An engineered Escherichia coli producing five types of crocin and its application

Abstract

The present invention discloses an Escherichia coli engineered bacterium producing five types of crocins and its application. The present invention belongs to the field of cell engineering technology. The deposit number of the engineered bacterium is CGMCC: NO.29364, and related genes and applications are provided. The present invention can realize the biosynthesis of five types of crocins from glucose, including crocin III, crocin IV and crocin V which are difficult to extract from plants or have very low plant content, and will provide support for the acquisition of five types of crocins and promote the development of new drugs for five types of crocins.

Description

Technical Field

[0001] The invention relates to the technical field of cell engineering, and more specifically to an Escherichia coli engineering bacterium capable of producing five types of crocin and application thereof. Background Art

[0002] Crocin is an apocarotenoid compound, which can be divided into five types according to the number and position of sugar groups connected at both ends of its molecule. Crocin is mainly found in the stigma of the Chinese medicine Crocus sativus and the ripe fruit of Gardenia jasminoides. Modern pharmacological studies have shown that it has pharmacological effects such as anti-cancer, anti-inflammatory, and anti-depressant, but its high price and resource shortage have greatly limited the new drug development and clinical application of crocin. At present, the source of crocin mainly depends on plant chemical extraction and separation, which has the disadvantages of low yield and long time consumption, so it is of great significance to study and develop other effective ways to synthesize crocin.

[0003] Based on multi-omics research, the biosynthetic pathway of gardenia crocin has been clearly elucidated, and its downstream biosynthetic pathway is divided into three steps: first, C40-carotenoid precursor compounds are cleaved by carotenoid cleavage dioxygenase (GjCCD4a) to produce crocetin dialdehyde; then, crocetin dialdehyde is oxidized to crocetin by acetaldehyde dehydrogenase (GjALDH2C3); finally, crocetin is generated into five types of crocin under the action of two glycosyltransferases (GjUGT74F8 and GjUGT94E13). The identification of these key gene components has laid a solid foundation for the study of crocin synthetic biology.

[0004] E. coli chassis cells are easy to culture, have high heterologous protein expression, short cultivation cycle, and low requirements for human and material resources. However, they lack the function of post-translational modification of proteins, which makes it difficult to express eukaryotic genes. In addition, it is challenging to integrate multiple genes into the same E. coli chassis cell and express them heterologously. Experimenters need to conduct a lot of experimental exploration to explore the appropriate conditions for the simultaneous expression of multiple genes.

[0005] Therefore, providing an engineered Escherichia coli that produces five types of crocin and its application is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0006] In view of this, the present invention provides an Escherichia coli engineered bacterium that produces five types of crocin and its application. The present invention uses 11 genes to construct the crocin metabolic pathway into the Escherichia coli chassis cells, realizing the heterologous synthesis of five types of crocin from glucose, laying the foundation for the large-scale industrial production of crocin;

[0007] Preservation information: E579-Crocins, classified and named Escherichia coli; deposited in the General Microbiology Center of China Culture Collection Administration, the preservation address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the preservation number is CGMCC: NO.29364, and the preservation date is December 21, 2023.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] An engineered Escherichia coli producing five types of crocins, with a deposit number of CGMCC: NO.29364.

[0010] The present invention also provides a microbial agent, comprising the above-mentioned engineered Escherichia coli bacteria that produce five types of crocin.

[0011] The present invention also provides a microbial fermentation liquid, which is obtained by fermenting the above-mentioned Escherichia coli engineering bacteria that produce five types of crocin.

[0012] The present invention also provides the use of the above-mentioned engineered bacteria, the above-mentioned microbial agent or the above-mentioned fermentation liquid in fermentation preparation or drug research and development.

[0013] Preferably, heterologous synthesis of crocin from glucose is achieved, wherein crocin includes crocin III, crocin IV and crocin V.

[0014] Preferably, the induction conditions include: 1.0 mM IPTG, FM medium with a final glucose concentration of 5%, and induction at 16° C. for 84 h.

[0015] The present invention also provides a mutant gene N212 or N212m of GjCCD4a, wherein the nucleotide sequence of N212 is shown as SEQ ID No.1; and the nucleotide sequence of N212m is shown as SEQ ID No.2.

[0016] The present invention also provides an expression vector of the mutant gene.

[0017] The present invention also provides a method for constructing an engineered Escherichia coli that produces five types of crocins, comprising sequentially transforming pACCAR25ΔcrtX, p2C3-N212m, pSLB208-Pgm-T7-GalU and pCDF-Duet-1-UGT74F8-UGT94E13 into competent Escherichia coli.

[0018] Preferred: Construction method of p2C3-N212m: using pET-Duet-1 as a vector, using the primer sets p-2C3-XF and pX-2C3-R, construct GjALDH2C3 into the multiple cloning site 1 of the pET-Duet-1 vector, and using the primer sets p2C3-N212m-F and p2C3-N212m-R, construct N212m into the multiple cloning site 2 of the pET-Duet-1 vector;

[0019] The nucleotide sequence of N212m is shown in SEQ ID No. 2;

[0020] The nucleotide sequence of GjALDH2C3 is shown in SEQ ID No. 3;

[0021] The nucleotide sequence of p-2C3-XF is shown in SEQ ID No. 4;

[0022] The nucleotide sequence of pX-2C3-R is shown in SEQ ID No. 5;

[0023] The nucleotide sequence of p2C3-N212m-F is shown in SEQ ID No.6;

[0024] The nucleotide sequence of p2C3-N212m-R is shown in SEQ ID No.7.

[0025] It can be seen from the above technical scheme that compared with the prior art, the present invention discloses an Escherichia coli engineered bacterium that produces five types of crocins and its application. The technical effect achieved is that the present invention can realize the biosynthesis of five types of crocins from glucose, including crocin III, crocin IV and crocin V, which are difficult to extract from plants or have very low plant content. It will provide support for the acquisition of five types of crocins and promote the progress of new drug research and development of five types of crocins. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0027] Figure 1 The accompanying drawings are diagrams showing the construction and activity evaluation of the highly efficient GjCCD4a mutant obtained through transformation provided by the present invention, wherein: Figure 1Middle A: Downstream biosynthesis pathway of gardenia crocin, GjCCD4a is responsible for cleaving the carbon-carbon double bonds at positions 7,8 (7′,8′) of lycopene, β-carotene and zeaxanthin to produce crocetin dialdehyde; GjALDH2C3 is responsible for oxidizing crocetin dialdehyde to produce crocetin; GjUGT74F8 and GjUGT94E13 are responsible for the generation of crocetin from crocetin to five crocins; Figure 1 Middle B: Schematic diagram of GjCCD4a (4a) gene truncation, taking N2 as an example, which is obtained by deleting the 4th to 99th amino acids at the 5' end (N-terminus) of the GjCCD4a gene; Figure 1 Middle C: 5' end (N-terminal) truncated body catalytic function liquid phase detection diagram, Zea represents the engineered strain producing zeaxanthin, crocetin dialdehyde represents crocetin dialdehyde; such as: Zea-4a, represents the liquid phase detection diagram of the zeaxanthin product cleaved by the original sequence of GjCCD4a; Figure 1 Middle D: Liquid phase detection of catalytic function of 3' end (C-terminal) truncated form; Figure 1 Middle E: Activity evaluation diagram of GjCCD4a mutants; GjCCD4a is the original sequence; N1-N8 are 5' end (N-terminal) truncation mutants; C1-C2 are 3' end (C-terminal) truncation mutants; N212 is GjCCD4a-N-96bp-F153L-S415A mutant; N212m is

[0028] MBP·Tag-GjCCD4a-N-96bp-F153L-S415A mutant.

[0029] Figure 2 The accompanying drawings are diagrams showing the construction of the N212m and GjALDH2C3 gene co-expression vector and the results of activity screening provided by the present invention; wherein, Figure 2 Middle A: Schematic diagram of the construction of crocetin-producing engineered strains, which include 4 modules: Module 1 is responsible for overexpressing glycosyl donor pathway genes, including pSLB208-Pgm-T7-GalU plasmid; Module 2 is responsible for converting FPP produced endogenously by E. coli to zeaxanthin, including pACCAR25ΔcrtX plasmid; Module 3 is responsible for cleavage and oxidation of zeaxanthin to produce crocetin, including pET-Duet-1-X plasmid; Module 4 is responsible for glycosylation of crocetin, including pCDF-Duet-1-UGT74F8-UGT94E13 plasmid; Figure 2 Middle B: Schematic diagram of pET-Duet-1-X plasmid, including pN212m-2C3 and p2C3-N212m (the difference between the two vectors is the different gene connection order); Figure 2Middle C: Schematic diagram of E2 and E5 engineered strains, where E2 includes Module 2 / 3 (Module 3 is pN212m-2C3) and E5 includes Module 2 / 3 (Module 3 is p2C3-N212m); Figure 2 Middle D: indicates the detection of catalytic products of E2 and E5 engineered strains in LB medium; Figure 2 E in the middle represents the detection of catalytic products of E2 and E5 engineered strains in FM medium; Figure 2 F in the figure shows the comparison results of the catalytic efficiency of E2 and E5 engineered strains in LB and FM medium.

[0030] Figure 3 The accompanying drawings are product detection and optimal reaction condition exploration experiments of the Escherichia coli engineered bacteria E579-Crocins provided by the present invention, wherein: Figure 3 Middle A: indicates the construction of E579 Crocins project and liquid phase detection of catalytic products. E579-Crocins contains Module 1 / 2 / 3 / 4 (Module 1 is pSLB208-Pgm-T7-GalU, Module 2 is pACCAR25ΔcrtX, Module 3 is p2C3-N212m, and Module 4 is pCDF-Duet-1-UGT74F8-UGT94E13); Figure 3 B in the middle represents the mass spectrometry detection results of crocin I, crocin II and crocin IV; Figure 3 Middle C: represents the mass spectrometry detection results of crocin V and crocin III. Figure 3 Middle D: Statistical graph showing the product accumulation of E579 Crocins strain at different induction temperatures. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The embodiment of the invention discloses an engineered Escherichia coli bacteria producing five types of crocins and application thereof.

[0033] Example 1

[0034] Construction and activity evaluation of GjCCD4a high-efficiency mutants

[0035] Experimental methods

[0036] Based on the nucleotide sequence of gene GjCCD4a (GenBankKY631925), GjCCD4a was synthesized between the KpnI and EcoRI restriction enzyme sites of pET32a vector by gene chemical synthesis to form pET32a-GjCCD4a vector. MBP·Tag nucleotide sequence was synthesized between the NcoI and NdeI restriction enzyme sites of pET28a vector by gene chemical synthesis to form pET28a-MBP·Tag vector.

[0037] Using the truncated primers in Table 1:

[0038] Table 1. GjCCD4a truncation primers

[0039]

[0040] The MutExpress II Fast Mutagenesis Kit V2 of Novogene was used. According to the instructions of the kit, the C-terminus (3′ end) of the GjCCD4a gene sequence in the pET32a-GjCCD4a vector was truncated by 48 bp (to obtain pET32a-GjCCD4a-C-48 bp, where GjCCD4a-C-48 bp is referred to as C1) and 81 bp (to obtain pET32a-GjCCD4a-C-81 bp, where GjCCD4a-C-81 bp is referred to as C2), and its N-terminus (5′ end) was truncated by 48 bp p (obtained pET32a-GjCCD4a-N-48bp, of which GjCCD4a-N-48bp is referred to as N1), 96bp (obtained pET32a-GjCCD4a-N-96bp, of which GjCCD4a-N-96bp is referred to as N2), 150bp (obtained pET32a-GjCCD4a-N-150bp, of which GjCCD4a-N-150bp is referred to as N3), 198bp (obtained pET32a-GjCCD4a-N-48bp, of which GjCCD4a-N-48bp is referred to as N1), 96bp (obtained pET32a-GjCCD4a-N-96bp, of which GjCCD4a-N-96bp is referred to as N2), 150bp (obtained pET32a-GjCCD4a-N-150bp, of which GjCCD4a-N-150bp is referred to as N3), jCCD4a-N-198bp, of which GjCCD4a-N-198bp is referred to as N4), 246bp (to obtain pET32a-GjCCD4a-N-246bp, of which GjCCD4a-N-246bp is referred to as N5), 297bp (to obtain pET32a-GjCCD4a-N-297bp, of which GjCCD4a-N-297bp is referred to as N6), 348bp (to obtain pET32a-GjCCD4a- N-348bp, of which GjCCD4a-N-348bp is referred to as N7), 396bp (to obtain pET32a-GjCCD4a-N-396bp, of which GjCCD4a-N-396bp is referred to as N8), the constructed mutant expression vector was verified by Sanger sequencing; the gel recovery experiment was carried out using Tiangen ordinary agarose gel DNA recovery kit (centrifugal column type); the plasmid extraction experiment was carried out using Tiangen plasmid mini extraction kit (centrifugal column type).

[0041] The mutant N2 with the most significant improvement in catalytic activity was selected, and the mutant primers in Table 2 were used:

[0042] Table 2. N2 mutation primers

[0043]

[0044] Based on the Mut Express II Fast Mutagenesis Kit V2 of Novagen, referring to the instructions of the kit, N2 was mutated so that the 153rd F of the amino acid sequence corresponding to GjCCD4a-N-96bp was mutated to L, and the 415th S was mutated to A, thereby obtaining the mutant expression vector pET32a-GjCCD4a-N-96bp-F153L-S415A, wherein GjCCD4a-N-96bp-F153L-S415A is referred to as N212; in order to further improve the catalytic activity, based on the primers in Table 3:

[0045] Table 3. MBP fusion protein primers

[0046]

[0047] The GjCCD4a-N-96bp-F153L-S415A gene sequence was ligated into the EcoRI and XhoI restriction enzyme sites of the pET28a-MBP·Tag vector using gene cloning and enzyme ligation techniques to obtain the mutant expression vector pET28a-MBP·Tag-GjCCD4a-N-96bp-F153L-S415A, where MBP·Tag-GjCCD4a-N-96bp-F153L-S415A was abbreviated as N212m.

[0048]

[0049]

[0050] The catalytic activity verification method of each mutant is as follows: first, the pACCAR25ΔcrtX plasmid is transformed into Escherichia coli BL21 (DE3) to construct an Escherichia coli engineered strain producing zeaxanthin (the engineered strain is a conventional strain, for example, see: "Structure and Functional Analysis of a Marine Bacterial Carotenoid Biosynthesis Gene Cluster and Astaxanthin Biosynthetic Pathway Proposed at the Gene Level". DOI: 10.1128 / jb.177.22.6575-6584.1995); then, the mutant expression vector is transformed into the Escherichia coli engineered strain producing zeaxanthin by the calcium chloride transformation method, and the resistance to ampicillin (50 μg / mL) and chloramphenicol (34 μg / mL) is used for screening, and the positive colonies obtained by screening are cultured overnight, 2 mL of the overnight bacterial solution is taken and added to 40 mL LB (ampicillin and chloramphenicol resistance), and activated at 37°C for about 3 h (until OD 600 value to about 0.6), add 0.3mM IPTG for induction, and culture at 16℃ for 24h; centrifuge 40mL culture solution (12000rpm, 5min) to remove the supernatant to obtain bacterial precipitation, add 800μL acetone, and treat with 100W ultrasound for 20min, extract until the bacteria are colorless, centrifuge and take the supernatant through 0.22μm filter membrane, and use UPLC for chemical detection.

[0051] Conditions for UPLC chemical detection: instrument model, Thermo Ultimate 3000; injection volume: 5 μL; chromatographic column: Waters Acquity BEH C18 column, 1.7 μm, 100×2.1 mm; column temperature: 35°C; detection wavelength: UV 440 nm; mobile phase elution conditions: (A): 0.1% formic acid-acetonitrile solution, (B): 0.1% formic acid-water, flow rate: 0.2 mL / min, elution program: 0-8 min, 10% A linearly increased to 50% A; 8-12 min, 50% A linearly increased to 90% A; 12-13 min, 90% A linearly increased to 100% A, 100% A continued for 4 min; 17-22 min, 100% A linearly decreased to 10% A, 10% A continued for 4 min.

[0052] Results and Analysis

[0053] According to the analysis of UPLC liquid phase detection results, among all truncations, the N2 truncations had the most obvious improvement in catalytic activity; in the mutation study, the catalytic activity of N212 was increased by 21.81 times compared with the GjCCD4a original protein; after the addition of the MBP tag, N212m was obtained, and its catalytic activity was increased by 25.08 times compared with the GjCCD4a original protein ( Figure 1 ).

[0054] Example 2

[0055] Construction of co-expression vector of GjCCD4a mutant N212m and GjALDH2C3 gene and activity screening

[0056] Experimental methods

[0057]

[0058] Table 4. Primers for constructing pN212m-2C3 and p2C3-N212m expression vectors

[0059]

[0060]

[0061] The N212m and GjALDH2C3 genes were constructed into the multiple cloning site of the pET-Duet-1 vector using the Mut Express II Fast Mutagenesis Kit V2 from Novagen, and two expression vectors were constructed, named: pN212m-2C3 and p2C3-N212m (GjALDH2C3 was constructed into the multiple cloning site 1 of the pET-Duet-1 vector using the primer set p-2C3-XF and pX-2C3-R, and N212m was constructed into the multiple cloning site 2 of the pET-Duet-1 vector using the primer set p2C3-N212m-F and p2C3-N212m-R). The constructed vectors were respectively transferred into the zeaxanthin-producing Escherichia coli engineering strains for catalytic efficiency testing using the calcium chloride transformation method, and were screened using ampicillin (50 μg / mL) and chloramphenicol (34 μg / mL) resistance.

[0062] The culture media used in the study included LB (5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride) and FM (5% glucose concentration, 1.7 g / L citric acid, 10.5 g / L potassium dihydrogen phosphate trihydrate, 6.0 g / L diammonium hydrogen phosphate and 1% trace metal solution, pH = 8.0, each liter of trace metal solution includes 10.0 g FeSO4·7H2O, 5.25 g ZnSO4·7H2O, 3.0 g CuSO4·5H2O, 0.5 g MnSO4·4H2O, 0.23 g Na2B4O7·10H2O, 2.0 g CaC12, and 0.1 g (NH4)6Mo7O2) culture media. The positive colonies obtained above were cultured overnight, and 2 mL of the overnight bacterial solution was taken and added to 40 mL LB (ampicillin and chloramphenicol resistance), and activated at 37°C for about 3 h (until OD 600 When the OD value is about 0.6), 1.0 mM IPTG is added and the cells are induced at 16°C for 24 h. When FM medium is used for culture, the cells are activated at 37°C for about 3 h (when the OD value is about 0.6). 600 After the value reaches about 0.6), the culture solution needs to be centrifuged at 2500g for 10 min to obtain the bacterial pellet, which should be resuspended in an equal volume of FM medium before induction.

[0063] After induction, 3 M HCl was added to 40 mL of bacterial solution to adjust the pH value of the bacterial solution to 3, and then 6 mL of ethyl acetate was added thereto. The mixture was ultrasonically treated at 100 W for 15 min and centrifuged (6000 g, 5 min). 2 mL of the upper yellow transparent liquid was aspirated and concentrated to dryness under reduced pressure. 500 μL of methanol was added for resuspending, and the supernatant was centrifuged and filtered through a 0.22 μm filter membrane for chemical detection using UPLC.

[0064] Mobile phase elution conditions: (A): 0.1% formic acid-acetonitrile solution, (B): 0.1% formic acid-water, flow rate: 0.2 mL / min, elution program: 0-7 min, 10% A linearly increased to 50% A; 7-11 min, 50% A linearly increased to 90% A; 11-12 min, 90% A linearly increased to 100% A, 100% A continued for 3 min; 15-16 min, 100% A linearly decreased to 10% A, 10% A continued for 4 min.

[0065] Results and Analysis

[0066] p2C3-N212m was transformed into zeaxanthin-producing Escherichia coli to construct the engineered strain E5, and pN212m-2C3 was transformed into zeaxanthin-producing Escherichia coli to construct the engineered strain E2. According to the analysis of the UPLC liquid phase detection results, after induction culture, the production of crocetin by the E5 strain was greater than that of E2, so it can be concluded that the catalytic efficiency of p2C3-N212m is higher than that of pN212m-2C3. In addition, FM medium is more conducive to the accumulation of catalytic product crocetin than LB medium ( Figure 2 ).

[0067] Example 3

[0068] Construction, product detection and optimal reaction conditions of Escherichia coli engineered strain E579-Crocins

[0069] Experimental methods

[0070] Based on the above-mentioned E5 strain, pCDF-Duet-1-UGT74F8-UGT94E13 (plasmid is a conventional plasmid, for example, see: "In Vivo Production of Five Crocins in the Engineered Escherichia coli". DOI: 10.1021 / acssynbio.0c00039) and pSLB208-Pgm-T7-GalU (plasmid is a conventional plasmid, for example, see: "Systems Metabolic Engineering of Escherichia coli to Enhance the Production of Flavonoid Glucuronides》.DOI:10.1039 / C6RA03304K) plasmid was transformed into E5 engineering strain by calcium chloride transformation method, and the engineering bacteria were screened with ampicillin (50μg / mL), kanamycin (50μg / mL), streptomycin sulfate (50μg / mL) and chloramphenicol (34μg / mL) antibiotics to obtain Escherichia coli engineered bacteria E579-Crocins, which were preserved under patent procedure.

[0071] For the E. coli engineered bacteria E579-Crocins, a screening study on induction temperature, time and glucose concentration was conducted. The induction temperature included: 37°C, 28°C and 16°C; the induction time was 84h; the final concentration of exogenous glucose was 5%. The screening study process is as follows: the E. coli engineered bacteria E579-Crocins obtained above was cultured overnight, 2mL of the overnight bacterial solution was taken and added to 40mL LB (ampicillin, kanamycin, streptomycin sulfate and chloramphenicol resistance), and activated at 37°C for about 3h (wait until OD 600 value to about 0.6), centrifuged at 2500g for 10min, obtained bacterial precipitate, resuspended with FM medium (5% glucose concentration), added with 1.0mMIPTG for induction, and induced at 16-37℃ for 84h. Product extraction was performed after 84h. The product extraction method is as follows: add 5mL of water-saturated n-butanol to 40mL of culture solution, ultrasonically treat at 100W for 15min, centrifuge (6000g, 5min), absorb 200μL of the upper yellow transparent liquid, concentrate to dryness under reduced pressure, resuspend with 200μL of methanol, centrifuge (centrifuge at 12000rpm for 30min), take the supernatant through a 0.22μm filter membrane, perform chemical detection by UPLC, and perform qualitative analysis of the product by MS / MS.

[0072] Mobile phase elution conditions: (A): 0.1% formic acid-acetonitrile solution, (B): 0.1% formic acid-water, flow rate: 0.25 mL / min, elution program: 0-7 min, 10% A linearly increased to 50% A; 7-11 min, 50% A linearly increased to 90% A; 11-12 min, 90% A linearly increased to 100% A, 100% A continued for 3 min; 15-16 min, 100% A linearly decreased to 10% A, 10% A continued for 4 min.

[0073] The reaction products were qualitatively analyzed using Agilent Technologies 1290 Infinity II and 6545Q-TOF high-resolution mass spectrometers. The mass spectrometry conditions were as follows: drying gas at 350°C, 8.0 L / min; sheath gas at 350°C; carrier gas flow rate at 11.0 L / min; nebulizer at 35 psig; VCap at 3500 V; and data analysis using MassHunter (version 10.0) software.

[0074] In order to measure the yield of the engineered strain, crocin I purchased from Dalian Meilun Biotechnology Co., Ltd. was used as a standard to prepare standard samples of different concentrations (0.01 mg / L, 0.02 mg / L, 0.05 mg / L, 0.08 mg / L, 0.1 mg / L). The prepared standard samples were used to establish a standard curve using the above-mentioned UPLC method, and each concentration sample was repeated three times; the crocin yield was calculated with reference to the standard curve.

[0075] Results and Analysis

[0076] According to the analysis of UPLC liquid phase detection results, the production of five types of crocins can be detected in the E. coli engineered bacteria E579-Crocins at 28°C and 16°C after FM (5% glucose concentration) induction culture for 84 hours; and when cultured at 16°C, the production of five types of crocins is higher than that when cultured at 28°C. In addition, when cultured at 37°C, the production of five types of crocins could not be detected.

[0077] Among them, the standard curve y=304.47x-1.0703 (R 2 =0.9994), the yields of five crocins produced by the E. coli engineered bacteria E579-Crocins at 16°C and FM (5% glucose concentration) induction culture for 84 hours were calculated as follows: crocin I 1.71 mg / L, crocin II 2.04 mg / L, crocin III 3.11 mg / L, crocin IV 0.91 mg / L, crocin V 0.66 mg / L. ( Figure 3 )

[0078] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0079] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An engineered Escherichia coli producing five types of crocin, characterized in that: The deposit number is CGMCC: NO.29364.

2. A microbial agent, characterized in that: It comprises the engineered Escherichia coli bacteria producing five types of crocins as described in claim 1.

3. A microbial fermentation broth, characterized in that: The method is obtained by fermenting the engineered Escherichia coli bacteria producing five types of crocins as described in claim 1.

4. Use of the engineered bacteria according to claim 1, the microbial agent according to claim 2 or the fermentation liquid according to claim 3 in fermentation preparation or drug development; The application is to realize heterologous synthesis of crocin from glucose, wherein the crocin includes crocin III, crocin IV and crocin V.

5. The use according to claim 4, characterized in that: The induction conditions included: 1.0 mM IPTG, FM medium with a final glucose concentration of 5%, and induction at 16°C for 84 h.

6. A mutant gene N212 or N212m of GjCCD4a, characterized in that: The nucleotide sequence of N212 is shown in SEQ ID No.1; the nucleotide sequence of N212m is shown in SEQ ID No.

2.

7. An expression vector comprising the mutant gene according to claim 6.

8. A method for constructing an engineered Escherichia coli that produces five types of crocin, characterized in that: The process comprises sequentially transforming pACCAR25ΔcrtX, p2C3-N212m, pSLB208-Pgm-T7-GalU and pCDF-Duet-1-UGT74F8-UGT94E13 into competent Escherichia coli; The construction method of p2C3-N212m is as follows: using pET-Duet-1 as a vector, using p-2C3-XF and pX-2C3-R primer sets, constructing GjALDH2C3 into the multiple cloning site 1 of the pET-Duet-1 vector, and using p2C3-N212m-F and p2C3-N212m-R primer sets, constructing N212m into the multiple cloning site 2 of the pET-Duet-1 vector; The nucleotide sequence of N212m is shown in SEQ ID No.2; The nucleotide sequence of GjALDH2C3 is shown in SEQ ID No. 3; The nucleotide sequence of p-2C3-XF is shown in SEQ ID No. 4; The nucleotide sequence of pX-2C3-R is shown in SEQ ID No. 5; The nucleotide sequence of p2C3-N212m-F is shown in SEQ ID No.6; The nucleotide sequence of p2C3-N212m-R is shown in SEQ ID No.7.

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