CN105441496A - Method for improving yield of cadaverine produced by utilizing microorganisms for saccharides fermentation - Google Patents
Method for improving yield of cadaverine produced by utilizing microorganisms for saccharides fermentation Download PDFInfo
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Abstract
The invention relates to a method for improving yield of cadaverine produced by utilizing microorganisms for saccharides fermentation. According to the method, key regulation and control sites in a metabolic pathway for synthesizing cadaverine through microorganisms are modified, and preferably, the key regulation and control sites are lysine transport protein gene lysE site and/or D-glucose-6-phosphateketol-isomerase gene pgi site, or the key regulation and control sites are regulation and control sites for regulating and controlling expression of the above genes. The modification method for the regulation and control sites comprises deleting lysine transport protein gene lysE and/or D-glucose-6-phosphateketol-isomerase gene pgi, or weakening expression or enzyme activity of lysine transport protein gene lysE and/or D-glucose-6-phosphateketol-isomerase gene pgi, and therefore the yield of cadaverine is effectively improved, saccharides utilization rate is increased, and production cost is reduced.
Description
Technical field
The invention belongs to genetic engineering bacterium technical field, transform especially by the key regulatory site utilizing carbohydrate fermentation to synthesize in the pathways metabolism of cadaverine microorganism, thus effectively a kind of of output of raising cadaverine improves the method that microorganism utilizes carbohydrate fermentation production cadaverine.
Background technology
Cadaverine (Cadaverine) is a polyamine species, namely 1,5-pentamethylene diamine (abbreviation pentamethylene diamine), is generated by Methionin decarboxylation in vivo, be extensively be present in, in organism, there is bioactive nitrogenous base, but be also present in septic matter as a kind of ptomaine.Cadaverine is the important source material of synthesizing new material polymeric amide-54 (being formed by cadaverine and succsinic acid condensation) and polymeric amide-56 (being formed by cadaverine and oxalic acid condensation), has important industrial use.
The method of current synthesis cadaverine has chemical synthesis and enzyme transforming process.Chemical synthesis condition is harsh, contaminate environment, and enzyme transforming process process is complicated, cost is higher.Utilizing genetic engineering technique to construct metabolic engineering bacteria, directly necessary for human product is prepared in mass-producing is most economical, environmental protection and the most promising method, is direction and the focus of metabolic engineering research.
Production by Microorganism Fermentation cadaverine is exactly that microorganism utilizes carbohydrate to ferment, and directly synthesizes cadaverine in a large number by metabolism, this method is simple, economy, environmental protection and efficient.Although intestinal bacteria, corpse bacillus, hafnia alvei etc. directly can synthesize cadaverine, also carried out extensive research to cadaverine synthesis adjustment, the output of cadaverine has much room for improvement.
JP2002223770 and some papers show to improve cadaverine produce by lysine decarboxylase gene and/or Methionin-cadaverine antiporter gene cadB being incorporated into Corynebacterium glutamicum or intestinal bacteria.CN200780006903.9 discloses knocking out, suppress or strengthening of series of genes in cadaverine route of synthesis, but these documents and patent all do not find to knock out or weaken the output that Methionin transporter gene lysE and glucose-6-phosphate isomerase gene pgi effectively can improve cadaverine.
Therefore, by contrast, there are the different of essence in patent application of the present invention and above-mentioned open source literature.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art part, there is provided a kind of by finding the new regulatory site that can improve fermentable carbohydrate production cadaverine, and the phraseology changing these locus genes improves to improve a kind of of cadaverine output the method that microorganism utilizes carbohydrate fermentation production cadaverine.
To achieve these goals, the technical solution adopted in the present invention is as follows:
Improving microorganism utilizes carbohydrate fermentation to produce a method for cadaverine, is transformed by the key regulatory site in the pathways metabolism to Microbe synthesis cadaverine.
And described key regulatory site is Methionin transporter gene lysE site and glucose-6-phosphate isomerase gene pgi site, or regulate and control the regulatory site of these two genes that above-mentioned key regulatory locus gene is expressed.
And, knock out Methionin transporter gene lysE and/or glucose-6-phosphate isomerase gene pgi, and/or, by changing their promotor, ribosome bind site or carrying out transgenation and weaken their expression or the activity of weakening enzyme.
And described microorganism is intestinal bacteria, Corynebacterium glutamicum, honeycomb Hough Buddhist nun or their recombinant bacterial strain.
And the concrete steps of transformation are as follows:
By enterobacteria lysine decarboxylase gene cadA is cloned on the PCK gene pepck site of Corynebacterium glutamicum, builds and produce cadaverine Corynebacterium glutamicum recombinant bacterium CAD1;
The Methionin transporter gene lysE of recombinant bacterial strain CAD1 is knocked out, constructs the Corynebacterium glutamicum recombinant bacterium CAD2 of cadaverine output increased;
The glucose-6-phosphate isomerase gene pgi of recombinant bacterial strain CAD2 is knocked out, constructs the Corynebacterium glutamicum recombinant bacterium CAD3 of cadaverine output increased.
The advantage that the present invention obtains and positively effect:
1, present method is transformed by utilizing the pathways metabolism of carbohydrate fermentation synthesis cadaverine to microorganism, the microorganism being produced cadaverine by transformation utilizes carbohydrate fermentation to produce the regulatory site of cadaverine, these regulatory sites comprise Methionin transporter gene lysE and glucose-6-phosphate isomerase gene pgi, by knocking out and/or weaken Methionin transporter gene lysE and glucose-6-phosphate isomerase gene pgi, effectively can improve the output of cadaverine, thus improve the utilization ratio of carbohydrate, reduce production cost.
2, Methionin transporter gene lysE knocks out and Methionin-pentamethylene diamine antiporter gene cadB is integrated into lysE gene locus, stops their expression by present method, suppresses the secretion of Methionin, accelerates pentamethylene diamine to be transported to outside born of the same parents simultaneously; Acetyl transferase gene Ncgl1469 is pounded out the degraded that can prevent cadaverine; Glucose-6-phosphate isomerase gene pgi is knocked out, cuts off glycometabolic glycolytic pathway, thus effectively improve the output of cadaverine.
Accompanying drawing explanation
Fig. 1 is the Yield mapping of different recombinant bacterial strain cadaverine in the present invention.
Embodiment
Below in conjunction with embodiment, the present invention is further described, and following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
The method used in the present invention, if no special instructions, is the ordinary method in this area; The reagent used in the present invention, if no special instructions, is the common agents in this area.
The implication of technical term involved in the present invention:
" cadaverine " be i.e. 1,5-pentamethylene diamine.
" recombinant bacterial strain " refers to non-wild type strain, comprises the non-wild type strain obtained by selection by mutation, genetic engineering breeding or other any method.
" regulatory site " refers to other gene and the position thereof of Methionin transporter gene lysE and glucose-6-phosphate isomerase gene pgi and position and all these genetic expressions of regulation and control.
" gene knockout " to refer to knock out or suddenly change the regulatory gene of goal gene, the ribosome bind site of goal gene, the promotor of goal gene or goal gene, goal gene can not be expressed or can not be expressed as activated protein (enzyme).
" gene reduction " refers to that the expression level of goal gene or enzymic activity are lower than microorganism expression level before operation or enzymic activity.Comprise the promotor by changing them, ribosome bind site or carry out transgenation and other any method weakens their expression or the activity of weakening enzyme.
The technique means that the present invention uses:
It is all disclosed sequence that gene order of the present invention comprises lysine decarboxylase gene, Methionin-cadaverine albumen antiport gene, Methionin transporter gene lysE and glucose-6-phosphate isomerase gene pgi sequence.The detection etc. of plasmid pK18mobsacB, molecule manipulation technology, microbial culture technique and cadaverine is all known for a person skilled in the art.
Embodiment 1
A kind ofly improve microorganism and utilize carbohydrate fermentation to produce the method for cadaverine, key regulatory site for Microbe synthesis cadaverine in described method is transformed, such as, described key regulatory site is Methionin transporter gene lysE site and/or glucose-6-phosphate isomerase gene pgi site, or described key regulatory site is the regulatory site that regulation and control said gene is expressed.
More preferably, the described key regulatory site for Microbe synthesis cadaverine transform as: knock out Methionin transporter gene lysE and/or glucose-6-phosphate isomerase gene pgi, and/or, by changing their promotor, ribosome bind site or carrying out transgenation and weaken their expression or the activity of weakening enzyme.
More preferably, described microorganism is intestinal bacteria, Corynebacterium glutamicum, honeycomb Hough Buddhist nun or their recombinant bacterial strain.
Above-mentioned raising microorganism utilizes carbohydrate fermentation to produce the method for cadaverine, and concrete steps are as follows:
(1) produce the structure of cadaverine Corynebacterium glutamicum recombinant bacterium CAD1
Primers near the intestinal bacteria lysine decarboxylase gene cadA provided according to NCBI and PCK gene pepck, amplify the upper of cadA and PCK gene pepck respectively, downstream homology arm sequence pckL and pckR, then with cadA, pckL and pckR is template, by Overlap extension PCR, these three fragments are linked into pckL-cadA-pckR fragment, this fragment by HindIII and BamHI enzyme cut rear clone to pK18mobsacB identical restriction enzyme site between, construct plasmid pK18-pckL-cadA-pckR.With this Plastid transformation Corynebacterium glutamicum C.glutamicumATCC13032, and filter out positive colony by bacterium colony PCR.The positive colony filtered out is inoculated into sucrose plate (the LB substratum of 10% sucrose), is inoculated into respectively after growing bacterium colony containing kantlex with not containing on the LB flat board of kantlex.LB flat board containing kantlex does not grow and the bacterium colony of LB grow on plates not containing kantlex is the recombinant bacterial strain that possible cadA gene is inserted into pepck gene locus, and then carry out bacterium colony PCR checking, if amplify the band of object clip size, be and produce cadaverine Corynebacterium glutamicum recombinant bacterium CAD1.
Primer sequence designed by structure recombinant bacterium CAD1 is as table 1:
Table 1 builds primer table needed for recombinant bacterium CAD1 for the present invention
(2) knock out the structure of the Corynebacterium glutamicum recombinant bacterium CAD2 of Methionin transporter gene lysE
Primers near the Methionin-cadaverine antiporter gene cadB provided according to NCBI and Methionin transporter gene lysE, amplify the upper of cadB and Methionin transporter gene lysE respectively, downstream homology arm sequence lysEL and lysER, then with cadB, lysEL and lysER is template, by Overlap extension PCR, these three fragments are linked into lysEL-cadB-lysER fragment, this fragment by HindIII and BamHI enzyme cut rear clone to pK18mobsacB identical restriction enzyme site between, obtain pK18-lysEL-cadB-lysER carrier, with this vector to product cadaverine Corynebacterium glutamicum recombinant bacterium CAD1, adopt screening method above, obtain the product cadaverine Corynebacterium glutamicum recombinant bacterium CAD2 that lysE gene is knocked.
Primer sequence designed by structure recombinant bacterium CAD2 is as table 2:
Table 2 is for building primer table needed for recombinant bacterium CAD2
(3) knock out the structure of the Corynebacterium glutamicum recombinant bacterium CAD3 of glucose-6-phosphate isomerase gene pgi
According to the primers of the glucose-6-phosphate isomerase gene pgi that NCBI provides, amplify the upper of glucose-6-phosphate isomerase gene pgi respectively, downstream homology arm sequence pgiL and pgiR, then with pgiL and pgiR for template, by Overlap extension PCR, these two fragments are linked into pgiL-pgiR fragment, this fragment by HindIII and BamHI enzyme cut rear clone to pK18mobsacB identical restriction enzyme site between, obtain pK18-pgiL-pgiR carrier, with the product cadaverine Corynebacterium glutamicum recombinant bacterium CAD2 that this vector is pounded out to lysE gene, adopt screening method above, obtain the product cadaverine Corynebacterium glutamicum recombinant bacterium CAD3 that lysE and pgi gene is all knocked.
Primer sequence designed by structure recombinant bacterium CAD3 is as table 3:
Table 3: build primer needed for recombinant bacterium CAD3
(4) the shake flask fermentation of recombinant bacterial strain CAD1, CAD2 and CAD3 and the mensuration of cadaverine output
On flat board, picking one ring mono-clonal recombinant bacterial strain is inoculated into containing 5mLLBG seed culture medium (containing the LB substratum of 0.5% glucose, above-mentioned percentage ratio is quality percent by volume g/mL) middle cultivation 14-16h, inoculum size with 2% is linked into and 50mL fermention medium (glucose 5%, (NH is housed
4)
2sO
43%, yeast powder 0.5%, K
2hPO
42.5g/L, MgSO
47H
2o0.75g/L, CaCl
22H
2o50mg/L, FeSO
47H
2o50mg/L, above-mentioned percentage ratio is quality percent by volume g/mL) 250mL triangular flask in, 30 DEG C, 200r/min cultivates 36h.The centrifugal 5min of fermented liquid 12000r/min, gets supernatant liquor 4 DEG C preservation, obtains the fermented supernatant fluid containing cadaverine.
Get 1mL fermented supernatant fluid to add in 5mL tool plug scale test tube, add 500 μ L2mol/LNaOH solution, 10 μ L Benzoyl chlorides, 37 DEG C of water-bath jolting 20min, every 5min vortex vibration 30s, add 0.5gNaCl, 1mL ether vibration 30s stratification.Upper strata ether is taken in another centrifuge tube, treats ether volatilization completely, add 500 μ L dissolve with methanol, as HPLC detection sample after mistake film.Column temperature: 20 DEG C; Moving phase: 100% acetonitrile, 0.02mol/L ammonium acetate; Gradient: 0 ~ 5min acetonitrile volume fraction is 30%, 5 ~ 10min acetonitrile volume fraction be 75%, 10 ~ 15min acetonitrile volume fraction is 30%.Meanwhile, get different concns cadaverine standard substance working solution and carry out same process, and formulate typical curve.According to the content of the cadaverine in the different sample of standard curve determination.Each sample does three repetitions, asks the mean value of cadaverine output.Result is as Fig. 1.
As can be seen from Figure 1, after knocking out lysE and/or pgi gene, cadaverine output significantly improves, and wherein recombinant bacterial strain CAD2 is than the cadaverine output increased about 77.43% of CAD1; Recombinant bacterial strain CAD3, than the cadaverine output increased about 304.87% of CAD1, than the cadaverine output increased about 128.18% of CAD2, illustrates that lysE and pgi gene is the critical sites regulating cadaverine output, knocks out the output that cadaverine effectively can be improved in these sites.
Embodiment 2
Improving microorganism utilizes carbohydrate fermentation to produce a method for cadaverine, is transformed by the key regulatory site in the pathways metabolism to Microbe synthesis cadaverine.
More preferably, described key regulatory site is Methionin transporter gene lysE site and glucose-6-phosphate isomerase gene pgi site, or regulates and controls the regulatory site of these two genes that above-mentioned key regulatory locus gene is expressed.
More preferably, describedly to transform as: knock out Methionin transporter gene lysE and/or glucose-6-phosphate isomerase gene pgi, and/or, by changing their promotor, ribosome bind site or carrying out transgenation and weaken their expression or the activity of weakening enzyme.
More preferably, described microorganism is intestinal bacteria, Corynebacterium glutamicum, honeycomb Hough Buddhist nun or their recombinant bacterial strain.
The concrete steps of transformation are as follows:
By being cloned on the PCK gene pepck site of Corynebacterium glutamicum by enterobacteria lysine decarboxylase gene cadA, build and produce cadaverine Corynebacterium glutamicum recombinant bacterium CAD1, cadaverine output is 2.26g/L.
Knocked out by the Methionin transporter gene lysE of recombinant bacterial strain CAD1, construct the Corynebacterium glutamicum recombinant bacterium CAD2 of cadaverine output increased, its cadaverine output is 4.01g/L, improves 77.43% than CAD1.
Knocked out by the glucose-6-phosphate isomerase gene pgi of recombinant bacterial strain CAD2, construct the Corynebacterium glutamicum recombinant bacterium CAD3 of cadaverine output increased, its cadaverine output is 9.15g/L.Recombinant bacterial strain CAD3 than the cadaverine output increased about 304.87% of CAD1, than the cadaverine output increased about 128.18% of CAD2.
Claims (5)
1. improving microorganism utilizes carbohydrate fermentation to produce a method for cadaverine, it is characterized in that: transformed by the key regulatory site in the pathways metabolism to Microbe synthesis cadaverine.
2. raising microorganism according to claim 1 utilizes carbohydrate fermentation to produce the method for cadaverine, it is characterized in that: described key regulatory site is Methionin transporter gene lysE site and glucose-6-phosphate isomerase gene pgi site, or regulate and control the regulatory site of these two genes that above-mentioned key regulatory locus gene is expressed.
3. raising microorganism according to claim 1 utilizes carbohydrate fermentation to produce the method for cadaverine, it is characterized in that: described in transform as: knock out Methionin transporter gene lysE and/or glucose-6-phosphate isomerase gene pgi, and/or, by changing their promotor, ribosome bind site or carrying out transgenation and weaken their expression or the activity of weakening enzyme.
4. the raising microorganism according to any one of claims 1 to 3 utilizes carbohydrate fermentation to produce the method for cadaverine, it is characterized in that: described microorganism is intestinal bacteria, Corynebacterium glutamicum, honeycomb Hough Buddhist nun or their recombinant bacterial strain.
5. the raising microorganism according to any one of claims 1 to 3 utilizes carbohydrate fermentation to produce the method for cadaverine, it is characterized in that: the concrete steps of transformation are as follows:
By enterobacteria lysine decarboxylase gene cadA is cloned on the PCK gene pepck site of Corynebacterium glutamicum, builds and produce cadaverine Corynebacterium glutamicum recombinant bacterium CAD1;
The Methionin transporter gene lysE of recombinant bacterial strain CAD1 is knocked out, constructs the Corynebacterium glutamicum recombinant bacterium CAD2 of cadaverine output increased;
The glucose-6-phosphate isomerase gene pgi of recombinant bacterial strain CAD2 is knocked out, constructs the Corynebacterium glutamicum recombinant bacterium CAD3 of cadaverine output increased.
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