KR101413143B1 - Method for production of ribose with metabolically engineered E. coli in medium with optimum ratio of xylose and glucose - Google Patents

Abstract

The present invention relates to a method for producing ribose in a medium in which xylose and glucose are optimally mixed using metabolically modified E. coli. When ribose is produced using recombinant Escherichia coli transformed so that the enzymatic functions of transketolase A, transketolase B and glucose transphase (PtsG) of the present invention are lost, xylose and glucose In the ratio of 3: 5 to 5: 3 was used, the yield, yield and productivity of ribose were improved.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing ribose in a medium in which xylose and glucose are optimally mixed using metabolic engineering modified E. coli,

The present invention relates to a method for producing ribose using metabolically engineered Escherichia coli. More particularly, the present invention relates to a method for producing ribose by using metabolically engineered Escherichia coli (Escherichia coli) in an optimum ratio of xylose and glucose ribose) with high productivity.

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, no technology has been disclosed specifically for producing ribose using E. coli in the past. However, the inventors of the present invention have filed a patent application on Nov. 18, 2011 in Korean Patent Application No. 10-2011-0116023, entitled " Method for Producing Ribose Using Metabolically Modified Escherichia coli. "

However, in the patents filed by the inventors of the present invention, the optimum ratio of xylose and glucose required for increasing the productivity of ribose has not been considered.

Therefore, in the present invention, in the production of ribose using metabolic engineering modified E. coli, the optimum ratio of xylose and glucose affecting the productivity of ribose is determined, and on the basis of this, ribose is produced with high productivity .

The present invention relates to a method for producing ribosomes from xylose by inoculating Escherichia coli into a medium containing xylose and glucose, , Transketolase A, transketolase B and glucose phosphate transferase (PtsG) in the medium containing recombinant Escherichia coli, and the medium containing xylose and glucose , And the ratio of xylose to glucose is 3: 5 to 5: 3.

On the other hand, in the ribosome production method of the present invention, the medium containing xylose and glucose preferably has a ratio of xylose to glucose of 5: 5. This is because it shows optimal ribose productivity at this ratio.

Meanwhile, in the ribosome production method of the present invention, enzyme function loss of transketolase A or transketolase B or glucose phosphate transferase (PtsG) is preferably inhibited by transketolase A or It is preferable that a part of the gene encoding transketolase B or the transglucosyltransferase (PtsG) is broken or all of the gene is removed. It is because the disruption of a part of the gene or the removal of the whole part is a way to completely remove the activity of the enzyme rather than inhibiting the activity of the enzyme in the cell by other methods.

Hereinafter, the contents of the present invention will be described in more detail.

Escherichia coli, also called coli, coli ) is an industrially used strain, but it has not yet been known how to produce ribose from xylose There is no bar. Because, in the case of wild-type Escherichia coli, metabolic engineering does not produce ribose using xylose as a substrate.

Therefore, in order to produce ribose from xylose using E. coli, In the experiment of the present invention, it was confirmed that ribose was produced by eliminating the enzymatic function of transketolase A, transketolase B, and glucose phosphate transferase (PtsG) in metabolic engineering transformation. The metabolic pathway for the production of ribose is established in E. coli by the disappearance of transketolase A and transketolase B, and the production of glucose and xylose by the loss of glucose phosphate transferase (PtsG) Simultaneous inflow is possible.

Escherichia coli exhibits glucose repression that does not consume other carbon sources until glucose is consumed, when glucose is present with other carbon sources. Therefore, in the presence of glucose in the medium, xylose, which is a substrate for ribose production, can not be introduced into cells, resulting in a great problem of increasing time for production of ribose, that is, lowering of productivity. In the present invention, such a problem can be solved through the loss of the PtsG enzyme function.

In the present invention, in the production of ribose using recombinant Escherichia coli transformed such that the enzymatic functions of transketolase A, transketolase B and glucose transphase (PtsG) having the above-mentioned characteristics are lost, , The amount of production (concentration of production), the composition of the medium which can further improve the production yield and productivity, and more specifically, the content of xylose and glucose. As shown in Example 1 of the present invention, when the addition ratio of xylose and glucose was 3: 5 to 5: 3, the initial concentration of xylose and glucose was varied. Production density, production yield and productivity were all excellent. Especially, it was highest at 5: 5.

When producing ribose using transformed recombinant Escherichia coli so that the enzymatic functions of transketolase A, transketolase B and glucose transphosphorylase (PtsG) are lost, xylose and glucose are mixed in a ratio of 3: 5 to 5: 3 showed improved productivity, production yield and productivity of ribose compared with the other cases.

FIG. 1 is a schematic diagram of a final culture solution of SGK016 'in which both the PtsG coding gene and the transketolase B coding gene are deleted, and SGK016' in which both the PtsG coding gene and the transketolize A coding gene and the transketolase B coding gene are deleted ≪ / RTI >
FIG. 2 is an HPLC chromatogram of a final culture of Escherichia coli SGK016 'and a ribose and ribulose standard material in which both the PtsG coding gene, the transketolize A coding gene and the transketolase B coding gene are deleted.
FIG. 3 shows results of analysis of various analytical parameters of the culture broth after fermentation of Escherichia coli SGK016 in a basic medium supplemented with 5 g / L xylose and 5 g / L glucose through HPLC chromatogram.

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

[ Manufacturing example  1: Preparation of recombinant Escherichia coli for ribose production]

 (One) ptsG  Gene deletion

For the production of the ribose, but are not essential, Physicochemical substance inhibiting (catabolite repression) the release was deleted by the ptsG gene coding for the wild state in the E. coli MG1655 PtsG to allow simultaneous inflow cell within the agarose by geulrukoohseugwa characters.

(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.

Furthermore, since the plasmid pCP20 has a temperature-sensitive replication origin and a high temperature inducible promoter, the FRT sequence-specific recombinase induces expression at a high temperature. 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)

After introducing the plasmid pKD46 into the mutant Escherichia coli SGK014 prepared above, a competent cell was prepared, and the kanamycin cassette for deletion of the tktA gene 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 Name of the primer Primer sequences (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

[ Experimental Example  One: Manufacturing example  Production of ribose using recombinant Escherichia coli produced in 1]

The mutant strain Escherichia coli SGK016 produced in Preparation Example 1 was used to confirm the ability to produce ribose.

A culture medium containing 5 g / L of glucose, 5 g / L of xylose, 5 g / L of yeast extract, 10 g / L of tryptone and 10 g / L of sodium chloride was used for culturing. After culturing at 37 DEG C and 200 rpm for 12 hours using a 500 mL baffled flask, the concentration of cells, ribose, xylose and glucose were measured by taking the final culture solution.

The cell concentration was measured by measuring absorbance at a wavelength of 600 nm, and ribose, xylose, glucose and ribulose concentrations were 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.

On the other hand, in this experiment, mutant strain Escherichia coli SGK014 in which only the activity of PtsG and transketolase B was lost was used as a control. Culture and analysis were carried out under the same conditions as above.

As a result of the experiment, ribose was not detected in the culture medium of the mutant Escherichia coli SGK014 in which the activity of PtsG and transketolase B was deleted as shown in Fig. 1, and ribose was detected only in the mutant Escherichia coli SGK016 in which both PtsG, transketolize A and transketolize B were lost. Was produced.

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 of the experiment, the newly produced material in the culture medium of mutant Escherichia coli SGK016 showed the same residence time as the ribose standard material and showed different residence time with LiBuRoS.

Therefore, it was confirmed that the substance newly produced in the culture solution of the mutant Escherichia coli SGK016 of the present invention is ribose, which is not ribulose (Fig. 2).

[ Example  1: for ribose production Glucose Xylose  Determine Optimal Ratio]

 (1) E. coli for ribose production

In this Example, Escherichia coli strain SGK016, in which the ability to produce ribose was confirmed through the above Experimental Example 1, was used as a fermentation strain. Escherichia coli strain SGK016 is a strain in which the transketolase A and transketolase B genes are deleted for ribose biosynthesis and the PtsG gene is further deleted so that glucose and xylose can be co- to be.

 (2) Basic medium composition for ribose production

For the pre-culture or main culture, a medium of 5 g / L of glucose, 5 g / L of xylose, 5 g / L of yeast extract, 10 g / L of tryptone and 10 g / L of sodium chloride was used (Table 2). In this culture, however, the change was observed while various ratios of glucose and xylose were controlled.

The composition of the basic culture medium of the ribose producing strain ingredient Concentration (g / L) Yeast extract 5 Tryptone 10 Sodium chloride 10 Xylose 5 Glucose 5

 (3) Pre-culture method

Escherichia coli SGK016, which was stored in a 10% glycerol solution, was slowly dissolved in a cryogenic refrigerator (-80 ° C) at room temperature. Then, the cells were inoculated into the above basic culture medium and cultured at 37 ° C for 12 hours with shaking at 200 rpm.

 (4) The present culture method

The pre-cultured Escherichia coli SGK016 was inoculated in a 5 L fermenter containing 2 L of medium at 1% of the total volume, and cultured at 37 ° C, 500 rpm, 1 vvm, and the pH was not adjusted. The medium used was a medium having the same composition as that of the pre-culture, and only the ratio of xylose to glucose was 1/5, 3/5, 5/5, 5/3, 5/1 (xylose concentration / glucose concentration) Respectively.

 (4) Analysis method

The cell concentration was measured by measuring the absorbance at a wavelength of 600 nm, and the concentration of ribose, xylose and glucose 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 third distilled water, and the column temperature was 25 ° C at a flow rate of 0.4 mL / min.

 (5) The added xylose Wow  Effect of ratio of glucose

In this example, the ratio of xylose to glucose was varied using a fermenter and its effect was examined. The culture medium used was the above-mentioned basic medium in which the concentrations of xylose and glucose were varied at 1/5, 3/5, 5/5, 5/3, and 5/1 (xylose concentration / glucose concentration) Respectively.

As a result of experiments, ribose concentration, yield, and productivity were superior to other ratios in the ratio of 3/5 to 5/3 of xylose and glucose. In particular, when cultured at a ratio of 5/5, the results were the best in terms of ribose concentration, yield, and productivity. In addition, lowering of the glucose ratio resulted in decreased cell growth, decreased ribose concentration, yield and productivity. Glucose was consumed in all experimental groups at the end of fermentation, and xylose was no longer converted to ribose after glucose consumption (Fig. 3). The overall test results are shown in Table 3 and FIG. 3 below.

Effect of ratio of xylose and glucose added Xylose / Glucose (g / L) O.D (600 nm) Ribose (g / L) Residual xylose (g / L) Yield (%) Productivity (g / L · hr) 1/5 7.935 0.48 0 37.63 0.01 3/5 7.62 1.435 0.555 51.11 0.03 5/5 6.165 2.72 1.005 75.98 0.06 5/3 4.97 2.185 2.15 71.14 0.045 5/1 2.365 0.87 2.355 35.52 0.02

Claims (3)

In producing a ribose from xylose by inoculating Escherichia coli into a medium containing xylose and glucose,
The Escherichia coli was transformed with recombinant E. coli transformed to eliminate the enzymatic functions of transketolase A, transketolase B and glucose phosphate transferase (PtsG)
Wherein the medium containing xylose and glucose has a ratio of xylose to glucose of from 3: 5 to 5: 3.
The method according to claim 1,
The culture medium containing xylose and glucose may contain,
Wherein the ratio of xylose to glucose is 5: 5.
The method according to claim 1,
The enzymatic loss of the transketolase A or transketolase B or glucose phosphate transferase (PtsG)
Wherein a part of a gene encoding transketolase A or transketolase B or a transglucosyltransferase (PtsG) is cleaved or all of the gene is removed.

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