CN103555646B - Genetic engineering bacterium for co-expressing L-arabinose isomerase gene and mannose-6-phosphate isomerase - Google Patents

Abstract

The invention discloses a genetic engineering bacterium co-expressing L-arabinose isomerase gene and mannose-6-phosphate isomerase. The strain contains the nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:2. The genetically engineered bacterium of the present invention can produce L-ribose by catalyzing L-arabinose, one-step conversion, process is simple, and conversion rate is high, through the genetically engineered bacterium of the present invention, the conversion rate of L-ribose reaches 25～50%, has good industrialization prospects.

Description

A co-expression of L-arabinose isomerase gene and mannose-6-phosphate isomerase genetically engineered bacteria

technical field

The invention belongs to the technical field of bioengineering, and in particular relates to the construction and application of a genetic engineering bacterium co-expressing L-arabinose isomerase gene and mannose-6-phosphate isomerase gene.

Background technique

Ribose (Ribose, C ₅ H ₁₀ O ₅ ) is a typical five-carbon sugar, which is a component of various ribonucleic acid (RNA), nucleotide coenzymes, ATP, and NADP, and is closely related to biological genetics. The physiological activity of organisms plays an important role in regulation. Ribose in nature mainly exists in the form of D-ribose, which widely exists in plant and animal cells in the form of furanose. D-ribose is also a variety of vitamins, coenzymes and certain antibiotics, such as neomycin A, B and The composition of paromomycin; L-ribose is the chiral enantiomer opposite to D-ribose, which does not exist in nature and organisms, and is an extremely expensive rare sugar. Pure L-ribose is white crystal or white crystal powder with sweet taste; soluble in water, ethanol, methanol, insoluble in ether, benzene and acetone. L-ribose has good anti-tumor and anti-virus capabilities and has little toxic side effects on normal cells. L-ribose is an important drug synthesis intermediate, widely used in the synthesis of nucleoside compounds with antiviral activity, and has shown strong potential in anti-AIDS and antiviral intermediates. L-ribose can also produce 2-deoxy-L-ribose through reductive deoxyhydrolysis and other processing. The nucleoside derivatives formed by this intermediate and organic bases such as adenine also have great potential in the treatment of cancer, hepatitis B, hepatitis C and other diseases. application potential.

With the continuous expansion of the application of L-ribose, the global demand for L-ribose is increasing year by year, and people are increasingly interested in the preparation and development of methods suitable for industrial production of L-ribose. With the continuous expansion of the application of L-ribose, the global demand for L-ribose is increasing year by year, and people are increasingly interested in the preparation and development of methods suitable for industrial production of L-ribose.

Although the chemical synthesis of L-ribose has been greatly improved in terms of synthesis steps and yields, there are still complex synthetic process routes, cumbersome reaction steps, expensive reagents, low overall yields, and the need to use a large amount of organic compounds. Problems such as solvents and harmful by-products are difficult to adapt to the requirements of industrial production. The production of L-ribose by biotransformation by microorganisms or enzymes has become a research hotspot at home and abroad. The synthesis of L-ribose by biotransformation has the characteristics of good stereoselectivity, mild reaction conditions, and less pollution. According to different raw materials, it can be divided into two categories: ribitol as raw material and L-arabinose as raw material. It is worth mentioning that the transformation of L-arabinose into L-ribose requires two steps in recent years. For catalysis, many researchers are committed to using L-arabinose isomerase and mannose-6-phosphate isomerase as an enzyme catalytic system to realize the one-step catalysis of L-arabinose to L-ribose by the two enzymes.

With the rapid development of genetic engineering technology, molecular cloning and exogenous expression technology can greatly increase the expression of certain industrial enzymes in host microorganisms. The genetically engineered strains constructed by this method have enzymes that are difficult for ordinary microorganisms. Catalytic efficiency; Utilize the technique of co-expression genetic engineering bacterial strain to produce L-ribose at home and do not have report yet, the present invention selects self-screening Lactobacillus fermentum and Thermus thermophilus as the starting strain of molecular biology operation, from this by PCR technology The gene encoding L-arabinose isomerase, araA, and the gene encoding mannose-6-phosphate isomerase, manA, were amplified from the genome of the strain. Using Escherichia coli as the host, a strain capable of efficiently co-expressing L-arabinoisomerase was successfully constructed. Genetically engineered bacteria for sugar isomerase and mannose-6-phosphate isomerase.

Contents of the invention

The technical problem to be solved by the present invention is to provide a genetically engineered bacterium with simple production process, suitable conditions and greatly reduced production cost.

The technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned genetically engineered bacteria.

The final technical problem to be solved by the present invention is to provide the application of the above-mentioned genetically engineered bacteria.

In order to solve the problems of the technologies described above, the technical scheme adopted in the present invention is as follows:

An exogenous co-expression of L-arabinose isomerase and mannose-6-phosphate isomerase genetically engineered bacteria is characterized in that it is introduced into the nucleotide sequence SEQ ID NO: 1 (L-arabinose isomerase The recombinant Escherichia coli with the gene sequence of mannose-6-phosphate isomerase, araA) and the nucleotide sequence of SEQ ID NO: 2 (the gene sequence of mannose-6-phosphate isomerase, manA).

The construction method of above-mentioned genetic engineering bacterium, it comprises the following steps:

(1) Construction of expression vectors containing L-arabinose isomerase gene and mannose-6-phosphate isomerase gene:

According to the L-arabinose isomerase gene and mannose-6-phosphate isomerase gene derived from Lactobacillus fermentum (Lactobacillus fermentum, deposit number: CGMCC2921) and Thermus thermophilus HB8 (ATCC 27634) published by Genbank The sequence of enzyme gene, use Vector NTI software to design following primers:

araA-up: 5'GGAATTCCATATGCGTAAGATGCAAG3';

araA-down: 5'GCGGTACCCTACTTGATGTTGAT3';

manA-up: 5'ATATATCCATGGGTGGGGCCCCGGGTA3';

manA-down: 5'AGAATTCTCACGCCCCCCTCCTT3';

The genomic DNA of Lactobacillus fermentum and Thermus thermophilus in the logarithmic growth phase were extracted respectively as templates, and PCR amplification was performed to obtain the L-arabinose isomerase gene and mannose-6-phosphate isomerase gene respectively PCR amplified product; reclaim the PCR amplified product of described L-arabinose isomerase gene and described mannose-6-phosphate isomerase gene, the L-arabinose isomerase gene is subjected to restriction endonuclease Digest with enzymes Nde I and Kpn I, and connect with the plasmid pETDuet-1 that has undergone the same double digestion under the action of T4 ligase to obtain the recombinant plasmid pETDuet-araA; transform the recombinant plasmid pETDuet-araA into competent Escherichia coli In BL21(DE3), spread LB solid medium containing 100 μg/mL ampicillin and culture at 37°C for 12-16 hours to obtain a single clone;

(2) Positive clones were obtained by screening with resistant medium:

Single clones were picked and cultured overnight in 5 mL of LB liquid medium containing 100 μg/mL ampicillin at 37°C and 200 rpm to obtain L-arabinose isomerase genetically engineered bacteria;

(3) Carry out plasmid extraction to L-arabinose isomerase genetically engineered bacteria:

The mannose-6-phosphate isomerase gene recovered in (1) was double-digested with restriction endonucleases Nco I and EcoR I, and then combined with the same double-digested plasmid pETDuet-araA in the role of T4 ligase The connection was carried out to obtain the co-expression plasmid pETDuet-araA-manA;

(4) Transform the co-expression plasmid pETDuet-araA-manA into host cells:

Transform the co-expression plasmid pETDuet-araA-manA into competent Escherichia coli BL21(DE3), spread LB solid medium containing 100 μg/mL ampicillin, and culture at 37°C for 12-16 hours to obtain a single clone;

(5) Positive clones were obtained by screening with resistant medium:

Single clones were picked and cultured overnight in 5 mL LB liquid medium containing 100 μg/mL ampicillin at 37 °C and 200 rpm to obtain genetic engineering that co-expresses L-arabinose isomerase and mannose-6-phosphate isomerase bacteria.

Among them, the PCR amplification system is: genomic DNA 2 μL, araA-up/manA-up and araA-down/manA-down 2 μL each, dNTP 4 μL, 10×Taq buffer 5 μL, Taq enzyme 1 μL, ddH ₂ O 34 μL;

The PCR reaction program was as follows: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 2 min, and 35 cycles; final extension at 72°C for 10 min.

Application of the above-mentioned genetically engineered bacteria in the preparation of L-ribose.

Utilize above-mentioned genetically engineered bacterium, the process of preparing L-ribose with L-arabinose as substrate is as follows:

(1) Induced expression of genetically engineered bacteria: the genetically engineered bacteria were inoculated in LB liquid medium and cultivated overnight, then transferred into LB medium with an inoculum size of 0.5-10% (v/v), 20-10% (v/v) Ferment at 40°C for 1-3 hours, then add lactose with a final concentration of 0.1-10g/L or isopropyl-β-D-thiogalactopyranoside with a final concentration of 0.1-1.5mM, and place at 20-40°C Induce the expression for 3-48 hours, and collect the bacteria by centrifugation;

Or inoculate the genetically engineered bacterium in LB liquid medium and cultivate overnight, then transfer it into the fermentation medium with an inoculum size of 0.5-10% (v/v), and directly induce the expression of 3-3 48h, centrifuge to collect the bacteria, wherein the fermentation medium contains lactose, peptone or yeast powder and NaCl in a mass ratio of 2:1:2, the pH value is 4-10, and is sterilized by high-pressure moist heat;

(2) Transformation reaction: use 25-100g/L L-arabinose as the substrate, add the genetically engineered bacteria induced in (1) to carry out the transformation reaction, the dosage is 10-100g/L in terms of wet bacteria, and the pH value is 5~12, the reaction temperature is 40~80°C, the conversion time is 12~48h, after the reaction is completed, the liquid phase detects the content of L-ribose;

Alternatively, use 25-100g/L L-arabinose as a substrate, add 1-15mmol/L Mn ²⁺ ions and 0.2-5mmol/L Co ²⁺ ions, and then add the induced genetically engineered bacteria in (1) to carry out For the transformation reaction, the dosage is 10-100g/L in terms of wet bacteria, the pH value is 5-12, the reaction temperature is 40-80°C, and the transformation time is 12-48h. After the reaction, the content of L-ribose is detected by liquid phase.

The above LB medium contains peptone, yeast powder and NaCl in a mass ratio of 2:1:2.

Beneficial effects: the co-expression genetically engineered bacteria of L-arabinose isomerase and mannose-6-phosphate isomerase of the present invention can produce L-ribose by catalyzing L-arabinose under suitable conditions, one-step transformation, saving reaction time , high conversion rate, through the genetically engineered bacteria of the present invention, the conversion rate of L-ribose reaches 25-60%, has good industrialization prospects, and the addition of low-concentration Mn ²⁺ and Co ²⁺ ions can greatly improve the activity of the enzyme Enzyme activity, after adding 1-15mmol/L Mn ²⁺ ions and 0.2-5mmol/L Fe ²⁺ ions, the enzyme activity of the control group without adding metal ions increased by 58%.

Description of drawings

Figure 1 agarose gel electrophoresis verification of araA product PCR, lanes 1 to 2 are araA product PCR, 3 is the standard DNA molecular weight, and the bands from top to bottom are (kb): 15.0, 10.0, 7.5, 5.0, 2.5, 1.0.

Figure 2 Agarose gel electrophoresis verification of manA product PCR, lane 1 is the standard DNA molecular weight, the bands from top to bottom are (kb): 15.0, 10.0, 7.5, 5.0, 2.5, 1.0, lanes 2 to 3 are manA Product PCR.

Figure 3 Nucleic acid gel verification of the recombinant plasmid pETDuet-araA, in which lanes 1 to 4 are the PCR products of recombinant plasmids 1 to 4 for araA, and lane 5 is the standard DNA molecular weight, and the bands from top to bottom are (kb): 15.0 , 10.0, 7.5, 5.0, 2.5, 1.0. Lane 6 is the NdeI single-cut product of empty plasmid pETDuet-1, and lanes 7-10 are the NdeI single-cut products of recombinant plasmids 1-4.

Fig. 4 Schematic diagram of the construction of the co-expression plasmid pETDuet-araA-manA.

Figure 5 PCR verification of co-expression plasmid pETDuet-araA-manA Lanes 1-6 are PCR products of co-expression plasmids 1-6 for araA, lanes 7-12 are PCR products of co-expression plasmids 1-6 for manA, and lane 13 It is the standard DNA molecular weight, and the bands from top to bottom are (kb): 15.0, 10.0, 7.5, 5.0, 2.5, 1.0.

Figure 6 Double enzyme digestion verification of the co-expression plasmid pETDuet-araA-manA, lane 1 is the standard DNA molecular weight, and the bands from top to bottom are (kb): 15.0, 10.0, 7.5, 5.0, 2.5, 1.0. Swimming lanes 2 to 3 are the double cut products of Nde I and Kpn I, Nco I and EcoR I of co-expression plasmid No. 2, respectively, and lanes 4 to 5 are Nde I, Kpn I, Nco I and EcoR I of co-expression plasmid No. 3, respectively double cut product.

Fig. 7 SDS-PAGE diagram of induced expression of L-arabinose isomerase and mannose-6-phosphate isomerase co-expression strains, lane M is standard protein molecular weight, and lanes 1-2 are supernatant and whole cells of co-expression strains respectively.

The liquid phase detection spectrum of Fig. 8 product mixed sample.

detailed description

The present invention can be better understood from the following examples. However, those skilled in the art can easily understand that the content described in the embodiments is only for illustrating the present invention, and should not and will not limit the present invention described in the claims.

Example 1: Construction of a co-expression plasmid vector containing L-arabinose isomerase gene and mannose-6-phosphate isomerase gene.

According to the L-arabinose isomerase gene and mannose-6-phosphate derived from Lactobacillus fermentum (Lactobacillus fermentum, deposit number: CGMCC2921) and Thermus thermophilus HB8 (ATCC 27634) reported on Genbank The sequence of the isomerase gene uses Vector NTI software to design primers, and the primer sequences are as shown in Table 1:

Table 1 Primer Sequence

According to the instructions provided by the manufacturer, the Genomic DNA Purification Kit (Takara, Dalian) was used to extract Lactobacillus fermentum NXTag1 (Lactobacillus fermentum, deposit number: CGMCC2921, which was disclosed in the patent document ZL200910025982. Genomic DNA of Thermus thermophilus (Thermus thermophilus, HB8, ATCC 27634), and the obtained genomic DNA was detected by 1% (10g/L) agarose gel electrophoresis, and the extracted Lactobacillus fermentum genomic DNA and Genomic DNA of Thermus thermica was used as a template for PCR amplification.

The amplification system of PCR (polymerase chain reaction) is: genomic DNA 2pL, primer araA-up (manA-up) and primer araA-down (manAdown) 2 μL each, dNTP 4 μL, 10×Taq buffer 5 μL, Taq enzyme 1 μL , ddH ₂ O 34 μL;

The PCR reaction program was: pre-denaturation at 94°C for 2 minutes; denaturation at 94°C for 30 seconds, then annealing at 55°C for 30 seconds, extension at 72°C for 2 minutes, and 35 cycles; finally, extension at 72°C for 10 minutes;

The PCR product was verified by 1% (10 g/L) agarose gel electrophoresis, and the results are shown in Fig. 1 and Fig. 2 . After DNA bands corresponding to the expected molecular weight (1424bp and 900bp) were found, they were recovered with the Axygen company's column type rubber tapping recovery kit.

The PCR product araA obtained in (2) was digested with the restriction endonuclease corresponding to the restriction site in the primer sequence designed in advance. The restriction enzymes used were EcoRI and Hind III. The digestion system is: 12.5 μL of PCR product, 1 μL of Nde I, 1 μL of Kpn I, 2.5 μL of 10×M buffer, 8 μL of ddH ₂ O, and a total volume of 25 μL. The digested PCR product was purified by a DNA purification kit.

The pETDuet-1 plasmid was digested with the same restriction endonuclease and restriction enzyme system, because the two selected restriction sites were separated from each other on the multiple cloning site (Multiple Cloning Site, MCS) of the pETDuet-1 plasmid It is very close (about 20bp), so the digested plasmid only needs to pass through the DNA purification kit to achieve the purpose of purification.

The purified PCR product was ligated with pETDuet-1 plasmid. The ligation reaction system was: 6 μL of digested and purified PCR product, 2 μL of digested and purified pETDuet-1 plasmid, 1 μL of T4 ligase, and 1 μL of 10×T4 ligase buffer. The recombinant plasmid pETDuet-araA can be obtained after ligation at 37°C for 2 hours.

The recombinant plasmid pETDuet-araA is used to transform competent E. coli cells using the calcium chloride method:

(1) Take 10 μL of the recombinant plasmid pETDuet-araA in 50 μL of Escherichia coli BL21 (DE3) competent cells, and place in ice bath for 30 minutes.

(2) Heat shock in a water bath at 42°C for 90 seconds, and quickly place on ice for 2-3 minutes.

(3) Add 800 μL of fresh LB liquid medium, shake and culture at 37° C. for 45 minutes.

(4) Take 200 μL of bacteria and spread on the surface of LB solid medium containing 100 μg/mL ampicillin. Cultivate at 37°C for 12-16 hours until a single colony appears.

Identification of the recombinant pETDuet-araA: inoculate a single colony in LB liquid medium containing ampicillin (100 μg/mL) and culture overnight at 37°C and extract the plasmids respectively, perform colony PCR verification on each plasmid, and then follow the "restriction enzyme Digestion system and conditions in "Digestion Reaction, Purification and Ligation Reaction" Use Nde I to perform single enzyme digestion on the empty plasmid pETDuet-1 and recombinant plasmid pETDuet-araA respectively, and perform agarose gel electrophoresis on the colony PCR product and the digested product Identification, the results are shown in Figure 3. The experimental results show that the obtained recombinant plasmid pETDuet-araA is correct.

The results of electrophoresis confirmed that the positive cloned colony contained a DNA fragment inserted into the plasmid pETDuet-araA, and the recombinant Escherichia coli containing the recombinant plasmid pETDuet-araA was the transformed recombinant Escherichia coli BL21-araA. Sequencing results showed that the insert contained a 1424bp open reading frame (Open Reading Frame, ORF). Restriction enzyme digestion reaction, purification and ligation reaction of manA gene: Use the restriction endonuclease corresponding to the restriction endonuclease in the primer sequence designed in advance to carry out enzyme digestion reaction on the obtained PCR product manA and recombinant plasmid pETDuet-araA. The restriction enzymes used were Nco I and EcoR I. The digestion system is: 12.5 μL of PCR product, 1 μL of Nco I, 1 μL of EcoR I, 2.5 μL of 10×M buffer, 8 μL of ddH ₂ O, and a total volume of 25 μL. The digested PCR products and recombinant plasmids were purified by a DNA purification kit.

The purified PCR product was ligated with the pETDuet-araA recombinant plasmid. The ligation reaction system was: 5 μL of digested and purified PCR product, 3 μL of digested and purified pETDuet-araA plasmid, 1 μL of T4 ligase, and 1 μL of 10×T4 ligase buffer. The co-expression plasmid pETDuet-araA-manA can be obtained after ligation at 37°C for 2 hours, and its main structure is shown in Figure 4.

The co-expression plasmid pETDuet-araA-manA was used to transform competent E. coli cells using the calcium chloride method, which was the same as the calcium chloride method used to transform competent E. coli cells with the above-mentioned recombinant plasmid pETDuet-araA.

Identification of the recombinant pETDuet-araA-lacZ: Inoculate a single colony in LB liquid medium containing ampicillin (100 μg/mL) and culture overnight at 37°C and extract the plasmids respectively, perform colony PCR verification on each plasmid, and then follow the above-mentioned Enzyme digestion system and conditions in restriction enzyme digestion, purification and ligation reactions Use Nde I and KpnI, NcoI and EcoRI to perform double enzyme digestion on the co-expression plasmid pETDuet-araA-manA, respectively, and carry out the colony PCR product and the double enzyme digestion product Identification by agarose gel electrophoresis. The identification results are shown in Figure 5 and Figure 6, and the experimental results show that the obtained co-expression plasmid pETDuet-araA-manA No. 3 is correct.

It was confirmed by electrophoresis results, as shown in Figure 5 and Figure 6, that the positive cloned colony contained a DNA fragment inserted into the plasmid pETDuet-araA-manA, and the recombinant Escherichia coli containing this co-expression plasmid pETDuet-araA-manA was the transformed recombinant Escherichia coli BL21-araA-manA. Sequencing results showed that the insert contained a 1424bp open reading frame (Open Reading Frame, ORF) and a 900bp open reading frame.

Example 2: Induced expression of genetically engineered bacteria.

Two ways are used to induce expression of the genetically engineered bacteria obtained in Example 1:

(1) Prepare 1 L of seed solution, the medium is LB liquid medium (peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, sterilized by high pressure at 121°C for 30min, and then put it into several 500mL wide-mouthed triangular flasks In. Use an inoculation needle to insert a ring of genetically engineered bacterial strains in Example 1 into the seed solution, and place it on a shaker at 37° C. to cultivate overnight at a speed of 200 rpm. The preparation contains 10 g/L of peptone, 5 g/L of yeast powder, and NaCl 1000mL of 10g/L LB culture medium was divided into wide-mouthed Erlenmeyer flasks with a capacity of 500mL. 1mL of the seed solution cultivated overnight, put the Erlenmeyer flask on a shaker at 37°C and cultivate it at a speed of 200rpm. After about 1.5h, add lactose with a final concentration of 0.1-10g/L or IPTG with a final concentration of 1mM (isopropyl-β -D-thiogalactopyranoside), and placed in a shaker at 37° C. for 6 hours at a speed of 200 rpm to obtain a co-expressed genetically engineered bacterium fermentation broth A, and the fermentation broth was collected by centrifugation to obtain a co-expressed genetically engineered bacterium cell A .

(2) Prepare 1 L of seed solution, the medium is LB liquid medium (peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, adjust the pH value to 7.0 with NaOH), after sterilizing by high pressure at 121°C for 30min Put it into several 500mL wide-mouth Erlenmeyer flasks. Inject a ring of genetically engineered strains into the seed solution with an inoculation needle, and place it on a shaker at 37°C at a speed of 200rpm for overnight cultivation. Prepare 1000 mL of fermentation medium containing 10 g/L of lactose, 5 g/L of peptone (or yeast powder), and 10 g/L of sodium chloride, and distribute it in a wide-mouthed conical flask with a capacity of 500 mL. The liquid volume of each bottle is 100 mL; The above fermentation culture was sterilized based on 121° C. high-pressure damp heat for 30 minutes. After cooling the culture medium, insert 1mL of the overnight cultured seed solution, place the flask in a shaker at 25°C and cultivate it at a speed of 200rpm, use lactose as both a carbon source and an inducer, and induce for 22 hours to obtain co-expressed genetically engineered bacteria Fermentation Broth B. The fermentation broth was collected by centrifugation to obtain co-expressed genetically engineered bacterial cell B, which was detected by SDS-PAGE, and the results are shown in FIG. 7 .

Example 3: Effects of the type and concentration of metal ions on the enzyme activity of a genetically engineered strain co-expressing L-arabinose isomerase and mannose-6-phosphate isomerase.

Add L-arabinose to 1 mL of 100 mM sodium phosphate buffer solution (pH 6.5) to a final concentration of 100 mM, then add 12 mg of co-expressed genetically engineered bacteria, and add the final concentration of each metal ion to 1 mM or 10 mM, and bathe in water at 65 ° C for 20 min , the generation amount of the L-ribose that generates by HPLC measurement, measurement result is as shown in table 2 (with no metal ion group as contrast, enzymatic activity is set as 100%).

Table 2 Effects of different metal ions and concentrations on the enzyme activity of co-expressed genetically engineered strains

As can be seen from the results, when the Co ²⁺ concentration is 1mM and the Mn ²⁺ concentration is 10mM, the enzyme activity is higher, and the added ion concentration is Co ²⁺ 1mM, Mn ²⁺ 10mM.

Example 4: Preparation of L-ribose (without adding metal ions) by co-expressing genetically engineered bacteria.

Take 1.2g of the co-expressed genetically engineered bacterial cell A collected by centrifugation and put it into 30g/L L-arabinose buffered with 100mM pH6.5 sodium phosphate buffer for catalytic reaction. The reaction conditions are temperature 65°C and shaker speed 180rpm. Samples were taken every 4 hours to measure the generated L-ribose content, and the reaction ended after 29 hours. The conversion rate of L-ribose was detected by HPLC to reach 17%.

Example 5: Preparation of L-ribose (adding metal ions) by co-expressing genetically engineered bacteria.

Take 1.2g of the co-expressed genetically engineered bacterial cell B collected by centrifugation, put the collected cell into 30g/L L-arabinose buffered with 100mM pH6.5 sodium phosphate buffer for catalytic reaction, the reaction conditions are temperature 50°C, add 10mM Mn ²⁺ and 1mM Co ²⁺ , the shaker rotates at 180rpm. Samples were taken every 4 hours to measure the generated L-ribose content, and the reaction ended after 29 hours. The conversion rate of L-ribose was detected by HPLC to reach 32%.

Embodiment 6:

Utilize the co-expression genetically engineered bacteria to prepare L-ribose, the method is the same as in Example 5, the difference is that the amount of co-expressed genetically engineered bacteria thalline B is 3.6g, the concentration of the substrate L-arabinose is 60g/L, and the reaction temperature is 65°C, the reaction time is 24h. The conversion rate of L-ribose was detected by HPLC to reach 43%, as shown in FIG. 8 .

Embodiment 7:

L-ribose was prepared by co-expressing genetically engineered bacteria, the method was the same as in Example 5, except that 3.6 g of co-expressing genetically engineered bacteria cell A collected by centrifugation was used, and the concentration of the substrate L-arabinose was 100 g/L. The conversion rate of L-ribose was detected by HPLC to reach 28%.

Embodiment 8:

The method for preparing L-ribose by co-expressing genetically engineered bacteria is the same as in Example 5, except that the reaction temperature is 65° C. and the reaction time is 25 hours. The conversion rate of L-ribose was detected by HPLC to reach 36%.

Example 9: Preparation of L-ribose by co-expressing genetically engineered bacteria, the method is the same as in Example 5, except that the concentration of the substrate L-arabinose is 100 g/L, and the reaction time is 36 hours. The conversion rate of L-ribose was detected by HPLC to reach 18%.

Example 10:

Utilize co-expression genetically engineered bacteria to prepare L-ribose, the method is the same as in Example 5, the difference is that the thalline used is co-expressed genetically engineered bacterium A, the reaction time is 30h, and the conversion rate of L-ribose detected by HPLC reaches 21%. .

Example 11:

Utilize co-expression genetically engineered bacteria to prepare L-ribose, the method is the same as in Example 5, the difference is that the thalline used is co-expression genetically engineered thalline A, the thalline amount is 3.6g, and the reaction time is 28h, HPLC detects L-ribose The conversion rate of ribose reaches 25%.

Claims (5)

1. coexpression L-arabinose isomerase gene and a genetic engineering bacterium for mannose-6-phosphate isomerase, its feature Being, it is to have imported nucleotide sequence SEQ ID NO:1 and the recombination bacillus coli of nucleotide sequence SEQ ID NO:2, its In, described SEQ ID NO:1 derives from lactobacillus fermenti, and SEQ ID NO:2 derives from thermus thermophilus, described genetic engineering The construction method of bacterium comprises the steps:

(1)

The L-arabinose isomerase gene deriving from lactobacillus fermenti and thermus thermophilus announced according to Genbank and sweet dew The sequence of sugar-6-phosphate isomerases gene, utilization Vector NTI Software for Design following primer:

AraA-up:5 ' GGAATTCCATATGCGTAAGATGCAAG3 '；

AraA-down:5 ' GCGGTACCCTACTTGATGTTGAT3 '；

ManA-up:5 ' ATATATCCATGGGTGGGGCCCCGGGTA3 '；

ManA-down:5 ' AGAATTCTCACGCCCCCTCCTT3 '；

Extract respectively and be in the lactobacillus fermenti genomic DNA of exponential phase and thermus thermophilus genomic DNA as template, Carrying out PCR amplification, the PCR amplification respectively obtaining L-arabinose isomerase gene and mannose-6-phosphate isomerase gene is produced Thing；Reclaim described L-arabinose isomerase gene and the pcr amplification product of described mannose-6-phosphate isomerase gene, will L-arabinose isomerase gene through restriction enzyme Nde I and Kpn I double digestion, with through as the plasmid of double digestion PETDuet-1 is attached under the effect of T4 ligase, obtains recombinant plasmid pETDuet-araA；By recombinant plasmid PETDuet-araA converts to competence e. coli bl21 (DE3), then coats containing 100 μ g/mL ampicillins LB solid medium, cultivate for 37 DEG C and 12～16h obtain monoclonal；

(2) positive colony is obtained through the screening of resistance culture base:

Picking monoclonal is in 5mL contains the LB fluid nutrient medium of 100 μ g/mL ampicillins respectively, 37 DEG C, 200rpm cultivation Overnight, it is thus achieved that L-arabinose isomerase genetic engineering bacterium；

(3) L-arabinose isomerase genetic engineering bacterium is carried out plasmid extraction, different to the Man-6-P reclaimed in (1) Structure enzyme gene restriction enzyme Nco I and EcoR I double digestion, then with through as the plasmid pETDuet-of double digestion AraA is attached under the effect of T4 ligase, obtains co-expression plasmid pETDuet-araA-manA；

(4) co-expression plasmid pETDuet-araA-manA is converted to host cell:

Co-expression plasmid pETDuet-araA-manA is converted in competence e. coli bl21 (DE3), coat containing The LB solid medium of 100 μ g/mL ampicillins, cultivates 12～16h for 37 DEG C and obtains monoclonal；

(5) positive colony is obtained through the screening of resistance culture base:

Picking monoclonal is in 5mL contains the LB fluid nutrient medium of 100 μ g/mL ampicillins respectively, 37 DEG C, 200rpm cultivation Overnight, it is thus achieved that coexpression L-arabinose isomerase and the genetic engineering bacterium of mannose-6-phosphate isomerase.

Genetic engineering bacterium the most according to claim 1, it is characterised in that wherein PCR amplification system is: genomic DNA 2 μ The each 2 μ L of L, araA-up/manA-up and araA-down/manA-down, dNTP 4 μ L, 10 × Taq buffer solution 5 μ L, Taq enzyme 1 μ L, ddH₂O 34μL；

PCR response procedures is: 94 DEG C of denaturations 2min；94 DEG C of sex change 30s, then 55 DEG C of annealing 30s, 72 DEG C extend 2min, follow Ring 35 times；Last 72 DEG C extend 10min.

3. the application in preparing L-ribose of the genetic engineering bacterium described in claim 1.

Application the most according to claim 3, it is characterised in that utilize genetic engineering bacterium, with Arabinose for substrate system The technique of standby L-ribose is as follows:

(1) abduction delivering of genetic engineering bacterium: described genetic engineering bacterium is inoculated in overnight incubation in LB fluid nutrient medium, then Transfer in LB culture medium with the inoculum concentration of 0.5～10% (v/v), 20～40 DEG C of fermented and cultured 1～3h, add final concentration eventually The lactose of concentration 0.1～10g/L or the isopropyl-beta D-thio galactopyranoside of 0.1～1.5mM, be placed in 20～40 Abduction delivering 3～48h at DEG C, centrifugal collection thalline；

Or described genetic engineering bacterium is inoculated in overnight incubation in LB fluid nutrient medium, then with 0.5～the connecing of 10% (v/v) The amount of kind is transferred in fermentation medium, abduction delivering 3～48h at 20～40 DEG C, centrifugal thalline of collecting, and wherein, described sends out Ferment culture medium comprises the lactose of mass ratio 2: 1: 2, peptone or dusty yeast and NaCl, and pH value is 4～10, through high pressure moist heat sterilization Process；

(2) conversion reaction: with 25～100g/L Arabinoses as substrate, adds the genetic engineering bacterium after inducing in (1) and carries out Conversion reaction, consumption is calculated as 10～100g/L with wet bacterium, and pH value is 5～12, reaction temperature 40～80 DEG C, transformation time 12～ 48h, after reaction terminates, the content of liquid phase detection L-ribose；

Or, with 25～100g/L Arabinoses as substrate, add 1～15mmol/L Mn²⁺Ion and 0.2～5mmol/L Co²⁺Ion, is subsequently adding the genetic engineering bacterium after inducing in (1) and carries out conversion reaction, and consumption is calculated as 10～100g/L with wet bacterium, PH value is 5～12, reaction temperature 40～80 DEG C, transformation time 12～48h, after reaction terminates, and the content of liquid phase detection L-ribose.

Application the most according to claim 4, it is characterised in that it is 2 that the LB culture medium described in step (1) comprises mass ratio : peptone, dusty yeast and the NaCl of 1: 2.