Method for Producing L-Citrulline by Using a Recombinant Corynebacterium crenatum Strain

Abstract

Disclosed is a method for producing L-citrulline using recombinant Corynebacterium crenatum cells as whole-cell biocatalysts. The present invention provides a recombinant C. crenatum that expresses an exogenous arginine deiminase gene from Lactobacillus brevis. The recombinant C. crenatum SDNN403 is used as biocatalysts for converting L-arginine to produce L-citrulline. Using the method of the invention, the concentration of L-citrulline reached 301.4 g/L after a 48 hr conversion reaction, and the molar conversion rate reached 99.9%.

Claims

1. A recombinant Corynebacterium crenatum SDNN403 strain with expression of an exogenous arginine deiminase gene, wherein the recombinant Corynebacterium crenatum SDNN403 strain is made by the following steps: ligating an arginine deiminase gene arcA to plasmid pXMJ19 to make a pXMJ19-arcA plasmid; and transferring the pXMJ19-arcA plasmid to Corynebacterium crenatum SDNN403 to obtain the recombinant Corynebacterium crenatum SDNN403 strain with expression of an exogenous arginine deiminase gene.

2. The method of claim 1, wherein said arginine deiminase gene is from Lactobacillus brevis, having a sequence of SEQ ID NO: 1.

3. A method for L-citrulline production comprises producing L-citrulline in a reaction system that uses L-arginine as a substrate and the recombinant cells of claim 1 as whole-cell biocatalysts.

4. The method of claim 3, wherein said reaction system comprises phosphate buffer at pH 6.0-7.0.

5. The method of claim 3, wherein concentration of the recombinant cells is at OD.sub.600=7-8 and concentration of L-arginine is 80-100 g/L.

6. The method of claim 3, wherein the reaction system has a temperature of 40-45 C.

7. The method of claim 3, wherein the L-arginine concentration in the reaction system is maintained at 60-100 g/L during the process of L-citrulline production.

8. The method of claim 3, wherein the reaction system contains 1.0 mM Mn.sup.2+ and/or 1.0 mM mg.sup.2+.

9. The method of claim 3, wherein the reaction system contains a pH 6.4 phosphate buffer, 100 g/L arginine, 1.0 mM Mn.sup.2+, and 1.0 mM Mg.sup.2+; and wherein the production process is performed under 40-45 C. with the concentration of L-arginine maintained at 60-100 g/L.

10. The method of claim 3, wherein the reaction system contains 100 g/L L-arginine, 1.0 mM Mn.sup.2+, and 1.0 mM Mg.sup.2+; and wherein the production process is performed at 45 C. with the concentration of L-arginine maintained at 60-100 g/L.

Description

DETAILED DESCRIPTION

Materials and Methods:

[0020] Assay for Determination of Arginine Deiminase Activity:

[0021] The principle of the assay: the activity of arginine deiminase is determined by the citrulline conversion from L-arginine. There is a good linear relationship between the enzyme activity and citrulline concentrations in solutions containing 0-20 mg/L citrulline. The carbamido group of citrulline reacts with diacetylmonoxime (DAM) under acid conditions and condensates to form red oxadiazole compounds, which is detected by a colorimetric method.

[0022] Method for enzyme activity determination: dissolve L-arginine in 0.2 M phosphate buffer, pH 6.5 to prepare a 0.2 M L-arginine solution. Mix 1.8 ml L-arginine solution and 0.2 ml enzyme solution to react under 37 C. for 10 min. Dilute the resulting solution for 200-2000 times, and mix 2 ml diluted solution with 3 ml iron-acid solution and 0.5 ml mixed solution of diacetylmonoxime and aminothiourea. Shake the mixture and immediately put it a boiling water bath for 10 min, followed by determining the absorbance at the wavelength of 530 nm.

[0023] Preparation of the mixed solution of diacetylmonoxime and aminothiourea: dissolve 1 g diacetylmonoxime and 60 mg aminothiourea in 100 ml water.

[0024] Preparation of the iron-acid solution: concentrated phosphoric acid 70 ml, concentrated sulfuric acid 160 ml and 5 ml 10 mg/ml FeCl.sub.3 are dissolved in water to obtain a solution with a final volume of 1000 ml.

[0025] Determination of L-arginine and L-citrulline By Chromatography:

[0026] (a) chromatographic column: Agilent TC-C18 column 250 mm4.6 mm5 m;

[0027] (b) column temperature: 40 C.;

[0028] (c) mobile phase: preparation of phase A: 8 g sodium acetate is dissolved in 1000 ml water, 225 ml triethylamine is then added, and the pH of the mixture is adjusted to 7.200.05 using 5% acetic acid. 5 ml tetrahydrofuran is then added and mixed. Preparation of phase B: 12 g sodium acetate is dissolved in 400 ml water. The pH of the solution is adjusted to 7.200.05 by 5% acetic acid, and the resulting solution is mixed with 800 ml acetonitrile and 800 ml of methanol.

[0029] (d) detector: the wavelength of the UV detector is 338 nm.

EXAMPLE 1

Primer Design for Cloning of Arginine Deiminase Gene

[0030] The forward primer F and the backward primer R for cloning an arginine deiminase gene were designed according to the arcA gene sequence of Lactobacillus brevis disclosed by NCBI GenBank database.

TABLE-US-00001 PrimerF(SEQIDNO:2): 5-ACCCGAAGCTTATGACAAGTCCGATTCACGTAATG- 3 (HindIII) PrimerR(SEQIDNO:3): 5-ACCGGAATTCTTAAAGGTCTTCTCGAACTAATGGC- 3 (EcoRI)

EXAMPLE 2

Cloning of the Arginine Deiminase Gene

[0031] The genome DNA of Lactobacillus brevis was used as a template for cloning the arginine deiminase gene. The primers designed in Example 1 were used for the PCR amplification. The PCR amplification conditions were: 5 min at 94 C.; 35 cycles of 1 min at 94 C., 1 min, at 56 C., 1 min at 72 C.; and 10 min at 72 C. PCR systems was carried out in a 50 Ill reaction containing: 1 l DNA template, 0.4 l each forward and backward primers, 4 l dNTP Mix, 5 L 10ExTaq Buffer, 37 l of sterile double distilled water, and 1 l ExTaq DNA polymerase. The resulting product was purified by a gel extraction kit, and preserved in a 1.5 ml EP at 20 C. for further use. The purified PCR product was ligated to pMD18-T cloning vector, and transformed to E. coli JM109 cells. The resulting cells were plated on a LB medium with Ampicillin and incubated at 37 C. overnight. The resulting colonies were picked and transferred to 10 ml liquid LB medium, and incubated at 37 C. overnight. The DNA of the cultured bacteria were extracted and verified by sequencing. The plasmids containing the correct arginine deiminase gene were named as pMD18-T-arcA, and conserved in 15%-20% (w/v) glycerol at 70 C.

EXAMPLE 3

Construction of Recombinant Plasmid pXMJ19-arcA

[0032] The recombinant plasmid pMD18-T-arcA and the pXMJ19 were digested by EcoRI and HindIII. The resulting DNA fragment were purified by a gel extraction kit and ligated in a 10 l ligation system containing 1 l digestion product from pXMJ19, 7 l digestion product from pMD18-T-arcA, 1 l T4 DNA ligase buffer, and 1 l T4 DNA ligase. The ligation was performed under 16 C. for overnight. The ligated recombinant plasmid pXMJ9-arcA was transformed into E. coli JM 109 competent cells, and incubated in LB culture medium containing chloroamphenical for 10 hr. The positive colonies were picked and transferred to 10 ml LB liquid medium. After incubated at 37 C. for overnight, the plasmid was extracted and named as pXMJ9-arcA. After the sequence verification, the correct plasmid pXMJ9-arcA was preserved in 15%-20% (w/v) glycerol at 70 C.

EXAMPLE 4

Construction of Recombinant C. crenatum SDNN 403/pXMJ9-arcA

[0033] Competent preparation: C. crenatum SDNN403 were picked and inoculated into a 10 ml LBG (LB+0.5% glucose) medium, and incubated at 30 C. overnight. 500 l culture broth were transferred into 50 ml LB medium containing 3% glycine and 0.1% Tween-80 to make the initial cell concentration at 0.3 (OD.sub.600 value), and then incubated at 30 C., 200 r/min until OD.sub.600 reached 0.9. The culture was cooled for 15 min after the fermentation finished, the culture was then centrifuged and washed for 4 times using precooled 10% glycerol. After that, the cells were dispensed in 0.2 ml 10% glycerol, and the resulting cell suspension was conserved at 80 l/1.5 ml EP tube.

[0034] Electrotransformation: the electrotransformation was carried out at 1800V for 5 ms. The resulting cell suspension was transferred to 800 l LBG medium and incubated at 30 C. for 2-3 hr.

[0035] The harvest of recombinant C. crenatum SDNN 403/pXMJ9-arcA: the electrotransformed cells were plated on LGB medium containing chloramphenicol, and incubated at 30 C. Positive colonies were picked, and the plasmid DNAs of the picked colonies were extracted and verified by sequencing. The correct colony was named recombinant C. crenatum SDNN403/pXMJ19-arcA.

EXAMPLE 5

Determination of Arginine Deiminase Activity in Recombinant C. crenatum SDNN403/pXMJ19-arcA

[0036] The recombinant C. crenatum SDNN403/pXMJ19-arcA was inoculated into LBG medium containing 10 ml chloramphenicol, incubated at 30 C. overnight, and then transferred at 1% inoculation rate to 50 ml LBG medium. The cells were incubated at 30 C. for 8 hr until the OD.sub.600 reached to 0.9. The cells were then added with 0.7 mmol/L (final concentration) IPTG and incubated at 30 C. for 8 hr. The cells were harvested by centrifugation at 10000 rpm for 10 min, and then washed by Tris-HCl buffer for 3 times before resuspended in 5 ml Tris-HCl buffer (pH7.0). The cell suspension was sonicated to obtain the crude enzyme preparation.

[0037] The C. crenatum SDNN403 was used as control and treated according to the same procedure above.

[0038] 1.8 ml 0.2 M L-arginine (pH 6.4, dissolved in 0.2 M phosphate buffer) and 0.2 ml crude enzyme solution was mixed and incubated at 37 C. for 10 min. The resulting solution was diluted to 200-2000-fold, and 2 ml diluted reaction solution was mixed with 3 ml iron-acid solution and 0.5 ml mixed solution of diacetylmonoxime and aminothiourea. The mixed solution was then immediately incubated in a boiling water bath for 10 min, followed by the determination of absorbance values at the wavelength of 530 nm.

[0039] Result showed that the arginine deiminase produced by recombinant C. crenatum SDNN403/pXMJ19-arcA was 2.56 U/mg cell mass, while no arginine deiminase activity was detected in C. crenatum SDNN 403.

EXAMPLE 6

Methods for L-citrulline Production Using Whole-Cell Biocatalysts

[0040] The recombinant C. crenatum SDNN403/pXMJ19-arcA cells were inoculated in 200 ml ADI-producing medium (g/L: glucose, 40; yeast extract 8; KH.sub.2PO.sub.4, 2.5; K.sub.2HPO.sub.4, 2.3; MgSO.sub.4, 0.5) and cultured for 8 hr. The cells were cultured for another 8 hr with addition of 0.7 mM IPTG for induction of arginine deiminase expression, and the cells were collected by centrifugation.

[0041] Cells were washed twice by pH 7.0 Tris-HCl and resuspended in 200 ml 0.2 M phosphate buffer (pH 6.4) containing 100 g/L L-arginine, at a cell concentration at OD.sub.600=7-8.

[0042] The whole-cell catalytic reaction was carried out under different temperatures between 20 to 60 C. (the interval was set to be 5 C.). The results showed that the molar conversion rate was only 45.7% of the maximum rate under the temperature of 20 C. The conversion rate increased when temperature rose from 20 to 45 C. When the temperature was higher than 45 C., the conversion rate reduced sharply as the temperature increases. The conversion rate was only 29.3% of the maximum rate at the temperature of 60 C.

[0043] The whole-cell catalytic reaction was carried out under different pHs. An acetate buffer (pH 3.5-5.0), a Na.sub.2HPO.sub.4-citric acid buffer (pH 5.0-6.0), a phosphate-buffered saline (pH 6.0-8.0), a Tris-HCl buffer (pH 8.0-9.0), and a carbonate buffer (pH 9.0-10.5) were used as the testing buffer solutions. The results showed that the conversion rate was the relatively high under pH 6.0-7.0 with the conversion rate being the highest at pH 6.4. The conversion rate dropped to 76.3% of the maximum rate at pH 4.0. The conversion efficiency were relatively slow under alkaline conditions, indicating that the conversion reaction favors slightly acidic conditions. Different metal ions were added into whole-cell catalytic reaction system, including 1 mM Cu.sup.2+, Ca.sup.2+, Co.sup.2+, La.sup.3+, K.sup.+, Fe.sup.2+, Mg.sup.2+, Ba.sup.2+, Mn.sup.2+, Fe.sup.3+, Al.sup.3+, Zn.sup.2+, and Na.sup.+. The results showed that supplement of Mn.sup.2+ and Mg.sup.2+ ions significantly increased the conversion rate. The conversion rate were increased to 183.2% and 167.4% for Mn.sup.2+ and Mg.sup.2+ supplements, respectively. Other metal ions showed less activating effect on the conversion process.

[0044] The whole-cell catalytic reaction was carried out in 0.2 mol/L phosphate buffer (pH 6.4) containing 100 g/L L-arginine, 1.0 mM Mn.sup.2+, and 1.0 mM Mg.sup.2+ with the recombinant cell concentration of OD600=7-8. The conversion process was performed under a temperature of 45 C. with the L-arginine concentration maintained at 60-100 g/L. After 48 hr conversion, high performance liquid chromatography (HPLC) was used to determine the content of L-arginine and L-citrulline. The results showed that, 301.4 g/L L-citrulline was detected in the resulting solution, indicating a mole conversion rate from L-arginine to L-citrulline was 99.9%.

[0045] While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention. All figures, tables, appendices, patents, patent applications and publications, referred to above, are hereby incorporated by reference.