CN101613669B - Colibacillus engineering strain for aerobic fermentation - Google Patents
Colibacillus engineering strain for aerobic fermentation Download PDFInfo
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Abstract
The invention discloses a colibacillus engineering strain for aerobic fermentation. The strain is named as (Escherichia coli) Q10, the preservation code thereof is CCTCC M209113, the gene type thereof is MG1655 delta ptsG delta poxB delta pta:: Cm. Taking a wild Escherichia coli as a contrast, a result of culturing shows that the secreting quantity of the acetate of the engineering strain reduces by 90%, at the same time, the biomass of the strain almost improves by twice. The invention provides a base for building applications of fermentation ways of succinic acid, lactic acid, glutamic acid, 5-aminolevulinic acid, 3-hydracrylicacid, mevalonic acid or PHB as a platform, and has important application prospects and values.
Description
Technical field
The present invention relates to the colibacillus engineering strain E.coli Q10 (CCTCC M 209113) that a strain is used for aerobic fermentation, belong to genetically engineered and microbial fermentation field.
Background technology
Intestinal bacteria are as a kind of model animals, because characteristics such as its genetic background is clear, operative technique maturation have been widely used in numerous areas such as heredity, metabolic engineering.The success of many extrinsic proteins is in expression in escherichia coli and production.Through genetic engineering modified intestinal bacteria, successfully make up such as approach such as succsinic acid fermentation, poly-hydroxy fatty acid fermentation, glutamic acid fermentation and lactobacillus ferments.
Though intestinal bacteria are widely used, there are many shortcomings.Be exactly one like the secretion of acetate and utilize the problem that usually faces in the Escherichia coli fermentation.When having the carbon sources such as glucose of high density in the external environment, intestinal bacteria secrete a large amount of acetates owing to metabolism is uneven.The secretion of acetate has not only caused the waste of carbon source, and acetate has restraining effect to the growth of thalline simultaneously, thereby the secretion of acetate does not utilize the normal growth of thalline and the carrying out of fermentation.
Exist two main acetate to generate approach in the intestinal bacteria.Article one, be the catalysis generation acetate that pyruvic acid passes through PoxB (pyruvic oxidase); Another approach then is that acetyl-CoA generates acetate through Pta (phosphotransacetylase base enzyme) and ackA (E.C. 2.7.2.1), generates ATP simultaneously.Above-mentioned two approach action times are different, intestinal bacteria on space-time through regulating the generating rate that relevant enzymes is regulated acetate.The PoxB approach mainly plays a role after intestinal bacteria get into stationary phase; The Pta-ackA approach then mainly is that intestinal bacteria play a role when logarithmic phase.
Current have two kinds of methods to be applied to reduce the secretion of acetate.A kind of is optimization to zymotechnique, promptly reduces the secretion of acetate through the concentration of carbon sources such as control glucose.Though this method can reduce the generation of acetate, has prolonged fermentation period.Another kind of method then is through the metabolic engineering means, promptly blocks acetate and generates the excretory direct way, reduces the generation of acetate.Yet the correlative study document in past is just studied separately to indivedual acetate genes involveds.And the research and the patent that on the basis of intestinal bacteria pathways metabolism and control methods thereof, make up the engineering strain that lacks ptsG, poxB and pta gene are not simultaneously also appeared in the newspapers.
Summary of the invention
To existing acetate secretion problem in the Escherichia coli fermentation process, the present invention seeks to through genetically engineered, utilize gene knockout to make up the colibacillus engineering strain that a strain is applied to aerobic fermentation.Simultaneously, utilize engineering bacillus strain accumulating poly hydroxy fatty acid (PHA), the present invention is the application that example is set forth engineering bacillus strain with the representative poly-beta-hydroxy-butyrate (PHB) in the PHA family.
The colibacillus engineering strain that is used for aerobic fermentation according to the invention is characterized in that: this bacterial strain is called dust Xi Shi intestinal bacteria Q10, is intestinal bacteria Q10 (Escherichia coli Q10); Bacterial strain has been deposited on May 27th, 2009 that " Chinese typical culture collection " center ", deposit number is CCTCC M 209113.
Above-mentioned colibacillus engineering strain Q10 profile is shaft-like, and size is 0.4～0.6 micron * 1～3 micron, no brood cell.Ordinary pilus and sex fimbria are arranged, belong to gram negative bacillus.This bacterium anabolism ability is strong, well-grown on the ordinary culture medium that contains inorganic salt, amine salt, glucose.Optimum growth temperature is 37 ℃, under 42-44 ℃ of condition, still can grow, and growth temperature range is 15-46 ℃.The genotype of bacterial strain is MG1655 Δ ptsG Δ poxB Δ pta::Cm.
The starting strain of above-mentioned colibacillus engineering strain is a kind of among intestinal bacteria MG1655, DH5a, JM109, W3110, the XL1-Blue, and wherein preferred strain is intestinal bacteria MG1655.Said MG1655, JM109 and W3110 buy in ATCC (U.S. typical case DSMZ); DH5a and X11-blue buy in DSMZ (German microbial preservation center).Alcaligenes eutrophus (Alcaligenes eutrophus) is bought in CICC (Chinese industrial microbial strains preservation administrative center).
Above-mentioned colibacillus engineering strain is to make up through knocking out ptsG, poxB and pta gene, and its genotype can further be transformed acquisition by MG1655 Δ ptsG, MG1655 Δ poxB, MG1655 Δ pta, MG1655 Δ ptsG Δ poxB, MG1655 Δ poxB Δ pta or MG1655 Δ ptsG Δ pta genotype.
The structure of above-mentioned colibacillus engineering strain and detection step are:
(1) the .ptsG gene knocks out
In intestinal bacteria MG1655, knock out gene ptsG (phosphotransferase system II) through the Red recombination system.The intestinal bacteria MG1655 Δ ptsG::Cm called after E.coli Q8 of gained disappearance ptsG gene.
(2) the .poxB gene knocks out
In intestinal bacteria E.coli Q8, knock out gene poxB (pyruvic oxidase) through the Red recombination system.The intestinal bacteria MG1655 Δ ptsG Δ poxB::Cm called after E.coli Q9 of gained disappearance poxB gene.
(3) the .pta gene knocks out
In intestinal bacteria E.coli Q9, knock out gene pta (phosphotransacetylase base enzyme) through the Red recombination system.The intestinal bacteria MG1655 Δ ptsG Δ poxB Δ pta::Cm called after E.coli Q10 of gained disappearance pta gene, this bacterial strain on May 27th, 2009 were preserved in that " Chinese typical culture collection " center ", deposit number is CCTCC M 209113.
Above-mentioned Red recombination system is to express lambda particles phage recombinase Gam through plasmid pKD46, Bet and Exo, and the primer amplification that has a homology arm through design has the reorganization segment of selection markers kan (kalamycin resistance gene) or Cm (chloramphenicol resistance gene) gene.Through the electroporation apparatus electric shock, the segment of will recombinating changes over to expresses lambda particles phage recombinase Gam, in the intestinal bacteria of Bet and Exo then.The reorganization segment the effect of recombinase under and genome on goal gene recombinate, thereby original gene substitution is got off.And resistant gene cuts away from genome through expressing the FLP restriction endonuclease.
(4). the growth curve of engineering strain in the LB substratum is relatively
For the meliority of the constructed engineering strain E.coli of comparative descriptions Q10 (deposit number is CCTCC M 209113), we are bacterial strain MG1655, E.coli LR1010, and E.coli QZ1011 and E.coli QZ1100 cultivate in the LB substratum.Method is following: the mono-clonal in the flat board is chosen to the test tube that LB substratum 3-5ml is housed, placed 30-40 ℃ to cultivate 10-16h down, the inoculum size according to 1-3% changes in the triangular flask of the 250ml that 30-50ml is housed then.As for placing 30-40 ℃, rotating speed is 200-250 commentaries on classics/min.Every interval 2-4h sampling detects bacterium liquid absorbance value then under the 600nm wavelength.Be about to bacterium liquid and place under the room temperature 10,000-12,000 commentaries on classics/min, centrifugal 2-5min.Supernatant is outwelled, utilized the NaCl solution of 0.4-0.6M to wash twice then, resuspended, detect absorbancy.
(5). engineering strain LB add in 1% glucose living weight and acetate rate ratio
Under situation, cultivating the used carbon source of thalline is cheap glucose.So we utilize the LB substratum to add glucose bacterial strain has been carried out cultivating relatively.Bacterial strain MG1655, E.coli Q8, E.coli Q9 and E.coli Q10 cultivate in LB adds the substratum of 1% glucose.Method is following: the mono-clonal in the flat board is chosen to the test tube that LB substratum 3-5ml is housed, placed 30-40 ℃ to cultivate 10-16h down, the inoculum size according to 1-3% changes in the triangular flask of the 250ml that 30-50ml is housed then.As for placing 30-40 ℃, rotating speed is 200-250 commentaries on classics/min.Every interval 2-4h sampling detects bacterium liquid absorbance value then under the 600nm wavelength.Be about to bacterium liquid and place under the room temperature 10,000-12,000 commentaries on classics/min, centrifugal 2-5min.Supernatant is outwelled, utilized the NaCl solution of 0.4-0.6M to wash twice then, resuspended, detect absorbancy.
HPLC (HPLC) analysis is adopted in the meta-bolites analysis.Method: obtaining sample is placed 10,000-12,000 changes centrifugal 2-5min.To utilize the aperture be the membrane filtration of 0.2 μ m to supernatant then.If the untimely detection of filtered sample then place-20 ℃ to preserve weeks down.Glucose, acetate adopt the differential detector, and pyruvic acid then adopts UV-detector to detect, and the detection wavelength is 210nm.Before the test sample, glucose, acetate and pyruvic acid standard model carry out gradient and detect and the drawing standard curve.Sample detects again then.
(6) under the .pH7.0 condition, engineering strain LB add in 1% glucose living weight and acetate rate ratio
Based on (5), we have carried out the constant cultivation of pH to bacterial strain, promptly through regulating the pH of nutrient solution, prevent that thalline from stopping growing because pH crosses low.Thereby can analyze the growth metabolism characteristics of more constructed bacterial strain.Cultural method: the mono-clonal in the flat board is chosen to the test tube that LB substratum 3-5ml is housed, placed 30-40 ℃ to cultivate 10-16h down, the inoculum size according to 1-3% changes shaking in bottle cabinet of 200ml over to then.As for placing 30-40 ℃, rotating speed is 150-250rpm, and pH adopts 2M NaOH to regulate.Every interval 2-4h sampling detects bacterium liquid absorbance value then under the 600nm wavelength.Be about to bacterium liquid and place under the room temperature 10,000-12,000 commentaries on classics/min, centrifugal 2-5min.Supernatant filtered centrifugally be used to detect meta-bolites, and thalline utilizes the NaCl solution of 0.4-0.6M to wash twice, resuspended, detect absorbancy.
The colibacillus engineering strain that is used for aerobic fermentation according to the invention is as the application of platform in the fermentation approach that makes up succsinic acid, lactic acid, L-glutamic acid, 5-amino-laevulic acid, 3-hydroxy-propionic acid, RS-Mevalonic acid or PHB.
Further preferred mode is: the said colibacillus engineering strain that is used for aerobic fermentation is used to prepare PHB as the PHB fermentation approach of platform construction, and its fermentation condition is: 37 ± 1 ℃ of temperature, shaking speed 250 ± 30 commentaries on classics/min, fermentation time 48 ± 2h; Fermention medium consists of: Na for the M9 substratum of improvement
2HPO
46g/L, KH
2PO
43g/L, NaCl 0.5g/L, NH
4Cl 1g/L, YE 2g/L, MgSO
42mmol/L, CaCl
20.1mmol/L, glucose 20g/L.
PHB is the application that example is set forth colibacillus engineering strain according to the invention with accumulation, and concrete steps are following:
(1) structure of .PHB fermentation approach
PHB expression vector pSK-phbCAB is converted among the engineering strain E.coli Q10 (deposit number CCTCC M209113).Simultaneously, MG1655 also changes pSK-phbCAB over to as control strain.
(2) .PHB fermentative prodn
Engineering strain E.coli Q10/pSK-phbCAB and E.coli MG1655/pSK-phbCAB mono-clonal are chosen to the test tube of the LB that 3-5ml is housed.Place 30-40 ℃ to cultivate down 10-16h, then thalline is placed under the room temperature 10,000-12,000 commentaries on classics/min, centrifugal 2-5min.Utilize M9 substratum washing 2 times then, add the resuspended somatic cells of M9 substratum of 3-5ml then, the inoculum size according to volume ratio 1-3% changes in the triangular flask of the 1L that 100-200ml improvement M9 substratum is housed then.The M9 substratum of improvement adds 2% glucose.Place 30-40 ℃ to cultivate 10-16h down bacterium liquid.Inoculum size according to volume ratio 5-10% changes the 5L fermentation cylinder for fermentation that 2.5-3.5L is housed over to then.The induced concentration of IPTG is 0.1-0.2mol/L.Wherein bacterium liquid pH adopts 2M NaOH to regulate automatically, and dissolved oxygen (DO) is controlled at more than the 30-50 through regulating rotating speed.Every interval 4-8h sampling once.Centrifugal 5-10ml fermented liquid with the thalline lyophilize, is the membrane filtration of 0.2 μ m and supernatant utilizes the aperture.If the untimely detection of filtered sample then place-20 ℃ to preserve weeks down.
Said pSK
-Plasmid is the escherichia coli cloning plasmid, available from Invitrogen company.Promotor is an IPTG inductive lac promotor.The phbCAB gene source is in alcaligenes eutrophus (Alcaligenes eutrophus).
The present invention is on the basis of analyzing intestinal bacteria pathways metabolism and control methods thereof; Design and disappearance ptsG, poxB and pta engineering strain when having made up a strain and be applied to aerobic fermentation, and set forth this aerobic fermentation engineering strain with the example that is changed to of acetate secretory volume, living weight.The structure that is configured to other purpose product fermentation approach of aerobic fermentation bacterial strain of the present invention lays the foundation, and has important Practical significance.
Description of drawings
Intestinal bacteria according to the invention (Escherichia coli) Q10 has been deposited on May 27th, 2009 that " Chinese typical culture collection " center ", deposit number is CCTCC M 209113.
The aerobic fermentation approach platform that Fig. 1 makes up for the present invention.
Fig. 2 is the growth curve of the constructed bacterial strain of the present invention in the LB substratum.
Fig. 3 adds the biomass yield in 1% dextrose culture-medium for the constructed bacterial strain of the present invention at LB.
Fig. 4 adds the acetate output in 1% dextrose culture-medium for the constructed bacterial strain of the present invention at LB
Fig. 5 is under constant pH 7.0 conditions, and the constructed bacterial strain of the present invention adds the biomass yield in 1% dextrose culture-medium at LB.
Fig. 6 is under constant pH 7.0 conditions, and the constructed bacterial strain of the present invention adds the acetate output in 1% dextrose culture-medium at LB.
Fig. 7 is the PHB fermentation result of the constructed engineering strain of the present invention.
Embodiment
(a) knocking out of .ptsG gene:
Bacterial classification: intestinal bacteria MG1655
Said LB substratum is: peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, penbritin, 100mg/L, kantlex 50mg/L.
Said ammonia benzyl mycin resistant panel is the penbritin that contains 100mg/L, the LB solid medium of 1.5% agar powder.
Said chlorampenicol resistant is dull and stereotyped for containing the paraxin of 50mg/L, the LB solid medium of 1.5% agar powder.
Said SOC substratum is: peptone 2g/L, yeast powder 0.5g/L, NaCl 0.0585g/L, KCl 0.0186g/L, MgCl
20.203g, MgSO
40.246g/L, glucose 20mmol/L.
(1) the pulsating clone of homologous recombination
Utilize the Red recombination system that goal gene is knocked out.The ptsG gene order of announcing according to Genbank designs primer:
pKD-ptsG?F：
5′-ACGTAAAAAAAGCACCCATACTCAGGAGCACTCTCAATTGTGTAGGCTGGAGCTGCTTC-3′
pKD-ptsGR：
5′-AGCCATCTGGCTGCCTTAGTCTCCCCAACGTCTTACGGAATGGGAATTAGCCATGGTCC-3′
Obtain to have the reorganization segment of chlorampenicol resistant through PCR (polymerase chain reaction) amplification in vitro with pKD3.The PCR reaction system is following: (primer concentration is 20 μ mol/L)
10 * damping fluid, 5 μ l;
25mmol/LMgCl2?4μl；
Four kinds of dNTP mixed solutions of 10mmol/L, 1 μ l;
Each 1 μ l of upstream and downstream primer;
TaqDNA polysaccharase 0.5 μ l;
The PCR reaction conditions: 97 ℃ of preparatory sex change 10min, 94 ℃ of sex change 60s, 58 ℃ of annealing 30s, 72 ℃ are extended 90s, and 30 circulations are extended 10min, 4 ℃ of preservations for back 72 ℃.After the digestion of DpnI restriction endonuclease, reclaim purifying and concentrate the homologous recombination segment.
(2) preparation of electric transformed competence colibacillus cell
(I) picking has the intestinal bacteria MG1655 of pKD46 plasmid, changes in the LB substratum, adds 0.2% pectinose simultaneously, cultivates OD
600To 0.5;
(II) ice bath 15min, centrifugal thalline utilizes 10% glycerine washing three times then;
(III) glycerine of adding 10% is concentrated into 50 times, the packing competence.
(3) electricity transforms, the screening recon
(I) the homologous recombination segment of absorption 8 μ g/l adds in the competent cell of 100 μ l mixing.Regulate electroporation apparatus, 2.5Kv, electric shock;
(II) the SOC substratum of adding 900 μ l, 37 ℃, 150 commentaries on classics/min cultivate 1h;
(III) the coating chlorampenicol resistant is dull and stereotyped, transfers the recon utilization
ptsG?test?F：5′-CCTGTACACGGCGAGGCTCT-3′
ptsG?test?R：5′-AATAACACCTGTAAAAAAGGCAGCC-3′
Carry out PCR and detect, confirm further that through the order-checking of PCR product the poxB gene is changed by chloramphenicol resistance gene.
(IV) PLP site-specific reorganization
Change pCP20 over to chlorampenicol resistant clone, cultivate 8h for 30 ℃, after be increased to 42 ℃ and spend the night, thermal induction FLP recombinase is expressed, plasmid is also lost gradually.Utilize transfering loop to dip in to get bacterium liquid and on the non-resistant substratum, rule; With the mono-clonal that grows change over to simultaneously non-resistant dull and stereotyped with the kalamycin resistance flat board on cultivate, on the non-resistant flat board, grow and being deleted by the FlP recombinase of on the kalamycin resistance flat board, not growing.Utilize detection primer poxB test F and poxB test R further to identify.
(V) obtain engineering strain MG1655 Δ ptsG::Cm, called after E.coli Q8.
(b) knocking out of .poxB gene:
Bacterial classification: intestinal bacteria E.coli Q8
Said LB substratum is: peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, penbritin, 100mg/L, kantlex 50mg/L.
Said ammonia benzyl mycin resistant panel is the penbritin that contains 100mg/L, the LB solid medium of 1.5% agar powder.
Said chlorampenicol resistant is dull and stereotyped for containing the paraxin of 50mg/L, the LB solid medium of 1.5% agar powder.
Said SOC substratum is: peptone 2g/L, yeast powder 0.5g/L, NaCl 0.0585g/L, KCl 0.0186g/L, MgCl
20.203g, MgSO
40.246g/L, glucose 20mmol/L.
(1) the pulsating clone of homologous recombination
Utilize the Red recombination system that goal gene is knocked out.The poxB gene order of announcing according to Genbank designs primer:
pKD-poxB?F：
5′-AAACTTGTTACCGTTATCACATTCAGGAGATGGAGAACCGTGTAGGCTGGAGCTGCTTC-3′
pKD-poxB?R：
5′-CATGGCATGTCCTTATTATGACGGGAAATGCCACCCTTTATGGGAATTAGCCATGGTCC-3′
Obtain to have the reorganization segment of chlorampenicol resistant through PCR (polymerase chain reaction) amplification in vitro with pKD3.The PCR reaction system is following: (primer concentration is 20 μ mol/L)
10 * damping fluid, 5 μ l;
25mmol/LMgCl2?4μl；
Four kinds of dNTP mixed solutions of 10mmol/L, 1 μ l;
Each 1 μ l of upstream and downstream primer;
TaqDNA polysaccharase 0.5 μ l;
The PCR reaction conditions: 97 ℃ of preparatory sex change 10min, 94 ℃ of sex change 60s, 58 ℃ of annealing 30s, 72 ℃ are extended 90s, and 30 circulations are extended 10min, 4 ℃ of preservations for back 72 ℃.After the digestion of DpnI restriction endonuclease, reclaim purifying and concentrate the homologous recombination segment.
(2) preparation of electric transformed competence colibacillus cell
(I) picking has the intestinal bacteria E.coli Q8 of pKD46 plasmid, changes in the LB substratum, adds 0.2% pectinose simultaneously, cultivates OD
600To 0.5;
(II) ice bath 15min, centrifugal thalline utilizes 10% glycerine washing three times then;
(III) glycerine of adding 10% is concentrated into 50 times, the packing competence.
(3) electricity transforms, the screening recon
(I) the homologous recombination segment of absorption 8 μ g/l adds in the competent cell of 100 μ l mixing.Regulate electroporation apparatus, 2.5Kv, electric shock;
(II) the SOC substratum of adding 900 μ l, 37 ℃, 150 commentaries on classics/min cultivate 1h;
(III) the coating chlorampenicol resistant is dull and stereotyped, transfers the recon utilization
poxB?test?F：5′-TCCCCCTCCGTCAGATGA-3′
poxB?test?R：5′-GGTATCACTGCGTAAATCAA-3′
Carry out PCR and detect, confirm further that through the order-checking of PCR product the poxB gene is changed by chloramphenicol resistance gene.
(IV) PLP site-specific reorganization
Change pCP20 over to chlorampenicol resistant clone, cultivate 8h for 30 ℃, after be increased to 42 ℃ and spend the night, thermal induction FLP recombinase is expressed, plasmid is also lost gradually.Utilize transfering loop to dip in to get bacterium liquid and on the non-resistant substratum, rule; With the mono-clonal that grows change over to simultaneously non-resistant dull and stereotyped with the kalamycin resistance flat board on cultivate, on the non-resistant flat board, grow and being deleted by the FlP recombinase of on the kalamycin resistance flat board, not growing.Utilize detection primer poxB test F and poxB test R further to identify.
(V) obtain engineering strain MG1655 Δ ptsG Δ poxB::Cm, called after E.coli Q9.
(c) the pta gene knocks out
Bacterial classification: intestinal bacteria E.coli Q9
Said LB substratum is: peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, penbritin, 100mg/L, kantlex 50mg/L.
Said ammonia benzyl mycin resistant panel is the penbritin that contains 100mg/L, the LB solid medium of 1.5% agar powder.
Said chlorampenicol resistant is dull and stereotyped for containing the paraxin of 50mg/L, the LB solid medium of 1.5% agar powder.
Said SOC substratum is: peptone 2g/L, yeast powder 0.5g/L, NaCl 0.0585g/L, KCl 0.0186g/L, MgCl
20.203g, MgSO
40.246g/L, glucose 20mmol/L.
(1) the pulsating clone of homologous recombination
Utilize the Red recombination system that goal gene is knocked out.The pta gene order of announcing according to Genbank designs primer:
pKD-pta?F
5′-GTAACCCGCCAAATCGGCGGTAACGAAAGAGGATAAACCGTGTAGGCTGGAGCTGCTTC-3′
pKD-pta?R
5′-TCAGATATCCGCAGCGCAAAGCTGCGGATGATGACGAGAATGGGAATTAGCCATGGTCC-3′
Obtain to have the reorganization segment of chlorampenicol resistant through PCR (polymerase chain reaction) amplification in vitro with pKD3.The PCR reaction system is following: (primer concentration is 20 μ mol/L)
10 * damping fluid, 5 μ l;
25mmol/LMgCl2?4μl；
Four kinds of dNTP mixed solutions of 10mmol/L, 1 μ l;
Each 1 μ l of upstream and downstream primer;
TaqDNA polysaccharase 0.5 μ l;
The PCR reaction conditions: 97 ℃ of preparatory sex change 10min, 94 ℃ of sex change 60s, 58 ℃ of annealing 30s, 72 ℃ are extended 90s, and 30 circulations are extended 10min, 4 ℃ of preservations for back 72 ℃.After the digestion of DpnI restriction endonuclease, reclaim purifying and concentrate the homologous recombination segment.
(2) preparation of electric transformed competence colibacillus cell
(I) picking has the intestinal bacteria E.coli Q9 of pKD46 plasmid, changes in the LB substratum, adds 0.2% pectinose simultaneously, cultivates OD
600To 0.5;
(II) ice bath 15min, centrifugal thalline utilizes 10% glycerine washing three times then;
(III) glycerine of adding 10% is concentrated into 50 times, the packing competence.
(3) electricity transforms, the screening recon
(I) the homologous recombination segment of absorption 8 μ g/l adds in the competent cell of 100 μ l mixing.Regulate electroporation apparatus, 2.5Kv, electric shock;
(II) the SOC substratum of adding 900 μ l, 37 ℃, 150 commentaries on classics/min cultivate 1h;
(III) the coating chlorampenicol resistant is dull and stereotyped, transfers the recon utilization
Pta?test?F 5′-TCAGCTGGCGGTGCTGTTT-3′
Pta?test?R 5′-ACCGGAAATAGTGATTATTTCCGG-3′
Carry out PCR and detect, confirm further that through the order-checking of PCR product pta is changed by chloramphenicol resistance gene.
(IV) PLP site-specific reorganization
Change pCP20 over to chlorampenicol resistant clone, cultivate 8h for 30 ℃, after be increased to 42 ℃ and spend the night, thermal induction FLP recombinase is expressed, plasmid is also lost gradually.Utilize transfering loop to dip in to get bacterium liquid and on the non-resistant substratum, rule; With the mono-clonal that grows change over to simultaneously non-resistant dull and stereotyped with the kalamycin resistance flat board on cultivate, on the non-resistant flat board, grow and being deleted by the FlP recombinase of on the kalamycin resistance flat board, not growing.Utilize detection primer Pta test F and Pta test R further to identify.
(V) obtain engineering strain MG1655 Δ ptsG Δ poxB Δ pta::Cm, called after E.coli QZ10.Engineering bacillus strain preservation to " Chinese typical culture collection " center ", deposit number is CCTCC M 209113.
The carbon source pathways metabolism of constructed engineering strain is seen Fig. 1.In the glucose metabolism approach of the engineering strain E.coli QZ10 of transformation as shown in the figure (deposit number is CCTCC M 209113), the disappearance of ptsG will reduce the accumulation of pyruvic acid, thereby further reduce the carbon flow that flows to acetate; The disappearance of poxB and pta gene has been blocked the generation approach of acetate, thereby has reduced the generation of acetate.
Bacterial classification: E.coli MG1655, E.coli Q8, E.coli Q9 and E.coli Q10 (deposit number: CCTCC M209113)
Substratum: LB substratum: peptone 10g/L, yeast powder 5g/L, NaCl 10g/L.
Cultural method:
(deposit number: single bacterium colony CCTCC M 209113) goes to overnight cultures in the test tube that 4ml LB is housed to E.coli MG1655, E.coli Q8, E.coli Q9 and E.coli Q10 in the picking flat board respectively.Change the triangular flask of the 300ml that 50ml LB is housed over to according to the inoculum size of volume ratio 1%, 250 commentaries on classics/min cultivate down for 37 ℃.Every interval 2h sampling 1ml.Sample places 12,000 commentaries on classics/min under the room temperature, centrifugal 2min.Remove supernatant then, utilize 0.5M NaCl solution to clean twice thalline, utilize the 0.5M NaCl solution re-suspended cell of 1ml then.The optical density(OD) OD of thalline is that wavelength is the light absorption value at 600nm place.
Draw growth curve, relatively the growth differences of each strain bacterium.Comparative result is seen Fig. 2.As shown in Figure 2, engineering strain E.coli Q10 (deposit number: CCTCC M 209113) compare with the intestinal bacteria MG1655 of wild-type and have bigger growth velocity.
Embodiment 3, engineering strain LB add in 1% glucose living weight and acetate rate ratio
Substratum LBG: peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, glucose 20g/L.
Bacterial classification: E.coli MG1655, E.coli Q8, E.coli Q9 and E.coli Q10 (deposit number: CCTCC M209113)
(1) cultural method
(I) seed liquor must prepare: with E.coli MG1655, E.coli Q8, E.coliQ9 and the E.coli Q10 (deposit number: CCTCC M 209113) insert in the triangular flask of the 300ml that 50ml LB substratum is housed on the transfering loop difference picking flat board; Paraxin to final concentration is respectively 50mg/L in 37 ℃; 250 commentaries on classics/min cultivate 12h.
(II) shake flask fermentation: the inoculum size of seed liquor according to volume ratio 1% inserted in the triangular flask of the 300ml that 50ml LBG substratum is housed.(deposit number: CCTCC M209113) adding paraxin to final concentration in the strain cultures is 50mg/L for E.coli Q8, E.coli Q9 and E.coli Q10 during inoculation.Culture temperature is made as 37 ℃, and shaking speed is made as 250 commentaries on classics/min.Cultivate 48h.
(2) meta-bolites analysis
(I) detection of glucose: get bacterium liquid 12, the 000 commentaries on classics/min that fermentation finishes, centrifugal 2min.Get supernatant,, utilize glucose biological sensor to detect the concentration of glucose then with the membrane filtration of 0.2 μ m.HPLC (HPLC) detects.Testing conditions is: test column: waters C
18, moving phase: 1 ‰ formic acid, detector: differential detector.
(II) detection of acetate: get bacterium liquid 12, the 000 commentaries on classics/min that fermentation finishes, centrifugal 2min.Get supernatant,, utilize HPLC (HPLC) to detect then with the membrane filtration of 0.2 μ m.Testing conditions is: test column: watersC
18, moving phase: 1 ‰ formic acid, detector: differential detector.The acetate detected result is seen Fig. 3, and (deposit number: CCTCC M 209113) amount of excretory acetate is minimum for engineering strain E.coli Q10 as shown in Figure 3.
(3) living weight analysis
Different strains institute sample thief is placed 12,000 commentaries on classics/min under the room temperature, centrifugal 2min.Remove supernatant then, utilize 0.5M NaCl solution to clean twice thalline, utilize the 0.5M NaCl solution re-suspended cell of 1ml then.The optical density(OD) OD of thalline is that wavelength is the light absorption value at 600nm place.
Draw the living weight curve, comparing difference.The result sees Fig. 4.As shown in Figure 4, (deposit number: the living weight that CCTCC M 209113) accumulates is 4 times of wild-type E.coli MG1655 to engineering strain E.coli Q10.
Under embodiment 4, the pH7.0 condition, engineering strain LB add in 1% glucose living weight and acetate rate ratio
Substratum LBG: peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, glucose 20g/L.
Bacterial classification: E.coli MG1655, E.coli Q8, E.coli Q9 and E.coli Q10 (deposit number: CCTCC M209113)
(1) cultural method
(I) seed liquor must prepare: transfer E.coli MG1655, E.coli Q8, E.coli Q9 and the E.coli Q10 (deposit number: CCTCC M 209113) insert in the triangular flask of the 300ml that 50ml LB substratum is housed on the flat board with transfering loop; Paraxin to final concentration is respectively 50mg/L in 37 ℃; 250 commentaries on classics/min cultivate 12h.
(II) shaking bottle cabinet cultivates: seed liquor is inserted shaking in bottle cabinet of 1L that 200ml LBG substratum is housed according to 1% inoculum size.Nutrient solution utilizes the NaOH of 2M to regulate pH automatically, and pH is set at 7.0.Adding paraxin to final concentration in MG1655 Δ ptsG::Cm, E.coli QZ1011 and the E.coli QZ1100 strain cultures during inoculation is 50mg/L.Culture temperature is made as 37 ℃, and shaking speed is made as 250 commentaries on classics/min.Cultivate 48h.
(2) meta-bolites analysis
(I) detection of glucose: get bacterium liquid 12, the 000 commentaries on classics/min that fermentation finishes, centrifugal 2min.Get supernatant,, utilize glucose biological sensor to detect the concentration of glucose then with the membrane filtration of 0.2 μ m.HPLC (HPLC) detects.Testing conditions is: test column: waters C
18, moving phase: 1 ‰ formic acid, detector: differential detector.
(II) detection of acetate: get bacterium liquid 12, the 000 commentaries on classics/min that fermentation finishes, centrifugal 2min.Get supernatant,, utilize HPLC (HPLC) to detect then with the membrane filtration of 0.2 μ m.Testing conditions is: test column: waters C
18, moving phase: 1 ‰ formic acid, detector: differential detector.
(3) living weight analysis
Different strains institute sample thief is placed 12,000 commentaries on classics/min under the room temperature, centrifugal 2min.Remove supernatant then, utilize 0.5M NaCl solution to clean twice thalline, utilize the 0.5M NaCl solution re-suspended cell of 1ml then.The optical density(OD) OD of thalline is that wavelength is the light absorption value at 600nm place.
Biomass accumulation difference and acetate secretory volume result see Fig. 5, Fig. 6.(deposit number: living weight CCTCC M 209113) is maximum, and its acetate secretory volume is minimum simultaneously for E.coli Q10 as shown in Figure 5.
Fermention medium (improvement M9 substratum):
Na
2HPO
46g/L KH
2PO
43g/L NaCl 0.5g/L NH
4Cl 1g/L YE 2g/LMgSO
42mmol/L CaCl
20.1mmol/L glucose 20g/L.
Method:
(1) structure of PHB fermentation approach
(I) preparation of competent cell
Intestinal bacteria E.coli MG1655 in the picking LB flat board and E.coli Q10 (deposit number: CCTCC M209113), overnight cultures; With the intestinal bacteria MG1655 of overnight cultures and E.coli Q10 (deposit number: CCTCC M209113) change over to respectively in the triangular flask of the 300ml that 50ml LB is housed according to 1% inoculum size and cultivate, under 37 ℃, 225 commentaries on classics/min, be cultured to OD
600Be 0.4.Stop to cultivate, put 20 minutes on ice, 4 ℃, 4000 commentaries on classics/min, centrifugal 10 minutes, abandon supernatant, add the CaCl of ice-cold 0.1mol/L
2Solution suspends, and leaves standstill on ice 30 minutes.Centrifugal concentrating.Obtain competent cell.Be put in-70 ℃ of preservations.
(II) structure of PHB fermentation approach
PHB route of synthesis expression plasmid is converted into intestinal bacteria E.coli MG1655 and E.coli Q10 (deposit number: CCTCC M 209113).Thereby obtain PHB fermentation strain E.coli MG1655/pBluescript SK
-PhbCAB and E.coli Q10 (deposit number: CCTCC M 209113)/pBluescript SK
-PhbCAB.
(2) PHB fermentation
PHB recombination bacillus coli E.coli MG1655/pBluescript SK in the picking LB flat board
-PhbCAB and E.coli Q10 (deposit number: CCTCC M 209113)/pBluescript SK
-PhbCAB; Under aseptic condition, use inoculation articulating 1 to encircle in 50ml and be added with in the M9 improved culture medium of final concentration as the penbritin of 100mg/L respectively and (add 2% glucose); Under 37 ℃ of conditions, 250 commentaries on classics/min shaking table shaking culture 12 hours make seed liquor; Transfer in the 1000ml triangular flask that the 200ml substratum is housed with the inoculum size of volume ratio 2% respectively again.The IPTG induced concentration is 0.1mmol/L.Fermentation condition is 37 ℃, 250 commentaries on classics/min.Wherein control group is recombination bacillus coli E.coliMGl655/pBluescript SK
-PhbCAB, fermentation time are 48h, every interval 4h sampling.
Said plasmid pBluescript SK
--phbCAB is the pBluescript SK that has the phbCAB gene
-Plasmid is expressed through inducing of IPTG.
The PHB detection of fermenting
Collecting cell: with the fermentation broth sample of being got at 4,000 commentaries on classics/min, centrifugal 20min, the collecting precipitation cell, use distilled water wash cell 2 times after, 4,000 commentariess on classics/min, centrifugal 20min collecting cell, oven dry back its dry weight of title.
PHB detects: with the dry mycelium of gained, added in the vitriol oil boiling water bath of 150 microlitres heating 60 minutes, utilize 15% ammoniacal liquor to regulate pH to 2.5 then, behind filtering with microporous membrane, HPLC (HPLC) detects PHB content.Testing conditions is: analytical column C18; 1 ‰ formic acid is made moving phase; UV-detector; Ultraviolet wavelength is 210nm.Detected result is seen Fig. 7.As shown in Figure 7, and E.coli Q10 (deposit number: CCTCC M 209113)/pBluescriptSK
-The amount of phbCAB accumulation PHB is E.coli MG1655/pBluescript SK
-2 times of phbCAB accumulation volume.
Sequence table
< 110>Shandong University
< 120>one strains are used for the colibacillus engineering strain of aerobic fermentation
<141>2009-5-27
<160>12
<210>1
<211>59
<212>DNA
<213>pKD-ptsG?F
<400>1
acgtaaaaaa?agcacccata?ctcaggagca?ctctcaattg?tgtaggctgg?agctgcttc 59
<210>2
<211>59
<212>DNA
<213>pKD-ptsG?R
<400>2
agccatctgg?ctgccttagt?ctccccaacg?tcttacggaa?tgggaattag?ccatggtcc 59
<210>3
<211>20
<212>DNA
<213>ptsG?test?F
<400>3
cctgtacacg?gcgaggctct 20
<210>4
<211>25
<212>DNA
<213>ptsG?test?R
<400>4
aataacacct?gtaaaaaagg?cagcc 25
<210>5
<211>59
<212>DNA
<213>pKD-poxB?F
<400>5
aaacttgtta?ccgttatcac?attcaggaga?tggagaaccg?tgtaggctgg?agctgcttc 59
<210>6
<211>59
<212>DNA
<213>pKD-poxB?R
<400>6
catggcatgt?ccttattatg?acgggaaatg?ccacccttta?tgggaattagc?catggtcc 59
<210>7
<211>18
<212>DNA
<213>poxB?test?F
<400>7
tccccctccg?tcagatga 18
<210>8
<211>20
<212>DNA
<213>poxB?test?R
<400>8
ggtatcactg?cgtaaatcaa 20
<210>9
<211>59
<212>DNA
<213>pKD-pta?F
<400>9
gtaacccgcc?aaatcggcgg?taacgaaaga?ggataaaccg?tgtaggctgg?agctgcttc 59
<210>10
<211>59
<212>DNA
<213>pKD-pta?R
<400>10
tcagatatcc?gcagcgcaaa?gctgcggatg?atgacgagaa?tgggaattag?ccatggtcc 59
<210>11
<211>19
<212>DNA
<213>Pta?test?F
<400>11
tcagctggcg?gtgctgttt 19
<210>12
<211>24
<212>DNA
<213>Pta?test?R
<400>12
accggaaata?gtgattattt?ccgg 24
Claims (3)
1. a strain is used for the colibacillus engineering strain of aerobic fermentation, it is characterized in that: this bacterial strain is called intestinal bacteria (Escherichia coli) Q10; Bacterial strain has been deposited on May 27th, 2009 that " Chinese typical culture collection " center ", deposit number is CCTCC M 209113.
2. according to the said colibacillus engineering strain that is used for aerobic fermentation of claim 1, it is characterized in that: the genotype of said bacterial strain is MG1655 Δ ptsG Δ poxB Δ pta::Cm.
3. the said application that is used for the colibacillus engineering strain of aerobic fermentation at preparation PHB of claim 1, it is characterized in that: the fermentation condition that said engineering strain is produced PHB is: 37 ± 1 ℃ of temperature, shaking speed 250 ± 30 commentaries on classics/min, fermentation time 48 ± 2h; Fermention medium consists of: Na
2HPO
46g/L, KH
2PO
43g/L, NaCl 0.5g/L, NH
4Cl 1g/L, YE 2g/L, MgSO
42mmol/L, CaCl
20.1mmol/L, glucose 20g/L.
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Non-Patent Citations (4)
| Title |
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| Andrea Veit 等.Global gene expression analysis of glucose overflow.《Appl Microbiol Biotechnol》.2006,第74卷406-421. * |
| Marjan De Mey 等.Minimizing acetate formation in E. coli fermentations.《J Ind Microbiol Biotechnol》.2007,第34卷689-700. * |
| Mark A. Eiteman 等.Overcoming acetate in Escherichia coli.《TRENDS in Biotechnology》.2006,第24卷(第11期),530-536. * |
| 康振 等.好氧发酵生产琥珀酸工程菌株的构建.《生物工程学报》.2008,第24卷(第12期),2081-2085. * |
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