CN103146740A - Engineering bacteria for producing 1,3-propylene glycol and method for constructing same - Google Patents
Engineering bacteria for producing 1,3-propylene glycol and method for constructing same Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 39
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- 206010035717 Pneumonia klebsiella Diseases 0.000 claims description 23
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Abstract
The invention discloses an engineering bacteria for producing 1,3-propylene glycol and a method for constructing the same. The invention silences the phosphate transacetylase genes in a wild type strain producing 1,3-propylene glycol by utilizing a homologous recombination method and a gene insertional inactivation method so as to obtain a genetically engineered bacterium, of which the acetic acid metabolic pathway is blocked. When the 1,3-propylene glycol is fermented and produced by the engineering bacteria disclosed by the invention, the acetic acid production is largely reduced, the toxic action on cells caused by the by-product acetic acid is greatly decreased and the production rate of biomass per unit is enhanced. Moreover, the post-extracting process is simplified and the production cost is reduced as the species of the by-products are reduced. The experiments prove that the concentration of the 1,3-propylene glycol can reach more than 55g/L by fermenting the engineering bacteria provided by the invention for 28 hours according to a conventional method. The invention plays an important role in the industrial production for producing 1,3-propylene glycol by a microbiological fermentation method and has a wide application prospect.
Description
Technical field
The invention belongs to biological technical field, be specifically related to engineering bacteria and construction process thereof that 1,3-PD is produced in a strain.
Background technology:
1,3-PD (PDO) is a kind of important industrial chemicals, can be used as organic solvent, is applied to the industries such as withstand voltage high lubricant, dyestuff, printing ink, frostproofer.PDO can be used to synthesizing heterocyclic, pharmaceutical intermediate, polyester and urethane, especially can be used as the monomer of polyester PTT.PTT be continue the polyethylene terephthalate fifties (PET), the polybutylene terephthalate seventies (PBT) but after realize plant-scale new thread-forming polyester macromolecular material, be a kind of extremely rising new polyester material.PTT was chosen as one of six large petrochemical industry product innovations by the U.S. in 1998.PTT compares except the chemical resistant properties with polyester with PET, PBT, also has some other better characteristic.Recovery of elasticity as nylon; Need not to add the special chemical medicine in gamut and can present good continuous printing and dyeing characteristic; The tint permanence of uvioresistant, ozone and oxynitrides; Anti-internal stress; Low water absorption, low static and good biodegradable; Can follow and go back usability etc.Due to the above good characteristic of having of PTT, it has very widely in carpet industry, dress materials, engineering thermoplast and other various fields uses.
The key of producing ptt fiber is the source of raw material monomer PDO.The key that PTT dominates the market is price, and the price of PTT depends primarily on the price of PDO.Owing to can not cheapness making PDO, development and the market development of PTT have been restricted.Until the mid-90 PDO has realized suitability for industrialized production, make the PDO price greatly reduce, PTT just begins suitability for industrialized production and application.
Two transnational companys of Dupont and Shell once adopted chemical synthesis route, take oxyethane or propylene as raw material production PDO.The shortcoming that chemical synthesis is produced PDO is that by product is many, and selectivity and productive rate are lower, and operational condition needs High Temperature High Pressure, and facility investment is huge, and raw material is non-renewable; Because output is limited, the PDO price is higher for a long time simultaneously.
The production method of 1,3-PD is mainly microbe fermentation method at present.Compare with chemical synthesis, Production of 1,3-Propylenediol via Microbiological Fermentation Method has significant advantage: 1, utilizing lower-cost renewable resources (as glycerine, corn, starch) is raw material; 2, working condition is gentle, and is easy and simple to handle, do not need precious metal catalyst; 3, selectivity is good, and by product is less, is easy to separation and purification; 4, environmental pollution is little." green industry " to the strong challenge that traditional petrochemical complex proposes, has important practical significance, thereby more and more comes into one's own microbe fermentation method take biotechnology as feature.
Production of 1,3-Propylenediol via Microbiological Fermentation Method is to utilize microorganism disproportionation glycerine to produce.So far all found 1, ammediol is produced bacterial classification and is bacterium, wherein klebsiella (Klebsiella pneumoniae), citrobacter freundii (Citrobacter freudii) and clostridium butylicum (Clostridium butyricum) have higher by 1, ammediol transformation efficiency and 1, the ammediol production intensity, have higher DEVELOPMENT PROSPECT, thereby obtained more concern.
The klebsiella that is used at present to produce 1,3-PD mainly separates in edatope.Klebsiella can only utilize glycerine (can not utilize the cheap carbon sources such as carbohydrate) to produce 1,3-PD.In the process that produces 1,3-PD, glycerine generation disproportionation, the product in oxidative pathway is consistent with the carbohydrate fermentation product, and produces for the necessary ATP of Growth of Cells, discharges reducing power NADH when some product forms; The reduction approach consumes reducing power unnecessary in oxidative pathway, generates 1,3-PD.The first oxidized generation pyruvic acid of glycerine in oxidative pathway; Pyruvic acid is acetyl-CoA and formic acid by the pyruvate formate-lyase catalytic decomposition, and formic acid often can be decomposed into CO again
2And H
2Acetyl-CoA generates excessive ATP in the process that forms acetic acid through acetylphosphate, and will consume 2 moles of reducing powers in the reaction that forms ethanol through acetaldehyde; Pyruvic acid also may be converted into 2,3-butanediol, lactic acid and succsinic acid, and the process that generates lactic acid will consume 1 mole of reducing power.And the process that generates succsinic acid will consume 2 moles of reducing powers.The reduction approach comprises two-step reaction: the first step, and by depending on coenzyme B
12The glycerol dehydratase dehydrating glycerin with catalyst generate 3-HPA; Second step generates 1,3-PD by the reduction of dhaT catalysis 3-HPA, and this process consumes 1 mole of reducing power.
Produce in the process of 1,3-PD at the klebsiella ferment glycerin, the growth of cell is subject to the restraining effect of substrate glycerine and multi-products.In by product, maximum with the output of lactic acid, more than in the process of wild-type klebsiella ferment glycerin generation 1,3-PD, lactic acid production can reach 40g/L; By-product acetic acid is the strongest to the restraining effect of fermentation, and the acetic acid of 6g/L is bacteria growing inhibiting and fermentation significantly, and research reports that 7.6g/L acetic acid can reduce by 50% bacterial growth, 15g/L acetic acid bacteria growing inhibiting fully in substratum.Not only cell growth is unfavorable in the generation of a large amount of by products, also can cause the waste of substrate glycerine.For example, because the approach that produces lactic acid can be fought for reducing power, the generation of lactic acid causes the 1,3-PD yield reducation.Due to the generation of acetic acid, make substrate glycerine enter more glycolytic pathway, cause substrate conversion efficiency to reduce.In addition, the generation of by product makes the product extraction process complicated, and energy consumption cost increases.Therefore, reduce the generation of by product, to improving production level, reduce the 1,3-propanediol produced by microbial fermentation cost, have great importance.For by product lactic acid, the research report knocks out lactic acid metabolism pathway key encoding gene lactate dehydrogenase gene, can make the synthetic significantly minimizing of lactic acid, and 1,3-PD throughput is enhanced.But the report that also there is no inactivation acetate metabolism pathway key encoding gene at present, acetate metabolism approach inactivation is unknown on the impact of klebsiella producing 1,3-propanediol through fermentation.
Summary of the invention:
The purpose of this invention is to provide a kind of engineering bacteria of producing 1,3-PD-knock out engineering bacteria and construction process and the application of acetate metabolism pathway gene.
The engineering bacteria that knocks out the acetate metabolism pathway gene of the present invention builds by the following method, will produce the engineering bacteria that obtains after the phosphotransacetylase gene knockout of wild type strain of 1,3-PD.
The wild type strain of described 1,3-PD is preferably the bacterium of Klebsiella (Klebsiella), more preferably Klebsiella pneumonia (Klebsiella pneumoniae).
Described phosphotransacetylase gene knockout with klebsiella preferably knocks out by the following method:
The portion homologous sequence of the phosphotransacetylase gene of a, pcr amplification klebsiella is connected it with suicide vector, and then in the introductive crossing F+strain;
B, the portion homologous sequence that carries the phosphotransacetylase gene that step a is obtained and donor bacterium and the klebsiella of suicide vector carry out this hybridization of parents, utilize homologous recombination, gene disruption, obtain the phosphotransacetylase gene by the klebsiella that knocks out the acetate metabolism pathway gene of silence after screening.
The portion homologous sequence of the phosphotransacetylase gene of the pcr amplification klebsiella of described step a is which partial sequence of phosphotransacetylase gene is unimportant, so long as homologous sequence gets final product.When described 1, when the wild type strain of ammediol is Klebsiella pneumonia (Klebsiella pneumoniae), the portion homologous sequence preference of the phosphotransacetylase gene of the pcr amplification klebsiella of described step a be genomic dna take Klebsiella pneumonia as template, the primer that forms with upstream primer PTA-F:TACCCGGGTACCAGCGTAGGTCTGACCAGCGTC and downstream primer PAT-R:GACCCGGGTTACTTCTGCTGCTGAGCCGATTG carries out the sequence after pcr amplification.
Described suicide vector can be suicide vector pGPCm, can buy from reagent company.
In the described suicide vector introductive crossing F+strain that will carry phosphotransacetylase Gene Partial homologous sequence, can transform hybridization donor bacterium by ordinary methods such as heat shock method, electrotransformation, joint conversion methods.
Described hybridization donor bacterium can be intestinal bacteria SM10 (λ pir).By this hybridization of parents, make the partial sequence and product l of phosphotransacetylase gene, the purpose bacterial strain generation homologous recombination of ammediol produces 1 thereby make, phosphotransacetylase gene disruption in the wild type strain of ammediol, thus acquisition knocks out the engineering bacteria of acetate metabolism pathway gene.
The present invention also provides the application of the engineering bacteria that knocks out the acetate metabolism pathway gene in producing 1,3-PD.
The present invention utilizes the method for homologous recombination, gene disruption to make to produce the phosphotransacetylase gene silencing in the wild type strain of 1,3-PD, thereby obtains the genetic engineering bacterium that the acetate metabolism approach is blocked.Carry out 1 with engineering bacteria of the present invention, the fermentative production of ammediol, the acetic acid production decrease, make by-product acetic acid greatly reduce the toxic action of cell, unit biomass production speed strengthens, in addition, and because the by product kind reduces, also simplify rear extraction process, reduced production cost.1,3-PD concentration experiment showed, engineering bacteria of the present invention was fermented 28 hours according to a conventional method, more than can reach 55g/L.The present invention will play a significant role in the suitability for industrialized production of Production of 1,3-Propylenediol via Microbiological Fermentation Method, have broad application prospects.
Description of drawings:
Figure l reclaims the also PTA purpose fragment figure of purifying pcr amplification
Fig. 2 is SmaI digested plasmid pT-PTA figure
Fig. 3 is the physical map of carrier pGPCm
Embodiment:
Following examples are to further illustrate of the present invention, rather than limitation of the present invention.
Embodiment 1: acetate metabolism pathway key gene-phosphotransacetylase gene is by the structure of the Klebsiella pneumonia mutant strain of inactivation.
(l), the clone of phosphotransacetylase gene PTA partial sequence
The DNA sequence dna complete according to phosphotransacetylase gene PTA (No. GenBank: YP_002920553.1) design primer, its portion homologous sequence of pcr amplification, primer sequence is as follows: upstream primer PTA-F:TACCCGGGTACCAGCGTAGGTCTGACCAGCGTC and downstream primer PAT-R:GACCCGGGTTACTTCTGCTGCTGAGCCGATTG.(be preserved in Chinese Typical Representative culture collection center with the wild-type Klebsiella pneumonia, deposit number is: CCTCC M 2011075) genomic dna is template, under the guiding of primer PTA-F and PAT-R, the partial sequence of pcr amplification phosphotransacetylase gene PTA, the pcr amplification condition is: first 95 ℃ of 3min; Then 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 2min, totally 33 circulations; Last 72 ℃ of 10min.After reaction finishes, pcr amplification product is carried out 1.0% agarose gel electrophoresis, recovery and purifying be the purpose fragment (Fig. 1) of 2000bp approximately, it is cloned into carrier pGEM-T easy(TaKaRa company) in, recombinant products is transformed intestinal bacteria Dh5 α competent cell, the screening positive transformant, the upgrading grain, carry out enzyme with Restriction enzyme Sma I and cut evaluation, cut the endonuclease bamhi (Fig. 2) that has obtained 2000bp through enzyme, show and obtained the correct recombinant vectors of insertion sequence, called after pT-PTA plasmid.
(2), the structure of phosphotransacetylase gene PTA suicide vector pGP-PTA
cut the pT-PTA plasmid with the Restriction enzyme Sma I enzyme, the length that reclaims also purification step (1) amplification is the PTA part DNA fragmentation of 2000bp, again with its with its physical map of carrier pGPCm(of cutting through the SmaI enzyme as shown in Figure 3) be connected with DNA ligase, to connect product and change intestinal bacteria SM10 (λ pir) competent cell over to, the screening positive transformant, the upgrading grain, obtain carrying the intestinal bacteria SM10 (λ pir) of the suicide vector that contains phosphotransacetylase gene PTA, extract plasmid, the plasmid called after pGP-PTA that obtains, it is that portion homologous sequence with phosphotransacetylase gene PTA is connected with suicide vector pGPCm.
(3), the phosphotransacetylase gene is by the structure of the Klebsiella pneumonia mutant strain of inactivation
The intestinal bacteria SM10 that carries carrier pGP-PTA (λ pir) (donor bacterium) and the wild-type Klebsiella pneumonia (CCTCC M 2011075) (recipient bacterium) of step (2) are carried out this hybridization of parents, and concrete grammar is: incubated overnight donor bacterium and recipient bacterium in containing the LB liquid nutrient medium of paraxin; Donor bacterium and recipient bacterium are mixed in the MgSO of 10mM than 3:1 ratio in quantity
4In solution, filter, filter membrane is placed on the LB flat board, cultivated 8-12 hour for 37 ℃; MgSO with l0mM
4Solution washes long lawn on filter membrane, coats after gradient dilution on the chlorampenicol resistant flat board, and 37 ℃ of cultivations are screened.Finally obtain having the recombinant bacterial strain of paraxin (Cm) resistance, be acetate metabolism pathway key gene-phosphotransacetylase gene by the Klebsiella pneumonia mutant strain of inactivation.
Embodiment 2: the phosphotransacetylase gene is detected by the activity of the Klebsiella pneumonia mutant strain of inactivation.
Phosphotransacetylase gene to embodiment 1 is detected by the activity that the Klebsiella pneumonia mutant strain of inactivation carries out phosphotransacetylase, and take the wild-type Klebsiella pneumonia as contrast, concrete grammar comprises the following steps:
(l) the phosphotransacetylase gene is inoculated in the 100mL substratum by the Klebsiella pneumonia mutant strain of inactivation and (contains glycerine 10g in every premium on currency, Tryptones 10g, yeast powder 5g, NaCl5g, pH7.0,120 ℃ of sterilization 20min) in, at 37 ℃ of lower shaking culture 6-12 hours, the every 2 hours centrifugal collection thalline of taking a sample;
(2) wash thalline 2 times with 100mL phosphoric acid buffer (0.1M, pH7.5) suspension;
(3) with 2.5mL phosphoric acid buffer (0.1M, pH7.5) suspension thalline;
(4) ultrasonic disruption thalline;
(5) the centrifugal 30min of 3000g gets supernatant mensuration enzyme and lives, and measuring method is for using ultraviolet spectrophotometer assaying reaction system at OD
233nmChanging value in the 1min of place, i.e. △ A
233nm/ min.Reaction conditions is: 25 ℃ of temperature, pH7.6, time 5min, wavelength 233nm, enzyme concn 0.002-0.003u/ml, concentration of substrate, acetyl phosphate 8mmol/L, coenzyme A 0.2mmol/L.Enzyme work is defined as: at pH7.6, under 25 ℃ of environment, make substrate with acetyl phosphate, every 1min catalysis 1.0umol coenzyme A changes into the unit (u) that the required enzyme amount of acetyl-CoA is decided to be phosphotransacetylase.
Result shows, the phosphotransacetylase gene that EXAMPLE l builds is the 3.7%-4.9% of wild type strain by the phosphotransacetylase enzymic activity of the Klebsiella pneumonia mutant strain of inactivation, shows that the phosphotransacetylase gene of EXAMPLE l structure is by the phosphotransacetylase enzyme deactivation of the Klebsiella pneumonia mutant strain of inactivation.
Embodiment 3: the phosphotransacetylase gene is by the Klebsiella pneumonia mutant strain producing 1,3-propanediol through fermentation of inactivation
(1) substratum
LB substratum (gL
-1): yeast powder 5, peptone 10, NaCl10, agar 10 is adjusted to pH7.0, is used for short term storage and the activation of klebsiella bacterial classification.Seed and fermention medium composition see Table 1:
Table 1: substratum forms
(2) training method
(i) seed activation: the phosphotransacetylase gene from the embodiment 1 of glycerine pipe preservation is seeded to LB medium slant activation by the Klebsiella pneumonia mutant strain of inactivation, cultivates 12 hours activated seeds at 37 ℃ of temperature.
(ii) seed culture: 9 layers of gauze sealing of 250mL triangular flask, liquid amount 100mL seed culture medium, access inclined-plane lawn (activated seed of a step I) ring carries out aerobic seed culture, 30 ℃ of temperature, rotating speed 150rmin in shaking table
-1
(iii) fermentation culture: produced 1 by the Klebsiella pneumonia mutant strain ferment glycerin of inactivation in order to investigate constructed inactivation phosphotransacetylase PTA gene pairs phosphotransacetylase gene, the impact of ammediol, phosphotransacetylase gene in the embodiment 1 by the Klebsiella pneumonia mutant strain of inactivation as experimental group, take the wild mushroom Klebsiella pneumonia that sets out as control group, carry out fed-batch fermentation, adopt Shimadzu LC-20A HPLC to analyze material composition in fermented liquid.
When carrying out in the 5L stirred fermentor, liquid amount 4L, inoculum size 1% passes into the 0.5vvm air and carries out the micro-aerobe fermentation cultivation, and mixing speed is 250rpm.Leavening temperature is constant in 37 ℃; NaOH regulates pH to 6.8, and in fermenting process, system pH adds 40% NaOH solution regulation and control by stream.Carry out the glycerine feed supplement after bacterial strain enters logarithmic phase, glycerol concentration is controlled at 1-50g/L.
(iv) fermentation results
Fermentation was carried out 28 hours, and 1,3-PD and by-product production situation the results are shown in Table 2.
Table 2: inactivation phosphotransacetylase PTA gene pairs Klebsiella pneumonia ferment glycerin produces the impact of 1,3-PD
Can learn from fermentation results, inactivation phosphotransacetylase PTA gene reduces the strain bio amount, but the acetic acid metabolic pathway of synthesizing is cut off, acetic acid resultant quantity decrease 66.04%, the synthetic 1,3-PD of unit thalline biomass increases by 9.4%, unit thalline biomass 1, the production intensity of ammediol increases by 9.4%, and unit thalline biomass producing 1,3-propanediol through fermentation yield also increases by 8.47%.
Claims (10)
1. a construction process that knocks out the engineering bacteria of acetate metabolism pathway gene, is characterized in that, is the engineering bacteria that obtains after the phosphotransacetylase gene knockout of wild type strain of 1,3-PD producing.
2. construction process according to claim 1, is characterized in that, the wild type strain of described product 1,3-PD is the bacterium of Klebsiella (Klebsiella).
3. construction process according to claim 2, is characterized in that, the wild type strain of described product 1,3-PD is Klebsiella pneumonia (Klebsiella pneumoniae).
4. construction process according to claim 2, is characterized in that, described phosphotransacetylase gene knockout with klebsiella specifically knocks out by the following method:
The portion homologous sequence of the phosphotransacetylase gene of a, pcr amplification klebsiella is connected it with suicide vector, and then in the introductive crossing F+strain;
B, the portion homologous sequence that carries the phosphotransacetylase gene that step a is obtained and donor bacterium and the klebsiella of suicide vector carry out this hybridization of parents, utilize homologous recombination, gene disruption, obtain the phosphotransacetylase gene by the klebsiella that knocks out the acetate metabolism pathway gene of silence after screening.
5. construction process according to claim 4, it is characterized in that, when described product 1, when the wild type strain of ammediol is Klebsiella pneumonia (Klebsiella pneumoniae), the portion homologous sequence of the phosphotransacetylase gene of the pcr amplification klebsiella of described step a is that genomic dna take Klebsiella pneumonia is as template, the primer that forms with upstream primer PTA-F:TACCCGGGTACCAGCGTAGGTCTGACCAGCGTC and downstream primer PAT-R:GACCCGGGTTACTTCTGCTGCTGAGCCGATTG carries out the sequence after pcr amplification.
6. according to claim 4 or 5 described construction processs, is characterized in that, described suicide vector is suicide vector pGPCm, and described hybridization donor bacterium is intestinal bacteria SM10 (λ pir).
7. one kind builds according to claim 1,2,3,4 or 5 described construction processs the engineering bacteria that knocks out the acetate metabolism pathway gene that obtains.
8. the application of engineering bacteria in producing 1,3-PD that knocks out the acetate metabolism pathway gene claimed in claim 7.
9. one kind builds the engineering bacteria that knocks out the acetate metabolism pathway gene that obtains according to construction process claimed in claim 6.
10. the application of engineering bacteria in producing 1,3-PD that knocks out the acetate metabolism pathway gene claimed in claim 9.
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