CN103555646A - Gene engineering bacterium for coexpressing L-arabinose isomerase gene and mannitose-6-phosphoric acid isomerase - Google Patents
Gene engineering bacterium for coexpressing L-arabinose isomerase gene and mannitose-6-phosphoric acid isomerase Download PDFInfo
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Abstract
The invention discloses a gene engineering bacterium for coexpressing L-arabinose isomerase gene and mannitose-6-phosphoric acid isomerase. The strain comprises nucleotide sequences disclosed as SEQ ID NO:1 and SEQ ID NO:2. The gene engineering bacterium can be used for producing L-ribose by catalyzing L-arabinose, and has the advantages of one-step conversion, simple technique and high conversion rate; by using the gene engineering bacterium, the conversion rate of L-ribose is up to 25-50%; and thus, the gene engineering bacterium has favorable industrialization prospects.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to construction of genetic engineering and the application thereof of a kind of coexpression L-arabinose isomerase gene and mannose-6-phosphate isomerase gene.
Background technology
Ribose (Ribose, C
5h
10o
5) be a kind of typical five-carbon sugar, be the moiety of various Yeast Nucleic Acid (RNA), nucleotide coenzyme and ATP, NADP, in close relations with biological heredity, has important regulating and controlling effect to the physiologically active of organism.The ribose of occurring in nature mainly exists with D-ribose form, and D-ribose is extensively present in plant and animal cell with furanose type, and D-ribose is also multivitamin, coenzyme and some microbiotic, as the composition of neamine, B and paromycin; L-ribose is the chirality enantiomer relative with D-ribose, in nature and organism, does not exist, and is very expensive rare saccharide.Pure L-ribose is white crystal or white crystalline powder, is with pleasantly sweet; Water soluble, ethanol, methyl alcohol, be insoluble to ether, benzene and acetone.L-ribose has good anti-tumor virus capable and very little to Normocellular toxic side effect.L-ribose is important medicinal intermediates, is widely used in the synthetic nucleoside compound with antiviral activity, show strong potential aspect anti-AIDS, antiviral intermediate.L-ribose also can be produced 2-deoxidation-L-ribose through processing such as reduction deoxidation hydrolysis, and the nucleoside derivates that the organic basess such as this intermediate and VITAMIN B4 form also has very large application potential aspect the treatment of the diseases such as cancer, hepatitis B, the third liver.
Along with the continuous expansion of L-ribose application surface, the demand of global L-ribose increases year by year, and people are day by day dense to the interest of preparation and exploitation adaptation suitability for industrialized production L-ribose method.Along with the continuous expansion of L-ribose application surface, the demand of global L-ribose increases year by year, and people are day by day dense to the interest of preparation and exploitation adaptation suitability for industrialized production L-ribose method.
Although adopt at present chemical method to synthesize L-ribose, at aspects such as synthesis step and yields, be all enhanced, but still exist synthesis route complicated, reactions steps is loaded down with trivial details, reagent is expensive, total recovery is low, needs to use a large amount of organic solvents and produces harmful problems such as by product, is difficult to adapt to the requirement of suitability for industrialized production.By microorganism or enzyme, carry out bio-transformation production L-ribose and become domestic and international study hotspot, the synthetic L-ribose of bio-transformation has the features such as stereoselectivity is good, reaction conditions is gentle, pollution is few.According to raw material difference, can be divided into two classes: take ribitol as raw material and take L-arabinose as raw material, wherein be worth should be mentioned that the more L-arabinose of take of Recent study needs two-stage catalysis as feedstock conversion as L-ribose, a lot of researchists are devoted to, using L-arabinose isomerase and two kinds of enzymes of mannose-6-phosphate isomerase as an enzyme catalysis system, to realize two kinds of enzyme one-step catalysis L-arabinose and produce L-ribose.
Along with developing rapidly of genetic engineering technique, utilize molecular cloning and heterogenous expression technology can improve significantly the expression amount of certain industrial enzyme in host microorganism, the engineering strain building by this method has the enzyme catalysis efficiency that common micro-organisms is difficult to reach; The technique of utilizing co-expression gene engineering strain to produce L-ribose is not still reported at home, the present invention selects the lactobacillus fermentum of oneself sieve and the starting strain that thermus thermophilus operates as molecular biology, by round pcr, from the genome amplification of this bacterial strain, the encoding gene araA of L-arabinose isomerase and the encoding gene manA of mannose-6-phosphate isomerase have been obtained, utilize intestinal bacteria as host, successfully built the efficiently genetic engineering bacterium of coexpression L-arabinose isomerase and mannose-6-phosphate isomerase.
Summary of the invention
Technical problem to be solved by this invention is to provide that a kind of production process is simple, condition is suitable, the genetic engineering bacterium that can significantly reduce production costs.
The technical problem that the present invention also will solve is to provide the construction process of said gene engineering bacteria.
The technical problem that the present invention finally will solve, is to provide the application of said gene engineering bacteria.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
A kind of external source coexpression L-arabinose isomerase and mannose-6-phosphate isomerase genetic engineering bacterium, it is characterized in that, it is the gene order that has imported nucleotide sequence SEQ ID NO:1(L-Arabinose isomerase, araA) and the gene order of nucleotide sequence SEQ ID NO:2(mannose-6-phosphate isomerase, recombination bacillus coli manA).
The construction process of said gene engineering bacteria, it comprises the following steps:
(1) structure of the expression vector that contains L-arabinose isomerase gene and mannose-6-phosphate isomerase gene:
That according to Genbank, announces derives from lactobacillus fermentum (Lactobacillus fermentum, preserving number: CGMCC2921) and thermus thermophilus (Thermus thermophilus HB8, ATCC27634) L-arabinose isomerase gene and the sequence of mannose-6-phosphate isomerase gene, use Vector NTI software design following primer:
araA-up：5’GGAATTCCATATGCGTAAGATGCAAG3’；
araA-down：5’GCGGTACCCTACTTGATGTTGAT3’；
manA-up：5’ATATATCCATGGGTGGGGCCCCGGGTA3’；
manA-down：5’AGAATTCTCACGCCCCCTCCTT3’；
Extract respectively lactobacillus fermentum genomic dna in logarithmic phase and thermus thermophilus genomic dna as template, carry out pcr amplification, obtain respectively the pcr amplification product of L-arabinose isomerase gene and mannose-6-phosphate isomerase gene; Reclaim the pcr amplification product of described L-arabinose isomerase gene and described mannose-6-phosphate isomerase gene, by L-arabinose isomerase gene through restriction enzyme Nde I and Kpn I double digestion, be connected under the effect of T4 ligase enzyme with the plasmid pETDuet-01 through same double digestion, obtain recombinant plasmid pETDuet-araA; Recombinant plasmid pETDuet-araA is converted in competence e. coli bl21 (DE3), and the LB solid medium that coating contains 100 μ g/mL penbritins, cultivates 12～16h for 37 ℃ and obtains mono-clonal;
(2) through the screening of resistance culture base, obtain positive colony:
In the LB liquid nutrient medium that picking mono-clonal contains 100 μ g/mL penbritins in 5mL respectively, 37 ℃, 200rpm overnight incubation, obtain L-arabinose isomerase genetic engineering bacterium;
(3) L-arabinose isomerase genetic engineering bacterium is carried out to plasmid extraction:
To restriction enzyme Nco I and EcoR I double digestion for the mannose-6-phosphate isomerase gene reclaiming in (1), be connected under the effect of T4 ligase enzyme with the plasmid pETDuet-araA through same double digestion again, obtain co-expression plasmid pETDuet-araA-manA;
(4) co-expression plasmid pETDuet-araA-manA is converted in host cell:
Co-expression plasmid pETDuet-araA-manA is converted in competence e. coli bl21 (DE3), and the LB solid medium that coating contains 100 μ g/mL penbritins, cultivates 12～16h for 37 ℃ and obtains mono-clonal;
(5) through the screening of resistance culture base, obtain positive colony:
In the LB liquid nutrient medium that picking mono-clonal contains 100 μ g/mL penbritins in 5mL respectively, 37 ℃, 200rpm overnight incubation, the genetic engineering bacterium of acquisition coexpression L-arabinose isomerase and mannose-6-phosphate isomerase.
Wherein, pcr amplification system is: genomic dna 2 μ L, each 2 μ L of araA-up/manA-up and araA-down/manAdown, dNTP4 μ L, 10 * Taq damping fluid, 5 μ L, Taq enzyme 1 μ L, ddH
2o34 μ L;
PCR response procedures is: 94 ℃ of denaturation 2min; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s then, 72 ℃ are extended 2min, circulate 35 times; Last 72 ℃ are extended 10min.
The application of above-mentioned genetic engineering bacterium in preparation L-ribose.
Utilize said gene engineering bacteria, the technique that the L-arabinose of take is prepared L-ribose as substrate is as follows:
(1) mutagenesis of genetic engineering bacterium is expressed: described genetic engineering bacterium is inoculated in to overnight incubation in LB liquid nutrient medium, then with the inoculum size of 0.5～10% (v/v), transfer in LB substratum, 20～40 ℃ of fermentation culture 1～3h, add again the lactose of final concentration 0.1～10g/L or sec.-propyl-β-D-sulfo-galactopyranoside of final concentration 0.1～1.5mM, and be placed in abduction delivering 3～48h at 20～40 ℃, centrifugal collection thalline;
Or described genetic engineering bacterium is inoculated in to overnight incubation in LB liquid nutrient medium, then with the inoculum size of 0.5～10% (v/v), transfer in fermention medium, direct abduction delivering 3～48h at 20～40 ℃, centrifugal collection thalline, wherein, the lactose that described fermention medium comprises mass ratio 2:1:2, peptone or yeast powder and NaCl, pH value is 4～10, through high pressure moist heat sterilization, processes;
(2) conversion reaction: take 25～100g/L L-arabinose as substrate, add the genetic engineering bacterium after induction in (1) to carry out conversion reaction, consumption is counted 10～100g/L with the bacterium that wets, pH value is 5～12,40～80 ℃ of temperature of reaction, transformation time 12～48h, after reaction finishes, the content of Liquid Detection L-ribose;
Or, take 25～100g/L L-arabinose as substrate, add 1～15mmol/L Mn
2+ion and 0.2～5mmol/L Co
2+ion, then adds the genetic engineering bacterium after induction in (1) to carry out conversion reaction, and consumption is counted 10～100g/L with the bacterium that wets, and pH value is 5～12,40～80 ℃ of temperature of reaction, and transformation time 12～48h, after reaction finishes, the content of Liquid Detection L-ribose.
Peptone, yeast powder and NaCl that above-mentioned LB substratum comprises mass ratio 2:1:2.
Beneficial effect: L-arabinose isomerase of the present invention and mannose-6-phosphate isomerase co-expression gene engineering bacteria can be produced L-ribose by catalysis L-arabinose under optimum conditions, one step transforms, save the reaction times, transformation efficiency is high, by genetic engineering bacterium of the present invention, the transformation efficiency of L-ribose reaches 25～60%, has good industrialization prospect, lower concentration Mn
2+and Co
2+the interpolation of ion can increase substantially the enzyme activity of enzyme, adds 1～15mmol/L Mn
2+ion and 0.2～5mmol/L Fe
2+after ion, than the control group enzyme that does not add metal ion, live and improve 58%.
Accompanying drawing explanation
The agarose gel electrophoresis checking of Fig. 1 araA product P CR, swimming lane 1～2 is araA product P CR, and 3 is standard DNA molecular weight, and top-down band is respectively (kb): 15.0,10.0,7.5,5.0,2.5,1.0.
The agarose gel electrophoresis checking of Fig. 2 manA product P CR, swimming lane 1 is standard DNA molecular weight, top-down band is respectively (kb): 15.0,10.0,7.5,5.0,2.5,1.0, swimming lane 2～3 is manA product P CR.
Fig. 3 recombinant plasmid pETDuet-araA nucleic acid glue checking, wherein swimming lane 1～4 is No. 1～4, the recombinant plasmid PCR product to araA, and swimming lane 5 is standard DNA molecular weight, and top-down band is respectively (kb): 15.0,10.0,7.5,5.0,2.5,1.0.Swimming lane 6 is the mono-product of cutting of Nde I of empty plasmid pETDuet-01, the mono-product of cutting of Nde I that swimming lane 7～10 is No. 1～4, recombinant plasmid.
The structure schematic diagram of Fig. 4 co-expression plasmid pETDuet-araA-manA.
The PCR checking swimming lane 1～6 of Fig. 5 co-expression plasmid pETDuet-araA-manA is No. 1～6, the co-expression plasmid PCR product to araA, swimming lane 7～12 is the PCR product of 1～6 couple of manA of co-expression plasmid, swimming lane 13 is standard DNA molecular weight, top-down band is respectively (kb): 15.0,10.0,7.5,5.0,2.5,1.0.
The double digestion checking of Fig. 6 co-expression plasmid pETDuet-araA-manA, swimming lane 1 is standard DNA molecular weight, top-down band is respectively (kb): 15.0,10.0,7.5,5.0,2.5,1.0.Swimming lane 2～3 is respectively Nde I and the Kpn I of No. 2, co-expression plasmid, the two products of cutting of Nco I and EcoR I, and swimming lane 4～5 is respectively Nde I and the Kpn I of No. 3, co-expression plasmid, the two products of cutting of Nco I and EcoR I.
Fig. 7 L-arabinose isomerase and mannose-6-phosphate isomerase coexpression bacterial strain inducing are expressed SDS-PAGE figure, and swimming lane M is standard protein molecular weight, and swimming lane 1～2 is respectively cleer and peaceful full cell on coexpression bacterial strain.
The Liquid Detection collection of illustrative plates of Fig. 8 product compound sample.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand, the described content of embodiment is only for the present invention is described, and should also can not limit the present invention described in detail in claims.
Embodiment 1: the structure of the co-expression plasmid carrier that contains L-arabinose isomerase gene and mannose-6-phosphate isomerase gene.
According to what reported on Genbank, derive from lactobacillus fermentum (Lactobacillus fermentum, preserving number: CGMCC2921) and thermus thermophilus (Thermus thermophilus HB8, ATCC27634) L-arabinose isomerase gene and the sequence of mannose-6-phosphate isomerase gene, use Vector NTI software design primer, primer sequence is as shown in table 1:
Table 1 primer sequence
The working instructions that provide according to manufacturer, with Genomic DNA Purification Kit(Takara, Dalian) extracting is in the lactobacillus fermentum NXTag1(Lactobacillus of logarithmic phase fermentum, preserving number: CGMCC2921, it is disclosed in patent documentation ZL200910025982.9) and thermus thermophilus (Thermus thermophilus, HB8, ATCC27634) genomic dna, and with 1%(10g/L) agarose gel electrophoresis detects obtained genomic dna, using the lactobacillus fermentum genomic dna that extracts and thermus thermophilus genomic dna respectively as template, carry out pcr amplification.
PCR(polymerase chain reaction) amplification system is: genomic dna 2 μ L, each 2 μ L of primer araA-up (manA-up) and primer araA-down (manAdown), dNTP4 μ L, 10 * Taq damping fluid, 5 μ L, Taq enzyme 1 μ L, ddH
2o34 μ L;
PCR response procedures is: 94 ℃ of denaturation 2min; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s then, 72 ℃ are extended 2min, circulate 35 times; Last 72 ℃ are extended 10min;
By PCR product in 1%(10g/L) agarose gel electrophoresis checking, the results are shown in Figure 1 and Fig. 2.After the DNA band of finding to conform to expection molecular weight (1424bp and 900bp) size respectively, reclaiming test kit respectively with the pillar rubber tapping of Axygen company reclaims.
With designing in advance the corresponding restriction enzyme of restriction enzyme site in primer sequence, the PCR product araA obtaining in (2) is carried out to endonuclease reaction.Restriction enzyme used is EcoRI and Hind III.The enzyme system of cutting is: PCR product 12.5 μ L, Nde I1 μ L, Kpn I1 μ L, 10 * M damping fluid, 2.5 μ L, ddH
2o8 μ L, cumulative volume 25 μ L.PCR product after enzyme is cut is through DNA purification kit purifying.
Using same restriction enzyme and the enzyme system of cutting to carry out enzyme to pETDuet-01 plasmid cuts, due to multiple clone site (the Multiple Cloning Site of two selected restriction enzyme sites at pETDuet-01 plasmid, MCS) upper close proximity (about 20bp), so enzyme is cut plasmid afterwards only need to can reach the object of purifying through DNA purification kit.
Purified PCR product is connected with pETDuet-01 plasmid.Ligation system is: enzyme is cut the PCR product 6 μ L of purifying, and enzyme is cut the pETDuet-01 plasmid 2 μ L of purifying, T4 ligase enzyme 1 μ L, 10 * T4 ligase enzyme damping fluid, 1 μ L.After 37 ℃ of connection 2h, can obtain recombinant plasmid pETDuet-araA.
Recombinant plasmid pETDuet-araA transformed competence colibacillus Bacillus coli cells is used Calcium Chloride Method:
(1) get 10 μ L recombinant plasmid pETDuet-araA in 50 μ L intestinal bacteria Escherichia coli BL21 (DE3) competent cells, ice bath 30min.
(2) 42 ℃ of water-bath heat shock 90s, are placed in 2～3min on ice fast.
(3) add fresh LB liquid nutrient medium 800 μ L, in 37 ℃ of shaking culture 45min.
(4) get 200 μ L thalline and coat the LB solid culture primary surface that contains 100 μ g/mL penbritins.Cultivate 12～16h to single bacterium colony appearance for 37 ℃.
The evaluation of recon pETDuet-araA: single colony inoculation 37 ℃ of incubated overnight in the LB liquid nutrient medium that contains penbritin (100 μ g/mL) are also extracted respectively to plasmid, each plasmid is carried out to bacterium colony PCR checking, according to the enzyme in " restriction enzyme digestion reaction; purifying and ligation ", cut system again and condition is carried out respectively single endonuclease digestion with Nde I to empty plasmid pETDuet-01 and recombinant plasmid pETDuet-araA, bacterium colony PCR product and enzyme are cut to product and carry out agarose gel electrophoresis evaluation, result as shown in Figure 3.The recombinant plasmid pETDuet-araA that experimental result explanation obtains is correct.
Through electrophoresis result, confirm, this positive colony bacterium colony contains DNA fragmentation and inserts plasmid pETDuet-araA, is the recombination bacillus coli BL21-araA of conversion containing the recombination bacillus coli of this recombinant plasmid pETDuet-araA.Sequencing result shows the open reading frame (Open Reading Frame, ORF) that Insert Fragment contains a long 1424bp.
The restriction enzyme digestion reaction of manA gene, purifying and ligation: the PCR product manA and the recombinant plasmid pETDuet-araA that obtain are carried out to endonuclease reaction with designing in advance the corresponding restriction enzyme of restriction enzyme site in primer sequence.Restriction enzyme used is Nco I and EcoR I.The enzyme system of cutting is: PCR product 12.5 μ L, Nco I1 μ L, EcoR I1 μ L, 10 * M damping fluid, 2.5 μ L, ddH
2o8 μ L, cumulative volume 25 μ L.PCR product after enzyme is cut and recombinant plasmid are through DNA purification kit purifying.
Purified PCR product is connected with pETDuet-araA recombinant plasmid.Ligation system is: enzyme is cut the PCR product 5 μ L of purifying, and enzyme is cut the pETDuet-araA plasmid 3 μ L of purifying, T4 ligase enzyme 1 μ L, 10 * T4 ligase enzyme damping fluid, 1 μ L.After 37 ℃ of connection 2h, can obtain co-expression plasmid pETDuet-araA-manA, its primary structure as shown in Figure 4.
Co-expression plasmid pETDuet-araA-manA transformed competence colibacillus Bacillus coli cells is used Calcium Chloride Method, the Calcium Chloride Method that method is used with above-mentioned recombinant plasmid pETDuet-araA transformed competence colibacillus Bacillus coli cells.
The evaluation of recon pETDuet-araA-lacZ: single colony inoculation 37 ℃ of incubated overnight in the LB liquid nutrient medium that contains penbritin (100 μ g/mL) are also extracted respectively to plasmid, each plasmid is carried out to bacterium colony PCR checking, according to the enzyme in above-mentioned restriction enzyme digestion, purifying and ligation, cut system again and condition is used respectively Nde I and KpnI, NcoI and EcoRI carry out double digestion to co-expression plasmid pETDuet-araA-manA, and bacterium colony PCR product and double digestion product are carried out to agarose gel electrophoresis evaluation.Qualification result is shown in Fig. 5 and Fig. 6, and the co-expression plasmid that experimental result explanation obtains is correct No. pETDuet-araA-manA3.
Through electrophoresis result, confirm, as shown in Figure 5 and Figure 6, this positive colony bacterium colony contains DNA fragmentation and inserts plasmid pETDuet-araA-manA, is the recombination bacillus coli BL21-araA-manA of conversion containing the recombination bacillus coli of this co-expression plasmid pETDuet-araA-manA.The open reading frame (Open Reading Frame, ORF) that sequencing result demonstration Insert Fragment contains a long 1424bp and the open reading frame of a long 900bp.
Embodiment 2: the abduction delivering of genetic engineering bacterium.
Adopt in two ways to the genetic engineering bacterium abduction delivering obtaining in embodiment 1:
(1) preparation seed liquor 1L, substratum is that (peptone 10g/L, yeast powder 5g/L, NaCl10g/L pack in several 500mL wide-mouth triangular flasks LB liquid nutrient medium after 121 ℃ of high pressure moist heat sterilization 30min.With inoculating needle, to seed liquor access one, encircle genetic engineering bacterium bacterial classification in embodiment 1, and be placed in 37 ℃ of shaking tables with the rotating speed incubated overnight of 200rpm.The LB substratum 1000mL that preparation contains peptone 10g/L, yeast powder 5g/L, NaCl10g/L, is sub-packed in the wide-mouth triangular flask of capacity 500mL, and the liquid amount of every bottle is 100mL; Above-mentioned LB is cultivated based on 121 ℃ of high pressure moist heat sterilization 30min.After substratum is cooling, access the seed liquor 1mL of incubated overnight, triangular flask is placed in to 37 ℃ of shaking tables to be cultivated with the rotating speed of 200rpm, after about 1.5h, adding final concentration is the lactose of 0.1～10g/L or IPTG(sec.-propyl-β-D-sulfo-galactopyranoside that final concentration is 1mM), and be placed in 37 ℃ of shaking tables and carry out inducing action 6h with 200rpm rotating speed, obtain co-expression gene engineering bacterium fermentation liquid A, the centrifugal collection of fermented liquid obtains co-expression gene engineering bacteria thalline A.
(2) preparation seed liquor 1L, substratum is LB liquid nutrient medium (peptone 10g/L, yeast powder 5g/L, NaCl10g/L, regulating pH value with NaOH is 7.0), packs in several 500mL wide-mouth triangular flasks after 121 ℃ of high pressure moist heat sterilization 30min.With inoculating needle, to seed liquor, access a prf gene engineering bacteria bacterial classification, and be placed in 37 ℃ of shaking tables with the rotating speed incubated overnight of 200rpm.The fermention medium 1000mL that preparation contains lactose 10g/L, peptone (or yeast powder) 5g/L, sodium-chlor 10g/L, is sub-packed in the wide-mouth triangular flask of capacity 500mL, and the liquid amount of every bottle is 100mL; By above-mentioned fermentation culture based on 121 ℃ of high pressure moist heat sterilization 30min.After substratum is cooling, access the seed liquor 1mL of incubated overnight, triangular flask is placed in to 25 ℃ of shaking tables and with the rotating speed of 200rpm, cultivates, lactose is not only made carbon source but also as inductor, carry out inducing action 22h, obtains co-expression gene engineering bacterium fermentation liquid B.The centrifugal collection of fermented liquid, obtains co-expression gene engineering bacteria thalline B, by SDS-PAGE, detects, and result as shown in Figure 7.
Embodiment 3: species of metal ion and concentration are on coexpression L-arabinose isomerase and the impact alive of mannose-6-phosphate isomerase engineering strain enzyme.
To the middle interpolation of 1mL100mM buffer solution of sodium phosphate (pH6.5) L-arabinose to final concentration, be 100mM, add again co-expression gene engineering bacteria thalline 12mg, the interpolation final concentration of each metal ion is 1mM or 10mM, 65 ℃ of water-bath 20min, by HPLC, measure the growing amount of the L-ribose generating, measurement result (take and do not add metal ion group as contrast, setting enzyme work is 100%) as shown in table 2.
The impact on co-expression gene engineering strain enzyme activity of table 2 different metal ion and concentration
From result, can find out, work as Co
2+concentration is 1mM, Mn
2+when concentration is 10mM, enzyme is alive higher, Gu add ionic concn when preparing L-ribose with co-expression gene engineering bacteria, is Co
2+1mM, Mn
2+10mM.
Embodiment 4: utilize co-expression gene engineering bacteria to prepare L-ribose (not adding metal ion).
The 30g/L L-arabinose that the co-expression gene engineering bacteria thalline A that gets the centrifugal collection of 1.2g puts into the configuration of 100mM pH6.5 sodium phosphate buffer carries out catalyzed reaction, and reaction conditions is 65 ℃ of temperature, shaking speed 180rpm.Sampling in every 4 hours, measures the L-Ribose concentration generating, and after 29h, reaction finishes.The transformation efficiency that HPLC detects L-ribose reaches 17%.
Embodiment 5: utilize co-expression gene engineering bacteria to prepare L-ribose (adding metal ion).
Get the co-expression gene engineering bacteria thalline B of the centrifugal collection of 1.2g, the 30g/L L-arabinose that the thalline of collection is dropped into the configuration of 100mM pH6.5 sodium phosphate buffer carries out catalyzed reaction, and reaction conditions is temperature 50 C, adds 10mM Mn
2+with 1mM Co
2+, shaking speed 180rpm.Sampling in every 4 hours, measures the L-Ribose concentration generating, and after 29h, reaction finishes.The transformation efficiency that detects L-ribose through HPLC reaches 32%.
Embodiment 6:
Utilize co-expression gene engineering bacteria to prepare L-ribose, method is with embodiment 5, different, and co-expression gene engineering bacteria thalline B consumption is 3.6g, and the concentration of substrate L-arabinose is 60g/L, and temperature of reaction is 65 ℃, and the reaction times is 24h.The transformation efficiency that detects L-ribose through HPLC reaches 43%, as shown in Figure 8.
Embodiment 7:
Utilize co-expression gene engineering bacteria to prepare L-ribose, method is with embodiment 5, different, gets the co-expression gene engineering bacteria thalline A of the centrifugal collection of 3.6g, and the concentration of substrate L-arabinose is 100g/L.The transformation efficiency that detects L-ribose through HPLC reaches 28%.
Embodiment 8:
Utilize co-expression gene engineering bacteria to prepare L-ribose, method is with embodiment 5, different, and temperature of reaction is 65 ℃, and the reaction times is 25h.The transformation efficiency that detects L-ribose through HPLC reaches 36%.
Embodiment 9: utilize co-expression gene engineering bacteria to prepare L-ribose, method is with embodiment 5, different, and the concentration of substrate L-arabinose is 100g/L, and the reaction times is 36h.The transformation efficiency that detects L-ribose through HPLC reaches 18%.
Embodiment 10:
Utilize co-expression gene engineering bacteria to prepare L-ribose, method is with embodiment 5, different, and thalline used is co-expression gene engineering thalline A, and the reaction times is 30h, and the transformation efficiency that HPLC detects L-ribose reaches 21%.
Embodiment 11:
Utilize co-expression gene engineering bacteria to prepare L-ribose, method is with embodiment 5, different, and thalline used is co-expression gene engineering thalline A, and biomass is 3.6g, and the reaction times is 28h, and the transformation efficiency that HPLC detects L-ribose reaches 25%.
Claims (6)
1. a genetic engineering bacterium for coexpression L-arabinose isomerase gene and mannose-6-phosphate isomerase, is characterized in that, it is the recombination bacillus coli that has imported nucleotide sequence SEQ ID NO:1 and nucleotide sequence SEQ ID NO:2.
2. the construction process of genetic engineering bacterium claimed in claim 1, is characterized in that, it comprises the following steps:
(1) structure of the expression vector that contains L-arabinose isomerase gene and mannose-6-phosphate isomerase gene:
The L-arabinose isomerase gene that derives from lactobacillus fermentum and thermus thermophilus of announcing according to Genbank and the sequence of mannose-6-phosphate isomerase gene, use Vector NTI software design following primer:
araA-up：5’GGAATTCCATATGCGTAAGATGCAAG3’；
araA-down：5’GCGGTACCCTACTTGATGTTGAT3’；
manA-up：5’ATATATCCATGGGTGGGGCCCCGGGTA3’；
manA-down：5’AGAATTCTCACGCCCCCTCCTT3’；
Extract respectively lactobacillus fermentum genomic dna in logarithmic phase and thermus thermophilus genomic dna as template, carry out pcr amplification, obtain respectively the pcr amplification product of L-arabinose isomerase gene and mannose-6-phosphate isomerase gene; Reclaim the pcr amplification product of described L-arabinose isomerase gene and described mannose-6-phosphate isomerase gene, by L-arabinose isomerase gene through restriction enzyme Nde I and Kpn I double digestion, be connected under the effect of T4 ligase enzyme with the plasmid pETDuet-01 through same double digestion, obtain recombinant plasmid pETDuet-araA; Recombinant plasmid pETDuet-araA is converted in competence e. coli bl21 (DE3), then coats the LB solid medium that contains 100 μ g/mL penbritins, cultivate 12～16h for 37 ℃ and obtain mono-clonal;
(2) through the screening of resistance culture base, obtain positive colony:
In the LB liquid nutrient medium that picking mono-clonal contains 100 μ g/mL penbritins in 5mL respectively, 37 ℃, 200rpm overnight incubation, obtain L-arabinose isomerase genetic engineering bacterium;
(3) L-arabinose isomerase genetic engineering bacterium is carried out to plasmid extraction:
To restriction enzyme Nco I and EcoR I double digestion for the mannose-6-phosphate isomerase gene reclaiming in (1), be connected under the effect of T4 ligase enzyme with the plasmid pETDuet-araA through same double digestion again, obtain co-expression plasmid pETDuet-araA-manA;
(4) co-expression plasmid pETDuet-araA-manA is converted in host cell:
Co-expression plasmid pETDuet-araA-manA is converted in competence e. coli bl21 (DE3), coats the LB solid medium that contains 100 μ g/mL penbritins, cultivate 12～16h for 37 ℃ and obtain mono-clonal;
(5) through the screening of resistance culture base, obtain positive colony:
In the LB liquid nutrient medium that picking mono-clonal contains 100 μ g/mL penbritins in 5mL respectively, 37 ℃, 200rpm overnight incubation, the genetic engineering bacterium of acquisition coexpression L-arabinose isomerase and mannose-6-phosphate isomerase.
3. the construction process of genetic engineering bacterium according to claim 2, is characterized in that, wherein pcr amplification system is: genomic dna 2 μ L, each 2 μ L of araA-up/manA-up and araA-down/manAdown, dNTP4 μ L, 10 * Taq damping fluid, 5 μ L, Taq enzyme 1 μ L, ddH
2o34 μ L;
PCR response procedures is: 94 ℃ of denaturation 2min; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s then, 72 ℃ are extended 2min, circulate 35 times; Last 72 ℃ are extended 10min.
4. the application of genetic engineering bacterium claimed in claim 1 in preparation L-ribose.
5. application according to claim 4, is characterized in that, utilizes genetic engineering bacterium, and the technique that the L-arabinose of take is prepared L-ribose as substrate is as follows:
(1) mutagenesis of genetic engineering bacterium is expressed: described genetic engineering bacterium is inoculated in to overnight incubation in LB liquid nutrient medium, then with the inoculum size of 0.5～10% (v/v), transfer in LB substratum, 20～40 ℃ of fermentation culture 1～3h, add again the lactose of final concentration final concentration 0.1～10g/L or sec.-propyl-β-D-sulfo-galactopyranoside of 0.1～1.5mM, and be placed in abduction delivering 3～48h at 20～40 ℃, centrifugal collection thalline;
Or described genetic engineering bacterium is inoculated in to overnight incubation in LB liquid nutrient medium, then with the inoculum size of 0.5～10% (v/v), transfer in fermention medium, abduction delivering 3～48h at 20～40 ℃, centrifugal collection thalline, wherein, the lactose that described fermention medium comprises mass ratio 2:1:2, peptone or yeast powder and NaCl, pH value is 4～10, through high pressure moist heat sterilization, processes;
(2) conversion reaction: take 25～100g/L L-arabinose as substrate, add the genetic engineering bacterium after induction in (1) to carry out conversion reaction, consumption is counted 10～100g/L with the bacterium that wets, pH value is 5～12,40～80 ℃ of temperature of reaction, transformation time 12～48h, after reaction finishes, the content of Liquid Detection L-ribose;
Or, take 25～100g/L L-arabinose as substrate, add 1～15mmol/L Mn
2+ion and 0.2～5mmol/L Co
2+ion, then adds the genetic engineering bacterium after induction in (1) to carry out conversion reaction, and consumption is counted 10～100g/L with the bacterium that wets, and pH value is 5～12,40～80 ℃ of temperature of reaction, and transformation time 12～48h, after reaction finishes, the content of Liquid Detection L-ribose.
6. application according to claim 5, is characterized in that, the LB substratum described in step (1) comprises peptone, yeast powder and the NaCl that mass ratio is 2:1:2.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105483108A (en) * | 2016-01-29 | 2016-04-13 | 南京工业大学 | L-arabinose isomerase and application thereof in production of L-ribulose |
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| CN105779425A (en) * | 2016-04-08 | 2016-07-20 | 南京工业大学 | L-ribose isomerase and application thereof for biologically preparing L-ribose |
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| CN106480006B (en) * | 2016-10-13 | 2019-09-10 | 南京林业大学 | A kind of L-arabinose isomerase and its application |
| CN108866120A (en) * | 2017-05-10 | 2018-11-23 | 韩国科学技术院 | The production method of L- ribose based on L-arabinose |
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