CN104560848A - Genetically engineered bacteria realizing high-density fermentation co-production of 2,3-butanediol as well as construction method and application thereof - Google Patents
Genetically engineered bacteria realizing high-density fermentation co-production of 2,3-butanediol as well as construction method and application thereof Download PDFInfo
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- CN104560848A CN104560848A CN201410559814.9A CN201410559814A CN104560848A CN 104560848 A CN104560848 A CN 104560848A CN 201410559814 A CN201410559814 A CN 201410559814A CN 104560848 A CN104560848 A CN 104560848A
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- butanediol
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Abstract
The invention discloses genetically engineered bacteria realizing high-density fermentation co-production of 2,3-butanediol as well as a construction method and an application thereof. The genetically engineered bacteria are constructed by integrating three key enzyme genes such as alpha-acetolactate synthetase encoding genes, alpha-acetolacetate decearboxylase encoding genes and 2,3-butanediol dehydrogenase encoding genes onto Escherichia coli chromosomes in a 2,3-butanediol synthesis path. According to the strain fermentation process, the content of the byproduct acetic acid is reduced, so that high-density fermentation can be realized, and 2,3-butanediol with high additional value is co-produced. In addition, the invention also discloses a method for realizing high-density fermentation co-production of other compounds and 2,3-butanediol genetically engineered bacteria and an application thereof. The 2,3-butanediol can be produced by virtue of high-density fermentation, and polyhydroxyalkanoates or functional proteins also can be co-produced, so that low-cost and high-efficiency co-production of the polyhydroxyalkanoates or functional proteins and the 2,3-butanediol is realized, and the genetically engineered bacteria have important industrial application values.
Description
Technical field
The invention belongs to technical field of biochemical industry, be specifically related to a kind of realize intestinal bacteria high density fermentation co-production 2,3-butanediol genetic engineering bacterium and construction process and application.
Background technology
Intestinal bacteria are as first for industrial bacterial strain, and simple by means of its structure, genetic background is clear, easy cultivation, fast growth, target gene expression level is high, the features such as culture cycle is short, and contamination resistance is strong, have become prokaryotic expression system the most conventional at present.Along with the research of metabolic engineering and field of fermentation engineering deepens continuously, Escherichia coli fermentation is utilized to produce the high molecular polymer of multiple high added value, as PHA and various functional protein, as proteolytic enzyme, Regular Insulin, Human-like Collagen etc. are widely used.
Concentration cultivation technology (high cell density cultivation, HCDC), i.e. high density fermentation technology, specifically refer under certain condition and culture system, utilize certain culture technique and device to improve the fermentation density of thalline, make cell density comparatively Nostoc commune Vanch be significantly increased, thus obtain maximum cell concentrations, obtain object product more or more efficiently thus, the final zymotechnique improving the specific production rate of product.Compared with traditional zymotic technique, high density fermentation not only can reduce volume of culture, and strengthening downstream separation is extracted, and can also shorten the production cycle, reduce facility investment, thus reduce production cost, enhance productivity, realize large-scale industrial production.
Along with the development of genetic engineering technique and high-density culture technology, there is the high molecular polymer of high added value, the output of recombinant protein and productive rate increase substantially, fermentation costs reduces greatly, and this produces for recombination bacillus coli large scale fermentation and provides more wide prospect.But in recombinant bacterial strain the high level expression of foreign gene, not only relate to host, mutual relationship between carrier and clone gene three, also closely bound up with the envrionment conditions residing for Host Strains, the factor affecting its growth and exogenous route expression is a lot, comprise and pass oxygen limited ability, substrate suppresses, metabolic by-prods suppression etc.Wherein the restraining effect of metabolic by-prods acetic acid is the topmost problem that high-density cultivation of Escherichia coli faces.The accumulation of acetic acid not only limit the growth of thalline, also can suppress the synthesis of product, and the formation of acetic acid also can cause the loss of carbon source and then reduce substrate conversion efficiency.Too high acetic acid concentration, by destroying the stability of intracellular protein, makes intracellular protein and DNA that irreversible degraded occur, thus causes lysis dead.
Utilizing in Escherichia coli fermentation production process, the generation of acetic acid is mainly due to the following aspects.First, in intestinal bacteria, glycolytic pathway and TCA circulation all can produce cofactor NADH, and under the condition of oxygen supply abundance, NADH can complete oxidation be NAD
+.But to take the photograph on oxygen in limited time when the wear rate of glucose and the synthesis speed of NADH have exceeded intestinal bacteria, NADH will start a large amount of accumulation.NADH is the specificity allosteric inhibitor of citrate synthase, can limit acetyl-CoA and oxaloacetic acid generation citric acid; Meanwhile, during TCA also can be suppressed to circulate, the ArcA regulation system of several gene is subject to the impact of born of the same parents' internal oxidition reduced state, thus acetic acid in fermenting process is accumulated in a large number.Secondly, in recombination bacillus coli process of high-density fermentation, all acetic acid can be produced when supplying hypoxgia or Growth of Cells is too fast.When oxygen supply abundance, the synthesis speed of acetic acid is relevant with cell oxygen uptake rate to the specific growth rate of the generation of acetic acid and cell.In Bacillus coli cells process of growth, along with the rising of specific growth rate, the corresponding increase of oxygen uptake rate, and when specific growth rate reaches certain value, the oxygen uptake capacity of cell reaches the upper limit, even if oxygen supply is abundant, the oxygen uptake rate of cell also can not increase again, thus the restrictive factor become in process of growth, cause the generation of acetic acid.In addition, in process of high-density fermentation, intestinal bacteria consume fast and utilize the glycogenetic more intermediate of grape can not in time for Growth of Cells and Product Expression, and excessive carbon source is gathered the acetyl-CoA in causing TCA to circulate and changed into acetic acid.
Scholars has significantly promoted the development of zymotechnique for the research that acetic acid suppresses in recent decades.In recombination bacillus coli process of high-density fermentation, for hypoxgia or Growth of Cells too fast time all can produce acetic acid; When oxygen supply abundance, the synthesis speed of acetic acid is directly related with cell growth rate; Meanwhile, the growth velocity of culture is determined, so acetic acid synthesis speed is also directly related with feed rate by the feed rate of restricted substrate.In early days, people are by changing nutrient media components, reducing the formation that the optimization fermentation process such as specific growth rate, dialysis culture, limiting glucose concentration reduce acetic acid.But, nutrient media components optimization can only slow down the negative effect of acetic acid to object Product Expression, the accumulation of acetic acid cannot be reduced, and the change of component often increases cost, also can have a negative impact to downstream purification process; Acetic acid can be made to reduce although reduce specific growth rate, the growth that simultaneously also can delay cell causes fermentation period to extend; Dialysis culture method is cured the symptoms, not the disease, and cannot avoid the formation of acetic acid, also can loss partial medium in removal acetic acid process.
Along with the continuous maturation of genetic engineering technique and metabolic engineering technology, utilize Molecular tools in the metabolism of gene level regulation and control engineering bacteria, thus control yield of acetic acid, be widely used.The genetic manipulation strategy controlling acetic acid generation employing can be divided into three kinds usually: the first strategy controls acetic acid by the consumption controlling glucose and generates, because the size of glucose consumption rate generates directly related with acetic acid, control glucose uptake speed thus directly control acetic acid to generate by knocking out genes involved bunch.The second strategy is cut off carbon source to flow to acetic acid generation or carbon source catalysis is generated the less material of murder by poisoning, and this strategy, mainly through knocking out the metabolic pathway key gene causing acetic acid to generate in glycolysis-and TCA circulation, reduces acetic acid and produces.Last a kind of strategy reduces the potential metabolic pathway causing acetic acid to generate, and as the accumulation by reducing NADH in cell, reduces the generation of acetic acid.
Summary of the invention
One is the object of the present invention is to provide to realize high density fermentation co-production 2, the genetic engineering bacterium of 3-butyleneglycol, when being fermenting substrate utilizing it with glucose, originally the metabolic fluxes flowing to acetate pathway can be consumed and generate the extremely low neutral products of toxicity 2, 3-butyleneglycol, thus the acetic acid generated reduces widely, simultaneously, due to 2, in 3-butyleneglycol building-up process, a large amount of NADH is consumed, reduce the generation of acetic acid more further, thus alleviate acetic acid suppression dramatically to the impact of fermentation, thus the high density fermentation of more high-biomass can be realized, also coproduction height adds 2 simultaneously, 3-butyleneglycol.
Another object of the present invention is to provide a kind of and build the above-mentioned method realizing the genetic engineering bacterium of intestinal bacteria high density fermentation co-production 2,3-butanediol.
In order to realize object of the present invention, described one realizes high density fermentation co-production 2,3-butyleneglycol genetic engineering bacterium, by 2, three key enzyme encoding genes: α-acetolactate synthestase encoding gene in 3-butyleneglycol route of synthesis, alpha-acetolactate decarboxylase encoding gene and 2,3-butanediol dehydrogenase coding genes are incorporated on escherichia coli chromosome, the recombinant bacterium built.
Described 2,3-butanediol dehydrogenase coding genes is (R, R)-2,3-butanediol dehydrogenase coding genes or meso-2,3-butanediol dehydrogenation enzyme coding gene.
The construction process of said gene engineering bacteria comprises concrete steps:
1) to increase respectively α-acetolactate synthestase encoding gene and alpha-acetolactate decarboxylase encoding gene from Klebsiella pneumonia, from subtilis amplification (R, R)-2, 3-butanediol dehydrogenation enzyme coding gene, recycle overlap extension pcr by above-mentioned three gene fragments according to α-acetolactate synthestase encoding gene, alpha-acetolactate decarboxylase encoding gene, (R, R)-2, the sequential concatenation assembling of 3-butanediol dehydrogenation enzyme coding gene obtains α-acetolactate synthestase encoding gene, alpha-acetolactate decarboxylase encoding gene and (R, R)-2, three gene fusion fragments of 3-butanediol dehydrogenation enzyme coding gene or directly from Klebsiella pneumonia amplification α-acetolactate synthestase encoding gene, alpha-acetolactate decarboxylase encoding gene and meso-2, the operon gene fragment of 3-butanediol dehydrogenation enzyme coding gene.
2) by two step methods of homologous recombination by step 1) in α-acetolactate synthestase encoding gene, α-acetylactis decarboxylation codase gene and (R, R)-2, the operon gene fragment of 3-butanediol dehydrogenation enzyme coding gene three gene fusion fragment or α-acetolactate synthestase encoding gene, alpha-acetolactate decarboxylase encoding gene and meso-2,3-butanediol dehydrogenation enzyme coding gene is incorporated in escherichia coli chromosome group.
Wherein, to the α-acetolactate synthestase encoding gene obtained that increases, alpha-acetolactate decarboxylase encoding gene, before the splicing of 2,3-butanediol dehydrogenase coding genes, first at α-acetolactate synthestase encoding gene, alpha-acetolactate decarboxylase encoding gene, ribosome bind site is introduced respectively in (R, R)-2,3-butanediol dehydrogenase coding genes upstream; Or directly at α-acetolactate synthestase encoding gene, ribosome bind site is introduced in the operon gene fragment upstream of alpha-acetolactate decarboxylase encoding gene and meso-2,3-butanediol dehydrogenation enzyme coding gene.Said gene upstream is introduced corresponding ribosome bind site and can improve corresponding gene protein translation speed, thus improve the expression amount of target protein, and then the output of 2,3-BD is increased.
Two described step methods of homologous recombination concrete steps are:
1) first the escherichia coli chromosome of pcr amplification knocked in by the technology of Overlap extension PCR one section of fragment containing 500-1000 base that one section of fragment containing 500-1000 base of site upstream sequence homology, resistance screening marker gene, sucrose levanase gene and escherichia coli chromosome knock in sites downstream sequence homology to merge, obtain recombinant fragment 1, recombinant fragment 1 is incorporated into knocking on site of genome of E.coli by the method re-using λ-Red homologous recombination.
2) first by the method for over-lap PCR, the escherichia coli chromosome of pcr amplification is knocked in one section of fragment containing 500-1000 base of site upstream sequence homology, α-acetolactate synthestase encoding gene, alpha-acetolactate decarboxylase encoding gene and (R, R)-2, 3-butanediol dehydrogenation enzyme coding gene three gene fusion fragment or α-acetolactate synthestase encoding gene, alpha-acetolactate decarboxylase encoding gene and meso-2, the operon gene fragment of 3-butanediol dehydrogenation enzyme coding gene, the one section of fragment containing 500-1000 base knocking in sites downstream sequence homology with escherichia coli chromosome merges, obtain recombinant fragment 2 or recombinant fragment 3, recombinant fragment 2 or recombinant fragment 3 are replaced above-mentioned steps 1 by the method re-using λ-Red homologous recombination) middle recombinant fragment 1.
Described resistance screening marker gene is the one in chloramphenicol resistance gene, kalamycin resistance gene, gentamicin resistance gene, tetracycline resistance gene.
It is lactic dehydrogenase enzyme coding gene site or 16SrDNA gene locus that described escherichia coli chromosome knocks in site, by 2, the gene integration that 3-butyleneglycol route of synthesis is correlated with is in intestinal bacteria process, lactic dehydrogenase enzyme coding gene site is selected to be that escherichia coli chromosome knocks in site, the gene that lactic acid route of synthesis is relevant is destroyed, and therefore, the genetic engineering bacterium that structure obtains during the fermentation, while acetic acid suppresses to be eased, by product lactic acid is also reduced.
Being of another object of the present invention provides a kind of method realizing high density fermentation coproduction 2,3-butanediol.For solving this technical problem, the present invention is that high density fermentation cultivates said gene engineering bacteria production 2,3-butanediol.
Wherein, high density fermentation cultivation seed culture medium (g/L) consists of: peptone 10; Yeast extract paste 5; NaCl 10; Fermention medium (g/L) consists of glucose 60-100; Yeast extract paste 0-40; K
2hPO
43H
2o 10.0-15.0; KH
2pO
42.0-6.0; (NH
4)
2hPO
42.0-6.0; (NH
4)
2sO
42.0-8.0; MgSO
47H
2o 0.1-0.3; FeSO
47H
2o 0.01-0.1; ZnSO
47H
2o 0.01-0.1; MnSO
4h
2o 0.01-0.1; CaCl
20.01-0.1; EDTA 0.01-0.1.
Without the need to mending alkali regulation and control fermented liquid pH value in this high density fermentation culturing process, simplify corresponding fed-batch fermentation strategy.
Another object of the present invention is to provide one to realize high density fermentation co-production poly-hydroxy fatty acid and 2,3-butanediol genetic engineering bacterium.For head it off, the present invention introduces exogenous plasmid above-mentioned realization in high density fermentation co-production poly-hydroxy fatty acid and 2,3-butanediol genetic engineering bacterium, and described exogenous plasmid is with the genes involved of poly-hydroxy fatty acid route of synthesis.
Described poly-hydroxy fatty acid is poly butyric ester or PHBV.
Another object of the present invention is to provide one to realize high density fermentation co-production poly-hydroxy fatty acid and 2,3-butanediol method.For head it off, high density fermentation of the present invention realizes high density fermentation co-production poly-hydroxy fatty acid and 2,3-butanediol gene engineering bacteria produces poly-hydroxy fatty acid and 2,3-butanediol.
Another object of the present invention is to provide one to realize high density fermentation co-production functional protein and 2,3-butanediol genetic engineering bacterium.For head it off, the present invention introduces exogenous plasmid above-mentioned realization in high density fermentation co-production poly-hydroxy fatty acid and 2,3-butanediol genetic engineering bacterium, and described exogenous plasmid is with the genes involved of functional protein route of synthesis.
Described functional protein is proteolytic enzyme, Interferon, rabbit, Regular Insulin or Human-like Collagen.
Another object of the present invention is to provide one to realize high density fermentation co-production functional protein and 2,3-butanediol method.For head it off, high density fermentation of the present invention realizes high density fermentation co-production functional protein and 2,3-butanediol gene engineering bacteria produces functional protein and 2,3-butanediol.
Beneficial effect of the present invention:
1, genetic engineering bacterium provided by the invention, by the external source 2 of man-made assembly, the genes involved of 3-butyleneglycol route of synthesis is incorporated on escherichia coli chromosome, genetic engineering bacterium is in synthesis 2, in 3-butyleneglycol process, consume the metabolic fluxes originally flowing to acetate pathway, generate the neutral products 2 that toxicity is extremely low, 3-butyleneglycol, reduce acetic acid widely to generate, simultaneously, due to 2, in 3-butyleneglycol building-up process, a large amount of NADH is consumed, thus comprehensive alleviation acetic acid suppresses during the fermentation on colibacillary impact, realize high density fermentation, the compound 2 of co-production high added value, 3-butyleneglycol.In addition because described genetic engineering bacterium achieves high density fermentation, not only reduce volume of culture, strengthening downstream separation is extracted, and also shortens the production cycle simultaneously, reduces facility investment, thus reduce production cost, further increase production efficiency.
2, the one that the present invention builds realizes other compounds of high density fermentation co-production and 2,3-butyleneglycol genetic engineering bacterium, decapacitation high density fermentation produces 2, outside 3-butyleneglycol, can also coproduction poly-hydroxy fatty acid or functional protein, thus realize poly-hydroxy fatty acid or functional protein, with 2,3-butyleneglycol low cost, high efficiency coproduction, have important industrial application value.
Specific embodiment
According to following embodiment, the present invention may be better understood.But those skilled in the art will readily understand, the content described by embodiment is only limitted to the present invention is described, and also should can not limit the present invention described in detail in claims.Biological material source in the present invention is as follows:
Klebsiella pneumonia CICC10011 bacterial strain (being abbreviated as K.pneumoniae bacterial strain): be purchased from Chinese microorganism strain preservation center; Subtilis 168 bacterial strain (being abbreviated as B.subtilis bacterial strain): be purchased from Chinese industrial Culture Collection;
Carrier pEASY-Blunt: commercial vector, is purchased from Beijing Quanshijin Biotechnology Co., Ltd;
Carrier pTrc99a: commercial vector, is purchased from Bei Nuo bio tech ltd, Shanghai;
Carrier pKD46: commercial vector, is purchased from Bei Nuo bio tech ltd, Shanghai;
Pk18mobsacB: commercial vector, is purchased from Bei Nuo bio tech ltd, Shanghai;
Intestinal bacteria MG1655 bacterial strain (being abbreviated as E.coli bacterial strain): be purchased from Bei Nuo bio tech ltd, Shanghai;
Embodiment 1 realizes the structure of the genetic engineering bacterium of high density fermentation co-production (R, R)-2,3-butanediol
The clone of 1, α-acetolactate synthestase encoding gene alsS, alpha-acetolactate decarboxylase Genes encode gene budA and (R, R)-2,3-butanediol dehydrogenase coding genes bdhA
(1) clone of α-acetolactate synthestase encoding gene alsS
According to alsS gene order design and synthesis primer in the Klebsiella pneumonia (K.pneumoniae) that Genebank announces:
P1：5’-GATATGGACAAACAGTATCCGGT-3’
P2：5’-GCGTTACAGAATCTGACTCAGAT-3’
With the genomic dna of K.pneumoniae CICC10011 for template, with primer P1 and P2, Takara high-fidelity enzyme
hS (Premix) pcr amplification alsS gene.
PCR reaction system:
hS (Premix) 25 μ L, primer P10.3 μ L, primer P20.3 μ L, K.pneumoniae CICC10011 genome 0.4 μ L, ddH
2o 24 μ L.
PCR reaction conditions: 98 DEG C of 10s, 62 DEG C of 15s, 72 DEG C of 1min45s, circulate 30 times; 72 DEG C of 7min, 4 DEG C of insulations.
Agarose gel electrophoresis with 0.8% detects PCR primer, and uses Takara glue to reclaim kits object fragment, then the fragment of purifying is cloned on carrier pEASY-Blunt.Clone's system: the fragment after 1 μ L pEASY-Blunt carrier, 4 μ L purifying, mixes gently; After 25 DEG C of reaction 20min, add in 50 μ L Trans1-T1 competent cells, ice bath 30min, 42 DEG C of heat shock 30s, are placed in 2min on ice immediately.Add rapidly 250 μ L LB substratum, 200rpm, 37 DEG C, cultivate 1h.Get 200 μ L bacterium liquid and be coated with the LB flat board containing penbritin, after incubated overnight, the LB liquid nutrient medium that picking 10 positive bacterium colonies connect containing penbritin is cultivated, extract positive colony plasmid and send sequence verification, alsS sequencing result as shown in SEQ ID NO:1, by the recombinant plasmid called after pEASY-Blunt-alsS obtained.
With recombinant plasmid pEASY-Blunt-alsS for template, with primer P3 and P4, Takara high-fidelity enzyme
hS (Premix) pcr amplification is containing the splicing sequence 1 of alsS gene fragment.
P3：5’-TAAGGAGGATATACATGATATGGACAAACAGTATCCGGT-3’
P4：5’-CATTCAGAGAGTGATTCATAGCGCGTTACAGAATCTGACTCAGAT-3’
PCR reaction system:
hS (Premix) 25 μ L, primer P30.3 μ L, primer P40.3 μ L, pEASY-Blunt-alsS 0.4 μ L, ddH
2o 24 μ L.
PCR reaction conditions: 98 DEG C of 10s, 61 DEG C of 15s, 72 DEG C of 1min45s, circulate 30 times; 72 DEG C of 7min, 4 DEG C of insulations.
Agarose gel electrophoresis with 0.8% detects PCR primer, and reclaim after purifying through glue, 4 DEG C of storages are stand-by.The sequence amplified comprises the 23bp sequence of RBS site, alsS gene order and end and budA gene front end homology, and the sequence in the RBS site of alsS gene order upstream is as shown in SEQ ID NO:2.
(2) clone of alpha-acetolactate decarboxylase Genes encode gene budA
According to budA gene order design and synthesis primer in the Klebsiella pneumonia (K.pneumoniae) that Genebank announces:
P5：5’-GCTATGAATCACTCTGCTGAATG-3’
P6：5’-GTCTTAACTTTCTACGGAACGGA-3’
With the genomic dna of K.pneumoniae CICC10011 for template, with primer P5 and P6, Takara high-fidelity enzyme
hS (Premix) pcr amplification budA gene.
PCR reaction system:
hS (Premix) 25 μ L, primer P50.3 μ L, primer P60.3 μ L, K.pneumoniae CICC10011 genome 0.4 μ L, ddH
2o 24 μ L.
PCR reaction conditions: 98 DEG C of 10s, 57 DEG C of 15s, 72 DEG C of 50s, circulate 30 times; 72 DEG C of 7min, 4 DEG C of insulations.
Agarose gel electrophoresis with 0.8% detects PCR primer, and uses Takara glue to reclaim kits object fragment, then the fragment of purifying is cloned on carrier pEASY-Blunt.Clone's system: the fragment after 1 μ L pEASY-Blunt carrier, 4 μ L purifying, mixes gently; After 25 DEG C of reaction 20min, add in 50 μ L Trans1-T1 competent cells, ice bath 30min, 42 DEG C of heat shock 30s, are placed in 2min on ice immediately.Add rapidly 250 μ L LB substratum, 200rpm, 37 DEG C, cultivate 1h.Get 200 μ L bacterium liquid and be coated with the LB flat board containing penbritin, after incubated overnight, the LB liquid nutrient medium that picking 10 positive bacterium colonies connect containing penbritin is cultivated, extract positive colony plasmid and send sequence verification, budA sequencing result as shown in SEQ IDNO:3, by the recombinant plasmid called after pEASY-Blunt-budA obtained
With recombinant plasmid pEASY-Blunt-budA for template, with primer P7 and P8, Takara high-fidelity enzyme
hS (Premix) pcr amplification is containing bdhA gene fragment splicing sequence 2.
P7:5’-ATCTGAGTCAGATTCTGTAACGCTAAGGAGGATATACATGCTATGAATCACTCTGCTGAATG-3’
P8：5’-TGCCATCTTGCTGCCTTCATGCGTCTTAACTTTCTACGGAACGGA-3
PCR reaction system:
hS (Premix) 25 μ L, primer P70.3 μ L, primer P80.3 μ L, pEASY-Blunt-alsS 0.4 μ L, ddH
2o 24 μ L.
PCR reaction conditions: 98 DEG C of 10s, 57 DEG C of 15s, 72 DEG C of 50s, circulate 30 times; 72 DEG C of 7min, 4 DEG C of insulations.
Agarose gel electrophoresis with 0.8% detects PCR primer, and reclaim after purifying through glue, 4 DEG C of storages are stand-by.The alsS gene fragment amplified comprises the 22bp sequence of the 23bp sequence of front end and alsS gene end homology, RBS binding site, budA gene order and end and bdhA gene front end homology.The sequence of the RBS binding site of budA gene order upstream is as shown in SEQ ID NO:4.
(3) clone of (R, R)-2,3-butanediol dehydrogenase coding genes bdhA
According to bdhA gene order design and synthesis primer in the subtilis (B.subtilis) that Genebank announces:
P9：5’-GCATGAAGGCAGCAAGATGGCA-3’
P10：5’-CGTTAGTTAGGTCTAACAAGGAT-3’
With the genomic dna of B.subtilis 168 for template, with primer P9 and P10, Takara high-fidelity enzyme
hS (Premix) pcr amplification bdhA gene.
PCR reaction system:
hS (Premix) 25 μ L, primer P90.3 μ L, primer P100.3 μ L, K.pneumoniae CICC10011 genome 0.4 μ L, ddH
2o 24 μ L.
PCR reaction conditions: 98 DEG C of 10s, 58 DEG C of 15s, 72 DEG C of 1min10s, circulate 30 times; 72 DEG C of 7min, 4 DEG C of insulations.
Agarose gel electrophoresis with 0.8% detects PCR primer, and uses Takara glue to reclaim kits object fragment, then the fragment of purifying is cloned on carrier pEASY-Blunt.Clone's system: the fragment after 1 μ L pEASY-Blunt carrier, 4 μ L purifying, mixes gently; After 25 DEG C of reaction 20min, add in 50 μ L Trans1-T1 competent cells, ice bath 30min, 42 DEG C of heat shock 30s, are placed in 2min on ice immediately.Add rapidly 250 μ L LB substratum, 200rpm, 37 DEG C, cultivate 1h.Get 200 μ L bacterium liquid and be coated with the LB flat board containing penbritin, after incubated overnight, the LB liquid nutrient medium that picking 10 positive bacterium colonies connect containing penbritin is cultivated, extract positive colony plasmid and send sequence verification, bdhA sequencing result as shown in SEQ ID NO:5, by the recombinant plasmid called after pEASY-Blunt-bdhA obtained.
With recombinant plasmid pEASY-Blunt-bdhA for template, with primer P11 and P12, Takara high-fidelity enzyme
hS (Premix) pcr amplification is containing the splicing sequence 3 of bdhA gene fragment.
P11:5’-TCCGTTCCGTAGAAAGTTAAGACTAAGGAGGATATACATGCATGAAGGCAGCAAGATGGCA-3’
P12：5’-CGTTAGTTAGGTCTAACAAGGAT-3’
PCR reaction system:
hS (Premix) 25 μ L, primer P110.3 μ L, primer P120.3 μ L, pEASY-Blunt-bdhA 0.4 μ L, ddH
2o 24 μ L.
PCR reaction conditions: 98 DEG C of 10s, 58 DEG C of 15s, 72 DEG C of 1min15s, circulate 30 times; 72 DEG C of 7min, 4 DEG C of insulations.
Agarose gel electrophoresis with 0.8% detects PCR primer, and reclaim after purifying through glue, 4 DEG C of storages are stand-by.The gene fragment amplified comprises the 23bp sequence of front end and budA gene end homology, RBS site and bdhA gene order.The RBS site of bdhA gene order upstream is as shown in SEQ ID NO:6.
2, over-lap PCR is utilized to splice assembling alsS, budA and bdhA three gene fragment
To splice sequence 1, splicing sequence 2 with splicing sequence 3 three gene for template, with primer P13 and P14, Takara high-fidelity enzyme
hS (Premix) anastomosing and splicing sequence 1, splicing sequence 2 and splicing sequence 3 three gene fragment.
P13：5’-ACCTATTGACAATTAAAGGCTAAAATGCTATAATTCCACAAATCTAAGGAGGATATACATGATATGG-3’
P14：5’-AAAAGGCCATCCGTCAGGATGGCCTTCTCGTTAGTTAGGTCTAACAAGGAT-3’
PCR reaction system:
hS (Premix) 25 μ L, primer P130.3 μ L, primer P140.3 μ L, alsS 0.6 μ L, bdhA 0.6 μ L, bdhA 0.6 μ L, ddH
2o 22.6 μ L.
PCR condition: 98 DEG C of 10s, 60 DEG C of 15s, 72 DEG C of 3min50s, circulate 30 times; 72 DEG C of 7min, 4 DEG C of insulations.The alsSbudAbdhA gene fragment amplified comprises: promotor, alsSbudAbdhA, terminator.
Agarose gel electrophoresis with 0.8% detects PCR primer, and uses Takara glue to reclaim kits object fragment, then the fragment of purifying is cloned on carrier pEASY-Blunt.Clone's system: the fragment after 1 μ L pEASY-Blunt carrier, 4 μ L purifying, mixes gently; After 25 DEG C of reaction 20min, add in 50 μ L Trans1-T1 competent cells, ice bath 30min, 42 DEG C of heat shock 30s, are placed in 2min on ice immediately.Add rapidly 250 μ L LB substratum, 200rpm, 37 DEG C, cultivate 1h.Get 200 μ L bacterium liquid and be coated with the LB flat board containing penbritin, after incubated overnight, the LB liquid nutrient medium that picking 10 positive bacterium colonies connect containing penbritin is cultivated, extract positive colony plasmid and send sequence verification, sequencing result is correct, by the recombinant plasmid called after pEASY-Blunt-alsSbudAbdhA obtained.
3, alsSbudAbdhA is incorporated in escherichia coli chromosome group by two step methods of homologous recombination.
(1) pcr amplification ldhA-1
With intestinal bacteria MG1655 genome for template, with primer ldhA-1-S and ldhA-1-A, Takara high-fidelity enzyme
hS (Premix) carries out pcr amplification, and the fragment amplified comprises one section of lactic dehydrogenase enzyme coding gene site upstream sequence homology containing the fragment of 996 bases and the 22bp fragment of end and Cm-sacB front end homology, by its called after ldhA-1.
ldhA-1-S：5’-AGTTCGCTGACTGTAAGTTGTTGCC-3’
ldhA-1-A：5’-TGCGAAGTGATCTTCCGTCACACCATACATAGTAAAGCCGGTCAGAC-3’
PCR reaction system:
hS (Premix) 25 μ L, primer ldhA-1-S 0.3 μ L, primer ldhA-1-A 0.3 μ L, intestinal bacteria MG1655 genome 0.4 μ L, ddH
2o 24 μ L.
PCR reaction conditions: 98 DEG C of 10s, 62 DEG C of 15s, 72 DEG C of 1min, circulate 30 times; 72 DEG C of 7min, 4 DEG C of insulations.
Agarose gel electrophoresis with 0.8% detects PCR primer, reclaims after test kit fragment purification through Takara glue, stand-by in 4 DEG C of storages.
Fragment ldhA-1 sequence verification: purified ldhA-1 fragment is cloned on carrier pEASY-Blunt and carries out sequence verification.Clone's system: the fragment after 1 μ L pEASY-Blunt carrier, 4 μ L purifying, mixes gently; After 25 DEG C of reaction 20min, add in 50 μ L Trans1-T1 competent cells, ice bath 30min, 42 DEG C of heat shock 30s, are placed in 2min on ice immediately.Add rapidly 250 μ L LB substratum, 200rpm, 37 DEG C, cultivate 1h.Get 200 μ L bacterium liquid and be coated with the LB flat board containing penbritin, after incubated overnight, the LB liquid nutrient medium that picking 10 positive bacterium colonies connect containing penbritin is cultivated, extract positive colony plasmid and send sequence verification, one section of sequencing fragment result containing 996 bases of lactic dehydrogenase enzyme coding gene site upstream sequence homology is as shown in SEQ ID NO:7.
With intestinal bacteria MG1655 genome for template, with primer ldhA-2-S and ldhA-2-A, Takara high-fidelity enzyme
hS (Premix) carries out pcr amplification, and the fragment amplified comprises one section of lactic dehydrogenase enzyme coding gene sites downstream sequence homology containing the fragment of 975 bases and the 22bp fragment of front end and Cm-sacB terminal homologous, by its called after ldhA-2.
ldhA-2-S：5’-GACAATTAACAGTTAACAAATAA TATCAGCGTACCCGTGATGCTAACT-3’
ldhA-2-A：5’-GTTCACCATTAGACAGTTTGCC-3’
PCR reaction system:
hS (Premix) 25 μ L, primer ldhA-2-S 0.3 μ L, primer ldhA-2-A0.3 μ L, intestinal bacteria MG1655 genome 0.4 μ L, ddH
2o 24 μ L.
PCR reaction conditions: 98 DEG C of 10s, 62 DEG C of 15s, 72 DEG C of 1min, circulate 30 times; 72 DEG C of 7min, 4 DEG C of insulations.
Agarose gel electrophoresis with 0.8% detects PCR primer, reclaims after test kit fragment purification through Takara glue, stand-by in 4 DEG C of storages.
Fragment ldhA-2 sequence verification: purified ldhA-2 fragment is cloned on carrier pEASY-Blunt and carries out sequence verification.Clone's system: the fragment after 1 μ L pEASY-Blunt carrier, 4 μ L purifying, mixes gently; After 25 DEG C of reaction 20min, add in 50 μ L Trans1-T1 competent cells, ice bath 30min, 42 DEG C of heat shock 30s, are placed in 2min on ice immediately.Add rapidly 250 μ L LB substratum, 200rpm, 37 DEG C, cultivate 1h.Get 200 μ L bacterium liquid and be coated with the LB flat board containing penbritin, after incubated overnight, the LB liquid nutrient medium that picking 10 positive bacterium colonies connect containing penbritin is cultivated, extract positive colony plasmid and send sequence verification, one section of fragment containing 975 bases of lactic dehydrogenase enzyme coding gene sites downstream sequence homology, sequencing result is as shown in SEQ ID NO:8.
(2) pcr amplification paraxin riddled basins and sucrose levanase gene C m-sacB
With plasmid pk18mobsacb for template, with primer Cm-sacB-S and Cm-sacB-A, Takara high-fidelity enzyme
hS (Premix) carries out pcr amplification, and the fragment amplified comprises: the 20bp sequence of front end and ldhA1 fragment ends homology, Cm-sacB sequence, and the sequence of the 20bp of end and ldhA2 fragment front end homology, by its called after Cm-sacB.
Cm-sacB-S：5’-TTCAATAACGTCGACCTTGACGTGTGACGGAAGATCACTTCGCA-3’
Cm-sacB-A：5’-TTTCAGAATGCGCAGCATCGCTTATTTGTTAACTGTTAATTGTCCT-3’
Answer system:
hS (Premix) 25 μ L, primer Cm-sacB-S 0.3 μ L, primer Cm-sacB-A0.3 μ L, intestinal bacteria MG1655 genome 0.4 μ L, ddH
2o 24 μ L.
Answer condition: 98 DEG C of 10s, 58 DEG C of 15s, 72 DEG C of 3min10s, circulate 30 times; 72 DEG C of 7min, 4 DEG C of insulations.Agarose gel electrophoresis with 0.8% detects PCR primer, reclaims after test kit fragment purification through Takara glue, stand-by in 4 DEG C of storages.
Fragment Cm-sacB sequence verification: purified Cm-sacB fragment is cloned on carrier pEASY-Blunt and carries out sequence verification.Clone's system: the fragment after 1 μ L pEASY-Blunt carrier, 4 μ L purifying, mixes gently; After 25 DEG C of reaction 20min, add in 50 μ L Trans1-T1 competent cells, ice bath 30min, 42 DEG C of heat shock 30s, are placed in 2min on ice immediately.Add rapidly 250 μ L LB substratum, 200rpm, 37 DEG C, cultivate 1h.Get 200 μ L bacterium liquid and be coated with the LB flat board containing penbritin, after incubated overnight, the LB liquid nutrient medium that picking 10 positive bacterium colonies connect containing penbritin is cultivated, and extract positive colony plasmid and send sequence verification, Cm-sacB measurement result is as shown in SEQ ID NO:9.
(3) method of over-lap PCR is utilized to build recombinant fragment 1,
With ldhA-1, Cm-sacB and ldhA-2 three gene for template, with primer ldhA-1-S and ldhA-2-A, Takara high-fidelity enzyme
hS (Premix) merges ldhA-1, Cm-sacB and ldhA-2 three gene fragment.
ldhA-1-S：5’-AAGGTCGACGTTATTGAAACC-3’
ldhA-2-A：5’-GTTCACCATTAGACAGTTTGCC-3’
Reaction system:
hS (Premix) 25 μ L, primer ldhA-1-S 0.3 μ L, primer ldhA-2-A0.3 μ L, ldhA-10.6 μ L, Cm-sacB 0.6 μ L, ldhA-20.6 μ L, ddH
2o 22.6 μ L.
PCR condition: 98 DEG C of 10s, 60 DEG C of 15s, 72 DEG C of 4min30s, circulate 30 times; 72 DEG C of 7min, 4 DEG C of insulations.
Agarose gel electrophoresis with 0.8% detects PCR primer, and uses Takara glue to reclaim kits object fragment, called after recombinant fragment 1, and in 4 DEG C of storages, for first time homologous recombination.
Fragment recombinant fragment 1 sequence verification: the fragment of purifying is cloned on carrier pEASY-Blunt.Clone's system: the fragment after 1 μ L pEASY-Blunt carrier, 4 μ L purifying, mixes gently; After 25 DEG C of reaction 20min, add in 50 μ L Trans1-T1 competent cells, ice bath 30min, 42 DEG C of heat shock 30s, are placed in 2min on ice immediately.Add rapidly 250 μ L LB substratum, 200rpm, 37 DEG C, cultivate 1h.Get 200 μ L bacterium liquid and be coated with the LB flat board containing penbritin, after incubated overnight, the LB liquid nutrient medium that picking 10 positive bacterium colonies connect containing penbritin is cultivated, and extract positive colony plasmid and send sequence verification, sequencing result is correct.
(4) plasmid pKD46 electricity transformation of E. coli MG1655.
1) intestinal bacteria MG1655 Electroporation-competent cells makes.The mono-bacterium colony of intestinal bacteria MG1655 that picking is fresh, is inoculated in the 50mL LB substratum 250mL shaking flask containing penbritin, 30 DEG C, 200rpm, shaking culture 14h.Getting overnight culture 1mL transfers containing in the 50mL LB substratum of penbritin, and 30 DEG C, 200rpm, is cultured to OD 600 and is about about 0.2, adds 1mL L-arabinose and makes its final concentration be 30mM.Continue 30 DEG C of 200rpm to be cultured to OD 600 and to be about 0.5-0.6.By bacterial cultures as quenching 15min in mixture of ice and water, by glycerine legal system for intestinal bacteria MG1655 Electroporation-competent cells.
2) plasmid pKD46 electricity transformation of E. coli MG1655.0.2cm Bio-Rad electric shock cup and intestinal bacteria MG1655 Electroporation-competent cells are placed on ice.100 μ L competent cells, add 1.5 μ L plasmid pKD46, place 5min on ice.Mixed solution is added the electric shock cup of precooling, ice bath 10min.Use MicroPulser (Bio-Rad) electroporation apparatus, shock parameters is 2.5KV, 200 Ω, 25uF.Add rapidly the LB substratum of 1mL 37 DEG C insulation after electric shock, by mixed solution sucking-off, transfer in the centrifuge tube of 1.5mL, 30 DEG C, 200rpm shaking culture 1h, the LB be coated with containing penbritin is dull and stereotyped.After incubated overnight, picking 10 single bacterium colonies, switching is containing the 5mL LB liquid nutrient medium of penbritin, and the checking of upgrading grain is correct.The bacterium colony that picking one is correct, by its called after LG-01.
(5) the electric transformation of E. coli LG-01 of recombinant fragment 1
1) intestinal bacteria LG-01 Electroporation-competent cells makes.The mono-bacterium colony of intestinal bacteria LG-01 that picking is fresh, is inoculated in the 50mL LB substratum 250mL shaking flask containing penbritin, 30 DEG C, 200rpm, shaking culture 14h.Getting overnight culture 1mL transfers containing in the 50mL LB substratum of penbritin, and 30 DEG C, 200rpm, is cultured to OD 600 and is about about 0.2, adds 1mL L-arabinose and makes its final concentration be 30mM.Continue 30 DEG C of 200rpm to be cultured to OD 600 and to be about 0.5-0.6.By bacterial cultures as quenching 15min in mixture of ice and water, by glycerine legal system for intestinal bacteria LG-01 Electroporation-competent cells.
2) the electric transformation of E. coli LG-01 of recombinant fragment 1.0.2cm Bio-Rad electric shock cup and intestinal bacteria LG-01 Electroporation-competent cells are placed on ice.100 μ L competent cells, add 1.5 μ L recombinant fragments 1, place 5min on ice.Mixed solution is added the electric shock cup of precooling, ice bath 10min.Use MicroPulser (Bio-Rad) electroporation apparatus, shock parameters is 2.5KV, 200 Ω, 25uF.Add rapidly the LB substratum of 1mL 30 DEG C insulation after electric shock, by mixed solution sucking-off, transfer in the centrifuge tube of 1.5mL, 30 DEG C, 200rpm shaking culture 1h, the LB be coated with containing penbritin and paraxin is dull and stereotyped.After incubated overnight, picking 10 single bacterium colonies, switching contains the 5mL LB liquid nutrient medium of penbritin and paraxin, proposes Genomic PCR checking correct (correct bacterium colony genomic amplified fragments size is at about 3000bp).The bacterium colony that picking one is correct, by its called after LG-02.
Checking primer:
Checking-S:5 '-TGAATTTTTCAATATCGCCATAGCT-3 '
Checking-A:5 '-GGCTACTTTCTTCATTGTGGTTCTC-3 '
PCR reaction system:
hS (Premix) 25 μ L, checking-S 0.3 μ L, checking-A 0.3 μ L, intestinal bacteria LG-02 genome 0.4 μ L, ddH
2o 24 μ L.
PCR reaction conditions: 98 DEG C of 10s, 60 DEG C of 15s, 72 DEG C of 3min, circulate 30 times; 72 DEG C of 7min, 4 DEG C of insulations.(6) pcr amplification homologous sequence ldhA-3, ldhA-4 and gene order alsSbudAbdhA
With intestinal bacteria MG1655 genome for template, with primer ldhA-3-S and ldhA-3-A, Takara high-fidelity enzyme
hS (Premix) carries out pcr amplification, the fragment amplified comprises one section of lactic dehydrogenase enzyme coding gene site upstream sequence homology containing the fragment of 996 bases and the 25bp fragment of end and alsSbdhAbdhA front end homology, by its called after ldhA-3.
ldhA-3-S：5’-CGTCAAGGTCGACGTTATTGAAACC-3’
LdhA-3-A:5 '-TTTTAGCCTTTAATTGTCAATAGGT CGTCAAGGTCGACGTTATTGAAACC-3 ' PCR reaction system:
hS (Premix) 25 μ L, primer ldhA-3-S 0.3 μ L, primer ldhA-3-A0.3 μ L, intestinal bacteria MG1655 genome 0.4 μ L, ddH
2o 24 μ L.
PCR reaction conditions: 98 DEG C of 10s, 62 DEG C of 15s, 72 DEG C of 1min, circulate 30 times; 72 DEG C of 7min, 4 DEG C of insulations.
Agarose gel electrophoresis with 0.8% detects PCR primer, reclaims after test kit fragment purification through Takara glue, stand-by in 4 DEG C of storages.
Fragment ldhA-3 sequence verification: purified ldhA-3 fragment is cloned on carrier pEASY-Blunt and carries out sequence verification.Clone's system: the fragment after 1 μ L pEASY-Blunt carrier, 4 μ L purifying, mixes gently; After 25 DEG C of reaction 20min, add in 50 μ L Trans1-T1 competent cells, ice bath 30min, 42 DEG C of heat shock 30s, are placed in 2min on ice immediately.Add rapidly 250 μ L LB substratum, 200rpm, 37 DEG C, cultivate 1h.Get 200 μ L bacterium liquid and be coated with the LB flat board containing penbritin, after incubated overnight, the LB liquid nutrient medium that picking 10 positive bacterium colonies connect containing penbritin is cultivated, and extract positive colony plasmid and send sequence verification, sequencing result is correct.
With intestinal bacteria MG1655 genome for template, with primer ldhA-4-S and ldhA-4-A, Takara high-fidelity enzyme
hS (Premix) carries out pcr amplification, the fragment amplified comprises one section of lactic dehydrogenase enzyme coding gene sites downstream sequence homology containing the fragment of 975 bases and the 22bp fragment of front end and alsSbdhAbdhA terminal homologous, by its called after ldhA-4.
ldhA-4-S：5’-CCATCCTGACGGATGGCCTTTTACCTATTGACAATTAAAGGCTAAAATGC-3’
ldhA-4-A：5’-AAAAGGCCATCCGTCAGGATGG-3’
PCR reaction system:
hS (Premix) 25 μ L, primer ldhA-4-S 0.3 μ L, primer ldhA-4-A0.3 μ L, intestinal bacteria MG1655 genome 0.4 μ L, ddH
2o 24 μ L.
PCR reaction conditions: 98 DEG C of 10s, 62 DEG C of 15s, 72 DEG C of 1min, circulate 30 times; 72 DEG C of 7min, 4 DEG C of insulations.
Agarose gel electrophoresis with 0.8% detects PCR primer, reclaims after test kit fragment purification through Takara glue, stand-by in 4 DEG C of storages.
Fragment ldhA-4 sequence verification: purified ldhA-4 fragment is cloned on carrier pEASY-Blunt and carries out sequence verification.Clone's system: the fragment after 1 μ L pEASY-Blunt carrier, 4 μ L purifying, mixes gently; After 25 DEG C of reaction 20min, add in 50 μ L Trans1-T1 competent cells, ice bath 30min, 42 DEG C of heat shock 30s, are placed in 2min on ice immediately.Add rapidly 250 μ L LB substratum, 200rpm, 37 DEG C, cultivate 1h.Get 200 μ L bacterium liquid and be coated with the LB flat board containing penbritin, after incubated overnight, the LB liquid nutrient medium that picking 10 positive bacterium colonies connect containing penbritin is cultivated, and extract positive colony plasmid and send sequence verification, sequencing result is correct.
With plasmid pEASY-Blunt-alsSbudAbdhA for template, with primer alsSbudAbdhA-S and alsSbdhAbdhA-A, Takara high-fidelity enzyme
hS (Premix) carries out pcr amplification, and the fragment amplified comprises: the 25bp sequence of front end and ldhA1 fragment ends homology, alsSbudAbdhA sequence, the sequence of the 25bp of end and ldhA2 fragment front end homology, and called after alsSbudAbdhA.
alsSbudAbdhA-S:5’-GGTTTCAATAACGTCGACCTTGACGACCTATTGACAATTAAAGGCTAAAATGC-3’
alsSbdhAbdhA-A:
5 '-AGTTAGCATCACGGGTACGCTGATAAAAAGGCCATCCGTCAGGATGG-3 ' PCR reaction system:
hS (Premix) 25 μ L, primer alsSbudAbdhA-S 0.3 μ L, primer alsSbdhAbdhA-A0.3 μ L, intestinal bacteria MG1655 genome 0.4 μ L, ddH
2o 24 μ L.
PCR reaction conditions: 98 DEG C of 10s, 58 DEG C of 15s, 72 DEG C of 3min45s, circulate 30 times; 72 DEG C of 7min, 4 DEG C of insulations.
Agarose gel electrophoresis with 0.8% detects PCR primer, reclaims after test kit fragment purification through Takara glue, stand-by in 4 DEG C of storages.
Fragment alsSbudAbdhA sequence verification: purified alsSbudAbdhA fragment is cloned on carrier pEASY-Blunt and carries out sequence verification.Clone's system: the fragment after 1 μ L pEASY-Blunt carrier, 4 μ L purifying, mixes gently; After 25 DEG C of reaction 20min, add in 50 μ L Trans1-T1 competent cells, ice bath 30min, 42 DEG C of heat shock 30s, are placed in 2min on ice immediately.Add rapidly 250 μ L LB substratum, 200rpm, 37 DEG C, cultivate 1h.Get 200 μ L bacterium liquid and be coated with the LB flat board containing penbritin, after incubated overnight, the LB liquid nutrient medium that picking 10 positive bacterium colonies connect containing penbritin is cultivated, and extract positive colony plasmid and send sequence verification, sequencing result is correct.
(7) Overlap extension PCR is utilized to build recombinant fragment 2
With ldhA-3, alsSbudAbdhA and ldhA-4 three gene for template, with primer ldhA-3-S and ldhA-4-A, Takara high-fidelity enzyme
hS (Premix) merges ldhA-3, alsSbudAbdhA and ldhA-4 three gene fragment.
ldhA-3-S：5’-AGTTCGCTGACTGTAAGTTGTTGCC-3’
ldhA-4-A：5’-TCAGTTCACCATTAGACAGTTTGCC-3’
PCR reaction system:
hS (Premix) 25 μ L, primer ldhA-3-S 0.3 μ L, primer ldhA-4-A0.3 μ L, ldhA-30.6 μ L, alsSbudAbdhA 0.6 μ L, ldhA-40.6 μ L, ddH
2o 22.6 μ L.
PCR condition: 98 DEG C of 10s, 60 DEG C of 15s, 72 DEG C of 5min30s, circulate 30 times; 72 DEG C of 7min, 4 DEG C of insulations.
Agarose gel electrophoresis with 0.8% detects PCR primer, and uses Takara glue to reclaim kits object fragment, called after recombinant fragment 2, and in 4 DEG C of storages, for second time homologous recombination.
Fragment recombinant fragment 2 sequence verification: the fragment of purifying is cloned on carrier pEASY-Blunt.Clone's system: the fragment after 1 μ L pEASY-Blunt carrier, 4 μ L purifying, mixes gently; After 25 DEG C of reaction 20min, add in 50 μ L Trans1-T1 competent cells, ice bath 30min, 42 DEG C of heat shock 30s, are placed in 2min on ice immediately.Add rapidly 250 μ L LB substratum, 200rpm, 37 DEG C, cultivate 1h.Get 200 μ L bacterium liquid and be coated with the LB flat board containing penbritin, after incubated overnight, the LB liquid nutrient medium that picking 10 positive bacterium colonies connect containing penbritin is cultivated, and extract positive colony plasmid and send sequence verification, sequencing result is correct.
(8) the electric transformation of E. coli LG-02 of recombinant fragment 2
1) intestinal bacteria LG-02 Electroporation-competent cells makes.The mono-bacterium colony of intestinal bacteria LG-02 that picking is fresh, is inoculated in the 50mL LB substratum 250mL shaking flask containing penbritin, 30 DEG C, 200rpm, shaking culture 14h.Getting overnight culture 1mL transfers containing in the 50mL LB substratum of penbritin, and 30 DEG C, 200rpm, is cultured to OD 600 and is about about 0.2, adds 1mL L-arabinose and makes its final concentration be 30mM.Continue 30 DEG C of 200rpm to be cultured to OD 600 and to be about 0.5-0.6.By bacterial cultures as quenching 15min in mixture of ice and water, by glycerine legal system for intestinal bacteria LG-02 Electroporation-competent cells.
2) the electric transformation of E. coli LG-02 of recombinant fragment 2.0.2cm Bio-Rad electric shock cup and intestinal bacteria LG-02 Electroporation-competent cells are placed on ice.100 μ L competent cells, add 1.5 μ L recombinant fragments 2, place 5min on ice.Mixed solution is added the electric shock cup of precooling, ice bath 10min.Use MicroPulser (Bio-Rad) electroporation apparatus, shock parameters is 2.5KV, 200 Ω, 25uF.Add rapidly the LB substratum of 1mL 37 DEG C insulation after electric shock, by mixed solution sucking-off, transfer in the centrifuge tube of 1.5mL, 37 DEG C, 200rpm shaking culture 3h, remove plasmid pKD46.Bacterium liquid is transferred to the salt-free LB liquid nutrient medium of 50mL containing 10% sucrose, 37 DEG C, after 200rpm shaking culture 18h, the salt-free LB solid medium of dilution spread 10%, 37 DEG C of 12h.Picking list bacterium colony, proposes Genomic PCR checking correct (correct bacterium colony genomic amplified fragments size is at about 3700bp).The bacterium colony that picking one is correct, by its called after LG-R.
Checking primer:
Checking-S:5 '-TGAATTTTTCAATATCGCCATAGCT-3 '
Checking-A:5 '-GGCTACTTTCTTCATTGTGGTTCTC-3 '
PCR reaction system:
hS (Premix) 25 μ L, checking-S 0.3 μ L, checking-A 0.3 μ L, intestinal bacteria LG-R genome 0.4 μ L, ddH
2o 24 μ L.
PCR reaction conditions: 98 DEG C of 10s, 60 DEG C of 15s, 72 DEG C of 4min, circulate 30 times; 72 DEG C of 7min, 4 DEG C of insulations.
Genetic engineering bacterium-recombination bacillus coli LG-R that embodiment 2 high density fermentation is implemented in profit 1 produces (R, R)-2,3-butanediol bacterial strain: intestinal bacteria MG1655, intestinal bacteria LG-R;
Seed culture medium (g/L): peptone 10; Yeast extract paste 5; NaCl 10.
Fermention medium (g/L): glucose 80; K
2hPO
43H
2o 13.7; KH
2pO
42.0; (NH
4)
2hPO
43.3; (NH
4)
2sO
46.6; MgSO
47H
2o 0.25; FeSO
47H
2o 0.05; ZnSO
47H
2o 0.01; MnSO
4h
2o 0.01; CaCl
20.01; EDTA 0.05.
Fermentation condition:
The preparation of seed liquor: the LB liquid nutrient medium that the strain transfer of glycerine pipe preservation is fresh, 37 DEG C, 200rpm activates 16h; The bacterial classification connecing 1 ring activation lines has corresponding antibiotic LB solid medium, cultivates 12h for 37 DEG C; From picking list bacterium colony fresh plate in the 250mL triangular flask that 50mL seed culture medium is housed, 37 DEG C, cultivate 12h, as first order seed under 200rpm condition; Again by primary seed solution with 5% inoculum size be connected to another 50mL/250mL seed culture medium, 37 DEG C, cultivate 12h, as secondary seed under 200rpm condition.
Ferment tank is cultivated: when utilizing 5L fermentor tank to carry out Batch fermentation, secondary seed solution accessed in fermentation tank culture medium with the inoculum size of 10% (v/v).During cultivation, 5L fermentor tank liquid amount is 3L, and initial pH is 7.0; Air flow and mixing speed are respectively 1.5vvm, 400rpm.Fermentation time is according to specific experiment and determining, and using glucose, depleted or concentration no longer reduces as fermentation termination.
Testing conditions:
Employing high performance liquid chromatography (HPLC) measures the meta-bolites in fermenting process.Japan Shimadzu high performance liquid chromatograph LC-20A, RID-10A differential refraction detector, Aminex HPX-87H chromatographic column, column temperature is 65 DEG C, and moving phase is 0.005M H
2sO
4, flow velocity is 0.6mL/min.
Result:
High-efficient liquid phase chromatogram technique analysis is adopted to measure intestinal bacteria MG1655 and recombination bacillus coli LG-R at fermentor tank stage each product.In original bacteria intestinal bacteria MG1655, yield of acetic acid is up to 23.45g/L, and the acetic acid content in recombination bacillus coli LG-R only has 0.21g/L, and (R, R)-2,3-butanediol content then has 33.7g/L.
Example 3 realizes high density fermentation co-production meso-2, the structure of the genetic engineering bacterium of 3-butyleneglycol
1, increase α-acetolactate synthestase encoding gene, the operon gene fragment alsSbudAbudC of alpha-acetolactate decarboxylase encoding gene and meso-2,3-butanediol dehydrogenation enzyme coding gene
With the genomic dna of K.pneumoniae CICC10011 for template, with primer P15 and P16, Takara high-fidelity enzyme
hS (Premix) carries out pcr amplification, and the fragment amplified comprises: promotor, RBS site, alsS, budA, budC and terminator.
P15：5’-ACCTATTGACAATTAAAGGCTAAAATGCTATAATTCCACAAATCGGAGGATATACATATGAATCATTCTGCTGAATG-3’
P16：5’-AAAAGGCCATCCGTCAGGATGGCCTTCTTTAGTTAAATACCATCCCGC-3’
PCR reaction system:
hS (Premix) 25 μ L, primer P150.3 μ L, primer P160.3 μ L, K.pneumoniae CICC10011 genome 0.4 μ L, ddH
2o 24 μ L.
PCR reaction conditions: 98 DEG C of 10s, 58 DEG C of 15s, 72 DEG C of 3min30s, circulate 30 times; 72 DEG C of 7min, 4 DEG C of insulations.
Agarose gel electrophoresis with 0.8% detects PCR primer, and uses Takara glue to reclaim kits object fragment, then the fragment of purifying is cloned on carrier pEASY-Blunt.Clone's system: the fragment after 1 μ L pEASY-Blunt carrier, 4 μ L purifying, mixes gently; After 25 DEG C of reaction 20min, add in 50 μ L Trans1-T1 competent cells, ice bath 30min, 42 DEG C of heat shock 30s, are placed in 2min on ice immediately.Add rapidly 250 μ L LB substratum, 200rpm, 37 DEG C, cultivate 1h.Get 200 μ L bacterium liquid and be coated with the LB flat board containing penbritin, after incubated overnight, the LB liquid nutrient medium that picking 10 positive bacterium colonies connect containing penbritin is cultivated, extract positive colony plasmid and send sequence verification, alsSbudAbudC sequencing result is as SEQ ID NO:10, the gene order in the RBS site of alsSbudAbudC upstream as shown in SEQ ID NO:11, by the recombinant plasmid called after pEASY-Blunt-alsSbudAbudC obtained.
2, alsSbudAbdhA is incorporated in escherichia coli chromosome group by two step methods of homologous recombination
(1) pcr amplification homologous sequence ldhA-5, ldhA-6
With intestinal bacteria MG1655 genome for template, with primer ldhA-5-S and ldhA-5-A, Takara high-fidelity enzyme
hS (Premix) carries out pcr amplification, and the fragment amplified comprises one section of lactic dehydrogenase enzyme coding gene site upstream sequence homology containing the fragment ends of 996 bases and the 25bp fragment of alsSbdhAbdhA front end homology, by its called after ldhA-5.
ldhA-5-S：5’-AGTTCGCTGACTGTAAGTTGTTGCC-3’
ldhA-5-A：5’-AACCTTTCAGAATGCGCAGCATCGCCGTCAAGGTCGACGTTATTGAAACC-3’
PCR reaction system:
hS (Premix) 25 μ L, primer ldhA-5-S 0.3 μ L, primer ldhA-5-A0.3 μ L, intestinal bacteria MG1655 genome 0.4 μ L, ddH
2o 24 μ L.
PCR reaction conditions: 98 DEG C of 10s, 62 DEG C of 15s, 72 DEG C of 30s, circulate 30 times; 72 DEG C of 7min, 4 DEG C of insulations.
Agarose gel electrophoresis with 0.8% detects PCR primer, reclaims after test kit fragment purification through Takara glue, stand-by in 4 DEG C of storages.
Fragment ldhA-5 sequence verification: purified ldhA-5 fragment is cloned on carrier pEASY-Blunt and carries out sequence verification.Clone's system: the fragment after 1 μ L pEASY-Blunt carrier, 4 μ L purifying, mixes gently; After 25 DEG C of reaction 20min, add in 50 μ L Trans1-T1 competent cells, ice bath 30min, 42 DEG C of heat shock 30s, are placed in 2min on ice immediately.Add rapidly 250 μ L LB substratum, 200rpm, 37 DEG C, cultivate 1h.Get 200 μ L bacterium liquid and be coated with the LB flat board containing penbritin, after incubated overnight, the LB liquid nutrient medium that picking 10 positive bacterium colonies connect containing penbritin is cultivated, and extract positive colony plasmid and send sequence verification, sequencing result is correct.
With intestinal bacteria MG1655 genome for template, with primer ldhA-6-S and ldhA-6-A, Takara high-fidelity enzyme
hS (Premix) carries out pcr amplification, and the fragment amplified comprises one section of lactic dehydrogenase enzyme coding gene sites downstream sequence homology containing the fragment of 975 bases and the 20bp fragment of front end and Cm-sacB terminal homologous, is named ldhA-6.LdhA-6-S:5 '-GCGGGATGGTATTTAACTAATATCAGCGTACCCGTGATGCTAACT-3 ' ldhA-6-A:5 '-TCAGTTCACCATTAGACAGTTTGCC-3 ' PCR reaction system:
hS (Premix) 25 μ L, primer ldhA-6-S 0.3 μ L, primer ldhA-6-A 0.3 μ L, intestinal bacteria MG1655 genome 0.4 μ L, ddH
2o 24 μ L.
PCR reaction conditions: 98 DEG C of 10s, 62 DEG C of 15s, 72 DEG C of 30s, circulate 30 times; 72 DEG C of 7min, 4 DEG C of insulations.
Agarose gel electrophoresis with 0.8% detects PCR primer, reclaims after test kit fragment purification through Takara glue, stand-by in 4 DEG C of storages.
Fragment ldhA-6 sequence verification: purified ldhA-6 fragment is cloned on carrier pEASY-Blunt and carries out sequence verification.Clone's system: the fragment after 1 μ L pEASY-Blunt carrier, 4 μ L purifying, mixes gently; After 25 DEG C of reaction 20min, add in 50 μ L Trans1-T1 competent cells, ice bath 30min, 42 DEG C of heat shock 30s, are placed in 2min on ice immediately.Add rapidly 250 μ L LB substratum, 200rpm, 37 DEG C, cultivate 1h.Get 200 μ L bacterium liquid and be coated with the LB flat board containing penbritin, after incubated overnight, the LB liquid nutrient medium that picking 10 positive bacterium colonies connect containing penbritin is cultivated, and extract positive colony plasmid and send sequence verification, sequencing result is correct.
(2) amplification gene sequence alsSbudAbudC
With plasmid pEASY-Blunt-alsSbudAbudC for template, with primer alsSbudAbudC-S and alsSbudAbudC-A, Takara high-fidelity enzyme
hS (Premix) carries out pcr amplification, the fragment amplified comprises: the 25bp sequence of front end and ldhA-5 fragment ends homology, alsSbudAbudC sequence, the sequence of the 25bp of end and ldhA-6 fragment front end homology, and called after alsSbudAbudC.
alsSbudAbudC-S：5’-GGTTTCAATAACGTCGACCTTGACG GGAGGATATACATATGAATCATTCT-3’
alsSbudAbudC-A：5’-AGTTAGCATCACGGGTACGCTGATATTAGTTAAATACCATCCCGC-3’
PCR reaction system:
hS (Premix) 25 μ L, primer alsSbudAbudC-S 0.3 μ L, primer alsSbudAbudC-A 0.3 μ L, intestinal bacteria MG1655 genome 0.4 μ L, ddH
2o 24 μ L.
PCR reaction conditions: 98 DEG C of 10s, 58 DEG C of 15s, 72 DEG C of 3min40s, circulate 30 times; 72 DEG C of 7min, 4 DEG C of insulations.
Agarose gel electrophoresis with 0.8% detects PCR primer, reclaims after test kit fragment purification through Takara glue, stand-by in 4 DEG C of storages.
Fragment alsSbudAbudC sequence verification: purified alsSbudAbudC fragment is cloned on carrier pEASY-Blunt and carries out sequence verification.Clone's system: the fragment after 1 μ L pEASY-Blunt carrier, 4 μ L purifying, mixes gently; After 25 DEG C of reaction 20min, add in 50 μ L Trans1-T1 competent cells, ice bath 30min, 42 DEG C of heat shock 30s, are placed in 2min on ice immediately.Add rapidly 250 μ L LB substratum, 200rpm, 37 DEG C, cultivate 1h.Get 200 μ L bacterium liquid and be coated with the LB flat board containing penbritin, after incubated overnight, the LB liquid nutrient medium that picking 10 positive bacterium colonies connect containing penbritin is cultivated, and extract positive colony plasmid and send sequence verification, sequencing result is correct.
(3) overlap extension pcr is utilized to build recombinant fragment 3
With ldhA-5, alsSbudAbudC and ldhA-6 three gene for template, with primer ldhA-5-S and ldhA-6-A, Takara high-fidelity enzyme
hS (Premix) merges ldhA-5, alsSbudAbudC and ldhA-6 three gene fragment, and called after recombinant fragment 3.
ldhA-5-S：5’-AGTTCGCTGACTGTAAGTTGTTGCC-3’
ldhA-6-A：5’-TCAGTTCACCATTAGACAGTTTGCC-3’
PCR reaction system:
hS (Premix) 25 μ L, primer ldhA-5-S 0.3 μ L, primer ldhA-6-A0.3 μ L, ldhA-50.6 μ L, alsSbudAbudC 0.6 μ L, ldhA-60.6 μ L, ddH
2o 22.6 μ L.
PCR condition: 98 DEG C of 10s, 60 DEG C of 15s, 72 DEG C of 4min30s, circulate 30 times; 72 DEG C of 7min, 4 DEG C of insulations.
Agarose gel electrophoresis with 0.8% detects PCR primer, and uses Takara glue to reclaim kits object fragment, called after recombinant fragment 3, and in 4 DEG C of storages, for first time homologous recombination.
Fragment recombinant fragment 3 sequence verification: the fragment of purifying is cloned on carrier pEASY-Blunt.Clone's system: the fragment after 1 μ L pEASY-Blunt carrier, 4 μ L purifying, mixes gently; After 25 DEG C of reaction 20min, add in 50 μ LTrans1-T1 competent cells, ice bath 30min, 42 DEG C of heat shock 30s, are placed in 2min on ice immediately.Add rapidly 250 μ L LB substratum, 200rpm, 30 DEG C, cultivate 1h.Get 200 μ L bacterium liquid and be coated with the LB flat board containing penbritin, after incubated overnight, the LB liquid nutrient medium that picking 10 positive bacterium colonies connect containing penbritin is cultivated, and extract positive colony plasmid and send sequence verification, sequencing result is correct.
(4) the electric transformation of E. coli LG-02 of recombinant fragment 3
1) intestinal bacteria LG-02 Electroporation-competent cells makes.The mono-bacterium colony of intestinal bacteria LG-02 that picking is fresh, is inoculated in the 50mL LB substratum 250mL shaking flask containing penbritin, 30 DEG C, 200rpm, shaking culture 14h.Getting overnight culture 1mL transfers containing in the 50mL LB substratum of penbritin, and 30 DEG C, 200rpm, is cultured to OD 600 and is about about 0.2, adds 1mL L-arabinose and makes its final concentration be 30mM.Continue 30 DEG C of 200rpm to be cultured to OD 600 and to be about 0.5-0.6.By bacterial cultures as quenching 15min in mixture of ice and water, by glycerine legal system for intestinal bacteria LG-02 Electroporation-competent cells.
2) the electric transformation of E. coli LG-02 of recombinant fragment 3.0.2cm Bio-Rad electric shock cup and intestinal bacteria LG-02 Electroporation-competent cells are placed on ice.100 μ L competent cells, add 1.5 μ L recombinant fragments 2, place 5min on ice.Mixed solution is added the electric shock cup of precooling, ice bath 10min.Use MicroPulser (Bio-Rad) electroporation apparatus, shock parameters is 2.5KV, 200 Ω, 25uF.Add rapidly the LB substratum of 1mL 37 DEG C insulation after electric shock, by mixed solution sucking-off, transfer in the centrifuge tube of 1.5mL, 37 DEG C, 200rpm shaking culture 3h, remove plasmid pKD46.Bacterium liquid is transferred to the salt-free LB liquid nutrient medium of 50mL containing 10% sucrose, 37 DEG C, after 200rpm shaking culture 18h, the salt-free LB solid medium of dilution spread 10%, 37 DEG C of 12h.Picking list bacterium colony, proposes Genomic PCR checking correct (correct bacterium colony genomic amplified fragments size is at about 3400bp).The bacterium colony that picking one is correct, by its called after LG-MS.
Checking primer:
Checking-S:5 '-TGAATTTTTCAATATCGCCATAGCT-3 '
Checking-A:5 '-GGCTACTTTCTTCATTGTGGTTCTC-3 '
PCR reaction system:
hS (Premix) 25 μ L, checking-S 0.3 μ L, checking-A 0.3 μ L, intestinal bacteria LG-MS genome 0.4 μ L, ddH
2o 24 μ L.
PCR reaction conditions: 98 DEG C of 10s, 60 DEG C of 15s, 72 DEG C of 4min, circulate 30 times; 72 DEG C of 7min, 4 DEG C of insulations.
The embodiment 4 high density fermentation genetic engineering bacterium-recombination bacillus coli LG-MS implemented in profit 3 produces meso-2,3-butyleneglycol bacterial strain: intestinal bacteria MG1655, intestinal bacteria LG-MS;
Seed culture medium (g/L): peptone 10; Yeast extract paste 5; NaCl 10.
Fermention medium (g/L): glucose 80, K
2hPO
43H
2o 13.7, KH
2pO
42.0, (NH
4)
2hPO
43.3, (NH
4)
2sO
46.6, MgSO
47H
2o 0.25, FeSO
47H
2o 0.05, ZnSO
47H
2o 0.01, MnSO
4h
2o 0.01, CaCl
20.01, EDTA 0.05.
Fermentation condition:
The preparation of seed liquor: the LB liquid nutrient medium that the strain transfer of glycerine pipe preservation is fresh, 37 DEG C, 200rpm activates 16h; The bacterial classification connecing 1 ring activation lines has corresponding antibiotic LB solid medium, cultivates 12h for 37 DEG C; From picking list bacterium colony fresh plate in the 250mL triangular flask that 50mL seed culture medium is housed, 37 DEG C, cultivate 12h, as first order seed under 200rpm condition; Again by primary seed solution with 5% inoculum size be connected to another 50mL/250mL seed culture medium, 37 DEG C, cultivate 12h, as secondary seed under 200rpm condition.
Ferment tank is cultivated: when utilizing 5L fermentor tank to carry out Batch fermentation, secondary seed solution accessed in fermentation tank culture medium with the inoculum size of 10% (v/v).During cultivation, 5L fermentor tank liquid amount is 3L, and initial pH is 7.0; Air flow and mixing speed are respectively 1.5vvm, 400rpm.Fermentation time is according to specific experiment and determining, and using glucose, depleted or concentration no longer reduces as fermentation termination.
Testing conditions:
Employing high performance liquid chromatography (HPLC) measures the meta-bolites in fermenting process.Japan Shimadzu high performance liquid chromatograph LC-20A, RID-10A differential refraction detector, Aminex HPX-87H chromatographic column, column temperature is 65 DEG C, and moving phase is 0.005M H
2sO
4, flow velocity is 0.6mL/min.
Result:
High-efficient liquid phase chromatogram technique analysis is adopted to measure intestinal bacteria MG1655 and recombination bacillus coli LG-R at fermentor tank stage each product.In original bacteria intestinal bacteria MG1655, yield of acetic acid is up to 23.55g/L, and the acetic acid content in recombination bacillus coli LG-R only has 0.53g/L, and (meso)-2,3-butanediol content then reaches 26.5g/L.
Example 5: the structure realizing the genetic engineering bacterium of high density fermentation coproduction PHB and (R, R)-2,3-butanediol
(document Spiekermann P is recorded in plasmid pBHR68, Rehm B H A, Kalscheuer R, Baumeister D, Steinb ü chel A.A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds [J] .Archives of microbiology, 1999, 171 (2): 73-80.) be template, PHB operon for synthesizing gene phbCAB is amplified with primer 5 '-TACGAGCTC (SacI) AAGGAGGATGGCGACCGGCAAACG-3 ' and primer 5'-CGCGGATCC (BamHI) CGGCAGGTCAGCCCATAT-3 ', after SacI and BamHI enzyme cuts process, be inserted between plasmid pTrcAlperO1 (buying in Jiangsu Tian Kai Bioisystech Co., Ltd) promotor multicloning sites downstream, obtain plasmid pTrcAlperO1-phbCAB.
Prepare intestinal bacteria MG1655 and intestinal bacteria LG-R Electroporation-competent cells respectively, by plasmid pTrcAlperO1-phbCAB respectively electricity be converted into intestinal bacteria MG1655 and intestinal bacteria LG-R.Electricity method for transformation: 0.2cm Bio-Rad electric shock cup and Electroporation-competent cells are placed on ice.100 μ L competent cells, add 1.5 μ L plasmid pTrcAlperO1-phbCAB, place 5min on ice.Mixed solution is added the electric shock cup of precooling, ice bath 10min.Use MicroPulser (Bio-Rad) electroporation apparatus, shock parameters is 2.5KV, 200 Ω, 25uF.Add rapidly the LB substratum of 1mL 37 DEG C insulation after electric shock, by mixed solution sucking-off, transfer in the centrifuge tube of 1.5mL, 37 DEG C, 200rpm shaking culture 1h, the LB be coated with containing penbritin is dull and stereotyped.After incubated overnight, picking 10 single bacterium colonies, switching is containing the 5mL LB liquid nutrient medium of penbritin, the screening of upgrading grain, sequence verification, phbCAB gene order, as shown in SEQ ID NO:12, obtains recombinant bacterial strain intestinal bacteria MG1655/pTrcAlperO1-phbCAB and intestinal bacteria LG-R/pTrcAlperO1-phbCAB.
Genetic engineering bacterium coproduction PHB in embodiment 6 high density fermentation embodiment 5 and (R, R)-2,3-butanediol
Bacterial strain: original bacteria intestinal bacteria MG1655/pTrcAlperO1-phbCAB and recombinant bacterial strain intestinal bacteria LG-R/pTrcAlperO1-phbCAB:
Seed culture medium (g/L): peptone 10; Yeast extract paste 5; NaCl 10; Penbritin 50mg/mL;
Fermention medium (g/L): glucose 80; KH
2pO
42.0; Na
2hPO
41.0; (NH
4)
2hPO
41.7; (NH
4)
2sO
44; MgSO
47H
2o 0.25; FeSO
47H
2o 0.05; ZnSO
47H
2o 0.001; CoCl
26H
2o 0.2; H
3bO
30.3; MnSO
4h
2o 0.001; CaCl
20.01; CuSO
45H
2o 0.01; NiCl6H
2o 0.02; Penbritin 50mg/mL.Fermentation condition:
Seed culture: the LB liquid nutrient medium that the strain transfer of glycerine pipe preservation is fresh, 37 DEG C, 200rpm activates 16h; The bacterial classification connecing 1 ring activation lines has corresponding antibiotic LB solid medium, cultivates 12h for 37 DEG C; From picking list bacterium colony fresh plate in the 250mL triangular flask that 50mL seed culture medium is housed, 37 DEG C, cultivate 12h, as first order seed under 200rpm condition; Again by primary seed solution with 5% inoculum size be connected to another 50mL/250mL seed culture medium, 37 DEG C, cultivate 12h, as secondary seed under 200rpm condition.
Fermentation culture:
Original bacteria: by secondary seed solution with the seed culture medium of inoculum size access 300mL/1000mL of 5%, 37 DEG C, cultivate 12h under 200rpm condition, be then transferred to be equipped with in 2.7L substratum 5L automatic fermenter with the inoculum size of 10%.During cultivation, the pH in thalli growth stage remains on 7.0, regulates with the ammoniacal liquor of 20%; During thalline accumulation PHB, pH can remain on 6.8, regulates with 10M KOH.Personal monitoring's glucose and sulphur ammonium concentration, during thalli growth, make glucose concn >1%, ammonium concentration >0.2%, and adopt 50% glucose solution and 25% ammonium sulfate solution to regulate when deficiency.Fermentation culture 72h, samples at interval of 4h.
Recombinant bacterial strain: by secondary seed solution with the seed culture medium of inoculum size access 300mL/1000mL of 5%, 37 DEG C, cultivate 12h under 200rpm condition, be then transferred to be equipped with in 2.7L substratum 5L automatic fermenter with the inoculum size of 10%.During cultivation, the pH of thalline starting stage controls 7.0, and during thalline accumulation PHB, pH can remain on 6.8.Personal monitoring's glucose and sulphur ammonium concentration, during thalli growth, make glucose concn >1%, ammonium concentration >0.2%, and adopt 50% glucose solution and 25% ammonium sulfate solution to regulate when deficiency.Fermentation culture 72h, samples at interval of 4h.
1. detection method:
(1) acetic acid and (R, R)-2,3-butanediol detection method: Japanese Shimadzu high performance liquid chromatograph LC-20A, RID-10A differential refraction detector, Aminex HPX-87H chromatographic column.Column temperature is 65 DEG C, and moving phase is 0.005M H
2sO
4, flow velocity is 0.6mL/min.
(2) PHB detection method: get the centrifugal 2min of fermented sample 12000rpm, collecting precipitation cell is with after distilled water wash 2 times, and the centrifugal 10min collecting cell of 6000rpm, dries and claim its dry weight.In the thalline of drying, add 150 μ L 98% sulfuric acid, after boiling water bath 60min, utilize 20% ammoniacal liquor to adjust pH to 2.5, through the membrane filtration of 0.22um, then utilize HPLC (high performance liquid chromatography) to detect.This Shimadzu high performance liquid chromatograph LC-20A, test column (C18, BioRadLabs); The formic acid of 1% makes moving phase; UV-detector (ultraviolet wavelength is 210nm).
(3) cell concentration measures: after the dilution of the suitable multiple of fermentation liquor, with ultraviolet spectrophotometer at λ=600nm place survey light absorption value, and itself and the product of extension rate and the cell concentration (OD600) of fermented liquid.
(4) dry cell weight (DCW) measures: get 30mL fermented liquid, collected by centrifugation, supernatant discarded.Thalline dries to constant weight in 105 DEG C, measures and is dry cell weight.
(5) glucose concentration determination: get the fermented liquid 1mL of specified phase in 1.5mL centrifuge tube, under room temperature, the centrifugal 2min of 12000rpm, get 100 μ L supernatants to 10mL volumetric flask, 10mL is settled to WAHAHA water, then utilize the glucose concn in biosensor assay fermented liquid, the unit recording numerical value is g/L.
2. result
Measure the cell density of original bacteria and recombination bacillus coli engineering bacteria respectively, the highest cell density of original bacteria is 82.5g/L, and the highest cell density of recombination bacillus coli engineering bacteria reaches 116.7g/L.Measure acetic acid and the accumulation volume of PHB in original bacteria and recombination bacillus coli engineering mycetocyte respectively.In original bacteria, yield of acetic acid is up to 20.95g/L, and in recombinant bacterium, yield of acetic acid only has 0.54g/L; In original bacteria, PHB reaches 34.5% of dry cell weight, and 40.9%, the PHB output that PHB reaches dry cell weight in recombinant bacterium adds 18.5%.Further, (R, R)-2,3-butanediol output of final coproduction reaches 27.5g/L.
Example 7 realizes the structure of the genetic engineering bacterium of high density fermentation coproduction Human-like Collagen and (R, R)-2,3-butanediol
With plasmid pMDHLC (buying in Jiangsu Tian Kai Bioisystech Co., Ltd) for template, Human-like Collagen synthetic gene HLC is amplified with primer 5 '-TACGAGCTC (SacI) ATGCTCCAGGGCACCTGCTCCGTGC-3 ' and primer 5 '-CGCGGATCC (BamHI) TTAAGGGTCTTGCGAGGTCATTCTG-3 ', it is after SacI and BamHI enzyme cuts process, be inserted between plasmid pTrcAlperO1 (buying in Jiangsu Tian Kai Bioisystech Co., Ltd) promotor multicloning sites downstream, obtain plasmid pTrcAlperO1-HLC.
Prepare intestinal bacteria MG1655 and intestinal bacteria LG-R Electroporation-competent cells respectively, by plasmid pTrcAlperO1-HLC respectively electricity be converted into intestinal bacteria MG1655 and intestinal bacteria LG-R.Electricity method for transformation: 0.2cm Bio-Rad electric shock cup and Electroporation-competent cells are placed on ice.100 μ L competent cells, add 1.5 μ L plasmid pTrcAlperO1-HLC, place 5min on ice.Mixed solution is added the electric shock cup of precooling, ice bath 10min.Use MicroPulser (Bio-Rad) electroporation apparatus, shock parameters is 2.5KV, 200 Ω, 25uF.Add rapidly the LB substratum of 1mL 37 DEG C insulation after electric shock, by mixed solution sucking-off, transfer in the centrifuge tube of 1.5mL, 37 DEG C, 200rpm shaking culture 1h, the LB be coated with containing penbritin is dull and stereotyped.After incubated overnight, picking 10 single bacterium colonies, switching is containing the 5mL LB liquid nutrient medium of penbritin, the screening of upgrading grain, sequence verification, HLC gene order, as shown in SEQ ID NO:13, obtains recombinant bacterial strain intestinal bacteria MG1655/pTrcAlperO1-HLC and intestinal bacteria LG-R/pTrcAlperO1-HLC.
Genetic engineering bacterium coproduction Human-like Collagen in embodiment 8 high density fermentation embodiment 7 and (R, R)-2,3-butanediol
Bacterial strain: original bacteria intestinal bacteria MG1655/pTrcAlperO1-HLC and recombinant bacterial strain intestinal bacteria LG-R/pTrcAlperO1-HLC.
Seed culture medium (g/L): peptone 10; Yeast extract paste 5; NaCl 10; Penbritin 50mg/mL.
Fermention medium (g/L): glucose 60; Yeast extract paste 60; K
2hPO
43H
2o 5.8; NaH
2pO
43.6; (NH
4)
2hPO
43.3; (NH
4)
2sO
44.2; MgSO
47H
2o 2.0; FeSO
47H
2o 0.6; ZnSO
47H
2o 0.2; MnSO
4h
2o 0.2; CaCl
20.3; EDTA 0.8; Penbritin 50mg/mL.
1. fermentation coproduction
Fermentation condition:
Seed culture: the LB liquid nutrient medium that the strain transfer of glycerine pipe preservation is fresh, 37 DEG C, 200rpm activates 16h; The bacterial classification connecing 1 ring activation lines has corresponding antibiotic LB solid medium, cultivates 12h for 37 DEG C; From picking list bacterium colony fresh plate in the 250mL triangular flask that 50mL seed culture medium is housed, 37 DEG C, cultivate 12h, as first order seed under 200rpm condition; Again by primary seed solution with 5% inoculum size be connected to another 50mL/250mL seed culture medium, 37 DEG C, cultivate 12h, as secondary seed under 200rpm condition.
Fermentation culture:
Original bacteria: secondary seed solution is accessed in fermentation tank culture medium with the inoculum size of 10% (v/v).During cultivation, 5L fermentor tank liquid amount is 3L, and culture temperature is 37 DEG C, and pH is regulated about 6.8 automatically by the ammoniacal liquor of 25%.When glucose is about to exhaust in tank, start flow feeding substratum, by changing mixing speed, air flow quantity and mending sugared speed, maintain specific growth rate (μ) at 0.1 ~ 0.2h
-1left and right, DO is 10% ~ 40% saturation ratio.After mixing speed, air flow quantity reach the upper limit, if dissolved oxygen rises fast, improve and mend sugared speed; If dissolved oxygen is lower than set(ting)value, reduces and mend sugared speed.Meanwhile, be aided with pH feedback, namely when sugared concentration is too low, cell utilizes nitrogenous source as carbon source, thus releases NH
4+, make fermented liquid pH increase, now improve and mend sugared speed, maintain normal pH.
Recombinant bacterial strain: secondary seed solution is accessed in fermentation tank culture medium with the inoculum size of 10% (v/v).During cultivation, 5L fermentor tank liquid amount is 3L, and culture temperature is 37 DEG C, and initial pH controls about 6.8.When glucose is about to exhaust in tank, start flow feeding substratum, by changing mixing speed, air flow quantity and mending sugared speed, maintain specific growth rate (μ) at 0.1 ~ 0.2h
-1left and right, DO is 10% ~ 40% saturation ratio.After mixing speed, air flow quantity reach the upper limit, if dissolved oxygen rises fast, improve and mend sugared speed; If dissolved oxygen is lower than set(ting)value, reduces and mend sugared speed.Meanwhile, be aided with pH feedback, namely when sugared concentration is too low, cell utilizes nitrogenous source as carbon source, thus releases NH
4+, make fermented liquid pH increase, now improve and mend sugared speed, maintain normal pH.
2. testing conditions
(1) acetic acid and (R, R)-2,3-butanediol detection method: Japanese Shimadzu high performance liquid chromatograph LC-20A, RID-10A differential refraction detector, Aminex HPX-87H chromatographic column.Column temperature is 65 DEG C, and moving phase is 0.005M H
2sO
4, flow velocity is 0.6mL/min.
(2) Human-like Collagen detection method: get 10mL fermented liquid, centrifugal, abandoning supernatant, after precipitation distilled water is washed 3 times, resuspension, ultrasonic disruption, measures Human-like Collagen content in broken supernatant liquor by light PA method.
(3) cell concentration measures: after the dilution of the suitable multiple of fermentation liquor, with ultraviolet spectrophotometer at λ=600nm place survey light absorption value, and itself and the product of extension rate and the cell concentration (OD600) of fermented liquid.
(4) dry cell weight (DCW) measures: get 30mL fermented liquid, collected by centrifugation, supernatant discarded.Thalline dries to constant weight in 105 DEG C, measures and is dry cell weight.
(5) glucose concentration determination: get the fermented liquid 1mL of specified phase in 1.5mL centrifuge tube, under room temperature, the centrifugal 2min of 12000rpm, get 100 μ L supernatants to 10mL volumetric flask, 10mL is settled to WAHAHA water, then utilize the glucose concn in biosensor assay fermented liquid, the unit recording numerical value is g/L.
3. result
Measure the cell density of original bacteria and recombination bacillus coli engineering bacteria respectively, the highest cell density of original bacteria is 60.8g/L, and the highest cell density of recombination bacillus coli engineering bacteria reaches 91.5g/L.Mensuration acetic acid and collagen protein are at original bacteria and the intracellular accumulation volume of recombination bacillus coli engineering bacteria respectively.In original bacteria, yield of acetic acid is up to 22.93g/L, and in recombinant bacterium, yield of acetic acid only has 0.69g/L; In original bacteria, collagen production is 11.5g/L, and in recombinant bacterium, collagen production reaches 16.9g/L, and collagen production adds 47.0%.Further, (R, R)-2,3-butanediol output of final coproduction reaches 20.3g/L.
Example 9 realizes the structure of high density fermentation coproduction TreP and (R, R)-2,3-butanediol genetic engineering bacterium
(document He G has been recorded in thermophilic sporangium T.fusca WSH03-11, Huo G, Liu L, Zhu Y, Du G, Chen J.Enhanced cutinase production of Thermobifida fusca by a two-stage batch and fed-batch cultivation strategy [J] .Biotechnology and Bioprocess Engineering, 2009, 14 (1): 46-51.) be template, trehalose synthesize enzyme gene TreS is amplified with primer 5 '-TACGAGCTC (SacI) ACCACACAGCCGGCTCCT-3 ' and primer 5 '-CGCGGATCC (BamHI) CAGGACCGCTGGGTCGGGTCG-3 ', after SacI and BamHI enzyme cuts process, be inserted between plasmid pTrcAlperO1 (buying in Jiangsu Tian Kai Bioisystech Co., Ltd) promotor multicloning sites downstream, obtain plasmid pTrcAlperO1-TreS.
Prepare intestinal bacteria MG1655 and intestinal bacteria LG-R Electroporation-competent cells respectively, by plasmid pTrcAlperO1-TreS respectively electricity be converted into intestinal bacteria MG1655 and intestinal bacteria LG-R.Electricity method for transformation: 0.2cm Bio-Rad electric shock cup and Electroporation-competent cells are placed on ice.100 μ L competent cells, add 1.5 μ L plasmid pTrcAlperO1-TreS, place 5min on ice.Mixed solution is added the electric shock cup of precooling, ice bath 10min.Use MicroPulser (Bio-Rad) electroporation apparatus, shock parameters is 2.5KV, 200 Ω, 25uF.Add rapidly the LB substratum of 1mL 37 DEG C insulation after electric shock, by mixed solution sucking-off, transfer in the centrifuge tube of 1.5mL, 37 DEG C, 200rpm shaking culture 1h, the LB be coated with containing penbritin is dull and stereotyped.After incubated overnight, picking 10 single bacterium colonies, switching is containing the 5mL LB liquid nutrient medium of penbritin, the screening of upgrading grain, sequence verification, TreS gene order, as shown in SEQ ID NO:14, obtains recombinant bacterial strain intestinal bacteria MG1655/pTrcAlperO1-TreS and intestinal bacteria LG-R/pTrcAlperO1-TreS.
The genetic engineering bacterium coproduction TreP of embodiment 10 high density fermentation embodiment 9 and (R, R)-2,3-butanediol
Bacterial strain: original bacteria intestinal bacteria MG1655/pTrcAlperO1-TreS and recombinant bacterial strain intestinal bacteria LG-R/pTrcAlperO1-TreS:
Seed culture medium (g/L): peptone 10; Yeast powder 5; NaCl 10; Penbritin 50mg/mL;
Fermention medium (g/L): glucose 70; KH
2pO
41.0; Na
2hPO
43.8; (NH
4)
2hPO
42.2; (NH
4)
2sO
43.6; MgSO
47H
2o 0.3; FeSO
47H
2o 0.1; ZnSO
47H
2o 0.001; CoCl
26H
2o 0.1; H
3bO
30.1; MnSO
4h
2o 0.01; CaCl
20.01; CuSO
45H
2o 0.01; NiCl6H
2o 0.01; Penbritin 50mg/mL.Fermentation condition:
The preparation of seed liquor: the LB liquid nutrient medium that the strain transfer of glycerine pipe preservation is fresh, 37 DEG C, 200rpm activates 16h; The bacterial classification connecing 1 ring activation lines has corresponding antibiotic LB solid medium, cultivates 12h for 37 DEG C; From picking list bacterium colony fresh plate in the 250mL triangular flask that 50mL seed culture medium is housed, 37 DEG C, cultivate 12h, as first order seed under 200rpm condition; Again by primary seed solution with 5% inoculum size be connected to another 50mL/250mL seed culture medium, 37 DEG C, cultivate 12h, as secondary seed under 200rpm condition.
High density fermentation:
Original bacteria: secondary seed solution is accessed in fermentation tank culture medium with the inoculum size of 10% (v/v).During cultivation, 5L fermentor tank liquid amount is 3L, leavening temperature 37 DEG C, makes dissolved oxygen percentage ratio keep being not less than 20% by regulating mixing speed and air velocity.PH is controlled about 7.0 by Feeding ammonia water.After batch culture 8h, carry out feeding culture.With constant flow velocity (stream rate of acceleration is for 80mL/h), stream adds 80% glucose.
Recombinant bacterial strain: secondary seed solution is accessed in fermentation tank culture medium with the inoculum size of 10% (v/v).During cultivation, 5L fermentor tank liquid amount is 3L, and leavening temperature 37 DEG C, initial pH controls about 7.0.Dissolved oxygen percentage ratio is made to keep being not less than 20% by regulating mixing speed and air velocity.After batch culture 8h, carry out feeding culture.With constant flow velocity (stream rate of acceleration is for 80mL/h), stream adds 80% glucose.
1. testing conditions
(1) acetic acid and (R, R)-2,3-butanediol detection method: Japanese Shimadzu high performance liquid chromatograph LC-20A, RID-10A differential refraction detector, Aminex HPX-87H chromatographic column.Column temperature is 65 DEG C, and moving phase is 0.005M H
2sO
4, flow velocity is 0.6mL/min.
(2) TreP detection method: Japanese Shimadzu high performance liquid chromatograph LC-20A, RID-10A differential refraction detector, mobile phase ratio: acetonitrile: water=65:35, column temperature: 30 DEG C, flow velocity: 1.0mL/min.Working time 15min.
Transformation efficiency measures: maltose reaction solution dilutes 25 times and crosses sample introduction after films.
Transformation efficiency calculates:
Wherein: for reacting the peak area of rear trehalose;
For reacting the peak area of front maltose.
(3) cell concentration measures: after the dilution of the suitable multiple of fermentation liquor, with ultraviolet spectrophotometer at λ=600nm place survey light absorption value, and itself and the product of extension rate and the cell concentration (OD600) of fermented liquid.
(4) dry cell weight (DCW) measures: get 30mL fermented liquid, collected by centrifugation, supernatant discarded.Thalline dries to constant weight in 105 DEG C, measures and is dry cell weight.
(5) glucose concentration determination: get the fermented liquid 1mL of specified phase in 1.5mL centrifuge tube, under room temperature, the centrifugal 2min of 12000rpm, get 100 μ L supernatants to 10mL volumetric flask, 10mL is settled to WAHAHA water, then utilize the glucose concn in biosensor assay fermented liquid, the unit recording numerical value is g/L.
2. result
Measure the cell density of original bacteria and recombination bacillus coli engineering bacteria respectively, the highest cell density of original bacteria is 52.3g/L, and the highest cell density of recombination bacillus coli engineering bacteria reaches 71.6g/L.Mensuration acetic acid and TreP are at original bacteria and the intracellular accumulation volume of recombination bacillus coli engineering bacteria respectively.In original bacteria, yield of acetic acid is up to 22.11g/L, and in recombinant bacterium, yield of acetic acid only has 0.66g/L; In original bacteria, TreP enzyme is lived as 2720nmol/min, and in recombinant bacterium, TreP enzyme is lived as 3680nmol/min, and TreP enzyme is lived and improve 35.3%.Further, (R, R)-2,3-butanediol output of final coproduction reaches 22.5g/L.
Embodiment 11 realizes high density fermentation coproduction PHB and meso-2, the structure of the genetic engineering bacterium of 3-butyleneglycol
(document Spiekermann P has been recorded in plasmid pBHR68, Rehm B H A, Kalscheuer R, Baumeister D, Steinb ü chel A.A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds [J] .Archives of microbiology, 1999, 171 (2): 73-80.) be template, PHB operon for synthesizing gene phbCAB is amplified with primer 5 '-TACGAGCTC (SacI) AAGGAGGATGGCGACCGGCAAACG-3 ' and primer 5 '-CGCGGATCC (BamHI) CGGCAGGTCAGCCCATAT-3 ', after SacI and BamHI enzyme cuts process, be inserted between plasmid pTrcAlperO1 (buying in Jiangsu Tian Kai Bioisystech Co., Ltd) promotor multicloning sites downstream, obtain plasmid pTrcAlperO1-phbCAB.
Prepare intestinal bacteria MG1655 and intestinal bacteria LG-MS Electroporation-competent cells respectively, by plasmid pTrcAlperO1-phbCAB respectively electricity be converted into intestinal bacteria MG1655 and intestinal bacteria LG-MS.Electricity method for transformation: 0.2cm Bio-Rad electric shock cup and Electroporation-competent cells are placed on ice.100 μ L competent cells, add 1.5 μ L plasmid pTrcAlperO1-phbCAB, place 5min on ice.Mixed solution is added the electric shock cup of precooling, ice bath 10min.Use MicroPulser (Bio-Rad) electroporation apparatus, shock parameters is 2.5KV, 200 Ω, 25uF.Add rapidly the LB substratum of 1mL 37 DEG C insulation after electric shock, by mixed solution sucking-off, transfer in the centrifuge tube of 1.5mL, 37 DEG C, 200rpm shaking culture 1h, the LB be coated with containing penbritin is dull and stereotyped.After incubated overnight, picking 10 single bacterium colonies, switching is containing the 5mL LB liquid nutrient medium of penbritin, and upgrading grain screens, sequence verification.Empirical tests is correct, obtains recombinant bacterial strain intestinal bacteria MG1655/pTrcAlperO1-phbCAB and intestinal bacteria LG-MS/pTrcAlperO1-phbCAB.
Example 12 high density fermentation embodiment 11 genetic engineering bacterium coproduction PHB and meso-2,3-butyleneglycol
Bacterial classification: original bacteria intestinal bacteria MG1655/pTrcAlperO1-phbCAB and recombinant bacterial strain intestinal bacteria LG-MS/pTrcAlperO1-phbCAB:
Seed culture medium (g/L): peptone 10; Yeast extract paste 5; NaCl 10; Penbritin 50mg/mL;
Fermention medium (g/L): glucose 80; KH
2pO
42.0; Na
2hPO
41.0; NH
4)
2hPO
41.7; (NH
4)
2sO
44; MgSO
47H
2o 0.25; FeSO
47H
2o 0.05; ZnSO
47H
2o 0.001; CoCl
26H
2o 0.2; H
3bO
30.3; MnSO
4h
2o 0.001; CaCl
20.01; CuSO
45H
2o 0.01; NiCl6H
2o 0.02; Penbritin 50mg/mL.
1. fermentation coproduction
Seed culture: the LB liquid nutrient medium that the strain transfer of glycerine pipe preservation is fresh, 37 DEG C, 200rpm activates 16h; The bacterial classification connecing 1 ring activation lines has corresponding antibiotic LB solid medium, cultivates 12h for 37 DEG C; From picking list bacterium colony fresh plate in the 250mL triangular flask that 50mL seed culture medium is housed, 37 DEG C, cultivate 12h, as first order seed under 200rpm condition; Again by primary seed solution with 5% inoculum size be connected to another 50mL/250mL seed culture medium, 37 DEG C, cultivate 12h, as secondary seed under 200rpm condition.
Fermentation culture:
Original bacteria: by secondary seed solution with the seed culture medium of inoculum size access 300mL/1000mL of 5%, 37 DEG C, cultivate 12h under 200rpm condition, be then transferred to be equipped with in 2.7L substratum 5L automatic fermenter with the inoculum size of 10%.During cultivation, the pH in thalli growth stage remains on 7.0, regulates with the ammoniacal liquor of 20%; During thalline accumulation PHB, pH can remain on 6.8, regulates with 10M KOH.Personal monitoring's glucose and sulphur ammonium concentration, during thalli growth, make glucose concn >1%, ammonium concentration >0.2%, and adopt 50% glucose solution and 25% ammonium sulfate solution to regulate when deficiency.Fermentation culture 72h, samples at interval of 4h.
Recombinant bacterial strain: by secondary seed solution with the seed culture medium of inoculum size access 300mL/1000mL of 5%, 37 DEG C, cultivate 12h under 200rpm condition, be then transferred to be equipped with in 2.7L substratum 5L automatic fermenter with the inoculum size of 10%.During cultivation, the thalline starting stage, pH controlled 7.0, and during thalline accumulation PHB, pH can remain on 6.8.Personal monitoring's glucose and sulphur ammonium concentration, during thalli growth, make glucose concn >1%, ammonium concentration >0.2%, and be regulate with 50% glucose solution and 25% ammonium sulfate solution in deficiency, fermentation culture 72h, samples at interval of 4h.
2. testing conditions
(1) acetic acid and meso-2,3-butyleneglycol detection method: Japanese Shimadzu high performance liquid chromatograph LC-20A, RID-10A differential refraction detector, Aminex HPX-87H chromatographic column.Column temperature is 65 DEG C, and moving phase is 0.005M H
2sO
4, flow velocity is 0.6mL/min.
(2) PHA detection method: get the centrifugal 2min of fermented sample 12000rpm, collecting precipitation cell is with after distilled water wash 2 times, and the centrifugal 10min collecting cell of 6000rpm, dries and claim its dry weight.In the thalline of drying, add 150 μ L 98% sulfuric acid, after boiling water bath 60min, utilize 20% ammoniacal liquor to adjust pH to 2.5, through the membrane filtration of 0.22um, then utilize HPLC (high performance liquid chromatography) to detect.This Shimadzu high performance liquid chromatograph LC-20A, test column (C18, BioRadLabs); The formic acid of 1% makes moving phase; UV-detector (ultraviolet waves is opened for 210nm)
(3) cell concentration measures: after the dilution of the suitable multiple of fermentation liquor, with ultraviolet spectrophotometer at λ=600nm place survey light absorption value, and itself and the product of extension rate and the cell concentration (OD600) of fermented liquid.
(4) dry cell weight (DCW) measures: get 30mL fermented liquid, collected by centrifugation, supernatant discarded.Thalline dries to constant weight in 105 DEG C, measures and is dry cell weight.
(5) glucose concentration determination: get the fermented liquid 1mL of specified phase in 1.5mL centrifuge tube, under room temperature, the centrifugal 2min of 12000rpm, get 100 μ L supernatants to 10mL volumetric flask, 10mL is settled to WAHAHA water, then utilize the glucose concn in biosensor assay fermented liquid, the unit recording numerical value is g/L.
3. result
Measure the cell density of original bacteria and recombination bacillus coli engineering bacteria respectively, the highest cell density of original bacteria is 81.4g/L, and the highest cell density of recombination bacillus coli engineering bacteria reaches 114.9g/L.Measure acetic acid and the accumulation volume of PHB in original bacteria and recombination bacillus coli engineering mycetocyte respectively.In original bacteria, yield of acetic acid is up to 21.16g/L, and in recombinant bacterium, yield of acetic acid only has 0.79g/L, and yield of acetic acid is extremely low; In original bacteria, PHB reaches 33.5% of dry cell weight, and 39.6%, the PHB output that PHB reaches dry cell weight in recombinant bacterium adds 18.2%.Further, the meso-2 of final coproduction, 3-butyleneglycol output reaches 24.1g/L.
Example 13 realizes the structure of the genetic engineering bacterium of high density fermentation coproduction Human-like Collagen and meso-2,3-butyleneglycol
With plasmid pMDHLC (buying in Jiangsu Tian Kai Bioisystech Co., Ltd) for template, Human-like Collagen synthetic gene HLC is amplified with primer 5 '-TACGAGCTC (SacI) ATGCTCCAGGGCACCTGCTCCGTGC-3 ' and primer 5 '-CGCGGATCC (BamHI) TTAAGGGTCTTGCGAGGTCATTCTG-3 ', after SacI and BamHI enzyme cuts process, be inserted between plasmid pTrcAlperO1 (buying in Jiangsu Tian Kai Bioisystech Co., Ltd) promotor multicloning sites downstream, obtain plasmid pTrcAlperO1-HLC.
Prepare intestinal bacteria MG1655 and intestinal bacteria LG-MS Electroporation-competent cells respectively, by plasmid pTrcAlperO1-HLC respectively electricity be converted into intestinal bacteria MG1655 and intestinal bacteria LG-MS.Electricity method for transformation: 0.2cm Bio-Rad electric shock cup and Electroporation-competent cells are placed on ice.100 μ L competent cells, add 1.5 μ L plasmid pTrcAlperO1-HLC, place 5min on ice.Mixed solution is added the electric shock cup of precooling, ice bath 10min.Use MicroPulser (Bio-Rad) electroporation apparatus, shock parameters is 2.5KV, 200 Ω, 25uF.Add rapidly the LB substratum of 1mL 37 DEG C insulation after electric shock, by mixed solution sucking-off, transfer in the centrifuge tube of 1.5mL, 37 DEG C, 200rpm shaking culture 1h, the LB be coated with containing penbritin is dull and stereotyped.After incubated overnight, picking 10 single bacterium colonies, switching is containing the 5mL LB liquid nutrient medium of penbritin, and upgrading grain screens, sequence verification.Empirical tests is correct, obtains recombinant bacterial strain intestinal bacteria MG1655/pTrcAlperO1-HLC and intestinal bacteria LG-MS/pTrcAlperO1-HLC.
Example 14 high density fermentation embodiment 13 engineering bacteria fermentation coproduction Human-like Collagen and meso-2,3-butyleneglycol
Bacterial strain: original bacteria intestinal bacteria MG1655/pTrcAlperO1-HLC and the equal intestinal bacteria LG-MS/pTrcAlperO1-HLC of restructuring:
Seed culture medium (g/L): peptone 10; Yeast extract paste 5; NaCl 10; Penbritin 50mg/mL.
Fermention medium (g/L): glucose 60; Yeast extract paste 60; K
2hPO
43H
2o 5.8; NaH
2pO
43.6; (NH
4)
2hPO
43.3; (NH
4)
2sO
44.2; MgSO
47H
2o 2.0; FeSO
47H
2o 0.6; ZnSO
47H
2o 0.2; MnSO
4h
2o 0.2; CaCl
20.3; EDTA 0.8; Penbritin 50mg/mL.
1. fermentation coproduction
Fermentation condition:
Seed culture: the LB liquid nutrient medium that the strain transfer of glycerine pipe preservation is fresh, 37 DEG C, 200rpm activates 16h; The bacterial classification connecing 1 ring activation lines has corresponding antibiotic LB solid medium, cultivates 12h for 37 DEG C; From picking list bacterium colony fresh plate in the 250mL triangular flask that 50mL seed culture medium is housed, 37 DEG C, cultivate 12h, as first order seed under 200rpm condition; Again by primary seed solution with 5% inoculum size be connected to another 50mL/250mL seed culture medium, 37 DEG C, cultivate 12h, as secondary seed under 200rpm condition.
Fermentation culture:
Original bacteria: secondary seed solution is accessed in fermentation tank culture medium with the inoculum size of 10% (v/v).During cultivation, 5L fermentor tank liquid amount is 3L, and culture temperature is 37 DEG C, and pH is regulated about 6.8 automatically by the ammoniacal liquor of 25%.When glucose is about to exhaust in tank, start flow feeding substratum, by changing mixing speed, air flow quantity and mending sugared speed, maintain specific growth rate (μ) at 0.1 ~ 0.2h
-1left and right, DO is 10% ~ 40% saturation ratio.After mixing speed, air flow quantity reach the upper limit, if dissolved oxygen rises fast, improve and mend sugared speed; If dissolved oxygen is lower than set(ting)value, reduces and mend sugared speed.Meanwhile, be aided with pH feedback, namely when sugared concentration is too low, cell utilizes nitrogenous source as carbon source, thus releases NH
4+, make fermented liquid pH increase, now improve and mend sugared speed, maintain normal pH.
Recombinant bacterial strain: secondary seed solution is accessed in fermentation tank culture medium with the inoculum size of 10% (v/v).During cultivation, 5L fermentor tank liquid amount is 3L, and culture temperature is 37 DEG C, and initial pH controls about 6.8.When glucose is about to exhaust in tank, start flow feeding substratum, by changing mixing speed, air flow quantity and mending sugared speed, maintain specific growth rate (μ) at 0.1 ~ 0.2h
-1left and right, DO is 10% ~ 40% saturation ratio.After mixing speed, air flow quantity reach the upper limit, if dissolved oxygen rises fast, improve and mend sugared speed; If dissolved oxygen is lower than set(ting)value, reduces and mend sugared speed.Meanwhile, be aided with pH feedback, namely when sugared concentration is too low, cell utilizes nitrogenous source as carbon source, thus releases NH
4+, make fermented liquid pH increase, now improve and mend sugared speed, maintain normal pH.
2. testing conditions
(1) acetic acid and meso-2,3-butyleneglycol detection method: Japanese Shimadzu high performance liquid chromatograph LC-20A, RID-10A differential refraction detector, Aminex HPX-87H chromatographic column.Column temperature is 65 DEG C, and moving phase is 0.005M H
2sO
4, flow velocity is 0.6mL/min.
(2) Human-like Collagen detection method: get 10mL fermented liquid, centrifugal, abandoning supernatant, after precipitation distilled water is washed 3 times, resuspension, ultrasonic disruption, measures Human-like Collagen content in broken supernatant liquor by light PA method.
(3) cell concentration measures: after the dilution of the suitable multiple of fermentation liquor, with ultraviolet spectrophotometer at λ=600nm place survey light absorption value, and itself and the product of extension rate and the cell concentration (OD600) of fermented liquid.
(4) dry cell weight (DCW) measures: get 30mL fermented liquid, collected by centrifugation, supernatant discarded.Thalline dries to constant weight in 105 DEG C, measures and is dry cell weight.
(5) glucose concentration determination: get the fermented liquid 1mL of specified phase in 1.5mL centrifuge tube, under room temperature, the centrifugal 2min of 12000rpm, get 100 μ L supernatants to 10mL volumetric flask, 10mL is settled to WAHAHA water, then utilize the glucose concn in biosensor assay fermented liquid, the unit recording numerical value is g/L.
3. result
Measure the cell density of original bacteria and recombination bacillus coli engineering bacteria respectively, the highest cell density of original bacteria is 61.1g/L, and the highest cell density of recombination bacillus coli engineering bacteria reaches 88.9g/L.Mensuration acetic acid and collagen protein are at original bacteria and the intracellular accumulation volume of recombination bacillus coli engineering bacteria respectively.In original bacteria, yield of acetic acid is up to 23.36g/L, and in recombinant bacterium, yield of acetic acid only has 0.96g/L; In original bacteria, collagen production is 11.1g/L, and in recombinant bacterium, collagen production reaches 16.3g/L, and collagen production adds 46.9%.Further, (meso)-2,3-butanediol output of final coproduction reaches 17.2g/L.
Example 15 realizes the structure of the genetic engineering bacterium of high density fermentation coproduction TreP and meso-2,3-butyleneglycol
(document He G has been recorded in thermophilic sporangium T.fusca WSH03-11, Huo G, Liu L, Zhu Y, Du G, Chen J.Enhanced cutinase production of Thermobifida fusca by a two-stage batch and fed-batch cultivation strategy [J] .Biotechnology and Bioprocess Engineering, 2009, 14 (1): 46-51.) be template, trehalose synthesize enzyme gene TreS is amplified with primer 5 '-TACGAGCTC (SacI) ACCACACAGCCGGCTCCT-3 ' and primer 5 '-CGCGGATCC (BamHI) CAGGACCGCTGGGTCGGGTCG-3 ', after SacI and BamHI enzyme cuts process, be inserted between plasmid pTrcAlperO1 (deriving from Jiangsu Tian Kai Bioisystech Co., Ltd) promotor multicloning sites downstream, obtain plasmid pTrcAlperO1-TreS.
Prepare intestinal bacteria MG1655 and intestinal bacteria LG-MS Electroporation-competent cells respectively, by plasmid pTrcAlperO1-TreS respectively electricity be converted into intestinal bacteria MG1655 and intestinal bacteria LG-MS.Electricity method for transformation: 0.2cm Bio-Rad electric shock cup and Electroporation-competent cells are placed on ice.100 μ L competent cells, add 1.5 μ L plasmid pTrcAlperO1-TreS, place 5min on ice.Mixed solution is added the electric shock cup of precooling, ice bath 10min.Use MicroPulser (Bio-Rad) electroporation apparatus, shock parameters is 2.5KV, 200 Ω, 25uF.Add rapidly the LB substratum of 1mL 37 DEG C insulation after electric shock, by mixed solution sucking-off, transfer in the centrifuge tube of 1.5mL, 37 DEG C, 200rpm shaking culture 1h, the LB be coated with containing penbritin is dull and stereotyped.After incubated overnight, picking 10 single bacterium colonies, switching is containing the 5mL LB liquid nutrient medium of penbritin, and upgrading grain screens, sequence verification.Empirical tests is correct, obtains recombinant bacterial strain intestinal bacteria MG1655/pTrcAlperO1-TreS and intestinal bacteria LG-MS/pTrcAlperO1-TreS.
The genetic engineering bacterium coproduction TreP of example 16 high density fermentation embodiment 15 and meso-2,3-butyleneglycol
Bacterial strain: original bacteria intestinal bacteria MG1655/pTrcAlperO1-TreS and recombinant bacterial strain intestinal bacteria LG-MS/pTrcAlperO1-TreS:
Seed culture medium (g/L): peptone 10; Yeast powder 5; NaCl 10; Penbritin 50mg/mL.
Fermention medium (g/L): glucose 70; KH
2pO
42.0; Na
2hPO
43.8; (NH
4)
2hPO
41.7; (NH
4)
2sO
44; MgSO
47H
2o 0.25; FeSO
47H
2o 0.05; ZnSO
47H
2o 0.001; CoCl
26H
2o 0.2; H
3bO
30.3; MnSO
4h
2o 0.001; CaCl
20.01; CuSO
45H
2o 0.01; NiCl6H
2o 0.02; Penbritin 50mg/L.
1. fermentation coproduction
The preparation of seed liquor: the LB liquid nutrient medium that the strain transfer of glycerine pipe preservation is fresh, 37 DEG C, 200rpm activates 16h; The bacterial classification connecing 1 ring activation lines has corresponding antibiotic LB solid medium, cultivates 12h for 37 DEG C; From picking list bacterium colony fresh plate in the 250mL triangular flask that 50mL seed culture medium is housed, 37 DEG C, cultivate 12h, as first order seed under 200rpm condition; Again by primary seed solution with 5% inoculum size be connected to another 50mL/250mL seed culture medium, 37 DEG C, cultivate 12h, as secondary seed under 200rpm condition.
High density fermentation:
Original bacteria: secondary seed solution is accessed in fermentation tank culture medium with the inoculum size of 10% (v/v).During cultivation, 5L fermentor tank liquid amount is 3L, leavening temperature 37 DEG C, makes dissolved oxygen percentage ratio keep being not less than 20% by regulating mixing speed and air velocity.PH is controlled about 7.0 by Feeding ammonia water.After batch culture 8h, carry out feeding culture.80% glucose is added with constant flow velocity (stream rate of acceleration is for 80mL/h) stream.
Recombinant bacterial strain: secondary seed solution is accessed in fermentation tank culture medium with the inoculum size of 10% (v/v).During cultivation, 5L fermentor tank liquid amount is 3L, and leavening temperature 37 DEG C, initial pH controls about 7.0.Dissolved oxygen percentage ratio is made to keep being not less than 20% by regulating mixing speed and air velocity.After batch culture 8h, carry out feeding culture.80% glucose is added with constant flow velocity (stream rate of acceleration is for 80mL/h) stream.
2. testing conditions
(1) acetic acid and meso-2,3-butyleneglycol detection method: Japanese Shimadzu high performance liquid chromatograph LC-20A, RID-10A differential refraction detector, Aminex HPX-87H chromatographic column.Column temperature is 65 DEG C, and moving phase is 0.005M H
2sO
4, flow velocity is 0.6mL/min.
(2) TreP detection method: Japanese Shimadzu high performance liquid chromatograph LC-20A, RID-10A differential refraction detector, mobile phase ratio: acetonitrile: water=65:35, column temperature: 30 DEG C, flow velocity: 1.0mL/min.Working time 15min.
Transformation efficiency measures: maltose reaction solution dilutes 25 times and crosses sample introduction after films.
Transformation efficiency calculates:
Wherein: for reacting the peak area of rear trehalose;
For reacting the peak area of front maltose.
(3) cell concentration measures: after the dilution of the suitable multiple of fermentation liquor, with ultraviolet spectrophotometer at λ=600nm place survey light absorption value, and itself and the product of extension rate and the cell concentration (OD600) of fermented liquid.
(4) dry cell weight (DCW) measures: get 30mL fermented liquid, collected by centrifugation, supernatant discarded.Thalline dries to constant weight in 105 DEG C, measures and is dry cell weight.
(5) glucose concentration determination: get the fermented liquid 1mL of specified phase in 1.5mL centrifuge tube, under room temperature, the centrifugal 2min of 12000rpm, get 100 μ L supernatants to 10mL volumetric flask, 10mL is settled to WAHAHA water, then utilize the glucose concn in biosensor assay fermented liquid, the unit recording numerical value is g/L.
3. result
Measure the cell density of original bacteria and recombination bacillus coli engineering bacteria respectively, the highest cell density of original bacteria is 51.4g/L, and the highest cell density of recombination bacillus coli engineering bacteria reaches 70.9g/L.Mensuration acetic acid and TreP are at original bacteria and the intracellular accumulation volume of recombination bacillus coli engineering bacteria respectively.In original bacteria, yield of acetic acid is up to 22.38g/L, and in recombinant bacterium, yield of acetic acid only has 0.82g/L, produces acetic acid hardly; In original bacteria, TreP enzyme is lived as 2680nmol/min, and in recombinant bacterium, TreP enzyme is lived as 3650nmol/min, and TreP enzyme is lived and improve 36.1%.Further, the meso-2 of final coproduction, 3-butyleneglycol output reaches 20.9g/L.
Claims (16)
1. one kind realizes high density fermentation co-production 2, the genetic engineering bacterium of 3-butyleneglycol, it is characterized in that, by 2, three key enzyme encoding genes: α-acetolactate synthestase encoding gene in 3-butyleneglycol route of synthesis, alpha-acetolactate decarboxylase encoding gene and 2,3-butanediol dehydrogenase coding genes are incorporated on escherichia coli chromosome, the recombinant bacterium built.
2. the genetic engineering bacterium realizing high density fermentation co-production 2,3-butanediol according to claim 1, is characterized in that, described 2,3-butanediol dehydrogenase coding genes is (R, R)-2,3-butanediol dehydrogenation enzyme coding gene or meso-2,3-butanediol dehydrogenation enzyme coding gene.
3. the construction process realizing the genetic engineering bacterium of high density fermentation co-production 2,3-butanediol according to right 1 or 2, comprises the steps:
1) to increase respectively α-acetolactate synthestase encoding gene and alpha-acetolactate decarboxylase encoding gene from Klebsiella pneumonia, from subtilis amplification (R, R)-2, 3-butanediol dehydrogenation enzyme coding gene, recycle overlap extension pcr by above-mentioned three gene fragments according to α-acetolactate synthestase encoding gene, alpha-acetolactate decarboxylase encoding gene, (R, R)-2, the sequential concatenation assembling of 3-butanediol dehydrogenation enzyme coding gene obtains α-acetolactate synthestase encoding gene, alpha-acetolactate decarboxylase encoding gene and (R, R)-2, three gene fusion fragments of 3-butanediol dehydrogenation enzyme coding gene or directly from Klebsiella pneumonia amplification α-acetolactate synthestase encoding gene, alpha-acetolactate decarboxylase encoding gene and meso-2, the operon gene fragment of 3-butanediol dehydrogenation enzyme coding gene.
2) by two step methods of homologous recombination by step 1) in α-acetolactate synthestase encoding gene, α-acetylactis decarboxylation codase gene and (R, R)-2, the operon gene fragment of 3-butanediol dehydrogenation enzyme coding gene three gene fusion fragment or α-acetolactate synthestase encoding gene, alpha-acetolactate decarboxylase encoding gene and meso-2,3-butanediol dehydrogenation enzyme coding gene is incorporated in escherichia coli chromosome group.
4. method according to claim 3, it is characterized in that, to the α-acetolactate synthestase encoding gene obtained that increases, alpha-acetolactate decarboxylase encoding gene, (R, R) before-2,3-butanediol dehydrogenase coding genes splicing, first at α-acetolactate synthestase encoding gene, alpha-acetolactate decarboxylase encoding gene, ribosome bind site is introduced respectively in (R, R)-2,3-butanediol dehydrogenase coding genes upstream.
5. method according to claim 3, it is characterized in that, by α-acetolactate synthestase encoding gene, alpha-acetolactate decarboxylase encoding gene and meso-2, before the operon gene fragment of 3-butanediol dehydrogenation enzyme coding gene is incorporated in escherichia coli chromosome group, first at α-acetolactate synthestase encoding gene, ribosome bind site is introduced in the operon gene fragment upstream of alpha-acetolactate decarboxylase encoding gene and meso-2,3-butanediol dehydrogenation enzyme coding gene.
6. the method according to the arbitrary claim of claim 3-5, is characterized in that two described step methods of homologous recombination concrete steps are:
1) first the escherichia coli chromosome of pcr amplification knocked in by the technology of Overlap extension PCR one section of fragment containing 500-1000 base that one section of fragment containing 500-1000 base of site upstream sequence homology, resistance screening marker gene, sucrose levanase gene and escherichia coli chromosome knock in sites downstream sequence homology to merge, obtain recombinant fragment 1, recombinant fragment 1 is incorporated into escherichia coli chromosome and knocks on site by the method re-using λ-Red homologous recombination;
2) first by the method for over-lap PCR, the escherichia coli chromosome of pcr amplification is knocked in one section of fragment containing 500-1000 base of site upstream sequence homology, α-acetolactate synthestase encoding gene, alpha-acetolactate decarboxylase encoding gene and (R, R)-2, 3-butanediol dehydrogenation enzyme coding gene three gene fusion fragment or α-acetolactate synthestase encoding gene, alpha-acetolactate decarboxylase encoding gene and meso-2, the operon gene fragment of 3-butanediol dehydrogenation enzyme coding gene, the one section of fragment containing 500-1000 base knocking in sites downstream sequence homology with escherichia coli chromosome merges, obtain recombinant fragment 2 or recombinant fragment 3, recombinant fragment 2 or recombinant fragment 3 are replaced above-mentioned steps 1 by the method re-using λ-Red homologous recombination) middle recombinant fragment 1.
7. method according to claim 6, is characterized in that described resistance screening marker gene is the one in chloramphenicol resistance gene, kalamycin resistance gene, gentamicin resistance gene, tetracycline resistance gene.
8. method according to claim 6, it is characterized in that described escherichia coli chromosome knocks in site is lactic dehydrogenase enzyme coding gene site or 16S rDNA gene locus.
9. realize a method for high density fermentation coproduction 2,3-butanediol, it is characterized in that, the genetic engineering bacterium that high density fermentation is cultivated described in claim 1 or 2 claim produces 2,3-butanediol.
10. according to claim 9ly realize high density fermentation coproduction 2,3-butanediol method, it is characterized in that: the seed culture medium (g/L) that high density fermentation is cultivated consists of: peptone 10, yeast extract paste 5, NaCl 10; Fermention medium (g/L) consists of: glucose 60-100; Yeast extract paste 0-40; K
2hPO
43H
2o 10.0-15.0; KH
2pO
42.0-6.0; (NH
4)
2hPO
42.0-6.0; (NH
4)
2sO
42.0-8.0; MgSO
47H
2o 0.1-0.3; FeSO
47H
2o 0.01-0.1; ZnSO
47H
2o 0.01-0.1; MnSO
4h
2o 0.01-0.1; CaCl
20.01-0.1; EDTA 0.01-0.1.
11. 1 kinds realize high density fermentation co-production poly-hydroxy fatty acid and 2, the genetic engineering bacterium of 3-butyleneglycol, it is characterized in that, in the genetic engineering bacterium described in claim 1 or 2, introduce exogenous plasmid, described exogenous plasmid is with the genes involved of poly-hydroxy fatty acid route of synthesis.
12. genetic engineering bacteriums realizing high density fermentation co-production poly-hydroxy fatty acid and 2,3-butanediol according to claim 11, is characterized in that, described poly-hydroxy fatty acid is poly butyric ester or PHBV.
13. 1 kinds of methods realizing high density fermentation coproduction poly-hydroxy fatty acid and 2,3-butanediol, is characterized in that, the genetic engineering bacterium that high density fermentation is cultivated described in claim 12 or 13 produces poly-hydroxy fatty acid and 2,3-butanediol.
14. 1 kinds realize high density fermentation co-production functional protein and (R, the genetic engineering bacterium of R)-2,3-butanediol, is characterized in that, in the genetic engineering bacterium described in claim 1 or 2, introduce exogenous plasmid, described exogenous plasmid is with the genes involved of functional protein route of synthesis.
15. one according to claim 14 realize high density fermentation co-production functional protein and (R, R)-2, the genetic engineering bacterium of 3-butyleneglycol 2,3-butanediol, is characterized in that described functional protein is proteolytic enzyme, Interferon, rabbit, Regular Insulin or Human-like Collagen.
16. 1 kinds of methods realizing high density fermentation coproduction functional protein and 2,3-butanediol, is characterized in that, the genetic engineering bacterium that high density fermentation is cultivated described in claims 14 or 15 produces functional protein and 2,3-butanediol.
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105505849A (en) * | 2016-01-22 | 2016-04-20 | 南京工业大学 | Genetically engineered bacterium for co-generating butanol and 2,3-butanediol and construction method and application thereof |
| CN105647953A (en) * | 2016-03-18 | 2016-06-08 | 黑龙江大学 | Construction method of klebsiella oxytoca gene engineering strains for producing 2,3-butanediol with high yield and fermentation method of klebsiella oxytoca gene engineering strains |
| CN105647954A (en) * | 2016-03-18 | 2016-06-08 | 黑龙江大学 | Construction method of klebsiella oxytoca gene engineering strains for producing 2,3-butanediol with high yield and fermentation method of klebsiella oxytoca gene engineering strains |
| CN105695499A (en) * | 2016-03-18 | 2016-06-22 | 黑龙江大学 | Construction method and fermentation method of klebsiella oxytoca gene engineering strain with BudAB gene overexpression |
| CN107058365A (en) * | 2017-03-03 | 2017-08-18 | 南京工业大学 | Isodynamic enzyme catalyzes and synthesizes genetic engineering bacterium and its construction method and the application of 2,3 butanediols altogether |
| CN107129959A (en) * | 2017-06-28 | 2017-09-05 | 广西科学院 | Produce the construction method and its application of (R) 3-hydroxy-2-butanone engineering strain |
| CN107177620A (en) * | 2017-06-28 | 2017-09-19 | 南宁中诺生物工程有限责任公司 | A kind of method that utilization cheap raw material produces Tetramethylpyrazine |
| CN107201375A (en) * | 2017-06-28 | 2017-09-26 | 南宁邦尔克生物技术有限责任公司 | Produce the construction method and its application of (R, R) 2,3 butanediol engineering strain |
| CN107201374A (en) * | 2017-06-28 | 2017-09-26 | 广西科学院 | The construction method of the butanediol high-yielding engineering bacterial strains of optical voidness meso 2,3 and application |
| CN107636151A (en) * | 2015-05-22 | 2018-01-26 | 杜邦营养生物科学有限公司 | Acetolactate decarboxylase |
| CN108570440A (en) * | 2018-03-23 | 2018-09-25 | 山东大学深圳研究院 | One plant of recombination bacillus coli and its application in producing optical voidness meso-2,3- butanediols |
| CN114075526A (en) * | 2021-11-22 | 2022-02-22 | 齐鲁工业大学 | Genetic engineering bacterium for efficiently producing (2S,3S) -2, 3-butanediol and construction method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101565685A (en) * | 2009-01-07 | 2009-10-28 | 山东大学 | Gene recombination bacterium and application thereof in preparing chiral pure acetoin and 2,3-butanediol |
| CN103243064A (en) * | 2013-05-28 | 2013-08-14 | 山东大学 | Escherichia coli engineered strain and application in succinic acid production through aerobic-microaerobic-anaerobic full-stage fermentation of Escherichia coli engineered strain |
| CN103805551A (en) * | 2014-02-14 | 2014-05-21 | 山东大学 | Genetically engineered bacterium for producing meso-2, 3-butanediol and application of genetically engineered bacterium |
| CN104046586A (en) * | 2014-07-02 | 2014-09-17 | 山东大学 | Genetically engineered bacteria and application of genetically engineered bacteria to production of (2R, 3R)-2,3-butanediol |
-
2014
- 2014-10-20 CN CN201410559814.9A patent/CN104560848B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101565685A (en) * | 2009-01-07 | 2009-10-28 | 山东大学 | Gene recombination bacterium and application thereof in preparing chiral pure acetoin and 2,3-butanediol |
| CN103243064A (en) * | 2013-05-28 | 2013-08-14 | 山东大学 | Escherichia coli engineered strain and application in succinic acid production through aerobic-microaerobic-anaerobic full-stage fermentation of Escherichia coli engineered strain |
| CN103805551A (en) * | 2014-02-14 | 2014-05-21 | 山东大学 | Genetically engineered bacterium for producing meso-2, 3-butanediol and application of genetically engineered bacterium |
| CN104046586A (en) * | 2014-07-02 | 2014-09-17 | 山东大学 | Genetically engineered bacteria and application of genetically engineered bacteria to production of (2R, 3R)-2,3-butanediol |
Non-Patent Citations (2)
| Title |
|---|
| 李正军等: "生产聚羟基脂肪酸酯的微生物细胞工厂", 《生物工程学报》 * |
| 沈梦秋等: "生物制造不同立体构型2, 3-丁二醇：合成机理与实现方法", 《催化学报》 * |
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