MICROORGANISM OF THE GENUS ESCHERICHIA PRODUCING O-PHOSPHOSERINE AND A METHOD FOR PRODUCING O-PHOSPHOSERINE OR L-CYSTEINE USING THE SAME

Abstract

The present application relates to a microorganism producing O-phosphoserine and a method for producing O-phosphoserine, cysteine or a cysteine derivative using same.

Claims

1. A KCCM11815P microorganism producing O-phosphoserine (OPS), wherein the microorganism has resistance to O-phosphoserine.

2. A method for producing O-phosphoserine, comprising: culturing the microorganism of claim 1 in a medium; and recovering O-phosphoserine from the microorganism or the medium.

3. A method for producing cysteine or a derivative thereof, comprising: a) producing O-phosphoserine by culturing the microorganism of claim 1 in a medium; and b) reacting the O-phosphoserine produced in step a) or a medium comprising the same with a sulfide in the presence of O-phosphoserine sulfhydrylase (OPSS) or a microorganism expressing OPSS.

4. The method of claim 3, wherein the sulfide is at least one selected from the group consisting of Na.sub.2S, NaSH, (NH.sub.4).sub.2S, H.sub.2S, and Na.sub.2S.sub.2O.sub.3.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0034] Hereinafter, the present disclosure will be described in detail through exemplary embodiments. However, these exemplary embodiments are provided for the purpose of illustration only and are not intended to limit the scope of the present disclosure.

Example 1: Selection of a Mutant Strain by a Method of Artificial Mutation

[0035] In order to obtain a mutant strain of a microorganism in which the productivity of O-phosphoserine (OPS) is enhanced, the method below was used to induce a mutation of the microorganism.

[0036] Specifically, Escherichia coli W3110 wild-type, a parent strain, was seeded in LB liquid medium and incubated for 12 hours at 37 C. Subsequently, 1 mL of the above culture was seeded into 100 mL of liquid medium and cultured for 5 hours and 30 minutes at 37 C., followed by recovery of 50 mL of the culture medium. The recovered culture was washed with 100 mM citric buffer, followed by addition of N-methyl-N-nitro-N-nitrosoguanidine (NTG) to make a final concentration of 200 mg/L and treatment for 45 minutes, and then the culture was washed with 100 mM phosphate buffer.

[0037] Subsequently, in order to obtain an OPS-producing strain, 20 g/L KH.sub.2PO.sub.4 and 10 g/L glucose were added to the M9 minimal medium, and these were solidified, followed by plating the strain treated with the NTG. In particular, the mortality rate in the M9 minimal medium was 97%, and after incubation at 37 C. for 12 hours, an OPS-producing mutant strain was obtained.

[0038] The mutant strain obtained with the above method was named Escherichia coli CA07-0348 and deposited at the Korean Culture Center of Microorganisms under Accession No. KCCM11815P on Feb. 26, 2016, under the Budapest treaty.

Composition of M9 Minimal Medium

[0039] Na.sub.2HPO.sub.4 6.78 g/L, KH.sub.2PO.sub.4 3 g/L, NH.sub.4Cl 1 g/L, NaCl 0.5 g/L, glucose 10 g/L, and agarose 15 g/L

Example 2: Investigation of OPS Productivity of OPS-Producing Mutant Strain

[0040] The following experiments were performed to confirm the OPS productivity of Escherichia coli CA07-90348, a mutant strain obtained from Example 1 above.

[0041] In order to enhance the OPS biosynthetic pathway, a pCL-Prmf-serA*(G336V)-(RBS)serC (Korean Patent No. 1381048) vector was transformed into a W3110 wild-type strain and the CA07-0348 strain above with a conventionally used electric pulse method. Each strain was plated on LB solid medium and cultured overnight in a 33 C. incubator. The strain cultured overnight in LB solid was seeded in 25 mL of the below titration medium, and incubated again at 34.5 C. at 200 rpm for 30 hours. After the incubation, the amount of OPS production was measured using high-performance liquid chromatography, and the OPS concentration in the culture medium for each strain tested is shown in Table 1 below.

[0042] The composition of the titration medium used in present Example 2 is as follows.

Titration Medium

[0043] Glucose 50 g/L, yeast extract 0.3 g/L, glycine 2.5 g/L, KH.sub.2PO.sub.4 6 g/L, (NH.sub.4).sub.2SO.sub.4 17 g/L, MgSO.sub.4.7H.sub.2O 1 g/L, FeSO.sub.4.7H.sub.2O 5 mg/L, MnSO.sub.4.4H.sub.2O 10 mg/L, and CaCO.sub.3 30 g/L

TABLE-US-00001 TABLE 1 Glucose Consumption O-Phosphoserine Yield Strain OD.sub.560 nm (g/L) (g/L) (%) W3110 21.9 40 0.0 0 CA07-0348 20.8 8.7 1.23 14 W3110/ 25.1 45.3 0.0 0 pCL-Prmf-serA*(G336V)-(RBS)serC CA07-0348/ 26.6 41.4 2.3 5.6 pCL-Prmf-serA*(G336V)-(RBS)serC

[0044] As can be seen in Table 1 above, the wild-type strain, W3110, never produced OPS, while the mutant strain of the present disclosure, CA07-0348, was confirmed to produce OPS at a concentration of 1.23 g/L. Additionally, in the case where the biosynthetic pathway was enhanced by transformation, the wild-type never produced OPS, while the CA07-0348 strain produced OPS at a concentration of 2.3 g/L, and it was confirmed that the amount of OPS increased before the transformation.

Example 3: Investigation of OPS Resistance of OPS-Producing Mutant Strain

[0045] In order to confirm the OPS resistance of Escherichia coli CA07-0348, the OPS-producing mutant strain obtained from Example 1 above, culture was performed with the following method.

[0046] Escherichia coli W3110, a parent strain, and the above mutant strain were seeded in a 15 mL disposable tube containing 2 mL of LB medium and incubated with shaking at 37 C. at 200 rpm for 12 hours, and then the seed culture was obtained. After washing the cultured strain once with phosphate-buffered saline, 100 t of the seed culture was seeded in a 50 mL disposable tube containing 5 mL of screening medium and incubated at 37 C. at 200 rpm for 20 hours.

[0047] The composition of the screening medium used in present Example 3 is as follows.

Screening Medium

[0048] Glucose 10 g/L, LB 100 g/L, H.sub.3PO.sub.4 1 g/L, OPS 100 g/L, KOH 0.27 M, and NaOH 0.27 M, pH 7.0

[0049] After the incubation, optical density (hereinafter referred to as OD) was measured by a spectrophotometer, and the OD of the culture medium for each strain tested is shown in Table 2 below.

TABLE-US-00002 TABLE 2 Comparison of OD of Escherichia coli CA07-0348 in screening medium W3110 CA07-0348 Optical density (OD.sub.560 nm) 0.20 0.39

[0050] As a result, as shown in Table 2 above, Escherichia coli W3110, a parent strain, showed a measured OD value of 0.20 when cultured in a medium (screening medium) containing 100 g/L of OPS for 20 hours, while the mutant strain of Escherichia coli CA07-0348 according to the present disclosure showed an OD value of 0.39, thereby confirming that the growth rate was about twice as fast.

[0051] The results above indicate that the mutant strain of Escherichia coli CA07-0348 showed resistance to 100 g/L of OPS, resulting in less inhibition of cell growth. Accordingly, the microorganism of the present disclosure can be advantageously used in the mass production of OPS.

[0052] From the foregoing, a skilled person in the art to which the present disclosure pertains will be able to understand that the present disclosure may be embodied in other specific forms without modifying the technical concepts or essential characteristics of the present disclosure. In this regard, the exemplary embodiments disclosed herein are only for illustrative purposes and should not be construed as limiting the scope of the present disclosure. On the contrary, the present disclosure is intended to cover not only the exemplary embodiments but also various alternatives, modifications, equivalents, and other embodiments that may be included within the spirit and scope of the present disclosure as defined by the appended claims.