CN110373435A

CN110373435A - A method of PHB is synthesized by knocking out rfaD gene efficient

Info

Publication number: CN110373435A
Application number: CN201910701822.5A
Authority: CN
Inventors: 王小元; 王建莉; 马文渐; 李烨
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2019-07-31
Filing date: 2019-07-31
Publication date: 2019-10-25
Anticipated expiration: 2039-07-31
Also published as: CN110373435B

Abstract

The method that the invention discloses a kind of to synthesize PHB by knocking out rfaD gene efficient, belongs to genetic engineering and field of fermentation engineering.The present invention knocks out and the encoding gene for synthesizing the enzyme of PHB is transformed into fermenting and producing PHB in bacterial strain after rfaD gene on genome of E.coli.It was found that the knockout of rfaD gene can significantly improve the ability of Escherichia coli W3110, DH5 α and JM109 synthesis PHB, the PHB volumetric production of WJW00/pDXW-8-phaCAB relatively control bacterium W3110/pDXW-8-phaCAB improves 3.95 and 3.66 times；WJD00/pDXW-8-phaCAB cell synthesis PHB accounts for dry cell weight ratio relatively control bacterium and improves about 80%, and conversion ratio improves about 1.9 times；WJJ00/pDXW-8-phaCAB cell synthesis PHB accounts for dry cell weight ratio relatively control bacterium and improves about 75%, and conversion ratio improves about 1.8 times.

Description

A method of PHB is synthesized by knocking out rfaD gene efficient

Technical field

The method that the present invention relates to a kind of to synthesize PHB by knocking out rfaD gene efficient belongs to genetic engineering and fermentation work Journey field.

Background technique

Polyhydroxyalkanoate (Polyhydroxyalkanoates, PHA) is that one kind can by the renewable of Microbe synthesis Degradation and the high molecular polymer with multicomponent material performance, before medicine, material and environment protection field have a wide range of applications Scape.Polyhydroxyalkanoate is widely present in microbial cell, mainly as carbon source and the storage carrier of energy.Growing environment C:N ratio is higher, is more conducive to the synthesis of PHA.PHA it is intracellular in the form of hydrophobic particle exist, under given conditions its Content can be more than the 90% of dry cell weight.PHA has according to the differences of monomeric species, polymerization methods from crisp hard of hard matter Plastics are to a series of multifarious materialogy characteristics such as soft elastomers.Since PHA can be Material synthesis by renewable resource, Can be degradable by the biology such as bacterium after into nature, substitution conventional oil base plastics can alleviate serious " white dirt Dye " problem, so as to cause the extensive attention of countries in the world scientific circles and industry.

Poly 3-hydroxy butyrate (PHB) is one of PHA, and Escherichia coli are usually used in the industrial production of PHB.PHB is one Kind intracellular product, PHB particle is carbon source stored substance intracellular, is present in cytoplasm in the form of insoluble micro-spherical particle shape In.PHB synthesis is much regulated and controled, and such as C:N ratio, when carbon is superfluous, nitrogen lacks, PHB is more easily-synthesized, when other sources C intracellular When being metabolized vigorous or when amino acid transport enzyme activity is low, PHB synthesis will receive influence.The key of Escherichia coli synthesis PHB is Equilibrium products and cell growth, should make cell grow, and enhance the metabolic fluxes of its route of synthesis again, and produce by control The expression of object forming feature improves yield.Mainly pass through optimization of fermentation conditions in existing report, optimization metabolic pathway, mention The means such as high coenzyme concentration intracellular and Optimal Expression plasmid improve the synthesis of PHB.But these methods make mentioning for PHB yield Height is still unable to satisfy the demand industrially produced.Therefore it provides a kind of new raising PHB synthetic method, for further The synthesis for improving PHB has very great meaning.

Summary of the invention

In order to solve above-mentioned technical problem, the present invention knocks out rfaD (also known as gmhD) base on genome of E.coli Cause, and after PHB synthetic gene cluster phaCAB is transformed into obtaining recombinant bacterium in bacterial strain, fermenting and producing PHB is carried out using recombinant bacterium, It is found surprisingly that the ability that the knockout of rfaD gene can significantly improve Escherichia coli synthesis PHB, PHB volumetric production is wild right According to 3.95 times of bacterium.

The first purpose of the invention is to provide the method that one kind efficiently synthesizes poly 3-hydroxy butyrate (PHB), the side Method is to knock out ADP-L- glycero-D-manno-heptose -6- epimerase RfaD gene on genome of E.coli, and by β -one The encoding gene of base thiolase, acetyl coenzyme A reductase and PHB synzyme is transformed into obtain recombinant bacterium in bacterial strain after, utilize weight Group bacterium carries out fermenting and producing.

In one embodiment, the Escherichia coli are Escherichia coli W3110, bacillus coli DH 5 alpha or Escherichia coli JM109。

In one embodiment, the ADP-L- glycero-D-manno-heptose -6- epimerase RfaD amino acid Sequence is as shown in SEQ ID NO.1.

In one embodiment, the protein ID of the beta-keto thiolase is QBK40993.1；The acetyl is auxiliary The protein ID of enzyme A reductase is QBK40994.1；The protein ID of the PHB synzyme is QBK40992.1.

In one embodiment, the conversion is connected to gene on plasmid pDXW-8, is then transformed into cell.

In one embodiment, the conversion is specifically that will contain beta-keto thiolase, acetyl coenzyme A reductase and The gene cluster phaCAB of PHB synthetase-coding gene is connected on plasmid pDXW-8, obtains recombinant plasmid pDXW-8-phaCAB, Then by recombinant plasmid transformed into cell.

In one embodiment, the NCBI accession number of the gene order of the gene cluster phaCAB is MH558939.1.

In one embodiment, the fermenting and producing is that the recombinant bacterium is inoculated into fermentation medium, with Portugal Grape sugar is substrate, carries out fermenting and producing.

In one embodiment, the composition of the fermentation medium are as follows: 20g/L glucose, 17.1g/L Na₂HPO₄· 12H₂O, 3g/L KH₂PO₄, 0.5g/L NaCl, addition 1mM MgSO₄, 0.1mM CaCl₂, 10mg/mL vitamin B1.

In one embodiment, the fermentation is specifically: the seed liquor of the recombinant bacterium is forwarded to fermentation medium, And it ferments after kanamycins and inducer IPTG (isopropylthiogalactoside) is added.

The present invention also provides the method medicine preparation, material or environment protection field application.

In one embodiment, the application is will to produce obtained PHB as high molecular polymer applied to described In field.

Beneficial effects of the present invention:

(1) knockout of rfaD gene can significantly improve the ability of Escherichia coli W3110 synthesis PHB, fermentation for 24 hours and 48h, The PHB volumetric production of WJW00/pDXW-8-phaCAB is increased to 2.24g/L and 2.13g/L, relatively compares bacterium W3110/pDXW-8- PhaCAB improves 3.95 and 3.66 times；

(2) knockout of rfaD gene can also significantly improve the ability of bacillus coli DH 5 alpha synthesis PHB, and shake flask fermentation is for 24 hours After 48h, for relatively compareing bacterium DH5 α/pDXW-8-phaCAB, WJD00/pDXW-8-phaCAB cell synthesizes PHB dry ratio About 80% is improved, conversion ratio improves about 1.9 times；

(3) knockout of rfaD gene can also significantly improve the ability of e. coli jm109 synthesis PHB, relatively compare bacterium For JM109/pDXW-8-phaCAB, WJJ00/pDXW-8-phaCAB cell synthesizes PHB dry ratio and improves about 75%, conversion Rate improves about 1.8 times.

Biomaterial

Wild-type e. coli W3110 is bought from ATCC, deposit number ATCC39936.

Detailed description of the invention

Fig. 1: the building of engineering bacteria WJW00.

Fig. 2: bacterial strain W3110/pDXW-8-phaCAB and WJW00/pDXW-8-phaCAB synthesize the laser copolymerization of PHB particle Pyrocellulose sem observation.DIC:differential interference contrast, i.e. differential interference contrast under white light conditions Degree；FITC:fluorescence contrast, the fluorescent contrast after Nile red dyeing under the conditions of exciting light 488nm； The superimposed effect of picture under the conditions of Merged:DIC condition and FITC, i.e., fold the imaging under the conditions of white light conditions and fluorescence Add.

The ultra-thin section of Fig. 3: bacterial strain W3110/pDXW-8-phaCAB and WJW00/pDXW-8-phaCAB synthesis PHB particle Electronic Speculum observation.

Fig. 4: Escherichia coli W3110/pDXW-8-phaCAB and WJW00/pDXW-8-phaCAB synthesize the shaking flask hair of PHB Ferment.

Fig. 5: recombinant bacterial strain DH5 α/pDXW-8-phaCAB, WJD00/pDXW-8-phaCAB, JM109/pDXW-8- The shake flask fermentation of phaCAB and WJJ00/pDXW-8-phaCAB cell synthesis PHB.

Specific embodiment

(1) the Electronic Speculum observation of Escherichia coli

1. cell violet staining and optical microphotograph sem observation

It is centrifuged fermentation liquid, and takes 0.5 μ L thallus to be coated on clean glass slide from pellet fraction, fixed drying, and drip One 0.1% violet staining 1min of drop, drips cedar oil after rinsing drying, carries out the observation of optical microscopy oil mirror, and take pictures.

2. laser co-focusing fiber sem observation

Fermentation thalli 0.5mL is taken, thallus is washed 2 times with the phosphate buffer of pH 7.4, prepares Nile red dye liquor (1 μ Mol Nile red is dissolved in the DMSO of 1 μ g/ μ L), and resuspended bacterium solution is placed in 37 DEG C of darkrooms and dyes 30min, with aluminium foil by EP Pipe wrap avoid it is light-exposed.After dyeing, again three times with the phosphate buffer washing thalline of the pH 7.4 of pre-cooling, exist as far as possible Opening operation under dark condition.10 μ L bacterium solutions are drawn in darkroom and are placed on glass slide and cover coverslip, with thumb press 5s More than, nail polish edge sealing is then used, carries out laser co-focusing fiber sem observation under dark condition of turning off the light after nail oil drying.First Slide is inverted on LCFM objective table, cell compartment is found under coarse adjustment, then alignment area is brought into focus and excitation wavelength 480nm is observed with LCFM amplification certain multiple.If observation during discovery cell have flow phenomenon if should sample preparation again, And pay special attention to the operation of edge sealing, the fixation degree of cell is better, and the better be imaged the more clear.Transmitting light is fixed, adjusts and puts Big multiple carries out more visuals field and takes pictures.

3. Electron Microscopic Observation

Take phosphotungstic acid negative staining.First by after microorganism collection, with PBS (pH 7.4) washing thalline, and with 2.5% penta Dialdehyde (PBS, pH7.4 that solvent is final concentration of 0.1M) is fixed, needs to add gloves and mask, fills it up with standing, use after 5min Toothpick provokes thallus group, so that fixer sufficiently fixes thallus, then fills it up with fixer, sealing prevents handle in transportational process Thallus is broken up.Then it is dehydrated, embedding and ultra-thin section, last negative staining carry out Electronic Speculum observation.

(2) fermentation parameter measuring method

1. OD and pH measurement

It is returned to zero with deionized water, adjustment extension rate to the OD surveyed in cuvette₆₀₀Between 0.2 to 0.8.To registration Reading is recorded after stabilization, multiplied by extension rate to get OD.

PH measurement is directly carried out to the fermented sample just taken, is directly read using pH meter measurement.

2. residual glucose

SBA-40C biosensor is to combine reflected electric signal to become by the receptor on glucose molecule and reaction film Change and carrys out quantifying glucose concentration；Measuring method is that sample 10000rpm is centrifuged 2min, and 20 μ l supernatants are added to totality It is to be in the system to be measured of 1000 μ l, and acutely concussion is uniform；Accurate 25 μ l standard specimens (containing 1g/L glucose) of drawing quickly are beaten Enter in sample holes, after waiting instrument balance, after lamp to be instructed does not flash, repeats 3 needles or more, it is almost the same, it is considered as and has calibrated At；Accurate 25 μ l samples of drawing quickly are squeezed into sample holes, register instrument display screen index, divided by 2 up to residual sugar numerical value.

3. the extraction and measurement of PHB

The PHB synthetic cell for taking the different fermentations time, is collected by centrifugation 5mL cell, is washed using the PBS buffer solution of pH 7.4 Twice, after being collected by centrifugation, cell is transferred in the centrifuge tube of preparatory correct amount, wraps preservative film, and prick out multiple duck eyes, Moisture is evaporated, and bacterium solution is avoided to spray, 48h is lyophilized in vacuum freeze drier to being completely dried, generally touches Tube bottom is normal temperature state, shows to parch completely, or flicks tube bottom with finger, and thallus can scatter and be detached from pipe easily Wall is then considered as thallus and is completely dried.It weighs, calculates dry weight.

The dry mycelium that 1-10mg is completely dried is weighed, while weighing PHB standard sample about 10mg and being transferred to accurate in advance claim In the esterification pipe of weight, accurately to calculate the example weight claimed.Then esterification operations are carried out, 2mL methanol is added and (contains 3% Sulfuric acid) and 2mL chloroform, in addition be esterified pipe lid and cover tightly, it during which can be ultrasonic, help sample to scatter, so that esterification is more thorough, Boiling water bath 6h or more.About after half an hour, that is, the generally fusing rapidly in powder form of PHB standard specimen can be observed.If not observing The metamorphosis of standard items and dissolution after half an hour, then should replace a whole set of reagent, and methanol and the rotten of chloroform can all cause to be esterified It can not be normally carried out, and the reagent for more renewing Kaifeng often can solve the above problem, and after six hours boiling water baths, divulge information It in cupboard after cooling sufficiently, carefully opens esterification pipe and 1mL deionized water simultaneously is added, at this moment need respectively to screw lid and fierceness is shaken It swings to system and cmpletely mixes, esterification pipe is placed in ventilation stands 3h or more with split-phase at this time, after split-phase, upper layer is water Phase, lower layer are organic phase, take it is appropriate it is organic be added in gas phase sample bottle, cover tightly lid and keep sealing, be stored in -80 DEG C In.

Gas-chromatography is the accurate sensitive means of quantitative PHB content.Gas phase quantitatively uses 2010 gas phase color of Shimadzu GC Spectrum, using Agilent DB WAX 30m-0.32mm gas chromatographic column and flame ionization detector, injector temperature is 250 DEG C.With Different amounts of commercialization PHB draws the standard curve of each standard sample as standard specimen.

The building of 1 engineering bacteria WJW00 of embodiment

Engineering bacteria WJW00 is in SCI paper " Construction and Characterization of an Escherichia coli Mutant Producing Kdo₂- Lipid A " in (publication date: on March 13rd, 2014) is disclosed. Specific building process is as follows:

(1) acquisition of rfaD gene knockout segment

RfaD gene knockout segment is obtained using the method for chemical fully synthetic or PCR substep amplification, both ends are rfaD base Because upstream and downstream homology arm, centre are the kan segment with the site FRT.The nucleotide sequence such as SEQ of rfaD gene knockout segment Shown in ID NO.2.RfaD gene knockout segment is cloned into plasmid pBlueScriptIISK (+), obtains recombinant plasmid pBlueScript IISK(+)-rfaDU-Fkan-rfaDD.Using the plasmid as template, acquisition can be expanded and knock out segment rfaDU-Fkan-rfaDD。

(2) preparation and electrotransformation of competence are knocked out

Inoculation recombinates helper plasmid pKD46 (DatsenkoKA, Wanner BL.One-Step with Red inactivation of chromosomal genes in Escherichia coli K-12using PCR Products.Proc Natl Acad Sci USA, 2000,97 (12): 6640-6645) Escherichia coli W3110 (ATCC39936) in the LB liquid medium containing 100 μ g/mL ampicillins, 30 DEG C, 200rpm is incubated overnight.It is connect by 2% Kind amount switching 100mL LB liquid medium, 30 DEG C, 200rpm is cultivated to OD₆₀₀L-arabinose (final concentration is added when=0.2 The expression that recombinase is induced for 30mmol/L) continues culture to OD₆₀₀=0.5, pre-cooling will be transferred to after culture solution ice bath half an hour 50mL centrifuge tube in, 4 DEG C, 8000rpm is centrifuged 10min and collects thallus, and 10% glycerol that precipitating is pre-chilled washs 3 times, finally It is suspended with 1mL10% glycerol, every 80 μ L of pipe is dispensed into the sterile EP tube of pre-cooling.

The rfaD of 500-1000ng is knocked out segment to be added in competent cell, is mixed, ice bath 15min, 1.5kv electric shock 5ms, 30 DEG C of incubation 2h are coated with the LB solid plate of 30 μ g/mL kanamycins, and 30 DEG C of cultures, picking transformant is in containing 30 μ g/mL It is cultivated in the LB liquid medium of kanamycins and 100 μ g/mL ampicillins, obtained Strain Designation is by PCR verifying WJW00-Fkan。

(3) removal of mutant strain resistance marker

By in positive LB liquid medium of the strain inoculated containing 30 μ g/mL kanamycins of PCR verifying, 42 DEG C are trained overnight It supports, by the LB solid plate of culture solution 30 μ g/mL kanamycins of scribing line, 37 DEG C of cultures, it is green to ammonia benzyl that picking single colonie verifies it The sensibility of mycin is W3110 △ rfaD::kan by resistance containing kanamycin and to the Strain Designation of ampicillin-sensitive And preservation.Competence is done as starting strain, is transferred to pCP20 plasmid (CherepanovPP, WackernagelW.Gene disruption in Escherichia coli:Tc R and Km Rcassettes with the option of Flp- catalyzed excision of the antibiotic-resistance determinant.Gene,1995,158(1): 9-14), FLP enzyme is expressed in 42 DEG C of heat shocks, and positive strain is cultivated in the LB liquid medium containing 100 μ g/mL ampicillins, The expression of L-arabinose (final concentration of 30mmol/L) induction Flp recombinase is added, mediates the recombination of FRT locus specificity, PCR Transformant is selected in verifying.The result is shown in Figure 1, the region the rfaD week in Escherichia coli WJW00, WJW00-Fkan and W3110 chromosome The DNA fragmentation size for enclosing amplification is respectively 552,1854 and 889bp.

PCR is verified into LB plate of crossing after the 42 DEG C of heat shocks of correct bacterial strain, picking single colonie verifies it to ampicillin And the sensibility of kanamycins, to two kinds of antibiotic, sensitive bacterial strain is E.coli W3110 △ rfaD, is named as WJW00 and preservation.

The building of embodiment 2 engineered strain W3110/pDXW-8-phaCAB and WJW00/pDXW-8-phaCAB

(1) building of plasmid

Plasmid pDXW-8-phaCAB is in article " Ma, W., Wang, J., Li, Y., et.al.Poly (3- hydroxybutyrate-co-3-hydroxyvalerate)co-produced with L-isoleucine in Corynebacterium glutamicum WM001[J].Microb Cell Fact,2018,17(1):93 10.1186/ S12934-018-0942-7 " in (publication date: 2018-12-31) is disclosed.Specific building process is as follows:

Using the true bacteria genome NC_008313.1 of Roche as template, expand to obtain using primer phaCAB-F/phaCAB-R (phaCAB gene cluster is shown in accession number https to phaCAB gene cluster: //www.ncbi.nlm.nih.gov/nuccore/ MH558939.1), PCR product, a kind of carrier pDXW-8 (patent: large intestine bar are digested with restriction enzyme EcoRI and XhoI Bacterium-coryneform shuttle type inducible expression carrier PDXW-8 and its construction method, publication date: 2010-04-14) also with EcoRI and XhoI digestion process, digestion products after purification, use the 22 DEG C of connections overnight of T4 ligase, conversion to bacillus coli DH 5 alpha, screening Correct transformant obtains correct transformant DH5 α/pDXW-8-phaCAB, extracts plasmid and obtains correct recombinant plasmid pDXW-8- phaCAB。

2 primer sequence table of table

(2) preparation of competence

It is inoculated with the engineering bacteria WJW00 constructed in Escherichia coli W3110 (ATCC39936) and embodiment 1 respectively in LB In fluid nutrient medium, 37 DEG C, 200rpm is incubated overnight.50mL LB liquid medium is transferred to by 2% inoculum concentration, 37 DEG C, 200rpm is cultivated to OD₆₀₀=0.5, it will be transferred in the 50mL centrifuge tube of pre-cooling after culture solution ice bath half an hour, 4 DEG C, 8000rpm It is centrifuged 10min and collects thallus, the CaCl of the precipitating 0.01M of pre-cooling₂The CaCl of 1mL 0.01M is finally used in washing 3 times₂It suspends, 30% glycerol of 1m is added to mix, every 200 μ L of pipe is dispensed into the sterile EP tube of pre-cooling.

(3) it converts

The plasmid pDXW-8-phaCAB of 100-200ng is added in competent cell, is mixed, ice bath 30min, 42 DEG C of heat 90s, 2~3min of ice bath are hit, the recovery of 1mL LB culture medium is added, 37 DEG C of incubation 2h are coated with the LB solid of 30 μ g/mL kanamycins Plate, 37 DEG C of cultures, picking transformant cultivate seed liquor in the LB liquid medium containing 30 μ g/mL kanamycins to get arriving Recombinant bacterial strain W3110/pDXW-8-phaCAB and WJW00/pDXW-8-phaCAB.

The qualitative observation of 3 engineering bacterium fermentation of embodiment production PHB

LB culture medium composition: yeast powder 5g/L, tryptone 10g/L and NaCl 10g/L.

The composition of M9G culture medium: 20g/L glucose (Glucose), 17.1g/L disodium hydrogen phosphate dodecahydrate (Na₂HP O₄·12H₂O), 3g/L potassium dihydrogen phosphate (KH₂PO₄), 0.5g/L sodium chloride (NaCl) adds 1mM magnesium sulfate (M gSO₄), 0.1mM calcium chloride (CaCl₂), 10mg/mL vitamin B1 (VB₁)。

(1) seed liquor culture

Recombinant bacterium W3110/pDXW-8-phaCAB and WJW00/pDXW-8-phaCAB1 ring lawn is picked them separately to 25mL In LB culture medium, and 30 μ g/mL kanamycins are added, 37 DEG C, 200rpm culture 5h to mid-log phase.

(2) fermentation synthesis PHB

Cultural method: by seed liquor (OD₆₀₀=1.8 or so) according to initial OD₆₀₀=0.25 is forwarded to conventional PHB fermentation training Base M9G is supported, and kanamycins is added to make its final concentration of 30 μ g/mL, 37 DEG C, 200rpm, ferment 48h.

Inductive condition: the IPTG of final concentration of 0.5mM is added within 0 hour.

(3) qualitative observation synthesis PHB particle intracellular

Situation is synthesized using a variety of electron microscopies observation fermented cells PHB particle intracellular.Cell is dyed by Nile red, is being swashed It shines as green fluorescence can be issued under the laser confocal microscope of 488nm, fluorescence is more, and it is more to represent PHB content.Such as figure Shown in 2, control bacterium W3110/pDXW-8-phaCAB contains seldom green fluorescence, illustrates that synthesized PHB particle is less, and WJW00/pDXW-8-phaCAB cell has synthesized many PHB particles, and nearly all cell has green fluorescence, and cell body Product becomes larger.

Electron Microscopic Observation is further used, as shown in figure 3, the synthesis of WJW00/pDXW-8-phaCAB cell is more PHB particle, and cell volume obviously becomes larger, and about increases 5-10 times；WJW00/pDXW-8-phaCAB cell membrane walls thickness only 11nm Left and right, and compareing bacterium W3110/pDXW-8-phaCAB is 13nm or so, illustrates that the membranous wall flexibility of mutant strain WJW00 is stronger, has Conducive to PHB particle in intracellular accumulation.

The quantitative determination of 4 engineering bacterium fermentation of embodiment production PHB

Fermentation process same as Example 3 is taken, to recombinant bacterium W3110/pDXW-8-phaCAB and WJW00/pDXW- 8-phaCAB ferments, and samples 2mL in different time points, measures corresponding fermentation parameter.

(1) ferment OD₆₀₀It is measured with dry cell weight

The result shows that: fermentation is for 24 hours and after 48h, WJW00/pDXW-8-phaCAB fermentation OD₆₀₀Reach 9.0 and 9.8, and it is right According to bacterium W3110/pDXW-8-phaCAB only 5.6 and 6.1 (Fig. 4 a), dry cell weight improves 47.9% and 67.0% (Fig. 4 b), still Cell consumption sugar increases only 30.4% and 34.3% (Fig. 4 c).

(2) PHB determination of yield

The result shows that: the raising of dry cell weight mainly due to WJW00/pDXW-8-phaCAB PHB particle accumulation intracellular more It is more, for 24 hours and 48h, WJW00/pDXW-8-phaCAB can synthesize that PHB is up to dry cell weight respectively 67.8% and 63.4%, And wild control bacterium W3110/pDXW-8-phaCAB is only 22.5% and 20.5% (Fig. 4 d)；Make volumetric production also bright simultaneously It is aobvious to rise to 2.24g/L and 2.13g/L, 3.95 and 3.66 times (Fig. 4 e) is improved compared with W3110/pDXW-8-phaCAB；Glucose turns The transformation ratio for turning to PHB reaches 0.224g/g and 0.185g/g, and the transformation ratio of W3110/pDXW-8-phaCAB is only 0.063g/g and 0.053g/g (Fig. 4 f).As it can be seen that the knockout of rfaD gene can significantly improve Escherichia coli W3110 synthesis PHB Ability.

3 engineering bacteria WJW00/pDXW-8-phaCAB fermenting and producing PHB of table

Other Escherichia coli fermentation production PHB are transformed in embodiment 5

(1) building of engineering bacteria WJD00, WJJ00

Referring to the construction method with engineering bacteria WJW00 in embodiment 1, knock out in bacillus coli DH 5 alpha and JM109 respectively RfaD gene obtains rfaD mutant strain WJD00 and WJJ00.

(2) building of engineered strain DH5 α/pDXW-8-phaCAB, WJD00/pDXW-8-phaCAB

Referring to the method for embodiment 2, the plasmid pDXW-8-phaCAB containing PHB synthesis related gene is transformed into respectively In wild-type strain DH5 α and mutant bacteria WJD00, recombinant bacterium DH5 α/pDXW-8-phaCAB, WJD00/pDXW-8- are obtained phaCAB。

(3) building of engineered strain JM109/pDXW-8-phaCAB, WJJ00/pDXW-8-phaCAB

Referring to the method for embodiment 2, the plasmid pDXW-8-phaCAB containing PHB synthesis related gene is transformed into respectively In wild-type strain JM109 and mutant bacteria WJJ00, recombinant bacterium JM109/pDXW-8-phaCAB, WJJ00/pDXW-8- are obtained phaCAB。

(4) strain fermentation produces PHB

According to the identical fermentation process of embodiment 3, recombinant bacterium obtained in step (2) and step (3) is subjected to fermentation life Produce PHB.Fermentation parameter measurement is carried out to the shake flask fermentation of each recombinant bacterial strain.

The fermentation of 4 recombinant bacterium of table produces PHB for 24 hours

5 recombinant bacterium of table fermentation 48h produces PHB

(1) recombinant bacterial strain WJD00/pDXW-8-phaCAB fermentation parameter measures

The result shows that: shake flask fermentation is for 24 hours and after 48h, the dry cell weight of recombinant bacterial strain WJD00/pDXW-8-phaCAB cell Reach 3.98g/L and 4.42g/L (Fig. 5 A), the PHB of synthesis reaches 78.3% and 78.6% (Fig. 5 B) of dry cell weight, glucose The transformation ratio for being converted into PHB is 0.37g/g and 0.39g/g (Fig. 5 C)；And DH5 α/pDXW-8-phaCAB dry cell weight is only 2.33g/L and 2.49g/L (Fig. 5 A), PHB yield are 39.4% and 43.3% (Fig. 5 B), and glucose is converted into the transformation system of PHB Number is 0.14g/g and 0.12g/g (Fig. 5 C).For control bacterium DH5 α/pDXW-8-phaCAB, WJD00/pDXW-8-phaCAB Cell synthesizes PHB dry ratio and improves about 80%, and conversion ratio improves about 1.9 times.

(2) recombinant bacterial strain WJJ00/pDXW-8-phaCAB fermentation parameter measures

For 24 hours and after 48h, the dry cell weight of recombinant bacterial strain WJJ00/pDXW-8-phaCAB cell reaches shake flask fermentation 4.93g/L and 5.51g/L (Fig. 5 A), the PHB of synthesis reach 84.3% and 84.8% (Fig. 5 B) of dry cell weight, glucose conversion Transformation ratio for PHB is 0.36g/g and 0.37g/g (Fig. 5 C)；And JM109/pDXW-8-phaCAB dry cell weight only 2.42g/ L and 2.57g/L (Fig. 5 A), PHB yield are 46.2% and 48.3% (Fig. 5 B), and the transformation ratio that glucose is converted into PHB is 0.15g/g and 0.13g/g (Fig. 5 C).For control bacterium JM109/pDXW-8-phaCAB, WJJ00/pDXW-8-phaCAB is thin Born of the same parents synthesize PHB dry ratio and improve about 75%, and conversion ratio improves about 1.8 times.

Although the present invention is disclosed as above with preferred embodiment, it is not intended to limit the invention, any to be familiar with this technology People can all do various change and modification, therefore protection scope of the present invention without departing from the spirit and scope of the present invention It should subject to the definition of the claims.

SEQUENCE LISTING

<110>Southern Yangtze University

<120>a kind of method for synthesizing PHB by knocking out rfaD gene efficient

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 310

<212> PRT

<213> E.coli W3110

<400> 1

Met Ile Ile Val Thr Gly Gly Ala Gly Phe Ile Gly Ser Asn Ile Val

1 5 10 15

Lys Ala Leu Asn Asp Lys Gly Ile Thr Asp Ile Leu Val Val Asp Asn

20 25 30

Leu Lys Asp Gly Thr Lys Phe Val Asn Leu Val Asp Leu Asn Ile Ala

35 40 45

Asp Tyr Met Asp Lys Glu Asp Phe Leu Ile Gln Ile Met Ala Gly Glu

50 55 60

Glu Phe Gly Asp Val Glu Ala Ile Phe His Glu Gly Ala Cys Ser Ser

65 70 75 80

Thr Thr Glu Trp Asp Gly Lys Tyr Met Met Asp Asn Asn Tyr Gln Tyr

85 90 95

Ser Lys Glu Leu Leu His Tyr Cys Leu Glu Arg Glu Ile Pro Phe Leu

100 105 110

Tyr Ala Ser Ser Ala Ala Thr Tyr Gly Gly Arg Thr Ser Asp Phe Ile

115 120 125

Glu Ser Arg Glu Tyr Glu Lys Pro Leu Asn Val Tyr Gly Tyr Ser Lys

130 135 140

Phe Leu Phe Asp Glu Tyr Val Arg Gln Ile Leu Pro Glu Ala Asn Ser

145 150 155 160

Gln Ile Val Gly Phe Arg Tyr Phe Asn Val Tyr Gly Pro Arg Glu Gly

165 170 175

His Lys Gly Ser Met Ala Ser Val Ala Phe His Leu Asn Thr Gln Leu

180 185 190

Asn Asn Gly Glu Ser Pro Lys Leu Phe Glu Gly Ser Glu Asn Phe Lys

195 200 205

Arg Asp Phe Val Tyr Val Gly Asp Val Ala Asp Val Asn Leu Trp Phe

210 215 220

Leu Glu Asn Gly Val Ser Gly Ile Phe Asn Leu Gly Thr Gly Arg Ala

225 230 235 240

Glu Ser Phe Gln Ala Val Ala Asp Ala Thr Leu Ala Tyr His Lys Lys

245 250 255

Gly Gln Ile Glu Tyr Ile Pro Phe Pro Asp Lys Leu Lys Gly Arg Tyr

260 265 270

Gln Ala Phe Thr Gln Ala Asp Leu Thr Asn Leu Arg Ala Ala Gly Tyr

275 280 285

Asp Lys Pro Phe Lys Thr Val Ala Glu Gly Val Thr Glu Tyr Met Ala

290 295 300

Trp Leu Asn Arg Asp Ala

305 310

<210> 2

<211> 1854

<212> DNA

<213>artificial synthesized

<400> 2

tccgttacac cttcagcaac ggttttgaac ggtttgtcgt aacccgccgc gcgcagattt 60

gtcagatctg cctgagtgaa cgcctgatag cggcctttca gtttatccgg gaacggaatg 120

tattcgatct ggcctttctt gtgataagcc agcgtagcat cagctaccgc ctggaaggat 180

tccgcacgac cagtaccgag attgaagatg ccggaaacgc cattttccag gaaccacaga 240

ttcacatcag ccacgaattc gtgtaggctg gagctgcttc gaagttccta tactttctag 300

agaataggaa cttcggaata ggaacttcaa gatcccctta ttagaagaac tcgtcaagaa 360

ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc 420

ggtcagccca ttcgccgcca agctcttcag caatatcacg ggtagccaac gctatgtcct 480

gatagcggtc cgccacaccc agccggccac agtcgatgaa tccagaaaag cggccatttt 540

ccaccatgat attcggcaag caggcatcgc catgggtcac gacgagatcc tcgccgtcgg 600

gcatgcgcgc cttgagcctg gcgaacagtt cggctggcgc gagcccctga tgctcttcgt 660

ccagatcatc ctgatcgaca agaccggctt ccatccgagt acgtgctcgc tcgatgcgat 720

gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc cgccgcattg 780

catcagccat gatggatact ttctcggcag gagcaaggtg agatgacagg agatcctgcc 840

ccggcacttc gcccaatagc agccagtccc ttcccgcttc agtgacaacg tcgagcacag 900

ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg cgctgcctcg tcctgcagtt 960

cattcagggc accggacagg tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca 1020

gccggaacac ggcggcatca gagcagccga ttgtctgttg tgcccagtca tagccgaata 1080

gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa 1140

acgatcctca tcctgtctct tgatcagatc ttgatcccct gcgccatcag atccttggcg 1200

gcaagaaagc catccagttt actttgcagg gcttcccaac cttaccagag ggcgccccag 1260

ctggcaattc cggttcgctt gctgtccata aaaccgccca gtctagctat cgccatgtaa 1320

gcccactgca agctacctgc tttctctttg cgcttgcgtt ttcccttgtc cagatagccc 1380

agtagctgac attcatccgg ggtcagcacc gtttctgcgg actggctttc tacgtgttcc 1440

gcttccttta gcagcccttg cgccctgagt gcttgcggca gcgtgagctt caaaagcgct 1500

ctgaagttcc tatactttct agagaatagg aacttcgaac tgcaggtcga cggatccccg 1560

gaattaattc tcatgtttga cagcttatca ctgatcagtg aattaatggc aagctttact 1620

tgccgtccca ctcggtggtg gaagagcacg cgccttcgtg gaaaatcgct tcgacatcgc 1680

cgaactcttc gccagccata atctggatca ggaagtcttc cttatccata tagtctgcga 1740

tattcagatc caccaggttc acaaacttgg tgccgtcttt caggttgtcc accaccagaa 1800

tatcggtgat gcctttatca ttcagggctt taacgatgtt gctgccgata aagc 1854

<210> 3

<211> 37

<212> DNA

<213>artificial synthesized

<400> 3

ctggaattca gaaggagaat caaatcatgg cgaccgg 37

<210> 4

<211> 28

<212> DNA

<213>artificial synthesized

<400> 4

ccgctcgaga ggtcagccca tatgcagg 28

Claims

1. the method that one kind efficiently synthesizes poly 3-hydroxy butyrate (PHB), which is characterized in that knock out on genome of E.coli ADP-L- glycero-D-manno-heptose -6- epimerase RfaD gene (also known as GmhD), and by beta-keto thiolase, acetyl is auxiliary The encoding gene of enzyme A reductase and PHB synzyme is transformed into obtain recombinant bacterium in bacterial strain after, carry out fermentation life using recombinant bacterium It produces.

2. the method according to claim 1, wherein the Escherichia coli are Escherichia coli W3110, Escherichia coli DH5 α or e. coli jm109.

3. the method according to claim 1, wherein ADP-L- glycero-D-manno-heptose -6- difference is to different The amino acid sequence of structure enzyme RfaD is as shown in SEQ ID NO.1.

4. the method according to claim 1, wherein the protein ID of the beta-keto thiolase is QBK40993.1；The protein ID of the acetyl coenzyme A reductase is QBK40994.1；The protein of the PHB synzyme ID is QBK40992.1.

5. the method according to claim 1, wherein it is described conversion be to be connected to gene on plasmid pDXW-8, Then it is transformed into cell.

6. according to the method described in claim 1, the fermenting and producing is that the recombinant bacterium is inoculated into fermentation medium, Using glucose as substrate, fermenting and producing is carried out.

7. according to the method described in claim 6, the composition of the fermentation medium are as follows: 20g/L glucose, 17.1g/L Na₂HPO₄·12H₂O, 3g/L KH₂PO₄, 0.5g/L NaCl, addition 1mM MgSO₄, 0.1mM CaCl₂, 10mg/mL vitamin B1。

8. according to the method described in claim 6, the fermentation is specifically: the seed liquor of the recombinant bacterium is forwarded to fermentation training Base is supported, and is fermented after kanamycins and inducer IPTG (isopropylthiogalactoside) is added.

9. any the method for claim 1~8 is in the application of medicine preparation, material or environment protection field.

10. application according to claim 9, which is characterized in that be that the PHB for obtaining production is answered as high molecular polymer For in the field.