction system is 100 μ L.
According to chloramphenicol resistance gene box sequential design amplimer ilvI2-F, ilvI2-R in plasmid pKD3, and with this
Plasmid is template amplification chloramphenicol resistance gene box fragment, and PCR condition is 94 DEG C 5min1 circulation, 94 DEG C of 30s, 55 DEG C of 30s,
72 DEG C 70s30 circulation, 72 DEG C 10min1 circulation, reaction system is 100 μ L.By PCR primer through 1.5% agarose gel electricity
Swimming rear cutout glue reclaims, and the fragment of acquisition is respectively designated as ilvI1, ilvI3 and ilvI2.
With ilvI1 and ilvI2 fragment as template, utilizing primer ilvI1-F and ilvI2R to carry out over-lap PCR, PCR condition is
94 DEG C 5min1 circulation, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C 100s30 circulations, 72 DEG C 10min1 circulation, reaction system is
100μL.PCR primer is reclaimed through 1.5% agarose gel electrophoresis rear cutout glue, named ilvI1-2.With ilvI1-2 and ilvI3
Fragment is template, utilizes primer ilvI1-F and ilvI3-R to carry out over-lap PCR, and PCR condition is 94 DEG C 5min1 circulation, 94 DEG C
30s, 55 DEG C of 30s, 72 DEG C 120s30 circulations, 72 DEG C 10min1 circulation, reaction system is 100 μ L.By PCR primer through 1.5%
Agarose gel electrophoresis rear cutout glue reclaims, it is thus achieved that ilvI knocks out fragment, named ilvIK.
IlvIK electricity is converted to the E.coli MG1655ilvB gene knockout strain competent cell containing pKD46 plasmid
(electricity converts voltage and the time is respectively 2500V and 5ms).Rapidly in 1mL SOC culture medium 37 DEG C, be coated with after 150rpm recovery 1h
It is distributed in containing on chloromycetin (25 μ g/mL) LB solid medium flat board.It is inverted after cultivating 24h to use and identifies that primer CH1 and CH2 passes through
The method of bacterium colony PCR identifies positive transformant, and chloramphenicol resistance gene box is successfully incorporated into its amplified fragments of bacterium colony of genome
About 1054bp.By pCP20 Plastid transformation to above-mentioned positive transformant to eliminate chloramphenicol resistance gene, through 42 DEG C of incubated overnight
After, screening can grow on non-resistant flat board and containing the single bacterium colony not grown on chloromycetin flat board and use qualification primer
ID3 and ID4 verifies, ilvI is by the bacterial strain amplified fragments about 233bp of successful knockout.
Building process is as it is shown on figure 3, Fig. 4 is PCR checking.
Primer sequence is shown in Table 1.
Wherein: LB solid medium (g/L): peptone 10, yeast extract 5, NaCl10, agar powder 15, distilled water is used
It is settled to 1000mL, 121 DEG C of high pressure steam sterilization 20min.
The structure of embodiment 3THRZ bacterial strain
According to thrL (GeneID:948283) gene 5 ' in E. coli MG1655 in ncbi database and 3 ' ends
400bp sequential design homology arm amplimer thrL1-F, thrL1-R, thrL3-F and thrL3-R, and with this bacterium genomic DNA
For template amplification homology arm fragment.
PCR condition is 94 DEG C 5min1 circulation, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C 40s30 circulations, 72 DEG C 10min1
Circulation, reaction system is 100 μ L.
PCR primer is reclaimed through 1.5% agarose gel electrophoresis rear cutout glue, the fragment of acquisition is respectively designated as thrL1,
thrL3。
According to chloramphenicol resistance gene box sequential design amplimer thrL2-F, thrL2-R in plasmid pKD3, and with this
Plasmid is template amplification chloramphenicol resistance gene box fragment.
PCR condition is 94 DEG C 5min1 circulation, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C 70s30 circulations, 72 DEG C 10min1
Circulation, reaction system is 100 μ L.
PCR primer is reclaimed through 1.5% agarose gel electrophoresis rear cutout glue, the named thrL2 of fragment that will obtain.
With thrL1 and thrL2 fragment as template, primer thrL1-F and thrL2R is utilized to carry out over-lap PCR.
PCR condition is 94 DEG C 5min1 circulation, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C 100s30 circulations, 72 DEG C of 10min1
Individual circulation, reaction system is 100 μ L.
PCR primer is reclaimed through 1.5% agarose gel electrophoresis rear cutout glue, named thrL1-2.With thrL1-2 and
ThrL3 fragment is template, utilizes primer thrL1-F and thrL3-R to carry out over-lap PCR, and PCR condition is 94 DEG C 5min1 circulation,
94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C 120s30 circulations, 72 DEG C 10min1 circulation, reaction system is 100 μ L.By PCR primer warp
1.5% agarose gel electrophoresis rear cutout glue reclaims, it is thus achieved that thrL knocks out fragment, named thrLK.
ThrLK electricity is converted ilvB, ilvI gene knockout strain competence obtained to the embodiment 2 containing pKD46 plasmid thin
In born of the same parents (electricity converts voltage and the time is respectively 2500V and 5ms).37 DEG C, 150rpm recovery 1h in 1mL SOC culture medium rapidly
After coat containing on chloromycetin (25 μ g/mL) LB solid medium flat board.It is inverted after cultivating 24h to use and identifies primer CH1 and CH2
Identifying positive transformant by the method for bacterium colony PCR, chloramphenicol resistance gene box is successfully incorporated into its amplification of bacterium colony of genome
Fragment about 1054bp.By pCP20 Plastid transformation to above-mentioned positive transformant to eliminate chloramphenicol resistance gene, overnight train through 42 DEG C
After Yanging, screening can grow on non-resistant flat board and containing the single bacterium colony not grown on chloromycetin flat board and use qualification primer
ID5 and ID6 verifies, thrL is by the bacterial strain amplified fragments about 246bp of successful knockout.
Building process is as it is shown in figure 5, Fig. 6 is PCR checking.The named THRZ of engineering strain that will successfully construct.
The process LAN of embodiment 4:ilvA gene
According to ilvA gene in escherichia coli in ncbi database (Escherichia coli) MG1655 (GeneID:
948287) primers ILA-F and ILA-R, with this bacterium genomic DNA for template amplification ilvA gene.
PCR condition is 94 DEG C 5min1 circulation, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C 130s30 circulations, 72 DEG C of 10min1
Individual circulation, reaction system is 100 μ L.
Take 10 μ L PCR primer after 1.5% agarose gel electrophoresis, obtain the band of 1.5-1.6kb, actual with gene big
Little consistent (1573bp).Xba I and BamH I is used to carry out double digestion, through 1.5% agar by remaining after 90 μ L amplified fragments reclaim
Sugar gel electrophoresis rear cutout glue reclaims and takes the recovery fragment employing T adapting to volume4Ligase is connected to the expression through identical enzyme action
Carrier pWSK29, uses CaCl2Method is converted to escherichia coli DH10 β competent cell.Picking can be containing ammonia benzyl mycin
In the LB solid culture of (100 μ g/mL) growth bacterium colony and utilize primer P29F and P29R to carry out bacterium colony PCR qualification.Extract warp
Be accredited as the positive transformant plasmid entrust Beijing Liuhe Huada Genomics Technology Co., Ltd check order, sequencing result with
Expection is consistent, shows plasmid construction success, by its named pW-ilvA.Building process such as Fig. 7, checking is such as Fig. 8.
Use CaCl2Recombiant plasmid pW-ilvA is converted ilvB, ilvI, thrL gene knockout obtained to embodiment 3 by method
Bacterium.Primer P29F and P29R is used to carry out bacterium colony after growing bacterium colony in the LB solid culture containing ammonia benzyl mycin (100 μ g/mL)
PCR identifies, the transformant being identified as the positive is genetic engineering bacterium of the present invention, named KBA.
Primer sequence is shown in Table 1.
Table 1 knocks out fragment and the primer sequence of process LAN gene for PCR amplification
Embodiment 5: the 10L ferment tank of genetic engineering bacterium KBA
(1) seed culture: with inoculating loop 3-5 propped up through the escherichia coli KBA of fresh slant activation be all seeded to equipped with
The 5L fermentation tank of 1L seed culture medium, stream add 25% (W/V) ammonia regulation fermentation liquid pH to 6.8-7.2, dissolved oxygen maintains 30-
50%, ventilation 3-5m3/ h, speed of agitator 200-600rpm, cultivate 6-8h for 32-37 DEG C.
(2) ferment tank: the inoculum of step (1) is connected to equipped with 6L fermentation medium with 5%-10% inoculum concentration
10L fermentation tank carry out send out tank cultivate, fermentation temperature 32-37 DEG C, ventilation 3-5m3/ h, speed of agitator 300-1000rpm, molten
Oxygen maintains 30-60%, and stream adds the glucose solution that concentration is 60-80% (W/V), and maintenance remaining sugar concentration is 0.1-0.5% (W/V),
Stream add 25% (W/V) ammonia regulation fermentation liquid pH to 6.8-7.2, fermentation period 20-24h.
(3) detection of α-one base butanoic acid in fermentation liquid: fermentation liquor 8000 × g takes supernatant and spends after being centrifuged 10min
After ionized water dilutes 5 times, UltiMate3000 (Thermo Scientific) high performance liquid chromatograph is used to measure α-one base fourth
The content of acid.Testing conditions is: chromatographic column REzex RoA-organic Acid H+, and flow phase 5mmol/L H2SO4, flow velocity
0.5mL/min, column temperature 30 DEG C, detect wavelength 215nm, sample size is 20 μ L.Testing result shows, when going out peak of α-one base butanoic acid
Between be about 12.5min, yield is 8.5-15.7g/L.
Wherein: seed culture based component is:
Sucrose 15-25g/L, Semen Maydis pulp 15-25mL/L, yeast powder 1-4g/L, (NH4)2SO41-4g/L, KH2PO40.5-
1.5g/L, MgSO40.1-0.5g/L, FeSO4·7H2O0.01-0.05g/L, MnSO4·H2O0.010.05g/L, pH7.0,
0.075MPa high pressure steam sterilization 15min.
Fermentation medium components is:
Glucose 15-45g/L, yeast powder 1-2g/L, soybean cake hydrolyzed solution 5-15mL/L, Semen Maydis pulp 5-15mL/L, KH2PO41-
5g/L, citric acid 0.5-2g/L, MgSO40.5-1g/L, FeSO4·7H2O0.1-0.5g/L, MnSO4·H2O0.1-0.5g/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 are all seeded to equipped with 1L through the escherichia coli KBA of fresh slant activation with inoculating loop
The 5L fermentation tank of seed culture medium, stream add 25% (W/V) ammonia regulation fermentation liquid pH to 7.0, dissolved oxygen maintains 35%, ventilation
3m3/ h, speed of agitator 200-600rpm, cultivate 6h for 37 DEG C.
(2) ferment tank: the inoculum of step (1) is connected to equipped with 6L fermentation medium with 8% inoculum concentration
10L fermentation tank carries out sending out tank and cultivates, fermentation temperature 37 DEG C, ventilation 4m3/ h, speed of agitator 300-1000rpm, dissolved oxygen maintains
40%, stream adds the glucose solution that concentration is 80% (W/V), and maintaining remaining sugar concentration is 0.15% (W/V), stream adds 25% ammonia of (W/V)
Water regulation fermentation liquid pH to 7.0, fermentation period 20h.
(3) detection of α-one base butanoic acid in fermentation liquid: fermentation liquor 8000 × g takes supernatant and spends after being centrifuged 10min
After ionized water dilutes 5 times, UltiMate3000 (Thermo Scientific) high performance liquid chromatograph is used to measure α-one base fourth
The content of acid.Testing conditions is: chromatographic column REzex RoA-organic Acid H+, and flow phase 5mmol/L H2SO4, flow velocity
0.5mL/min, column temperature 30 DEG C, detect wavelength 215nm, sample size is 20 μ L.Testing result shows, when going out peak of α-one base butanoic acid
Between be about 12.5min, yield is 11.6g/L.
Wherein: seed culture based component is:
Sucrose 15.5g/L, Semen Maydis pulp 15mL/L, yeast powder 2.5g/L, (NH4)2SO42.0g/L, KH2PO40.7g/L,
MgSO40.2g/L, FeSO4·7H2O0.02g/L, MnSO4·H2O0.01g/L, pH7.0,0.075MPa high pressure steam sterilization
15min。
Fermentation medium components is:
Glucose 20g/L, yeast powder 1.5g/L, soybean cake hydrolyzed solution 5mL/L, Semen Maydis pulp 8mL/L, KH2PO41.5g/L, Fructus Citri Limoniae
Acid 0.7g/L, MgSO40.5g/L, FeSO4·7H2O0.3g/L, MnSO4·H2O0.2g/L, pH7.0,0.075MPa high steam
Sterilizing 15min.
Embodiment 7: the 10L ferment tank of genetic engineering bacterium KBA
(1) seed culture: 4 are all seeded to equipped with 1L through the escherichia coli KBA of fresh slant activation with inoculating loop
The 5L fermentation tank of seed culture medium, stream add 25% (W/V) ammonia regulation fermentation liquid pH to 7.0, dissolved oxygen maintains 35%, ventilation
4m3/ h, speed of agitator 200-600rpm, cultivate 6h for 37 DEG C.
(2) ferment tank: the inoculum of step (1) is connected to equipped with 6L fermentation medium with 10% inoculum concentration
10L fermentation tank carries out sending out tank and cultivates, fermentation temperature 37 DEG C, ventilation 4m3/ h, speed of agitator 300-1000rpm, dissolved oxygen maintains
40%, stream adds the glucose solution that concentration is 80% (W/V), and maintaining remaining sugar concentration is 0.15% (W/V), stream adds 25% ammonia of (W/V)
Water regulation fermentation liquid pH to 7.0, fermentation period 24h.
(3) detection of α-one base butanoic acid in fermentation liquid: fermentation liquor 8000 × g takes supernatant and spends after being centrifuged 10min
After ionized water dilutes 5 times, UltiMate3000 (Thermo Scientific) high performance liquid chromatograph is used to measure α-one base fourth
The content of acid.Testing conditions is: chromatographic column REzex RoA-organic Acid H+, and flow phase 5mmol/L H2SO4, flow velocity
0.5mL/min, column temperature 30 DEG C, detect wavelength 215nm, sample size is 20 μ L.Testing result shows, when going out peak of α-one base butanoic acid
Between be about 12.5min, yield is 15.7g/L.
Wherein: seed culture based component is:
Sucrose 15.5g/L, Semen Maydis pulp 20mL/L, yeast powder 2.5g/L, (NH4)2SO42.0g/L, KH2PO40.7g/L,
MgSO40.2g/L, FeSO4·7H2O0.02g/L, MnSO4·H2O0.01g/L, pH7.0,0.075MPa high pressure steam sterilization
15min。
Fermentation medium components is:
Glucose 20g/L, yeast powder 1.5g/L, soybean cake hydrolyzed solution 6mL/L, Semen Maydis pulp 10mL/L, KH2PO42.5g/L, lemon
Lemon acid 0.7g/L, MgSO40.5g/L, FeSO4·7H2O0.1g/L, MnSO4·H2O0.25g/L, pH7.0,0.075MPa high pressure steams
Vapour sterilizing 15min.
Embodiment 8: genetic engineering bacterium KBA plasmid stability
Use plating dilutions counting method, by frozen genetic engineering bacterium KBA in-80 DEG C of glycerol pipes containing ammonia benzyl mycin
Rule on the LB solid plate of (100 μ g/mL), after 37 DEG C are inverted cultivation 48h, contain in 5ml with inoculating loop picking list colony inoculation
In the LB fluid medium of ammonia benzyl mycin (100 μ g/mL), after 37 DEG C of shaking table shaken cultivation 24h, proceed to 5ml with the inoculum concentration of 1%
In 37 DEG C of shaking table shaken cultivation in LB fluid medium.Once, inoculum concentration is 1% to every 24h transferred species, and continuous passage 50 times, every 5
Sub-sampling carries out the disappearance degree of plating dilutions detection plasmid.Detecting step is, takes bacterium solution lmL sterilized water stepwise dilution, takes
Three suitable gradients, are respectively coated containing on ammonia benzyl mycin (100 μ g/mL) and the LB solid plate of antibiotic-free, then in
Being inverted in 37 DEG C of incubators and cultivate 24h, calculate the clump count in each ware, and be calculated as follows plasmid loss rate, result is such as
Shown in Fig. 7, after 50 times pass on, plasmid loss rate is only 7.66%.
Claims (5)
1. the genetic engineering bacterium application in fermenting and producing α-one base butanoic acid, it is characterised in that described genetic engineering bacterium, be
Starting strain escherichia coli (Escherichia coli) MG1655 is carried out the bacterium of genetic engineering modified acquisition；Described gene work
Journey transform process LAN threonine dehydratase encoding gene ilvA as, and knocks out Acetohydroxyacid synthase I large subunit encoding gene
IlvB, Acetohydroxyacid synthase III large subunit encoding gene ilvI and and threonine operon leader peptide encoding gene
thrL；
Described application comprises the following steps:
1. seed culture: be seeded to the 5L fermentation tank equipped with 1L seed culture medium after activated for described genetic engineering bacterium, stream
The ammonia regulation fermentation liquid pH to 6.8-7.2 adding 25%, dissolved oxygen maintains 30-50%, ventilation 3-5m3/ h, speed of agitator
Cultivate 6-8h for 200-600rpm, 32-37 DEG C；
2. ferment tank: step inoculum 1. is connected to equipped with 6L fermentation medium with 5%-10% inoculum concentration
10L fermentation tank carries out fermentation culture, fermentation temperature 32-37 DEG C, ventilation 3-5m3/ h, speed of agitator 300-1000rpm, dissolved oxygen
Maintaining 30-60%, stream adds the glucose solution that concentration is 60-80%, and maintenance remaining sugar concentration is 0.1-0.5% (W/V), and stream adds
Ammonia regulation the fermentation liquid pH to 6.8-7.2, fermentation period 20-24h of 25%；
3. the detection of α-one base butanoic acid in fermentation liquid: fermentation liquor 8000 × g takes supernatant and uses deionized water after being centrifuged 10min
After diluting 5 times, UltiMate 3000Thermo Scientific high performance liquid chromatograph is used to measure containing of α-one base butanoic acid
Amount；
Testing conditions is: chromatographic column REzex RoA-organic Acid H+, and flow phase 5mmol/L H2SO4, flow velocity 0.5mL/
Min, column temperature 30 DEG C, detect wavelength 215nm, sample size is 20 μ L；
Described seed culture based component is: sucrose 15-25g/L, Semen Maydis pulp 15-25mL/L, yeast powder 1-4g/L, (NH4)2SO41-
4g/L, KH2PO40.5-1.5g/L, MgSO40.1-0.5g/L, FeSO4·7H2O 0.01-0.05g/L, MnSO4·
H2O0.010.05g/L, pH 7.0,0.075MPa high pressure steam sterilization 15min；
Described fermentation medium components is: glucose 15-45g/L, yeast powder 1-2g/L, soybean cake hydrolyzed solution 5-15mL/L, Semen Maydis
Slurry 5-15mL/L, KH2PO41-5g/L, citric acid 0.5-2g/L, MgSO40.5-1g/L, FeSO4·7H2O 0.1-0.5g/L,
MnSO4·H2O 0.1-0.5g/L, pH 7.0,0.075MPa high pressure steam sterilization 15min.
2. a genetic engineering bacterium, is that starting strain escherichia coli (Escherichia coli) MG1655 is carried out genetic engineering
The bacterium that transformation obtains；Described genetic engineering modified for process LAN threonine dehydratase encoding gene ilvA, and knock out acetyl hydroxyl
Acid enzyme I large subunit encoding gene ilvB, Acetohydroxyacid synthase III large subunit encoding gene ilvI and and threonine
Operon leader peptide encoding gene thrL；
Described engineering bacteria fermentation produces the method for α-one base butanoic acid, and step is as follows:
(1) seed culture: after described genetic engineering bacterium activation, be seeded to the 5L fermentation tank equipped with 1L seed culture medium, stream
Adding the ammonia regulation fermentation liquid pH to 6.8-7.2 of 25%W/V, dissolved oxygen maintains 30-50%, ventilation 3-5m3/ h, speed of agitator
Cultivate 6-8h for 200-600rpm, 32-37 DEG C；
(2) ferment tank: the inoculum of step (1) is connected to equipped with 6L fermentation medium with 5%-10% inoculum concentration
10L fermentation tank carries out fermentation culture, fermentation temperature 32-37 DEG C, ventilation 3-5m3/ h, speed of agitator 300-1000rpm, dissolved oxygen
Maintaining 30-60%, stream adds the glucose solution that concentration is 60-80%W/V, and maintenance remaining sugar concentration is 0.1-0.5%W/V, stream
Add ammonia regulation the fermentation liquid pH to 6.8-7.2, fermentation period 20-24h of 25%W/V；
Described seed culture based component is: sucrose 15-25g/L, Semen Maydis pulp 15-25mL/L, yeast powder 1-4g/L, (NH4)2SO41-
4, KH2PO40.5-1.5g/L, MgSO40.1-0.5g/L, FeSO4·7H2O 0.01-0.05g/L, MnSO4·
H2O0.010.05g/L, pH 7.0,0.075MPa high pressure steam sterilization 15min；
Described fermentation medium components is: glucose 15-45g/L, yeast powder 1-2g/L, soybean cake hydrolyzed solution 5-15mL/L, Semen Maydis
Slurry 5-15mL/L, KH2PO41-5g/L, citric acid 0.5-2g/L, MgSO40.5-1g/L, FeSO4·7H2O 0.1-0.5g/L,
MnSO4·H2O 0.1-0.5g/L, pH 7.0,0.075MPa high pressure steam sterilization 15min.
Genetic engineering bacterium the most according to claim 2, it is characterised in that: described threonine dehydratase encoding gene ilvA comes
From E. coli MG1655, GeneID:948287, nucleotide sequence such as the SEQ ID NO:1 in sequence table.
Genetic engineering bacterium the most according to claim 2, it is characterised in that: described Acetohydroxyacid synthase I large subunit is compiled
SEQ ID NO:2 in the nucleotide sequence such as sequence table of code gene ilvB, described Acetohydroxyacid synthase III large subunit encodes
SEQ ID NO:3 in the nucleotide sequence of gene ilvI such as sequence table, described threonine operon leader peptide encoding gene thrL
Nucleotide sequence such as sequence table in SEQ ID NO:4.
5. the method building genetic engineering bacterium described in claim 2, comprises the steps:
(1) the knocking out of ilvB gene
(1) use round pcr with escherichia coli MG1655 genome as template, according to ilvB in MG1655 (GeneID:
948181) gene 5 ' and 3 ' end 400bp sequential design homology arm primers, amplification obtains the upstream and downstream homology arm of ilvB gene；
(2) use round pcr with pKD3 plasmid as template, design primer, expand chloramphenicol resistance gene box fragment；
(3) amplified fragments obtained with step (1), (2) is that template passes through over-lap PCR acquisition ilvB gene knockout fragment, described
Gene knockout fragment by Acetohydroxyacid synthase I large subunit encoding gene ilvB upstream and downstream homology arm genetic fragment and
Chloramphenicol resistance gene box fragment forms；
(4) said gene is knocked out in the starting strain competent cell that fragment imports containing pKD46 plasmid, it is thus achieved that positive transformants
Son, obtains ilvB clpp gene after eliminating the chloramphenicol resistance gene in positive transformant degerming；
(2) the knocking out of ilvI gene
(1) ilvI (GeneID:947267) gene knockout fragment is built with the method that (1)-(3) in step () are identical, described
IlvI gene knockout fragment is made up of upstream and downstream homology arm genetic fragment and the chloramphenicol resistance gene box fragment of ilvI；
(2) the gene knockout fragment in (two)-(1) is imported the ilvB gene knockout bacterium competence cell containing pKD46 plasmid
Middle acquisition positive transformant, obtains ilvB, ilvI clpp gene after eliminating the chloramphenicol resistance gene in positive transformant degerming；
(3) the knocking out of thrL gene
(1) thrL (GeneID:948283) gene knockout fragment is built with the method that (1)-(3) in step () are identical；
(2) the gene knockout fragment in (three)-(1) is imported ilvB, ilvI gene knockout bacterium competence containing pKD46 plasmid
Cell obtains positive transformant, after eliminating the chloramphenicol resistance gene in positive transformant, obtains ilvB, ilvI, thrL gene
Knock out bacterium；
(4) process LAN of ilvA gene
Connect after ilvA gene and expression vector pWSK29 Xba I and BamH I thereof are carried out double digestion, connect product and convert
Degerming to above-mentioned ilvB, ilvI, thrL clpp gene, identify that the positive transformant obtained is described gene by bacterium colony PCR
Engineering bacteria.
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