Recombinant escherichia coli with high astaxanthin content as well as construction method and application thereof
By introducing a cyanobacterial acetyltransferase gene expression cassette into Escherichia coli, recombinant E. coli BW-ASTA-cKAT was constructed, solving the problem of high production cost of astaxanthin and achieving a significant increase in astaxanthin yield, reaching the economic threshold for industrial production.
Patent Information
- Application Number
- CN202511959001.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-24
- Publication Date
- 2026-03-20
AI Technical Summary
Existing astaxanthin production technologies suffer from high production costs, limited yields, long production cycles, and susceptibility to contamination, which restricts their widespread application.
A cyanobacterial acetyltransferase gene expression cassette was introduced into Escherichia coli, and its astaxanthin synthesis capacity was enhanced by inducing expression. Recombinant Escherichia coli BW-ASTA-cKAT was constructed to enhance its astaxanthin synthesis pathway.
It significantly increased the yield of astaxanthin to about four times that of the original strain, reduced production costs, and provided an efficient and stable industrial solution for microbial synthesis of astaxanthin.
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Figure CN121699896A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of metabolic engineering, specifically relating to a recombinant Escherichia coli with high astaxanthin content, its construction method, and its application. Background Technology
[0002] Astaxanthin, a potent antioxidant, is widely used in food, pharmaceuticals, and cosmetics, possessing significant commercial value. However, its high production cost limits its application. Currently, astaxanthin is primarily produced through chemical synthesis, microbial fermentation, and algal cultivation. Chemical synthesis is highly environmentally polluting, microbial fermentation (such as with Pharfogel's red yeast) has limited yields, and algal cultivation (such as with Haematococcus pluvialis) suffers from long production cycles, demanding cultivation conditions, and susceptibility to contamination. All these factors contribute to the persistently high unit production cost of astaxanthin.
[0003] In 2020, a synthetic biology team at MIT first reported using metabolic engineering to transfer the pathways required for astaxanthin synthesis into *E. coli*, resulting in the astaxanthin-synthesizing strain BW-ASTA. This was a core achievement of the "Microbial Astaxanthin Synthesis" project, and the research was published in *Nature Biotechnology*. The strain is already commercially available. However, the astaxanthin yield achieved using this strain is still far from reaching the economic threshold for industrial-scale production.
[0004] Therefore, it is necessary to further increase the production of astaxanthin on this basis in order to obtain recombinant Escherichia coli with higher astaxanthin synthesis capacity. Summary of the Invention
[0005] To address the above problems, this invention provides the application of cyanobacterial acetyltransferase in increasing the astaxanthin content in astaxanthin-producing Escherichia coli.
[0006] The present invention also provides a method for increasing the astaxanthin content in astaxanthin-producing Escherichia coli, comprising the step of introducing a cyanobacterial acetyltransferase gene expression cassette into the Escherichia coli.
[0007] In one specific embodiment, the protein sequence of the cyanobacterial acetyltransferase is shown in SEQ ID NO:2, or its encoding gene sequence is shown in SEQ ID NO:1.
[0008] In one specific implementation, the cyanobacterial acetyltransferase gene expression cassette is an inducible expression cassette.
[0009] In one specific implementation, the *E. coli* is an *E. coli* species that effectively expresses the astaxanthin synthesis pathway.
[0010] In one specific implementation, the Escherichia coli is strain BW-ASTA.
[0011] The present invention also provides recombinant Escherichia coli with high astaxanthin content prepared by the above method.
[0012] The present invention also provides the application of the above-mentioned recombinant Escherichia coli in the synthesis of astaxanthin.
[0013] The present invention also provides a method for synthesizing astaxanthin, comprising the step of synthesizing astaxanthin from the above-mentioned recombinant Escherichia coli.
[0014] In one specific implementation, the method includes the following steps: 1) Culture the *E. coli* to the logarithmic growth phase; 2) Add an inducer to induce the expression of the cyanobacterial acetyltransferase gene cassette, thereby further increasing the astaxanthin content in the recombinant Escherichia coli.
[0015] This invention amplifies the target gene cKAT from the genome of cyanobacteria, inserts cKAT into an Escherichia coli expression vector (either pET or pGEX series) using recombinant DNA technology, and transforms the constructed expression vector into the E. coli BW-ASTA strain to obtain recombinant E. coli. This increases the astaxanthin content in E. coli by approximately four times, providing a new, efficient, stable, and scalable microbial synthesis scheme for significantly reducing the production cost of astaxanthin. Attached Figure Description
[0016] Figure 1 Photographs of culture media containing recombinant Escherichia coli, BW-ASTA-cKAT, and basal bacteria BW-ASTA.
[0017] Figure 2 Western blotting was used to detect the protein expression of the cKAT gene in the recombinant strain BW-ASTA-cKAT.
[0018] Figure 3 The HPLC chromatograms are of BW-ASTA-cKAT recombinant bacteria, BW-ASTA control bacteria, and astaxanthin standard.
[0019] Figure 4 This is a bar chart showing the relative astaxanthin content of each strain, calculated based on HPLC peak area normalization. Detailed Implementation
[0020] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.
[0021] 1. Construct recombinant strains using Escherichia coli BW-ASTA as the substrate bacteria. The genome of Synechococcus PCC7002 was extracted, and the cKAT gene (nucleic acid sequence as shown in SEQ ID NO:1, protein sequence as shown in SEQ ID NO:2) was amplified using forward primer F: 5'-CCGGAATTCGTGAGTGCAGTTGCTTTAT and reverse primer R: 5'-CCGCTCGAGAGCATCGAGGGACCAAGAC-3'. The cKAT gene fragment was digested with EcoRI and XhoI enzymes at 37℃, and the digested cKAT gene fragment was ligated into the pGEX-4T-1 vector digested with EcoRI and XhoI using the Takara DNA ligation kit to construct the pGEX-cKAT expression vector.
[0022] The pGEX-cKAT plasmid was transformed into *E. coli* BW-ASTA competent cells via heat shock. 1 μL of the plasmid was added to the competent cells and incubated on ice for 20–30 min. The cells were then heat-shocked at 42°C for 90 s, and incubated in antibiotic-free LB broth at 37°C and 180 rpm for 1 h. The culture was then plated onto LB agar plates containing ampicillin (100 μg / mL) and chloramphenicol (25 μg / mL) and incubated overnight at 37°C. Single colonies were picked for colony PCR and plasmid sequencing verification to obtain the correctly transformed recombinant strain, named BW-ASTA-cKAT.
[0023] 2. Culture and induced expression of recombinant strain BW-ASTA-cKAT The recombinant strain BW-ASTA-cKAT was inoculated into a liquid culture medium containing ampicillin and chloramphenicol, and cultured at 37°C with shaking at 200 rpm until OD reached. 600 When the pH reached approximately 0.6, 0.6 mM IPTG was added to the culture medium to induce overexpression of the cKAT gene inserted in the recombinant *E. coli*, promoting more efficient astaxanthin synthesis. The temperature was then adjusted to 30°C and induction culture continued for 48 hours. *Bacillus thuringiensis* BW-ASTA was used as a control.
[0024] Photographs of the culture medium obtained after culturing are shown below. Figure 1 As shown, the recombinant strain BW-ASTA-cKAT is significantly darker in color than the chassis strain BW-ASTA.
[0025] Total protein was extracted from samples, and immunoblotting was performed using anti-GST antibody to detect protein expression. Results are as follows: Figure 2 As shown, the recombinant strain BW-ASTA-cKAT exhibits specific protein expression of approximately 45 kDa, which is consistent with the predicted size of cKAT-GST. This indicates that the recombinant strain was successfully constructed.
[0026] 3. Astaxanthin production detection of recombinant strain BW-ASTA-cKAT Collect the bacterial cells, freeze-dry them, and weigh an equal amount of dry weight. Add glass beads and organic extraction solution (dichloromethane:methanol = 1:3, v / v), vortex to break the cells, centrifuge to collect the supernatant, and filter through a membrane to obtain crude astaxanthin extract.
[0027] The astaxanthin content was determined by HPLC.
[0028] Chromatographic conditions: Agilent 1260 Infinity II system, ZORBAX SB-C18 column (4.6 × 250 mm, 5 μm); detection wavelength 476 nm; column temperature 25℃; mobile phase A: dichloromethane, B: methanol, C: acetonitrile, D: water; gradient program as follows: 0-8 min, flow rate 1.0 mL / min, A 5%, B 85%, C 5.5%, D 4.5%; 8-14 min, flow rate 1.0 mL / min, A linearly increases from 5% to 25%, B decreases from 85% to 28%, C linearly increases from 5.5% to 42.5%, D 4.5%; 14-38 min, flow rate 1.0 mL / min, A 25%, B 28%, C 42.5%, D 4.5%; 38-40 min, flow rate 1.0 mL / min. mL / min, A decreases from 25% to 5%, B increases from 28% to 85%, C decreases from 42.5% to 5.5%, D 4.5%; 40-45 minutes, flow rate 1.0 mL / min, A 5%, B 85%, C 5.5%, D 4.5%, injection volume 10 μL.
[0029] The results are as follows Figure 3 As shown, all samples exhibited a chromatographic peak at approximately 3 minutes, consistent with the retention time of the astaxanthin standard, confirming the product astaxanthin. The relative astaxanthin content in each strain was calculated by comparing the peak area of this characteristic peak and normalizing it to unit cell dry weight.
[0030] Astaxanthin calculation results are as follows Figure 4 As shown, the astaxanthin content of the BW-ASTA-cKAT recombinant strain was significantly higher than that of the original BW-ASTA, reaching approximately four times. This result conclusively demonstrates that overexpression of the cKAT gene can effectively enhance the astaxanthin synthesis pathway in the BW-ASTA strain, significantly increasing the yield of the target product.
[0031] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. Application of cyanobacterial acetyltransferase in increasing astaxanthin content in astaxanthin-producing Escherichia coli.
2. A method for increasing the astaxanthin content in astaxanthin-producing Escherichia coli, characterized in that, The step includes introducing a cyanobacterial acetyltransferase gene expression cassette into the *E. coli*.
3. The method according to claim 1 or claim 2, characterized in that, The protein sequence of the cyanobacterial acetyltransferase is shown in SEQ ID NO:2, or its encoding gene sequence is shown in SEQ ID NO:
1.
4. The method according to claim 2, characterized in that, The cyanobacterial acetyltransferase gene expression cassette is an inducible expression cassette.
5. The method according to claim 2, characterized in that, The *E. coli* strain mentioned is *E. coli* that effectively expresses the astaxanthin synthesis pathway.
6. The method according to claim 5, characterized in that, The Escherichia coli strain is BW-ASTA.
7. A recombinant Escherichia coli with high astaxanthin content, characterized in that, It is prepared by the method according to any one of claims 2-6.
8. The application of the recombinant Escherichia coli according to claim 7 in the synthesis of astaxanthin.
9. A method for synthesizing astaxanthin, characterized in that, The step includes culturing the recombinant Escherichia coli of claim 7 to synthesize astaxanthin.
10. The method according to claim 9, characterized in that, Includes the following steps: 1) Culture the *E. coli* to the logarithmic growth phase; 2) Add an inducer to induce the expression of the cyanobacterial acetyltransferase gene cassette, thereby further increasing the astaxanthin content in the recombinant Escherichia coli.