A method for synthesizing trans-crocetin I by multi-enzyme cascade in vitro

Abstract

The application discloses a method for synthesizing trans-crocins I in vitro by multi-enzyme level combination. The method takes zeaxanthin wrapped by cyclodextrin as a starting substrate, and solves the problem of inclusion body expression of key enzyme GjCCD4a-M1 in Escherichia coli by combining three kinds of solubilization strategies, and then successfully establishes an in-vitro four-enzyme cascade system based on purified enzymes to realize the synthesis of trans-crocins I. With 200 μM zeaxanthin as a substrate, the concentration of trans-crocins I in the product of the system reaches 168.5 μM, and the proportion in the total crocin product is 94.9%. The application solves the problem of low water solubility of zeaxanthin by introducing cyclodextrin wrapping technology, and avoids the dependence on high-priced crocetin substrate; the expression problem of GjCCD4a-M1 is solved by combining the solubilization strategy; the established in-vitro enzymatic system avoids the problems of low proportion of crocin I in the product of the cell factory and easy isomerization, and lays a foundation for developing a new sustainable industrial production platform.

Description

Technical Field

[0001] This invention relates to the field of biocatalytic synthesis, specifically to a method for the synthesis of crocin I via multi-enzyme-level synthesis. Background Technology

[0002] Crocin is a class of glycosylated carotenoid bioactive components mainly derived from Gardenia jasminoides and Crocus sativus. Among them, all-trans-crocin I (CAS No. 42553-65-1) has the highest content and is the most thoroughly studied (see attached chemical structure). Figure 1 Pharmacological studies have shown that all-trans-crocin I is as effective as some first-line clinical drugs in treating depressive symptoms.

[0003] However, the production of all-trans-crocin I currently mainly relies on extraction and separation from plants, which suffers from problems such as difficulty in raw material regeneration, complex purification, and high cost, necessitating the development of new sustainable production technologies. Researchers have attempted to synthesize crocin through chemical methods, but have only achieved the preparation of crocin V and crocin III, failing to achieve the preparation of all-trans-crocin I, see patent CN101514216A (publication date 2009-08-26).

[0004] Microbial and plant cell factory technologies have been used to produce crocin, but because the proportion of enzymes and reaction conditions in cell factories are difficult to control precisely, and crocin is prone to isomerization in host cells, the resulting products are usually a mixture of various crocin and their cis isomers, which leads to a low proportion of all-trans crocin I and difficulty in purification. See patent CN103764818A (publication date 2014-04-30).

[0005] In vitro enzymatic synthesis has also been applied to the production of crocin. Compared to cell factories, no obvious cis-isomerization phenomenon was observed in the in vitro catalytic synthesis of crocin, and the high controllability of the in vitro enzyme catalysis system itself is conducive to the synthesis of high-purity crocin I. However, because the starting substrate zeaxanthin of the natural biosynthetic pathway has extremely low solubility in the aqueous solution system required for enzyme catalysis, and the expression of carotenoid cleaving dioxygenases (CCDs) that catalyze the cleavage of zeaxanthin has poor solubility and is therefore difficult to purify, previous studies have usually bypassed the CCD step and directly used crocin acid, which has better water solubility but is more expensive, as the substrate, significantly increasing the cost of crocin production. A patent for the production of crocin using crocin acid as a substrate can be found in: KR20210033100A (publication date 2021-03-26). Summary of the Invention

[0006] To address the problems in existing technologies, such as the low proportion of all-trans crocin I in the product, its susceptibility to isomerization, and the poor water solubility of the low-cost substrate zeaxanthin and the poor solubility of the key enzyme CCD in in vitro enzymatic systems, this invention provides a method for synthesizing high-purity all-trans crocin I using zeaxanthin cyclodextrin inclusion complexes as substrates, employing a combined solubilization strategy to optimize CCD enzyme expression, and establishing an in vitro multi-enzyme cascade system (enzyme cascade reaction pathway is attached). Figure 2 ).

[0007] The technical solution adopted by this invention to solve its technical problem is: a method for the in vitro multi-enzyme cascade catalytic synthesis of all-trans crocin I, comprising the following steps:

[0008] S1. Preparation of purified enzyme solutions: Construct recombinant expression plasmids GjCCD4a-M1, GjALDH2C3, GjUGT74F8 and GjUGT94E13, transform them into the host to induce expression, and prepare purified enzymes GjCCD4a-M1, GjALDH2C3, GjUGT74F8 and GjUGT94E13;

[0009] S2. Preparation of substrate solution: Prepare zeaxanthin-cyclodextrin inclusion complex solution by mixing zeaxanthin solution and methyl-β-cyclodextrin solution;

[0010] S3. In vitro multi-enzyme synthesis of all-trans crocin I: Cofactors, auxiliary reagents, the zeaxanthin-methyl-β-cyclodextrin inclusion complex solution obtained in S2, and the four purified enzymes described in S1 were added sequentially to the buffer solution to achieve the synthesis of all-trans crocin I.

[0011] Furthermore, the vector of the GjCCD4a-M1 recombinant expression plasmid described in S1 is pMal-c5x, which contains a tac promoter (medium strength promoter) and a maltose binding protein (MBP) lysing tag on its backbone; the host into which the GjCCD4a-M1 recombinant expression plasmid is transformed is the Escherichia coli BL21(DE3) strain carrying the pG-Tf2 plasmid expressing molecular chaperones GroES, GroEL and trigger factor;

[0012] Further, the zeaxanthin solution mentioned in S2 is a tetrahydrofuran solution; the methyl-β-cyclodextrin solution is an anhydrous ethanol solution; the steps for preparing the inclusion complex are: mixing and stirring the two solutions to allow zeaxanthin and methyl-β-cyclodextrin to combine in the solvent, then evaporating the organic solvent, redissolving in water, and filtering through a filter membrane to obtain an aqueous solution of the zeaxanthin-cyclodextrin inclusion complex.

[0013] Furthermore, the cofactor described in S3 includes ferrous ions (Fe2+). 2+), oxidized coenzyme II (NADP) + The auxiliary reagents include tris(2-carboxyethyl)phosphine hydrochloride (TCEP), L-ascorbic acid, and catalase.

[0014] The beneficial effects of this invention are as follows:

[0015] This invention addresses the challenge of inclusion body expression of the key enzyme element GjCCD4a-M1 in *E. coli* by optimizing the expression strategy of the CCD enzyme (reducing promoter strength, fusion expression of the MBP solubilizing tag, and co-expression of molecular chaperones). Furthermore, the use of methyl-β-cyclodextrin to encapsulate zeaxanthin significantly enhances its water solubility, thereby increasing the maximum zeaxanthin loading in the reaction system. Based on this, a multi-enzyme cascade system using zeaxanthin as a substrate was successfully established using purified proteins of four key enzymes from the natural biosynthetic pathway of genistein, achieving the synthesis of high-purity all-trans crocin I. This lays the foundation for establishing a sustainable industrial production platform for all-trans crocin I. Attached Figure Description

[0016] Figure 1 Chemical structures of five major natural crocin glycosides found in saffron and gardenia;

[0017] Figure 2 This is a schematic diagram of the reaction pathway for the in vitro four-enzyme cascade catalytic synthesis of all-trans crocin I according to the present invention;

[0018] Figure 3 SDS-PAGE electrophoresis images of soluble expression of GjCCD4a-M1 in Escherichia coli under different expression strategies;

[0019] Figure 4 Comparison of UV absorption spectra of crocin I product catalyzed by in vitro multi-enzyme cascade system and all-trans crocin I standard.

[0020] Figure 5 A schematic diagram of the process for preparing zeaxanthin-methyl-β-cyclodextrin inclusion complex, along with UPLC-PDA analysis results and yield statistics after the inclusion complex was applied to an in vitro four-enzyme cascade reaction. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of protection of the present invention.

[0022] Example 1: Heterologous expression and purification of four key enzymes in Escherichia coli

[0023] 1. Constructing a recombinant vector and transforming it into Escherichia coli

[0024] The coding gene of GjCCD4a-M1 (SEQ ID NO.1) was cloned into the pET-28a(+)-MBP vector (an MBP tag was introduced into the pET-28a(+) vector backbone carrying a strong T7 promoter) and the pMal-c5x vector (carrying a moderately strong tac promoter and an MBP tag), respectively. To maintain consistency with the pET-28a(+)-MBP vector, a 6×His tag sequence was also introduced upstream of the MBP tag in the pMal-c5x vector. The resulting recombinant plasmids were named 4a-M1-28a-MBP and 4a-M1-pMal-c5x, respectively.

[0025] The coding gene for GjALDH2C3 (SEQ ID NO.2) was cloned into the pCold I vector, the coding gene for GjUGT74F8 (SEQ ID NO.3) was cloned into the pMal-c5x vector, and the coding gene for GjUGT94E13 (SEQ ID NO.4) was cloned into the pET32a vector.

[0026] The five recombinant plasmids were transformed into E. coli BL21(DE3) chemocompetent cells. The 4a-M1-pMal-c5x recombinant plasmid was additionally transformed into E. coli BL21(DE3) chemocompetent cells containing the pG-Tf2 plasmid (used to express three molecular chaperones: GroES, GroEL, and Trigger Factor). After adding antibiotic-free LB liquid medium and incubating at 37°C and 200 rpm for 1 hour, the bacterial culture containing the pG-Tf2 plasmid was spread onto LB solid medium containing 100 mg / L ampicillin sodium and 25 mg / L chloramphenicol. The other bacterial cultures were spread onto LB solid medium containing only 100 mg / L ampicillin sodium and incubated upside down at 37°C for approximately 12 hours.

[0027] 2. Bacterial selection, large-scale culture, and induction of expression

[0028] Single colonies were selected and positive clones were identified by PCR and Sanger sequencing. Positive clones containing the pG-Tf2 plasmid and transformed with 4a-M1-pMal-c5x were inoculated into 3 mL of LB liquid medium containing 100 mg / L ampicillin sodium and 25 mg / L chloramphenicol. Positive clones without the pG-Tf2 plasmid were inoculated into 3 mL of LB liquid medium containing 100 mg / L ampicillin sodium. The cultures were incubated overnight at 37°C and 200 rpm to obtain seed culture.

[0029] 1 mL of seed culture was inoculated into 300 mL of LB liquid medium containing the same antibiotic and cultured at 37 °C and 200 rpm until the OD600 reached 0.6-0.8. Isopropyl thiogalactoside (IPTG) was added to a final concentration of 0.5 mM and protein expression was induced at 16 °C and 160 rpm for 24 hours.

[0030] 3. Collect bacterial cells and purify proteins.

[0031] After induction, the cells of the six E. coli strains were collected by centrifugation at 7830 rpm and 4℃. The cells were resuspended in 100 mM HEPES buffer (pH 8.0), sonicated, and centrifuged again at 7830 rpm and 4℃. The supernatant crude enzyme solution was transferred to a new centrifuge tube, and the precipitate was resuspended in an equal volume of 100 mM HEPES buffer (pH 8.0). Samples were taken from the supernatant crude enzyme and the precipitate for SDS-PAGE analysis to compare the distribution of GjCCD4a-M1 protein in the supernatant and precipitate of E. coli under different expression strategies.

[0032] Subsequently, the crude enzymes in the supernatants of 4a-M1-pMal-c5x (expressed in E. coli carrying pG-Tf2) and GjUGT74F8 were purified by dextrin-agarose resin affinity chromatography, eluted with buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM maltose, and 1 mM TCEP, and concentrated using an Amicon Ultra-15 ultrafiltration tube (30 kDa MWCO). The supernatants expressing GjALDH2C3 and GjUGT94E13 proteins were purified by nickel affinity chromatography, with the elution buffer containing 50 mM HEPES (pH 8.0). The purified protein was prepared using 8.0), 300mM NaCl, and different concentrations of imidazole (20, 50, 200mM for GjUGT94E13; 20, 50, 100, 200, 500mM for GjALDH2C3). The purified protein was then concentrated by ultrafiltration and the buffer was changed to 25mM HEPES (pH 8.0) buffer containing 150mM NaCl.

[0033] Experimental results show that, compared with strategies using a strong promoter (T7) + MBP lysis tag or a moderate-strength promoter (tac) + MBP lysis tag, the co-expression strategy of the moderate-strength promoter (tac) + MBP lysis tag + molecular chaperone (pG-Tf2) described in this invention significantly increased the proportion of GjCCD4a-M1 in the crude enzyme supernatant (see appendix). Figure 3 This indicates that the strategy effectively alleviates the expression of GjCCD4a-M1 enzyme inclusion bodies in Escherichia coli.

[0034] Example 2: Preparation of zeaxanthin-methyl-β-cyclodextrin inclusion complex solution

[0035] Weigh 5 mg of zeaxanthin using a balance, add 0.5 mL of tetrahydrofuran, and vortex to dissolve. Separately weigh 250 mg of methyl-β-cyclodextrin, add 12 mL of anhydrous ethanol, and vortex to dissolve. Mix the two solutions by vortexing, heat and stir in an oil bath at 37°C for 24 hours, then transfer to a 50 mL centrifuge tube and centrifuge at 7830 rpm. Collect the supernatant and evaporate the solvent in a vacuum centrifuge at 45°C and 1400 rpm to obtain a bright yellow flaky solid. Weigh the solid and reconstitute it with water to a final concentration of 150 mg / mL, vortex to dissolve, and then filter through a 0.22 μm filter membrane to obtain an aqueous solution containing zeaxanthin-cyclodextrin inclusion complex. The concentration of zeaxanthin in the solution was determined to be 872 μM using UPLC-PDA.

[0036] Example 3: In vitro multi-enzyme cascade catalytic synthesis of all-trans crocin I

[0037] An in vitro multi-enzyme cascade reaction system was established using zeaxanthin-cyclodextrin inclusion complex and four purified enzymes: 1 mM TCEP, 0.5 mM L-ascorbic acid, and 2 mg / mL catalase were added sequentially to 100 mM M HEPES (pH 7.0) buffer, along with 10 mM NADP as a cofactor. + Add 40 mM UDPG, 200 μM zeaxanthin-cyclodextrin inclusion complex as substrate, four purified enzymes: 60 μM GjCCD4a-M1-pMal-c5x, 15 μM GjALDH2C3, 60 μM GjUGT74F8, and 0.45 μM GjUGT94E13, and finally add 8 mM Fe. 2+ The reaction was carried out at 35℃ for 5 hours. After the reaction was completed, methanol was added to terminate the reaction. The mixture was centrifuged at 15000 rpm and 4℃ for 20 min. The supernatant was filtered through a 0.22 μm filter membrane and then analyzed by UPLC-PDA.

[0038] The results showed that the crocin I obtained in the above reaction system had a UV spectrum that was basically consistent with that of the all-trans crocin I standard, and no characteristic absorption peak of the cis isomer was observed at ~326 nm (see attached image). Figure 4 This indicates that all-trans crocin I did not undergo significant isomerization in the in vitro multi-enzyme cascade system described in this invention; furthermore, the concentration of all-trans crocin I in the product reached 164.6 mg / L (168.5 μM), accounting for 94.9% of the total crocin product (see appendix). Figure 4 This indicates that the in vitro multi-enzyme cascade system described in this invention significantly increases the proportion of crocin I in the product, and has advantages in the production of crocin I.

Claims

1. A method for preparing crocin I via a multi-enzyme cascade reaction, characterized in that: Using zeaxanthin as a substrate, GjCCD4a-M1, GjALDH2C3, GjUGT74F8 and GjUGT94E13 enzymes were expressed in Escherichia coli. The purified proteins of the four enzymes were prepared to establish an in vitro one-pot reaction system. The main product was all-trans crocin I.

2. The method according to claim 1, characterized in that: When zeaxanthin is used as a substrate, it is first mixed with methyl-β-cyclodextrin to prepare a zeaxanthin-methyl-β-cyclodextrin inclusion complex solution.

3. The method according to claim 1, characterized in that: The enzymes GjCCD4a-M1, GjALDH2C3, GjUGT74F8, and GjUGT94E13 are key enzymes in the natural biosynthesis pathway of crocin in Gardenia jasminoides, and their amino acid sequences are shown in SEQ ID NO.1-4.

4. The method according to claims 1 and 3, characterized in that: The gene encoding GjCCD4a-M1 was constructed at the multiple cloning site of the pMal-c5x vector carrying the tac promoter and the maltose-binding protein (MBP) lysis tag.

5. The method according to claim 1, characterized in that: The host Escherichia coli that heterologously expresses the GjCCD4a-M1 enzyme is the BL21(DE3) strain, which carries the pG-Tf2 plasmid that can express GroES, GroEL, and trigger factor molecular chaperones.

6. The method according to claims 1, 3 and 5, characterized in that: The in vitro one-pot reaction system includes zeaxanthin-cyclodextrin inclusion complex, the four purified enzymes, and the cofactor ferrous ion (Fe). 2+ ), oxidized coenzyme II (NADP) + ) and uridine diphosphate glucose (UDPG) and auxiliary reagents tris(2-carboxyethyl)phosphine hydrochloride (TCEP), L-ascorbic acid and catalase.

7. The method according to claim 1, characterized in that: All-trans crocin I accounted for more than 90% of the total crocin products.

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