Variant of O-phosphoserine exporter and method of producing O-phosphoserine, cysteine, and its derivatives using the same

Abstract

The present disclosure relates to a novel polypeptide having O-phosphoserine (OPS) exporting activity, a polynucleotide encoding the polypeptide, a microorganism expressing the polypeptide, a method for producing OPS using the microorganism, and a method for producing cysteine or a derivative thereof comprising reacting the O-phosphoserine produced by the same with a sulfide, in the presence of O-phosphoserine sulfhydrylase (OPSS) or a microorganism expressing the same.

Claims

1. A polypeptide having O-phosphoserine (OPS) exporting activity, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5.

2. A polynucleotide encoding the polypeptide of claim 1.

3. The polynucleotide according to claim 2, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6.

4. A microorganism of the genus Escherichia producing O-phosphoserine, wherein the microorganism expresses the polypeptide of claim 1.

5. The microorganism according to claim 4, wherein an activity of phosphoserine phosphatase (SerB) is further weakened compared to its endogenous activity.

6. The microorganism according to claim 4, wherein an activity of phosphoglycerate dehydrogenase (SerA) or phosphoserine aminotransferase (SerC) is further enhanced compared to its endogenous activity.

7. The microorganism according to claim 4, wherein the microorganism of the genus Escherichia is Escherichia coli.

8. A method for producing O-phosphoserine (OPS) comprising culturing the microorganism of claim 4 in a medium.

9. A method for producing cysteine or a derivative thereof, comprising: culturing the microorganism of claim 4 in a medium to produce O-phosphoserine (OPS); and reacting said O-phosphoserine with a sulfide, in the presence of O-phosphoserine sulfhydrylase (OPSS) or a microorganism expressing the same.

10. The method according to claim 9, 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

MODE FOR INVENTION

(1) Hereinafter, the present disclosure will be described in more detail with reference to the following Examples. However, these Examples are for illustrative purposes only, and the disclosure is not intended to be limited by these Examples.

Example 1

Preparation of YhhS Major Facilitator Superfamily (MFS) Transporter Variants

(2) In order to improve the activity of the OPS exporter for the improvement of OPS-exporting activity in an OPS-producing strain, variants were prepared for the YhhS major facilitator superfamily (MFS) transporter (SEQ ID NO: 23), a newly identified OPS-exporter protein, and yhhS (SEQ ID NO: 24), a gene encoding the same. The detailed process is described herein below.

(3) First, a library of yhhS gene variants was constructed. To this end, a random mutagenesis PCR (JENA error-prone PCR) was performed using a gene-specific primer pair (SEQ ID NOS: 7 and 8) based on the genomic DNA of Escherichia coli K12_W3110 (ATCC27325) as a template. The thus-prepared gene fragments from the mutagenesis were cloned into a pCLPrhtB vector, wherein rhtB promoter (SEQ ID NO: 13), which was subjected to PCR using a gene-specific primer pair (SEQ ID NOS: 14 and 15), was inserted into the SacI-EcoRV site of a pCL1920 vector (GenBank No AB236930). Specifically, the pCLPrhtB vector was cut with EcoRV and PstI, and then the gene fragments from the mutagenesis were cloned thereinto using In-fusion Cloning Kit (Clontech Laboratories, Inc.). The cloning was performed at 50 C. for 10 minutes, thereby completing the construction of plasmid libraries of pCL PrhtB yhhS variants.

(4) The thus-constructed recombinant plasmid libraries were screened via high throughput screening (HTS). In particular, the platform strain used for screening was CA07-0012 (KCCM11121P), which is a recombinant microorganism modified to reduce the activity of endogenous phosphoserine phosphatase (SerB) in the wild-type E. coli strain W3110 (Korean Patent No. 10-1381048; US Patent Application Publication No. 2012-0190081).

(5) Subsequently, in order to obtain variants with improved OPS-exporting activity, the thus-constructed plasmid libraries were transformed into the platform strain CA07-0012 via electroporation, cultured in media containing an excess amount of OPS, and three colonies where the growth inhibition was released were selected. Then, plasmids were obtained from the three selected colonies and analyzed via sequencing technology.

(6) As such, three yhhS variants which are involved in the removal of growth inhibition under the OPS-added condition were selected, and these were named as yhhS M2, yhhS M25, and yhhS M45, respectively.

(7) Upon analysis of the nucleotide sequences of the yhhS M2, yhhS M25, and yhhS M45, it was confirmed that yhhS M2 has the amino acid sequence represented by SEQ ID NO: 1, yhhS M25 has that represented by SEQ ID NO: 3, and yhhS M45 has that represented by SEQ ID NO: 5.

Example 2

Confirmation of OPS-Exporting Activity by YhhS Variants in OPS-Producing Strain

(8) 2-1. Construction of a Strain with Enhanced YhhS MFS Transporter Using CA07-0012 and Evaluation of OPS-Producing Capability

(9) The plasmids containing the three different variants identified in Example 1 were respectively transformed into CA07-0012, the OPS-producing strain, by an electric-pulse method conventionally used in the art. As such, OPS-producing strains introduced with the yhhS variants, i.e., CA07-0012/pCL-PrhtB-yhhS M2, CA07-0012/pCL-PrhtB-yhhS M25, and CA07-0012/pCL-PrhtB-yhhS M45, were constructed, and these were named as Escherichia coli CA07-0345, Escherichia coli CA07-0344, and Escherichia coli CA07-0346, respectively. Additionally, the Escherichia coli CA07-0344, Escherichia coli CA07-0345, and Escherichia coli CA07-0346 strains were deposited with the Korean Culture Center of Microorganisms (KCCM), recognized as an international depositary authority under the Budapest Treaty, on Jul. 23, 2015, under the Accession Numbers of KCCM11736P, KCCM11737P, and KCCM11738P, respectively.

(10) The OPS-producing capabilities of the corresponding strains were evaluated.

(11) Specifically, each strain was plated out on a solid LB medium and cultured in a 33 C. incubator overnight. The strains cultured in the solid LB medium overnight were inoculated into a 25 mL titer medium shown in Table 1 below and cultured in a 34.5 C. incubator at a rate of 200 rpm for 48 hours. The results are shown in Table 2 below.

(12) TABLE-US-00001 TABLE 1 Composition Conc. (per 1 L) Glucose 50 g KH.sub.2PO.sub.4 6 g (NH.sub.4).sub.2SO.sub.4 17 g MgSO.sub.47H.sub.2O 1 g FeSO.sub.47H.sub.2O 5 mg MnSO.sub.44H.sub.2O 10 mg L-Glycine 2.5 g Yeast extract 3 g Calcium carbonate 30 g pH 6.8

(13) TABLE-US-00002 TABLE 2 OD Glucose O- 562 consumption Phosphoserine Strain nm (g/L) (g/L) CA07-0012/pCL-PrhtB-yhhS (wt) 23.9 40.5 1.38 CA07-0345 29.4 42.8 1.78 (CA07-0012/pCL-PrhtB-yhhS M2) CA07-0344 32.7 44.3 2.28 (CA07-0012/pCL-PrhtB-yhhS M25) CA07-0346 27.0 42.8 2.16 (CA07-0012/pCL-PrhtB-yhhS M45)

(14) As shown in Table 2 above, in the case of strains introduced with yhhS variants of the present disclosure, these showed an excellent result demonstrating an increase of OPS production by from 128% to 165%, compared to the strain introduced with a wild-type yhhS gene. Specifically, the yhhS M2 variant showed a 128% increase, the yhhS M25 variant showed a 165% increase, and the yhhS M45 variant showed a 156% increase compared to that of the wild-type, respectively.

(15) 2-2. Construction of a Strain with Enhanced YhhS MFS Transporter Using Strains with Enhanced SerA and SerC and Evaluation of OPS-Producing Capability

(16) In order to reconfirm the activity of yhhS variants of the present disclosure, CA07-0022/pCL-Prmf-serA*(G336V)-serC (KCCM11103P, Korean Patent No. 10-1381048), which, being an OPS-producing strain with improved OPS-producing capability, has enhanced activity of D-3-phosphoglycerate dehydrogenase (SerA) and 3-phosphoserine aminotransferase (SerC) as OPS biosynthesis pathways, was used.

(17) For the construction of a pCL-Prmf-serA(G336V)-serC PrhtB-genes vector, each of the yhhS variants was amplified using a primer pair (SEQ ID NOS: 9 and 10) based on the pCL-PrhtB-yhhS variant as a template, and the resultants were cloned into the HindIII restriction site of the pCL-Prmf-serA*(G336V)-serC vector.

(18) Specifically, the strains, where each of the plasmids was transformed by the conventionally used electric-pulse method, were plated out on a solid LB medium and then cultured in a 33 C. incubator overnight. The strains cultured in the solid LB medium overnight were inoculated into a 25 mL titer medium shown in Table 1 above and cultured in a 34.5 C. incubator at a rate of 200 rpm for 48 hours. The results are shown in Table 3 below.

(19) TABLE-US-00003 TABLE 3 OD Glucose O- 562 consumption Phosphoserine Strain nm (g/L) (g/L) CA07-0022/pCL-Prmf-serA*(G336V)- 28.9 40.5 3.79 (RBS)serC-PrhtB-yhhS(wt) CA07-0022/pCL-Prmf-serA*(G336V)- 28.2 42.8 4.96 (RBS)serC-PrhtB-yhhS M2 CA07-0022/pCL-Prmf-serA*(G336V)- 32.2 44.3 6.68 (RBS)serC-PrhtB-yhhS M25 CA07-0022/pCL-Prmf-serA*(G336V)- 30.7 42.8 5.01 (RBS)serC-PrhtB-yhhS M45

(20) As shown in Table 3 above, it was confirmed that when the yhhS variants of the present disclosure were introduced to an OPS-producing strain with an enhanced OPS biosynthesis gene, the OPS production was increased by from 130% to 176%. These results indicate that the yhhS variants of the present disclosure can be effectively used for OPS production.

(21) 2-3. Construction of a Strain with Enhanced YhhS MFS Transporter According to Promoter Strength on the Chromosome and Evaluation of OPS-Producing Capability

(22) Further to the above experiments, in order to confirm whether the OPS-exporting activity is improved when the yhhS variants are introduced on the chromosome, the self-promoter of the microorganism was substituted with the pCJ1 promoter (Korean Patent No. 10-0620092), and the strains introduced with the variants of the present disclosure were constructed and their OPS-producing capability evaluated. The introduction of the pCJ1 promoter and the variants into the chromosome was performed by the methods conventionally used in the art. First, for transformation, a recombinant vector was inserted into CA07-0022/pCL-Prmf-serA*(G336V)-serC (KCCM11103P, Korean Patent No. 10-1381048), the OPS-producing strain, by an electric-pulse method (Appl Microbiol Biotechnol. 1999 October; 52(4):541-5) as the primary step. Then, the strains inserted into the chromosome by recombination of homologous sequences were selected in a medium containing 25 mg/L kanamycin. The thus-selected primary strains were subjected to the secondary step of cross-over, and then the strains where the pCJ1 promoter and the variants were substituted and the vector was removed were selected.

(23) Finally, the presence of substitution of the promoter and the variants of the transformed strains were confirmed by performing a PCR using the primer pair (SEQ ID NOS: 11 and 12).

(24) Each of the strains was plated out on a solid LB medium and then cultured in a 33 C. incubator overnight. The strains cultured in the LB solid medium overnight were inoculated into a 25 mL titer medium shown in Table 1 above and cultured in a 34.5 C. incubator at a rate of 200 rpm for 40 hours. The results are shown in Table 4 below.

(25) TABLE-US-00004 TABLE 4 OD Glucose O- 562 consumption Phosphoserine Strain nm (g/L) (g/L) CA07-0022/pCL-Prmf- 30.0 37.1 1.7 serA*(G336V)-serC CA07-0022::Pcj1 yhhS (WT)/ 30.2 40.1 2.2 pCL-Prmf-serA*(G336V)-serC CA07-0022::Pcj1 yhhS M2/pCL- 39.9 43.2 2.9 Prmf-serA*(G336V)-serC CA07-0022::Pcj1 yhhS 35.3 44.1 3.8 M25/pCL-Prmf-serA*(G336V)- serC CA07-0022::Pcj1 yhhS 42.0 43.5 3.2 M45/pCL-Prmf-serA*(G336V)- serC

(26) As shown in Table 4 above, it was confirmed that when the activity of each protein variant was increased on the chromosome, the amount of OPS production by the protein variant showed a maximum of 172% compared to that of the strain introduced with the wild-type yhhS.

(27) 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.