A gene combination for synthesizing beta-cryptoxanthin glycoside, e. coli engineering bacteria and application thereof

Abstract

The application discloses a gene combination for synthesizing beta-cryptoxanthin glycoside, an escherichia coli engineering bacterium and application of the gene combination. The gene combination comprises a beta-carotene hydroxylase gene BCH derived from citrus and a carotenoid glycosyltransferase gene CrtX derived from bacteria. The BCH can catalyze beta-carotene to be hydroxylated to generate beta-cryptoxanthin, and the CrtX can catalyze the beta-cryptoxanthin to be glycosylated to generate beta-cryptoxanthin glycoside. The BCH and the CrtX are combined, and are introduced into escherichia coli BL21 (DE3) which has the ability of synthesizing beta-carotene to construct the escherichia coli engineering bacterium which can synthesize beta-cryptoxanthin glycoside. After cultivation, the engineering bacterium can take beta-cryptoxanthin glycoside as the main glycosylated carotenoid product, and make the beta-cryptoxanthin glycoside become the main carotenoid product. The application provides a key gene combination and an engineering strain basis for heterologous biosynthesis of beta-cryptoxanthin glycoside.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a gene combination for synthesizing β-cryptoxanthin glycosides, an engineered strain of Escherichia coli, and their applications. Background Technology

[0002] Carotenoids are a class of C40 natural terpene pigments composed of eight isoprene units. They are widely found in plants, algae, fungi, and some microorganisms, and typically appear yellow, orange, or red. Carotenoids play important roles in photosynthesis, photosensitivity, reactive oxygen species scavenging, and the coloring of fruits and floral organs. Furthermore, some dietary carotenoids are significant in maintaining visual function, enhancing the body's antioxidant capacity, and regulating immune function, thus possessing important applications in food, medicine, animal feed, and cosmetics.

[0003] β-cryptoxanthin is a monohydroxy carotenoid produced by the hydroxylation of β-carotene by β-carotene hydroxylase. It is also a key intermediate in the further hydroxylation of β-carotene to form zeaxanthin. β-cryptoxanthin is naturally abundant in most fruits and vegetables, accumulating mainly in a few plants such as citrus fruits, pumpkins, and peppers. Studies have shown that β-cryptoxanthin can serve as a vitamin A precursor and has potential nutritional value in areas such as bone health, anti-inflammation, and antioxidation, thus attracting widespread attention in the fields of functional foods, dietary supplements, and nutritional health.

[0004] However, β-cryptoxanthin and its esterified derivatives are highly hydrophobic, which limits their extraction, separation, processing, and utilization. Glycosylation is a common structural modification method in natural products, which can usually change the physicochemical properties of compounds and is expected to improve their solubility and stability. For carotenoids, glycosylation modification can provide an important pathway for the development of novel carotenoid derivatives.

[0005] Currently, glycosylated carotenoids can be obtained through chemical and biosynthetic methods. Chemical synthesis methods typically involve numerous reaction steps, stringent conditions, difficulties in selectivity control, and low recovery rates, hindering large-scale production. In contrast, biosynthesis using metabolically engineered microbial chassis offers advantages such as milder conditions, convenient genetic manipulation, and ease of further optimization, making it an important direction for obtaining glycosylated carotenoids.

[0006] Escherichia coli BL21 (DE3) is a commonly used prokaryotic expression host, with advantages such as simple culture conditions, rapid growth rate, short fermentation cycle, and mature genetic manipulation. By introducing carotenoid synthesis-related genes, Escherichia coli can serve as an important microbial substrate for the production of carotenoids and their derivatives.

[0007] While existing technologies have yielded some research on the biosynthesis of β-cryptoxanthin, reports on the efficient biosynthesis of β-cryptoxanthin glycosides (the main peak) are scarce, particularly lacking engineered bacterial systems that utilize microbial chassis to synthesize β-cryptoxanthin glycosides. Therefore, developing a gene combination, engineered bacteria, and its construction method capable of synthesizing β-cryptoxanthin glycosides in microorganisms is of great significance for the green preparation and subsequent research of β-cryptoxanthin and its glycosides. Summary of the Invention

[0008] To address the above problems, this invention provides a gene combination for synthesizing β-cryptoxanthin glycosides, an engineered Escherichia coli strain, and its applications.

[0009] The technical solution provided by this invention is as follows: 1. A gene combination for the synthesis of β-cryptoxanthin glycosides. The gene combination includes the β-carotene hydroxylase gene BCH and the carotenoid glycosyltransferase gene CrtX.

[0010] The BCH encodes a protein with β-carotene hydroxylase activity, which can catalyze the hydroxylation of β-carotene to generate β-cryptoxanthin.

[0011] The CrtX encodes a protein with carotenoid glycosyltransferase activity, which can catalyze the glycosylation modification of β-cryptoxanthin to generate β-cryptoxanthin glycoside.

[0012] Preferably, the BCH is derived from citrus fruits.

[0013] Preferably, the CrtX is derived from bacteria.

[0014] Preferably, the nucleotide sequence of the BCH is as shown in SEQ ID NO:1, or is a nucleotide sequence that has at least 90%, 95%, 98% or 99% sequence identity with SEQ ID NO:1 and encodes a protein with the same function.

[0015] Preferably, the nucleotide sequence of CrtX is as shown in SEQ ID NO:2, or is a nucleotide sequence that has at least 90%, 95%, 98% or 99% sequence identity with SEQ ID NO:2 and encodes a protein with the same function.

[0016] 2. A recombinant expression vector The recombinant expression vector contains the BCH gene and the CrtX gene.

[0017] The BCH and CrtX can be located in the same expression vector or in different expression vectors.

[0018] 3. An engineered strain of *Escherichia coli* that uses β-cryptoxanthin glycosides as the main glycosylated carotenoid product. The engineered Escherichia coli contains the BCH gene and the CrtX gene, and has the ability to synthesize β-carotene.

[0019] The engineered Escherichia coli strain can synthesize β-cryptoxanthin glycoside, and β-cryptoxanthin glycoside is the main glycosylated carotenoid product produced by the engineered strain.

[0020] Preferably, the engineered Escherichia coli strain is Escherichia coli BL21(DE3) with β-carotene synthesis ability as the chassis strain.

[0021] 4. A method for constructing engineered Escherichia coli strains The method includes the following steps: (1) Obtain the BCH gene and the CrtX gene; (2) The BCH gene and CrtX gene were constructed into the expression vector; (3) The expression vector was introduced into Escherichia coli with β-carotene synthesis ability; (4) Screening to obtain engineered Escherichia coli strains capable of synthesizing β-cryptoxanthin glycosides; (5) Detect the glycosylated carotenoid products produced by the engineered bacteria, and screen for engineered bacteria with β-cryptoxanthin glycoside as the main glycosylated carotenoid product.

[0022] 5. A method for producing β-cryptoxanthin glycosides using engineered Escherichia coli strains. The method includes the following steps: (1) Cultivate engineered Escherichia coli containing the BCH gene and CrtX gene; (2) Inducing BCH and CrtX expression; (3) The β-carotene in the engineered bacteria is catalyzed by BCH to generate β-cryptoxanthin, and further catalyzed by CrtX to generate β-cryptoxanthin glycoside; (4) Collect bacterial cells and extract β-cryptoxanthin glycosides; (5) The extract was tested, and β-cryptoxanthin glycoside was the main glycosylated carotenoid product.

[0023] The above-mentioned gene combinations or genetically engineered bacteria are used in the synthesis of β-cryptoxanthin glycosides.

[0024] The beneficial effects of this invention are as follows: This invention combines citrus BCH and bacteria CrtX and introduces them into *Escherichia coli* BL21(DE3), which has the ability to synthesize β-carotene, to construct an engineered *E. coli* strain capable of synthesizing β-cryptoxanthin glycosides. After cultivation, this engineered strain can use β-cryptoxanthin glycosides as the main glycosylated carotenoid product, making it the primary carotenoid product. This invention provides a key gene combination and engineered strain basis for the heterologous biosynthesis of β-cryptoxanthin glycosides, and has the potential to be applied to the biomanufacturing of carotenoid glycosides.

[0025] This invention utilizes BCH to catalyze the formation of β-cryptoxanthin from β-carotene, and further utilizes CrtX to catalyze the glycosylation modification of β-cryptoxanthin, thereby constructing a β-cryptoxanthin glycoside synthesis pathway.

[0026] The engineered Escherichia coli strain constructed in this invention can make β-cryptoxanthin glycoside the main glycosylated carotenoid product, indicating that the system has good target product orientation.

[0027] This invention does not only detect the formation of β-cryptoxanthin glycosides, but also achieves the major accumulation of β-cryptoxanthin glycosides in glycosylated carotenoid products, thereby increasing the relative proportion of the target product in the glycosylated products.

[0028] This invention establishes a method for synthesizing β-cryptoxanthin glycosides using an Escherichia coli chassis, providing a technical basis for the directed biosynthesis of glycosylated carotenoids and subsequent metabolic engineering optimization. Attached Figure Description

[0029] Figure 1 The structural formulas of β-cryptoxanthin and β-cryptoxanthin glycosides are shown. Figure 2 Photos of the bacterial culture after IPTG induction and after centrifugation; Figure 3 The chromatograms of BCH and CrtX detected by HPLC are shown from left to right as zeaxanthin, β-cryptoxanthin glycoside, β-cryptoxanthin and β-carotene, with peak times of 8.9 min, 10.4 min, 13.8 min and 17.6 min at a wavelength of 450 nm, respectively. Figure 4 The image shows the spectrum detected by HPLC. The left side corresponds to β-cryptoxanthin, and the right side corresponds to β-cryptoxanthin glycoside. Figure 5 This is a mass spectrum detected by LCMS. The left side corresponds to β-cryptoxanthin, and the right side corresponds to β-cryptoxanthin glycoside. Figure 6 Bar chart showing the yields of β-cryptoxanthin using BCH and the yields of glycosylated β-cryptoxanthin using BCH-CrtX. Detailed Implementation

[0030] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0031] Part 1: Obtaining the BCH and CrtX genes The β-carotene hydroxylase gene BCH and the carotenoid glycosyltransferase gene CrtX were obtained.

[0032] RNA was extracted from the peel of citrus fruits after 210 days and then reverse transcribed to obtain cDNA.

[0033] The hydroxylase gene BCH was obtained by TA cloning using citrus peel cDNA as a template.

[0034] Source Pantoea ananatis ATCC The CrtX gene of 19321 was synthesized by Qingke Biotechnology after codon optimization, and its sequence design references (Chen et al. Bioresources and Bioprocessing, 2021, 8, 67).

[0035] The BCH1 and BCH2 genes were used to predict chloroplast transport peptides using the TargetP-2.0 online website, and these peptides were removed during vector construction.

[0036] Among them, BCH encodes a protein with β-carotene hydroxylase activity, which can catalyze the formation of β-cryptoxanthin from β-carotene; CrtX encodes a protein with carotenoid glycosyltransferase activity, which can catalyze the formation of β-cryptoxanthin from β-cryptoxanthin glycosides.

[0037] Specifically, the CDS region sequence of BCH1 is shown below (the bolded part is the region with the signal peptide removed): ATGGCGGTCGGACTATTGGCCGCCGTAGTCCCGAAGCCCTTCTGTCTCCTCACAACAAAACTTCAACCCTCTTCGCTCCTCACAACAAAACCTGCTCCCCTTTTTGCCCCTCTCGGTACCCGCCACGGCTTCTTTAATGGCAAAAACCGAAGAAAAATCAACTCTTTCACCGTATGTTTTGTTTTAGAGGAGAAAAAACAAAGCACCCAGATCGAGACTTTCACGGAGGAGGAGGAGGAGGAGTCGGGTACCCAGATCTCGACTGCTGCCCGCGTGGCCGAGAAATTGGCGAGAAAGAGATCCGAGAGGTTCACTTATCTCGTTGCTGCCGTCATGTCTAGTTTTGGTATCACTTCCATGGCTGTCATGGCTGTTTATTACAGGTTCTGGTGGCAAATGGAGGGTGGAGAGGTGCCTTTAGCTGAAATGTTTGGCACATTTGCTCTCTCTGTTGGTGCTGCTGTGGGCATGGAGTTTTGGGCACGATGGGCTCATAAAGCTCTGTGGCATGCTTCTTTATGGCATATGCACGAGTCTCACCATCGACCAAGAGAGGGTCCTTTTGAGCTAAACGATGTGTTTGCCATAATCAACGCAGTTCCAGCCATAGCCCTTCTCTCTTTTGGCTTCTTCCACAAAGGCCTTGTACCTGGTCTCTGCTTTGGTGCTGGACTTGGCATTACGGTGTTTGGGATGGCCTACATGTTCGTCCACGATGGTCTCGTTCACAAAAGGTTCCCTGTGGGTCCCATTGCCGACGTGCCTTATTTCCGGAGAGTCGCTGCGGCTCACCAGCTTCACCACTCGGATAAATTCCACGGTGTTCCATATGGGCTCTTTCTCGGACCTAAGGAGCTTGAAGAAGTGGGGGGACTAGAAGAATTGGAGAAGGAGATCAGTAAGAGAATCAAATCATACAACAGGGTTCCAAAATAA The CDS sequence of BCH2 is shown below (the signal peptide region is removed in the bolded part): ATGGCAAGTGGAATGTCATCAGCCAATTCAAGTGTTTTGACATATCTTCTTGGTCGTAATACTTTCTTTACTGGAAAAGCAATATCCAAAGCGATAATTCCTCGATCATCCTTAAGGCAGGGAACCCAGAAGAGGAAACCGAAGAGCTTGAGTATGTGCTTTGTCACAGAGGATAAAAATGAAGGTACTACTAAAATTGAGAAGAAAACTTTTGTACAAGCACAGAATGAGATCAAATTTTTCTCGCCAGCAGAAGAGAGACAAGCAAGAAAAAGATCAGAGAGACAAACCTACCTTGTTGCAGCAATCGTGTCCAGTCTTGGCATCTCTTCAATGGCTGTTTTGGCCATACATCACAGATTTTCATGGCAAACAGAGGGTTGTGAGTTGCAGCTTCTAGAAATGTTCGGTACATTTGCACTGTCTGTTGGGGCAGCTGTAGGCATGGAGTTTTGGGCAAGGTGGGCTCATAGAGCTCTATGGCACGCTTCATTGTGGCACATGCATAAGTCTCATCACCAGCCGAGGGATGGTCCTTTCGAGTTGAACGATATTTTTGCAGTAATTAACGCGGTTCCTGCAATTGCTCTGCTTTCTTACGGCTTCTTCCACAAAGGCATCGTTCCAGGACTCTGTTTCGGCGCTGGTCTGGGAATTACAGTTTTCGGAATGGCCTACATGTTCGTTCACGATGGACTTGTCCACCGTCGATTTCCTGTGGGACCAATCGCTCACGTCCCTTATTTACGTAAAGTCGCTGCAGCCCATCAACTTCACCATTCGGAGAAATTTAATGGGTTGCCGTACGGGTTGTTCTTGGGACCTCAGGAGCTAGAAGAGGTGGAAGGCACGGAAGGATTAGATAAAGAAACGTAG The CDS sequence of CrtX is shown below (codon-optimized): II. Construction of Recombinant Expression Vectors BCH and CrtX were constructed separately or together into an E. coli expression vector to obtain a recombinant expression vector containing BCH and CrtX.

[0038] The vector PCDFDuet-CrtX was constructed by Qingke Biotechnology Co., Ltd. The vectors PCDFDuet-BCH1, PCDFDuet-BCH2, PCDFduet-BCH1-CrtX and PCDFDuet-BCH2-CrtX were constructed through homologous recombination.

[0039] 3. Construction of engineered bacteria for β-cryptoxanthin synthesis The recombinant expression vector containing BCH and CrtX was introduced into Escherichia coli BL21(DE3) with β-carotene synthesis ability, and positive transformants were obtained by screening, which are the β-cryptoxanthin glycoside synthesizing engineered bacteria.

[0040] 1. Preparation of β-carotene-producing engineered bacteria competent cells (named 16XBL21) The β-carotene engineered bacterial plasmid pACCAR16△crtX (which is available in our laboratory and includes the crtE, crtB, crtl, and crtY genes) was transferred into Escherichia coli BL21(DE3) competent cells.

[0041] Select healthy, orange-yellow monoclonal colonies and add them to 50 ml of LB liquid medium (containing 1:1000 chloramphenicol). Incubate at 37 °C and 200 rpm until the concentration reaches OD. 600 =0.4~0.5.

[0042] Transfer 1 ml of bacterial culture into a 1.5 ml centrifuge tube and centrifuge at 4000 rpm for 5 min at 4 °C.

[0043] Discard the supernatant and remove any remaining liquid with a pipette. Add 100 μL of 0.05 M CaCl2 solution and gently suspend the bacterial cells.

[0044] Centrifuge at 4000 rpm for 5 min at 4 ℃, wash the liquid with a pipette, add 100 μL of 0.05 M CaCl2 solution (containing 15% glycerol), gently suspend the cells, and store the obtained β-carotene competent cells at -80℃ for later use.

[0045] 2. Construction of engineered bacteria containing β-cryptoxanthin and its glycosides The plasmids PCDF-Duet-BCH1, PCDFDUet-BCH2, PCDSDuet-BCH1-CrtX, and PCDF-Duet-BCH2-CrtX were transferred into 16XBL21 competent cells that produce β-carotene.

[0046] Single clones were selected for PCR positive detection. The positive bacterial solutions were identified as β-cryptoxanthin engineered bacteria and β-cryptoxanthin glycoside engineered bacteria.

[0047] IV: Culture of engineered bacteria and detection of β-cryptoxanthin glycosides The engineered bacteria that synthesize β-cryptoxanthin glycosides were inoculated into a culture medium and cultured for induced expression. After culture, the bacterial cells were collected, and carotenoids and their glycosylated products were extracted using organic solvents and detected by high-performance liquid chromatography and other methods.

[0048] The test results showed that the engineered E. coli strains introduced with BCH and CrtX could produce β-cryptoxanthin glycosides. Further analysis indicated that β-cryptoxanthin glycosides were the main product peak among the glycosylated carotenoids produced by these engineered bacteria, suggesting that the engineered bacteria could accumulate β-cryptoxanthin glycosides as the main glycosylated carotenoid product.

[0049] In a 50 mL shake flask culture system, the yield of β-cryptoxanthin glycoside reached 0.145 mg / L.

[0050] 1. Cultivation and induction of engineered bacteria The preserved engineered bacterial culture was streaked in different areas, and three normally growing single clones were selected and added to 50 ml of 2YT liquid medium (containing 1:1000 chloramphenicol and 1:1000 spectinomycin) for overnight culture.

[0051] Take 500 μL of bacterial culture and inoculate it into 50 mL of 2YT liquid medium (containing 1:2000 chloramphenicol and 1:2000 spectinomycin), and incubate at 37 °C and 160 rpm until the concentration reaches OD. 600 It is around 0.5.

[0052] Add 5 μL of 1 M IPTG and induce in vivo enzyme expression at 16 °C and 160 rpm for 15 h.

[0053] 2. Extraction of carotenoids from engineered bacteria Transfer the induced bacterial culture to a 50 ml centrifuge tube and centrifuge at 4 ℃ and 5,000 rpm for 10 min.

[0054] Discard the supernatant, centrifuge at 5,000 rpm for 1 min at 4 ℃. Use a pipette to remove the liquid, add 2 ml of acetone, and vortex for 2 minutes to mix.

[0055] Centrifuge at 4 ℃, 5,000 rpm for 10 min.

[0056] Aspirate the supernatant using a 1 ml syringe, filter it through a 0.22 μm filter membrane, and transfer it to a 2 ml centrifuge tube.

[0057] Evaporate to dryness at 30 ℃ using a vacuum concentrator and store at -20 ℃ for later testing.

[0058] 3. High-performance liquid chromatography (HPLC) detection Dissolve the evaporated sample in 150 μl of ethyl acetate, mix well, centrifuge at 1000 rpm for 1 min, and transfer the supernatant through a 0.22 μm filter membrane into the inner tube of the sample vial for testing.

[0059] YMC C30 reversed-phase column (150 × 3 mm, μm) was used at a flow rate of 0.8 ml / min.

[0060] Mobile phase A: Methanol:Water:MTBE, 6:3:1, V:V;V. Mobile phase B: Methanol:MTBE = 1:1, V:V.

[0061] Program: 0 min: 70% A / 30%B; 19 min: 100%B; 24 min: 100%B; 25 min: 70% A / 30%B; 29min: 70% A / 30%B.

[0062] The peak times at a wavelength of 450 nm were 8.9 min for zeaxanthin, 13.8 min for β-cryptoxanthin, 17.6 min for β-carotene, and 10.4 min for glycosylated β-cryptoxanthin.

[0063] 4. Detection by liquid chromatography-mass spectrometry (LC-MS) The evaporated sample was dissolved in 150 μl of 90% acetonitrile, mixed well, centrifuged at 1000 rpm for 1 min, and the supernatant was passed through a 0.22 μm filter membrane into the inner tube of the sample vial for testing.

[0064] The chromatogram was run on an ACQUITY UPLC BEH C18 column (100 × 2.1 mm, 1.7 μm) with a C18 guard column (5 × 2.1 mm, 1.7 μm) at a flow rate of 0.2 mL / min. Mobile phase A: Acetonitrile:Water:Formic acid, 20:80:0.1, v:v:v. Mobile phase B: Acetonitrile:Isopropanol:Formic acid, 60:40:0.1, v:v:v.

[0065] Program: 0–1 min 100% A, 1–3 min 100–60% A, 3–8 min 60–20% A, 8–14 min 20–10% A and 14–15 min 10–0% A, then wash and balance with 100% B and 100% A respectively.

[0066] The elution time and molecular weight at a wavelength of 450 nm are 11.6 min and 569.43530, which are associated with zeaxanthin (C). 40 H 56 02), 14.4 min and 715.49321 are glycosylated β-cryptoxanthin (C 46 H 66 O6), 15.9 min and 553,44039 are β-cryptoxanthin (C 40 H 56 0) and 18.2 min are β-carotene.

[0067] 5. Results and Analysis Carotenoids were extracted from the induced bacterial culture and detected by HPLC and LC-MS. The results from the induced bacterial culture and after centrifugation showed that the β-carotene produced by the empty vector 16X-BL21 resulted in an orange-yellow color, while the PCDFDuet-BCH1-CrtX and PCDFDuet-BCH2-CrtX transformed into 16X-BL21 turned yellow. This result is consistent with the subsequent HPLC analysis of the substance content, as the yellow color is due to the accumulation of a large amount of β-cryptoxanthin glycosides.

[0068] HPLC results show that the PCDFDuet empty vector (EV) transformed with 16X-BL21 only produces the substrate β-carotene. PCDFDuet-BCH1 and PCDFDuet-BCH2 produce a small amount of β-carotene and a large amount of β-cryptoxanthin and zeaxanthin. PCDFDuet-BCH1-CrtX and PCDFDuet-BCH2-CrtX produce a new peak at 10.4 min, namely β-cryptoxanthin glycoside. It can be seen that the addition of a glycosyl group increases polarity, resulting in an earlier elution time than β-cryptoxanthin. Figure 2 , Figure 3 ).

[0069] The spectrum at 450 nm shows that its peak shape is the same as that of β-cryptoxanthin, which is consistent with the absorption spectrum characteristics of carotenoids. Figure 4 ).

[0070] The LC-MS mass spectrum shows that the molecular weight of this new peak is 715.49628, which is exactly the molecular weight of β-cryptoxanthin plus the molecular weight of glucose. Therefore, it can be determined that the CrtX and BCH genes can produce β-cryptoxanthin glycosides. Figure 1 , Figure 5 ).

[0071] The yields of BCH1-CrtX and BCH2-CrtX in 50 ml of E. coli BL21 reached 0.095 mg / L and 0.092 mg / L, respectively. Figure 6 ) 6. Calculation of β-cryptoxanthin and its glycoside yields Accurately weigh a certain amount of β-cryptoxanthin standard, dissolve it in ethyl acetate, dilute it by different factors, and detect the peak area of ​​different concentrations using HPLC. Plot a standard curve based on the standard concentration and peak area.

[0072] β-cryptoxanthin standard curve (Y is the sample concentration, μg / ml, X is the peak area) The peak area of ​​the sample of the test strain was determined by HPLC and substituted into the standard curve to obtain the corresponding concentration. Then, the concentration was multiplied by the volume of the sample dissolved and loaded, and then divided by the volume of the culture. The result is the yield of β-cryptoxanthin obtained by 50 ml of culture.

[0073] ;

[0074] For standards without β-cryptoxanthin glycosides, glycosylated β-cryptoxanthin is relatively quantified using a β-cryptoxanthin standard curve, and its content is expressed as β-cryptoxanthin equivalent.

[0075] The above description provides examples of the preferred embodiments of the present invention. Parts not detailed herein are common knowledge to those skilled in the art. The scope of protection of the present invention is determined by the claims. Any equivalent modifications based on the technical teachings of the present invention are also within the scope of protection of the present invention.

Claims

1. A gene combination for synthesizing β-cryptoxanthin glycosides, characterized in that, The gene combination includes the β-carotene hydroxylase gene BCH derived from citrus and the carotenoid glycosyltransferase gene CrtX derived from bacteria.

2. An engineered *Escherichia coli* strain that synthesizes β-cryptoxanthin glycosides, characterized in that... The engineered Escherichia coli strain is an Escherichia coli strain that expresses the citrus β-carotene hydroxylase gene BCH and the bacterial carotenoid glycosyltransferase gene CrtX and has the ability to synthesize β-cryptoxanthin glycosides. β-cryptoxanthin glycosides are the main glycosylated carotenoid products produced by the engineered Escherichia coli strain and become the main carotenoid products.

3. A method for constructing an engineered *Escherichia coli* strain that synthesizes β-cryptoxanthin glycosides, characterized in that, Includes the following steps: The β-carotene hydroxylase gene BCH and the carotenoid glycosyltransferase gene CrtX were constructed into an expression vector, and the expression vector was introduced into Escherichia coli with the ability to synthesize β-cryptoxanthin glycosides. Escherichia coli engineered strains that can use β-cryptoxanthin glycosides as the main glycosylated carotenoid products were screened to obtain the results.

4. A method for producing β-cryptoxanthin glycoside using the engineered Escherichia coli strain described in claim 2, characterized in that, The method includes the following steps: culturing the engineered Escherichia coli strain as described in claim 5, and obtaining β-cryptoxanthin glycoside from the engineered Escherichia coli strain.

5. The application of the gene combination as described in claim 1 or the engineered Escherichia coli as described in claim 2 in the synthesis of β-cryptoxanthin glycosides.

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