Recombinant microorganism for producing crocin and method for producing crocin using the same

Abstract

The present disclosure relates to a recombinant microorganism for producing crocin in which a gene (CCD2) encoding carotenoid cleavage dioxygenase, a gene (aldH) encoding crocetin dialdehyde dehydrogenase and a gene (UDP-glycosyltransftrase, UGT) encoding crocin biosynthesis enzyme are introduced, and a method for producing crocin using the same. Compared with the conventional method for producing crocin, which is produced in small amounts through a part of plants or callus, the production method using the recombinant microorganism of the present disclosure enables mass production of crocin.

Claims

1. A method for producing crocin, comprising culturing a recombinant microorganism, wherein the recombinant microorganism is prepared by introducing a gene encoding carotenoid cleavage dioxygenase 2 (CCD2) comprising the amino acid sequence of SEQ ID NO: 1, a gene encoding crocetin aldehyde dehydrogenase (aldH) comprising the amino acid sequence of SEQ ID NO: 2, and a gene encoding UDP-glycosyltransferase (UGT) comprising the amino acid sequence of SEQ ID NO: 3, into a microorganism which produces zeaxanthin.

2. The method of claim 1, further comprising recovering crocin from the cultured recombinant microorganism or its culture.

3. The method of claim 1, wherein the microorganism is from the genus Saccharomyces or the genus Escherichia.

4. The method of claim 3, wherein the microorganism of the genus Saccharomyces is Saccharomyces cerevisiae.

5. The method of claim 3, wherein the microorganism of the genus Escherichia is Escherichia coli.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A and 1B illustrate a schematic diagram of a vector introduced into a strain in which the crocin-5 biosynthetic metabolic pathway constructed in E. coli and the MEP metabolic pathway and the zeaxanthin biosynthetic pathway were advanced.

(2) FIGS. 2A and 2B illustrate the results of HPLC analysis of recombinant microorganisms introducing the CsCCD2 gene and the crocetin dialdehyde biosynthetic metabolic pathway constructed in E. coli (Peak 1: crocetin dialdehyde).

(3) FIGS. 3A-3C illustrate the results of HPLC analysis of recombinant microorganisms introducing the crocetin biosynthetic metabolic pathway constructed in E. coli, the CsCCD2 gene, and the aldH 7942 gene.

(4) FIGS. 4A and 4B illustrate mass spectral data (FIG. 4A) of crocetin and mass spectral data (FIG. 4B) of crocetin synthesized using a recombinant microorganism into which the CsCCD2 gene and the aldH 7942 gene are introduced.

(5) FIGS. 5A and 5B illustrate a schematic diagram of a recombinant microorganism introducing the crocin-5 biosynthetic metabolic pathway constructed in E. coli, the CsCCD2 gene, the aldH 7942 gene and the UGT-1 gene (FIG. 5A), and HPLC spectrum and mass spectrum analysis results of crocetin and crocin synthesized using the recombinant microorganism (FIG. 5B).

(6) FIGS. 6A and 6B illustrate crocin-3 biosynthetic metabolic pathway constructed in E. coli and a schematic diagram of transforming UGT-1 gene into a strain that can biosynthesize crocetin in order to biosynthesize crocetin (FIG. 6A), and an HPLC spectrum analysis result (FIG. 6B).

DETAILED DESCRIPTION

(7) 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 scope of the present disclosure is not limited only to these examples.

<Example 1> Preparation of Recombinant Microorganisms for Crocin Production

(8) 1-1. Advancement of MEP Metabolic Pathway and Zeaxanthin Biosynthetic Pathway

(9) In order to proceed with the biosynthesis of crocetin, which is a precursor of crocin in E. coli, it is necessary to advance the MEP metabolic pathway and the zeaxanthin biosynthetic metabolic pathway, which are precursors of crocetin, so it was inserted into the chromosome of Escherichia coli K12 MG1655 strain.

(10) Specifically, the advancement of MEP metabolic pathway was progressed in a way such that E. coli-derived ispA (geranyl diphosphate/famesyl diphosphate synthase), idi (isopentenyl-diphosphate A-isomerase), dxs (1-deoxy-D-xylulose-5-phosphate synthase), dxr (1-deoxy-D-xylulose 5-phosphate reductoisomerase) genes were expressed by constitutive expression lac promoters.

(11) Through the above process, the advancement of the zeaxanthin biosynthetic metabolic pathway in the strain with the strengthened MEP metabolic pathway was performed. Specifically, the zeaxanthin biosynthetic metabolic pathway was advanced so that the zeaxanthin synthetic genes CrtE, CrtB, CrtI, CrtY, CrtZ genes derived from Pantoea agglomerans were expressed by the systemic expression trc promoter. As a result, ZEA-1 strains which strengthened the MEP metabolic pathway and capable of producing zeaxanthin were obtained.

(12) 1-2. Introduction of Genes Related to Crocin Biosynthesis

(13) In order to proceed with the biosynthesis of crocetin dialdehyde to the strain obtained in Example 1-1 above, CsCCD2, which is a gene encoding a carotenoid cleavage dioxygenase derived from Crocus sativus, was subjected for gene synthesis by GenScript. After amplification by PCR, cloning was carried out at EcoRI and HindIII sites of pKK223-3 vector. Subsequently, subcloning was carried out at BglII and NotI sites of pSTVM vector.

(14) Subsequently, in order to proceed with the biosynthesis of crocetin, aldH, which is a gene encoding crocetin dialdehyde dehydrogenase, was amplified by PCR on chromosomal DNA of Synechococcus elongatus PCC 7942, and then cloning was carried out at XbaI and EcoRI sites of pUCM vector. Subsequently, subcloning was carried out at SalI and EcoRI sites of pBBR1MCS-2 vector.

(15) Subsequently, in order to proceed with the biosynthesis of crocin-5, UGT, which is a gene encoding crocin biosynthesis enzyme (UDP-glycosyltransferase) was subjected for gene synthesis by GenScript through the request of the UGT75L6(UGT-1) of Gardenia jasminoides. After amplification by PCR, cloning was carried out at EcoRI and PstI sites of pKK223-3 vector.

(16) As a result, as can be confirmed from FIGS. 1A and 1B, a recombinant microorganism for producing crocin into which carotenoid cleavage dioxygenase gene (CsCCD2), crocetin dialdehyde dehydrogenase gene (aldH_7942) and crocin biosynthesis enzyme (UDP-glycosyltransferase) gene (UGT-1) were introduced was produced.

(17) The microbial strain and the constructed plasmid used in the above process are shown in Table 1 below.

(18) TABLE-US-00001 TABLE 1 Strains and Plasmids Related characteristics Strains MG1655 (IlvG rfb-50 rph-1) ZEA-1 MG1655 (IlvGΔ::PLac-dxs glvC Δ::PLac-idl, yjblΔ::PLac-ispA, agaAV Δ::PLac-dxr, pfkAΔ::PLac-CrtE, atpl Δ::Ptrc-CrtYIB, ldhAΔ::Ptrc-YZ) Synechococcus elongatus PCC 7942 Plasmids pUCM Cloning vector modified from pUC19. pKK223-3 Constitutive lac promoter, Ap.sup.r tac promoter, Ap.sup.r pSTVM Plasmid vector is reconstructed with a replication origin of pACYC184, Cm.sup.r pKK_CaCCD2 Ap.sup.r, CsCCD2 cloned in EcoR1 and HindIII site of pkk223-3 pSTVM_C.sub.3CCD2 Cm.sup.r, CoCCD2 cloned in BgiIII and NotI site of pSTVM pUCM_aldH_7942 Ap.sup.r, aldH cloned in Xbal and EcoRI site of pUCM pBBR_aldH_7942 Km.sup.r, aldH cloned in SalI and EcoRI site of pBBR1MCS2 pKK_UGT-1 Ap.sup.r, CsCCD2 cloned in EcoRI and PstI site of pkk223-3

(19) The forward and reverse primers used in the polymerase chain reaction were prepared based on the result of comparing and analyzing the base sequence information of genes encoding enzymes associated with mevalonate biosynthetic pathways in each strain and the information shown in NCBI (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/). The base sequences of the forward and reverse primers used for amplification of each gene are shown in Table 2 below.

(20) TABLE-US-00002 TABLE 2 Enzyme Genes Primer Sequences Locations CsCCD2 F: 5′-CGGAATTCATGGCGAACAAAGAAGAGG (SEQ ID NO: 7) EcoRI R: 5′-CCCAAGCTTTTAGGTCTCCGCTTGATGC (SEQ ID NO: 8) HindIII aldH_7942 F: 5′-GCTCTAGAAGGAGGATTACAAAATGACTGCTGTCGTTCTCC (SEQ ID NO: 9) XbaI R: 5′-CGGAATTCCTAGAGCTTGCGGAAGAG (SEQ ID NO: 10) EcoRI sub_CCD2_F F: 5′-GGAAGATCTGCTGTGCAGGTCGTAAA (SEQ ID NO: 11) BglII sub_CCD2_R R: 5′-ATAAGAATGCGGCCGCGAAACGCAAAAAGGCCA (SEQ ID NO: 12) NotI sub_aldH_7942_F F: 5′-GTCGACCCGACTGGAAAGCG (SEQ ID NO: 13) SalI sub_aldH_7942_R R: 5′-CGGAATTCCTAGAGCTTGCGGAAGAG (SEQ ID NO: 14) EcoRI UGT-1_F F: 5′-CGGAATTCATGGTTCAGCAGCGTCACGT (SEQ ID NO: 15) EcoRI UGT-1_R R: 5′-AACTGCAGTTAGTTGCTCTCCGCTTGAT (SEQ ID NO: 16) PotI

<Example 2> Confirmation of Crocin Production Capacity of Recombinant Microorganisms

(21) In order to confirm the crocin production capacity of the strains prepared in Example 1 above, the biosynthesis of crocetin dialdehyde, crocetin and crocin of recombinant microorganisms in which the gene (CsCCD2) encoding the carotenoid cleavage enzyme, the gene (aldH_7942) encoding the crocetin biosynthesis enzyme, and the gene (UGT-1) encoding the crocin biosynthesis enzyme were sequentially introduced were confirmed sequentially.

(22) 2-1. Confirmation of Crocetin Dialdehyde Biosynthesis

(23) As explained in Example 1, in order to confirm the biosynthesis of crocetin dialdehyde of the recombinant microorganism transformed with the gene (CsCCD2) encoding the crocetin dialdehyde biosynthesis enzyme into strains into which the MEP metabolic pathway and zeaxanthin biosynthesis pathway were inserted, the incubation was carried out under the following incubation conditions.

(24) Specifically, the recombinant microorganism into which the plasmid of CsCCD2 was introduced was incubated for 48 hours at 250 rpm under aerobic conditions in a 100 ml medium using a 500 ml flask. In the case of the incubation temperature, the incubation was carried out at 30° C., and when OD600 became between 0.7 and 1.0, the temperature was converted into 20° C., and then the incubation was continued. As the medium composition, 50 μg/ml of chloramphenicol and 50 μg/ml of kanamycin were all added to a TB (Terrific broth) medium containing 5 g/L of glycerol as a carbon source.

(25) After the incubation process as described above, as a result of analyzing HPLC spectra after extraction, as can be confirmed in FIGS. 2A and 2B, it was confirmed that a peak representing crocetin dialdehyde was observed. Through this, it was possible to confirm the ability to produce crocetin dialdehyde of recombinant microorganisms in which a gene (CsCCD2) encoding the crocetin dialdehyde biosynthesis enzyme was introduced into strains in which the MEP metabolic pathway and zeaxanthin biosynthesis pathway were advanced.

(26) 2-2. Confirmation of Crocetin Biosynthesis

(27) As explained in Example 1, in order to confirm the biosynthesis of crocetin of the recombinant microorganism transformed with the gene (CsCCD2) encoding the crocetin dialdehyde biosynthesis enzyme and the gene (aldH_7942) encoding the crocetin biosynthesis enzyme into strains into which the MEP metabolic pathway and zeaxanthin biosynthesis pathway were inserted, the incubation was carried out under the incubation conditions as described in Example 2-1 above.

(28) 100 ml of medium incubated for extraction and separation of crocetin was all taken, and centrifuged at 4000 rpm for 20 minutes, and all supernatant was discarded. The obtained cells were washed with 0.9% NaCl solution and centrifuged under the same conditions once again. The cells thus obtained were repeatedly extracted with 5 ml or 10 ml of acetone until the color completely disappeared. The extracted solution was concentrated using a vacuum centrifuge (EZ-2 plus, Genevec), 5 ml of ethyl acetate was added to the concentrated solution, mixed, and 5N NaCl solution was added to separate the solution layer. After separating the upper layer containing the color, it was washed twice with tertiary water to remove the remaining water, and dried completely using a vacuum centrifuge. 100-200 μl of ethyl acetate was added to the completely dried sample, dissolved and used for later analysis.

(29) The structure of crocetin obtained by the culture and extraction methods was confirmed by HPLC retention time, UV-Vis spectrum, and mass spectrometry analysis.

(30) Specifically, HPLC analysis was performed using 10-20 μl of prepared samples, and HPLC spectra were analyzed using A: 100% MeOH (25 mM formic acid) and B: 100% DDW (25 mM formic acid) as mobile phases. As a gradient condition, the solvent A was 50% of up to 50 minutes, the solvent A was 80% of up to 60 minutes, and the solvent A was 100% of up to 80 minutes. Zorbax eclipse XDB-C18 column (4.6×150 mm or 250 mm, 5 μm; Agilent Technology) was analyzed as a fixed phase at a flow rate of 0.8 ml/min. HPLC retention time, absorption spectrum and mass spectrum were compared and analyzed for structural analysis. Mass spectra were monitored for both positive and negative modes using a Varian 1200L LC/MS system, and the atmosphere pressure chemical ionization (APCI) module was used for ionization.

(31) After the incubation process as described above, as a result of analyzing HPLC spectra after extraction, as can be confirmed in FIGS. 3A-3C, it was confirmed that a peak representing crocetin was observed. In addition, as a result of analyzing the mass spectrum, as can be confirmed in FIGS. 4A-4B, it was confirmed that a high concentration of crocetin was produced. Through this, recombinant microorganisms in which a gene (CsCCD2) encoding crocetin dialdehyde biosynthesis enzyme and a gene (aldH_7942) encoding crocetin biosynthesis enzyme were introduced into strains in which the MEP metabolic pathway and zeaxanthin biosynthesis pathway were advanced could produce crocetin with high efficiency.

(32) 2-3. Confirmation of Crocin-5 Biosynthesis

(33) As explained in Example 1, in order to confirm the biosynthesis of crocin-5 of the recombinant microorganism transformed with the gene (CsCCD2) encoding the crocetin dialdehyde biosynthesis enzyme, the gene (aldH_7942) encoding the crocetin biosynthesis enzyme, and the gene (UGT-1) encoding the crocin biosynthesis enzyme into strains which the MEP metabolic pathway and zeaxanthin biosynthesis pathway were inserted, the incubation was carried out under the same condition as the incubation condition described in Example 2-1 above. After incubation, crocin was extracted and separated in the same manner as described in Example 2-2 above. The structure of the cultured and extracted crocin was confirmed by HPLC retention time, UV-Vis spectrum, and mass spectrum analysis in the same manner as described in Example 2-2 above.

(34) As a result, as can be confirmed in FIGS. 5A-5B, it was confirmed that a peak (peak-1) representing crocin-5 was observed. In addition, as a result of analyzing the mass spectrum, it was confirmed that a high concentration of crocin-5 was produced. Through this, recombinant microorganisms in which a gene (CsCCD2) encoding crocetin dialdehyde biosynthesis enzyme, a gene (aldH_7942) encoding crocetin biosynthesis enzyme, and a gene (UGT-1) encoding crocin biosynthesis enzyme into strains in which the MEP metabolic pathway and zeaxanthin biosynthesis pathway were advanced could produce crocin-5 with high efficiency.

(35) 2-4. Confirmation of Crocin-3 Biosynthesis

(36) As explained in Example 1, the biosynthesis of crocin-3 of the recombinant microorganism transformed with the gene (CsCCD2) encoding the crocetin dialdehyde biosynthesis enzyme, the gene (aldH_7942) encoding the crocetin biosynthesis enzyme, and the gene (UGT-1) encoding the crocin biosynthesis enzyme into strains which the MEP metabolic pathway and zeaxanthin biosynthesis pathway were inserted was confirmed.

(37) Specifically, the recombinant microorganism was incubated at 250 rpm under aerobic conditions in a 100 ml TB medium using a 500 ml flask. In the case of the incubation temperature, the incubation was carried out at 30° C., and when OD600 became between 0.7 and 1.0, the temperature was converted into 20° C., and then the incubation was continued. As the medium composition, 50 μg/ml of chloramphenicol, 100 μg/ml of ampicillin and 50 μg/ml of kanamycin were all added to a TB medium containing 5 g/L of glycerol as a carbon source.

(38) 100 ml of medium with the cultured recombinant microorganisms for extraction and separation of crocin-3 was all taken, and centrifuged at 4000 rpm for 20 minutes, and the cells and the supernatant were separated and were all taken. Ethyl acetate and 5N NaCl were treated with the same volume in the obtained supernatant, and then reacted for 48 hours in a dark place indoors. Then, the ethyl acetate layer was taken. Water was removed using MgSO.sub.4. The extracted solution was added to 100-200 μl of ethyl acetate and used for later analysis.

(39) As a result, as can be confirmed in FIGS. 6A-6B, it was confirmed that a peak representing crocin-3 (peak-1) and a peak representing crocin-5 (peak-2) were observed. Through this, recombinant microorganisms in which a gene (CsCCD2) encoding crocetin dialdehyde biosynthesis enzyme, a gene (aldH_7942) encoding crocetin biosynthesis enzyme, and a gene (UGT-1) encoding crocin biosynthesis enzyme were introduced into strains in which the MEP metabolic pathway and zeaxanthin biosynthesis pathway were advanced could also produce crocin-3 with high efficiency.

<Example 3> Comparison of Crocin Production According to Origin of Introduced Genes

(40) In order to confirm the change in the efficiency of crocin biosynthesis according to the origin of the gene introduced into the recombinant microorganism, the production amount of crocetin of the case of introducing a gene (aldH_7942) encoding crocetin biosynthesis enzyme derived from Synechococcus elongatus PCC 7942 and the case of introducing a gene encoding a crocetin biosynthesis enzyme aldH 6803 derived from Synechococcus elongatus PCC 6803 was compared.

(41) As a result, as can be confirmed in Table 3 below, when the aldH 7942 gene of the present disclosure was introduced, it was confirmed that the production amount of crocetin increased by about 1.5 times. Through this, it was confirmed that the efficiency of biosynthesis of crocetin and crocin may vary significantly depending on the origin of the gene introduced into the recombinant microorganism for producing crocin.

(42) TABLE-US-00003 TABLE 3 ZEA- 1_pSTVM_CsCCD2 + Crocetin pBBR_aldHS (μg/L) μg/DCW μg/Glycerol aldH6803 698.66 ± 129.4 ± 67.23 ± 37.25 10.45 3.72 aldH7942 986.55 ± 196.35 ± 98.65 ± 41.65 1.36 4.17

(43) In the above results, it was confirmed that the carotenoid cleavage enzyme gene (CsCCD2), the crocetin biosynthesis enzyme gene (aldH_7942) and the crocin biosynthesis enzyme gene (UGT-1) were introduced into strains in which the MEP metabolic pathway and zeaxanthin metabolic pathway were advanced to produce a recombinant microorganism for producing crocin (FIGS. 1A and 1B). As the three genes were sequentially introduced, the recombinant microorganisms were able to biosynthesize crocetin dialdehyde, crocetin and crocin, respectively (FIGS. 2A, 2B, 3A-3C, 4A, 4B, 5A, 5B, 6A and 6B). Furthermore, it was confirmed that the biosynthetic efficiency of crocetin and crocin may vary significantly depending on the three genes derived (Table 3).

(44) From the above description, those skilled in the art will appreciate that the present disclosure can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, it should be understood that the embodiments described above are exemplary in all respects and not limiting. The scope of the present disclosure should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present disclosure.