KR20130050780A - Method for production of ribose with metabolically engineered escherichia coli - Google Patents

Abstract

PURPOSE: A method for preparing ribose using metabolically engineered E.coli is provided to obtain ribose from xylose using the E.coli by deleting an enzymatic function of transketolase-A and transketolase-B. CONSTITUTION: A method for preparing ribose is conducted from xylose using E.coli. The E.coli is transformed by deleting an enzymatic function of transketolase-A and transketolase-B. The deletion is performed by damaging a part of a gene encoding transketolase-A and transketolase-B, or removing the whole gene. The E.coli is also transformed to delete an enzymatic function of PtsG.

Description

Method for production of ribose with metabolically engineered Escherichia coli}

The present invention relates to a method for producing ribose, and more particularly, to a method for producing ribose from xylose using metabolic modified E. coli.

Ribose is a type of pentose and is used as a raw material for the synthesis of vitamin B2, antiviral agent and perfume enhancer. In nature, only D form exists, and it is a component of various coenzymes such as ATP and nicotinamide nucleotide.

Ribose is produced using chemical production methods or biological production methods.

As a chemical production method, a catalytic reaction using calcium gluconate was used (Steiger, Helv. Chim. Acta, 19, 189, (1936)) and ribose was produced by redox reaction using glucose Sowden, J. Am. Chem. Soc., 72, 808, (1950); Smith, Chem. Ind., 92, (1955)).

As a biological method, there is a method of producing ribose by a fermentation method using a strain of Bacillus subsp. Korean Registration No. 10-0490280 (registered on May 17, 2005) discloses a method in which "transketolase is lost The mutant strain Bacillus subtilis BS-197 was developed. The strain was grown in a basic culture medium containing xylose and glucose using the mutant strain Bacillus subtilis BS-197, and ribose production was performed. A method for culturing a strain in a fermentation medium after activation to produce ribose, and a method for biologically producing ribose by supplying a xylose and a glucose mixed medium in a fed-batch manner.

However, there is no known technique for producing ribose using E. coli.

The present invention, in the production of ribose (ribose) from xylose, Escherichia coli ) will be developed and provided.

In order to solve the above object, the present invention is Escherichia coli ( Esherichia In the production of ribose from xylose using coli ), the E. coli is E. coli transformed to lose the enzyme function of transketolase A and transketolase B. It provides a method for producing ribose, characterized in that.

Escherichia, also called E. coli coli ) is an industrially used strain, but it has not yet been known how to produce ribose from xylose There is no bar. Wild-type Escherichia coli cannot metabolically produce ribose with xylose as a substrate.

Therefore, to produce ribose from xylose using E. coli Metabolic transformation is required, and in the present invention, it was confirmed that ribose is produced by losing the enzymatic functions of transketolase A and transketolase B.

On the other hand, in the present invention, the loss of the enzyme function of transketolase A and transketolase B is preferably a part of the genes of each of the transketolase A and transketolase B It is preferred that this is done by disrupting or by removing all of the genes, which can be easily performed using known genetic engineering methods. Since some fragmentation of a particular gene or a method of removing all of the genes is well known in the art, a description thereof will be omitted.

On the other hand, in the present invention, the transformed E. coli is preferably further transformed so that the enzyme function of PtsG is lost, because it is possible to simultaneously enter the cells of glucose and xylose through this. At this time, it is preferable that the loss of the enzyme function of the PtsG is caused by the fragmentation of a part of the gene encoding PtsG or the removal of all of the genes. Since some of the fragments of a particular gene or a method of removing all of the genes are well known in the art, the description thereof will be omitted.

As in the present invention, when the enzyme functions of transketolase A and transketolase B are lost, ribose can be produced from E. coli using xylose as a raw material.

1 is an HPLC chromatogram of the final culture of E. coli SGK012, which only deleted the transketolase B coding gene, and SGK016, which deleted both the transketolase A coding gene and the transketolase B coding gene.
FIG. 2 is an HPLC chromatogram of the final culture of Escherichia coli SGK016 and the ribose and ribulose standards, which have deleted the transketolase A coding gene and the transketolase B coding gene.

Hereinafter, the present invention will be described in more detail in the following Examples. However, the scope of the present invention is not limited to the following embodiments, and includes modifications of equivalent technical ideas.

[ Example  1: Preparation of Transgenic Escherichia Coli for Ribose Production]

 (One) ptsG  Gene deletion

Although not essential for the production of ribose, the gene ptsG encoding PtsG in wild E. coli MG1655 was deleted to release catabolite repression to allow simultaneous influx of glucose and xylose into cells.

(PCR) using the plasmid pKD13 containing the kanamycin resistance gene as primers using the primers DEL-ptsG-F and DEL-ptsG-R (see Table 1 below) A kanamycin cassette for ptsG gene deletion was prepared which contained the 5 'untranslated region of ptsG (5' UTR) and the 3 'untranslated region (3' UTR) base sequence 39 bp, respectively.

Next, a plasmid pKD46 for lambda recombinase expression was introduced into wild-type Escherichia coli MG1655 for enhancing gene deletion efficiency through recombination. Then, a competent cell was prepared, and the kanamycin cassette for ptsG gene deletion prepared above was introduced Was introduced by electroporation.

The cells were plated in a solid medium containing kanamycin antibiotics and then cultured at 37 ° C to eliminate the plasmid pKD46 having a temperature-sensitive replication origin.

The kanamycin cassette for deletion of the ptsG gene introduced into the cell was recombined with the ptsG gene of the E. coli chromosome, so that the kanamycin resistance gene was inserted into the ptsG gene site and the mutant Escherichia coli lacking the ptsG gene could be selected from the solid medium containing kanamycin.

The mutant Escherichia coli selected ptsG gene by PCR and then introduced plasmid pCP20 expressing FRT sequence specific recombinase for the removal of kanamycin resistance gene. Both of the kanamycin resistance genes used in the present embodiment have an FRT sequence, and the kanamycin resistance gene can be removed by FRT sequence-specific recombinase.

In addition, since plasmid pCP20 has a temperature-sensitive replication origin and a high temperature inducible promoter, FRT sequence specific recombinases induce expression at high temperatures. Thus, by culturing at 43 캜, the plasmid pCP20 disappeared, and the ptsG gene deletion mutant Escherichia coli SGK012 in which the kanamycin resistance gene was removed was able to be produced.

 (2) ptsG  Following the gene Transketolase  The deletion of the B-encoding gene

The same method as described above was used to delete the gene tktB encoding the transketolase B in addition to the mutant Escherichia coli KSG012.

PCR was performed using the plasmids pKD13 containing the kanamycin resistance gene as primers using the primers DEL-tktB-F and DEL-tktB-R (see Table 1 below) , A kanamycin cassette for deletion of tktB gene, each containing a 5 'untranslated region (5'UTR) of tktB and a 39bp nucleotide sequence of 3' untranslated region (3 'UTR) was prepared.

After introducing the plasmid pKD46 into the mutant E. coli KSG012 prepared above, a competent cell was prepared, and the kanamycin cassette for deletion of the tktB gene prepared above was introduced by electroporation.

The cells were plated on a solid medium containing kanamycin antibiotics, and the plasmid pKD46 was abolished by incubation at 37 ° C. The mutant Escherichia coli, in which the kanamycin resistance gene was inserted into the tktB gene locus and deleted the tktB gene, was screened in a solid medium containing kanamycin.

The selected mutant Escherichia coli confirmed the deletion of the tktB gene by PCR and then introduced the plasmid pCP20 expressing the FRT sequence-specific recombinase to remove the kanamycin resistance gene. The mutant Escherichia coli into which the plasmid pCP20 was introduced was cultured at 43 占 폚 to thereby produce the tktB gene mutant Escherichia coli SGK014 in which the plasmid pCP20 disappeared and the kanamycin resistance gene was deleted.

 (3) ptsG  And Transketolase  B encoding gene Transketolase  Additional deletion of the A-encoding gene

The same method as described above was used to delete the transketolase A-encoding gene tktA in addition to mutant Escherichia coli SGK014.

PCR was performed using the plasmids pKD13 containing the kanamycin resistance gene as primers using the primers DEL-tktA-F and DEL-tktA-R (see Table 1 below) , A kanamycin cassette for deletion of tktA gene was prepared which contains the 5 'untranslated region (5' UTR) of tktA and the 39 base sequence of 3 'untranslated region (3' UTR)

Subsequently, plasmid pKD46 was introduced into the mutant strain E. coli KSG014, and then a gene for introduction of cells was prepared. The tktA gene deletion kanamycin cassette prepared above was introduced by electroporation. The cells were plated on a solid medium containing kanamycin antibiotics and cultured at 37 ° C to eliminate the plasmid pKD46.

The mutant E. coli, in which the kanamycin resistance gene was inserted into the tktA gene locus and deleted the tktA gene, could be selected from the solid medium containing kanamycin.

The selected mutant Escherichia coli confirmed the deletion of the tktA gene by PCR and then introduced the plasmid pCP20 expressing the FRT sequence specific recombinase gene for the removal of the kanamycin resistance gene. The plasmid pCP20-introduced mutant Escherichia coli was cultured at 43 ° C to produce a tktA gene mutant strain Escherichia coli SGK016 in which the plasmid pCP20 disappeared and the kanamycin resistance gene was removed.

The mutant strain Escherichia coli SGK016 in which the function of PtsG enzyme, transketolase A enzyme, and transketolase B enzyme disappeared was finally produced through the above process.

ptsG, tktA, tktB Base sequence of PCR primer for production of kanamycin cassette for gene deletion number Primer name Primer Sequence (5 '-> 3') One DEL-ptsG-F TAA AAA AAG CAC CCA TAC TCA GGA GCA CTC TCA ATT ATG GTG TAG 2 DEL-ptsG-R CAT CTG GCT GCC TTA GTC TCC CCA ACG TCT TAC GGA TTA ATT CCG GGG ATC CGT CGA CC 3 DEL-tktA-F AAG GGC GTG CCC TTC ATC ATC CGA TCT GGA GTC AAA ATG GTG TAG GCT GGA GCT GCT TC 4 DEL-tktA-R AGG TCG CCG AAG CGA CCT TTT TAC CCG AAA TGC TAA TTA ATT CCG GGG ATC CGT CGA CC 5 DEL-tktB-F TTG CCG CCA AAC TAT AAA CCA GCC ACG GAG TGT TAT ATG GTG TAG GCT GGA GCT GCT TC 6 DEL-tktB-R GCG TCG CAT CCG GCA ATC AGC ATC CGG CAA TCA CCA TCA ATT CCG GGG ATC CGT CGA CC

[ Example  2: Example  Production of Ribose Using Transgenic Escherichia Coli Produced in 1]

Using the mutant strain E. coli SGK016 produced in the above example was to determine the ribose production capacity.

For culture, medium of glucose 5g / L, xylose 5g / L, yeast extract 5g / L, tryptone 10g / L, sodium chloride 10g / L composition was used. After incubating for 12 hours at 37 ° C. and 200 rpm using a 500 mL baffled flask, the final culture medium was taken to measure the cell concentration, ribose, xylose, and glucose.

The cell concentration was measured by absorbance at a wavelength of 600 nm, and the concentration of ribose, xylose, glucose and ribulose was quantified by HPLC. At the time of quantitation, the correlation between the concentration and the peak area was determined using a standard solution having a known concentration, and the concentration of each substance was determined from the peak area using this.

In this experiment, Rezex RHM-Monosaccharide H + (8%) (Phenomenex) was used as the column and RI detector was used as the detector. At this time, the mobile phase was the third distilled water, and the column temperature was 25 ° C at a flow rate of 0.4 mL / min.

In the present experiment, the control strain E. coli SGK012 which lost only the activity of transketolase B was used. Culture and analysis were carried out under the same conditions as above.

As a result, as shown in FIG. 1, ribose was not detected in the culture medium of the mutant strain E. coli SGK012, which only lost the activity of transketolase B, and ribose was produced only in the mutant strain E. coli SGK016, which had lost both transketolase A and transketolase B. Confirmed.

On the other hand, it is known that the isomer of ribose, an intermediate of the ribose biosynthetic pathway, is produced from other microorganisms in which transketolase has been lost. In addition, it is known that it is structurally similar to ribose and exhibits the same retention time on HPLC. In this experiment, analysis was performed under the conditions of HPLC analysis in which two substances were separated.

As a result, the newly produced material in the culture of mutant Escherichia coli SGK012 showed the same retention time as the ribose standard and showed a different retention time from ribulose.

Therefore, it was confirmed that the newly generated material in the culture solution of the strain E. coli SGK016 of the present invention is ribose, not ribulose (FIG. 2).

Claims (4)

In producing ribose from xylose using Escherichia coli ,
The Escherichia coli is a method for producing ribose, characterized in that E. coli transformed to lose the enzyme function of transketolase A and transketolase B (transketolase) B.
The method of claim 1,
The loss of enzyme function of the transketolase A and transketolase B,
A method of producing ribose, characterized in that part of the gene encoding transketolase A and transketolase B is broken up or all of the genes are removed.
The method of claim 1,
The transformed E. coli,
The method of producing ribose, characterized in that further transformed so that the enzyme function of PtsG is lost.
5. The method of claim 4,
The loss of enzyme function of the PtsG,
A method of producing ribose, characterized in that part of the gene encoding PtsG is broken or all of the gene is removed.

Images