Construction and application of engineered bacteria based on beta-carotene isomerase dsbci from dunaliella salina

Abstract

This invention discloses a method based on Dunaliella salina β-carotene isomerase. DsBCI Construction and application of engineered bacteria. This invention discloses for the first time three Dunaliella salina β-carotene isomerases. DsBCI : DsBCI -1, DsBCI -2, DsBCI -3, the amino acid sequence is shown in SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, and the base sequence of the gene encoding it is shown in SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6. This invention relates to three β-carotene isomerases from Dunaliella salina. DsBCI Construction and application of [the enzyme / organism]. Among them, β-carotene isomerase... DsBCI The vector, based on pETduet-1 containing the T7 promoter and ampicillin resistance, was co-transformed with the W07 plasmid into *E. coli* BL21(DE3) for expression. Three *Dunaliella salina* β-carotene isomerases were verified by measuring the 9-cis-β-carotene content. DsBCI Function and effectiveness of heterologous expression.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and relates to the construction and application of engineered bacteria based on Dunaliella salina β-carotene isomerase DsBCI. Background Technology

[0002] Carotenoids are naturally occurring, fat-soluble pigments commonly found in plants and algae. The most important carotenoid is beta-carotene, considered a major source of vitamin A for the human body. Beta-carotene possesses strong antioxidant properties and can enhance immunity. According to a market research report by Mordor Intelligence, the global beta-carotene market is projected to reach $1.9 billion by 2030. Beta-carotene has two isomers: all-trans beta-carotene and 9-cis beta-carotene. 9-cis beta-carotene is highly oil-soluble and does not easily crystallize. Furthermore, research has found that the higher the proportion of 9-cis beta-carotene in beta-carotene, the stronger its antioxidant and anticancer activities.

[0003] However, existing research indicates that the abundance of 9-cis-β-carotene in nature is extremely limited, and its artificial synthesis faces significant challenges. Although Dunaliella salina (… Dunaliella salina This algae can naturally accumulate a high proportion of 9-cis-β-carotene (accounting for 40%-50% of total β-carotene), but its cultivation cycle is long, highly susceptible to environmental factors, and the extraction and purification process is complex, resulting in high production costs and a market price as high as €500,000 per gram, approximately eight times that of the all-trans isomer. Furthermore, chemical synthesis methods struggle to achieve selective synthesis of the 9-cis configuration, and the precise regulatory mechanism of the isomerization process remains unclear. Therefore, developing an efficient, stable, and directionally synthesizable engineered strain of 9-cis-β-carotene is of great significance for promoting the green biomanufacturing of highly physiologically active β-carotene isomers (Wu S, Wu X, Wen J, et al. Biosynthesis and biological activities of 9-cis beta-carotene from greenmicroalga Dunaliella. Algal Research. 2025;91:104270.).

[0004] Microalgae are considered an important source of β-carotene. Dunaliella salina (Dunaliella salina) Dunaliella salinaDunaliella salina is a single-celled green alga that can efficiently accumulate β-carotene. It can accumulate relatively large amounts of all-trans β-carotene and 9-cis β-carotene. The accumulation of β-carotene is closely related to the enzyme's conversion efficiency; therefore, discovering enzymes that can efficiently produce 9-cis β-carotene is of great significance for the industrial production of 9-cis β-carotene. Summary of the Invention

[0005] To address the difficulty of artificially synthesizing 9-cis-β-carotene, the main objective of this invention is to provide three Dunaliella salina β-carotene isomerases. DsBCI The amino acid sequence.

[0006] Another object of the present invention is to provide the above three Dunaliella salina β-carotene isomerases. DsBCI The base sequence.

[0007] Another object of the present invention is to provide a β-carotene isomerase based on the above-mentioned Dunaliella salina β-carotene isomerase. DsBCI Methods for constructing engineered bacteria.

[0008] Another object of the present invention is to provide a β-carotene isomerase based on Dunaliella salina. DsBCI Engineered bacteria.

[0009] Another object of the present invention is to provide the above-mentioned β-carotene isomerase based on Dunaliella salina. DsBCI The application of engineered bacteria.

[0010] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a Dunaliella salina (Dunaliella salina) Dunaliella salina β-Carotene isomerase DsBCI -1, whose amino acid sequence is shown in SEQ ID NO: 1.

[0011] This invention also provides a Dunaliella salina β-carotene isomerase. DsBCI -2, whose amino acid sequence is shown in SEQ ID NO: 3.

[0012] This invention also provides a Dunaliella salina β-carotene isomerase. DsBCI -3, whose amino acid sequence is shown in SEQ ID NO: 5.

[0013] The present invention also provides encoding the above-mentioned β-carotene isomerase. DsBCI The gene for -1 has a nucleotide sequence as shown in SEQ ID NO:2.

[0014] The present invention also provides encoding the above-mentioned β-carotene isomerase. DsBCIThe gene for -2 has the nucleotide sequence shown in SEQ ID NO:4.

[0015] The present invention also provides encoding the above-mentioned β-carotene isomerase. DsBCI The gene for -3 has the nucleotide sequence shown in SEQ ID NO:6.

[0016] This invention also provides a β-carotene isomerase based on Dunaliella salina. DsBCI The method for constructing engineered bacteria includes the following steps: (1) Dunaliella salina ( Dunaliella salina Using cDNA as a template, PCR amplification was performed with specific primer pairs, and the gene with the nucleotide sequence shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 was cloned. The specific primer pair for cloning the gene shown in SEQ ID NO:2 is as follows: Upstream primer: as shown in SEQ ID NO: 7, Downstream primer: as shown in SEQ ID NO: 8; The specific primer pair for cloning the gene shown in SEQ ID NO:4 is: Upstream primer: as shown in SEQ ID NO: 9, Downstream primer: as shown in SEQ ID NO: 10; The specific primer pair for cloning the gene shown in SEQ ID NO:6 is: Upstream primer: as shown in SEQ ID NO: 11, Downstream primer: as shown in SEQ ID NO: 12; (2) The gene is ligated into an expression vector to obtain a recombinant expression vector; (3) Transform the recombinant expression vector into host cells to obtain a β-carotene isomerase based on Dunaliella salina β-carotene. DsBCI Engineered bacteria.

[0017] Furthermore, the expression vector was pETduet-1; the host cell was Escherichia coli BL21(DE3).

[0018] This invention also provides a β-carotene isomerase based on Dunaliella salina constructed by the above method. DsBCI Engineered bacteria.

[0019] The present invention also provides the application of the above-mentioned engineered bacteria in the production of 9-cis-β-carotene.

[0020] Compared with the prior art, the present invention has the following advantages and effects: 1. This invention provides three β-carotene isomerases. DsBCI The gene sequence and amino acid sequence of Dunaliella salina β-carotene isomerase provided in this invention. DsBCI The enzyme is a novel isomer.

[0021] 2. The β-carotene isomerase provided by this invention DsBCI It can be effectively expressed in E. coli, demonstrating the efficiency of the enzyme.

[0022] 3. The three β-carotene isomerases provided by this invention DsBCI The yields of 9-cis-β-carotene synthesized by the engineered bacteria were 1.40 μg / mL, 3.00 μg / mL, and 3.92 μg / mL (WO7+). DsBCI -1, W07+ DsBCI -2, W07+ DsBCI -3).

[0023] In summary, the three β-carotene isomerases disclosed in this invention... DsBCI The enzyme can effectively synthesize 9-cis-β-carotene and has important application prospects. Attached Figure Description

[0024] Figure 1 pETduet- in Embodiment 2 of the present invention DsBCI -1 is the construction graph.

[0025] Figure 2 pETduet- in Embodiment 2 of the present invention DsBCI -2's construction graph.

[0026] Figure 3 pETduet- in Embodiment 2 of the present invention DsBCI -3 construction graph.

[0027] Figure 4 This is a map of the W07 plasmid in Example 3 of the present invention.

[0028] Figure 5 It includes a control strain (containing only the W07 plasmid) and three strains based on Dunaliella salina β-carotene isomerase. DsBCI Liquid phase diagram of engineered bacteria. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0030] Unless otherwise specified in the following implementation plan, the test conditions are generally as per standard test conditions or the test conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used are commercially available.

[0031] Example 1: Dunaliella salina β-carotene isomerase DsBCI Gene amplification Includes the following steps: 1. Dunaliella salina ( Dunaliella salina Culture of Dunaliella salina: The Dunaliella salina strain was CCAP 19 / 18, preserved by the team of Jiang Jianguo at South China University of Technology. The culture medium formula was: 1.5 M NaCl as the basal component, supplemented with the following essential nutrients: 0.5 ml / L Fe-EDTA stock solution, 1 ml / L A5 stock solution, 0.42 g / L Na2NO3, 1.23 g / L MgSO4•7H2O, 0.84 g / L NaHCO3, 0.015 g / L NaH2PO4•2H2O, 0.074 g / L KCl, and 0.044 g / L CaCl2•2H2O. The algal culture was placed in a light incubator at 25°C under 100 μmol photons / m² light from a cool white fluorescent lamp. -2 s -1 The culture was carried out under the following conditions: light intensity and light-dark cycle of 14 hours light and 10 hours darkness.

[0032] 2. The total RNA of Dunaliella salina obtained in step 1 was extracted from the algal solution using a plant RNA extraction kit (Sangon Biotech, Shanghai), and then reverse transcribed into cDNA using an All-in-One First-Strand Synthesis MasterMix (with dsDNase) kit (Xinkailai, Guangzhou).

[0033] 3. Search for Dunaliella salina β-carotene isomerase in the phytozome database (https: / / phytozome-next.jgi.doe.gov / ). DsBCI Primers were designed as shown in the table below. Using extracted Dunaliella salina cDNA as a template and PrimeSTAR® Max DNA Polymerase (Takara) as the PCR polymerase, PCR amplification was performed using the corresponding primers shown in Table 1. The PCR products were then recovered by 1.5% (g / mL) agarose gel electrophoresis. The PCR products were purified using the EZNATMGel Extraction Kit (Omega, USA), and sequencing yielded the enzyme encoding Dunaliella salina β-carotene isomerase. DsBCI The nucleotide sequence of the gene ( DsBCI-1: SEQ ID NO: 2; DsBCI -2: SEQ ID NO: 4; DsBCI -3: SEQ ID NO: 6), the amino acid sequence was obtained by translating according to the codon table ( DsBCI -1: SEQ ID NO: 1; DsBCI -2: SEQ ID NO: 3; DsBCI -3: SEQ ID NO: 5).

[0034] Table 1. Used for cloning DsBCI Primer sequences of genes

[0035] Example 2 Based on Dunaliella salina β-carotene isomerase DsBCI Carrier construction Includes the following steps: 1. Preparation of linearized vector: The plasmid pETduet-1 (Xinkailai, Guangzhou) was digested with restriction endonucleases NcoI and NotI (Xinkailai, Guangzhou) using a double digestion method. pETduet-1 contains the T7 promoter, ampicillin-resistant AmpR, etc. The digestion reaction system was as follows: 10× Buffer 5 μL, NcoI and NotI 1 μL each, pETduet-1 plasmid 10 μL and dH2O 32 μL. The reaction temperature was 37℃ and the reaction time was 3h.

[0036] The linearized vector was then recovered by 1% agarose gel electrophoresis. It was then purified using the EZNATMGel Extraction Kit (Omega, USA) and sequenced to obtain the pETduet-1 linearized vector.

[0037] 2. Amplification of the target fragment: Design of Dunaliella salina β-carotene isomerase DsBCI Primers with enzyme-binding sticky ends are shown in Table 2. The target gene obtained in Example 1 ( DsBCI -1; DsBCI -2; DsBCI Using -3) as a template, PrimeSTAR® Max DNA Polymerase (Takara) was used as the PCR polymerase for PCR amplification. The PCR products were then recovered by 1.5% agarose gel electrophoresis. The PCR products were further purified using the EZNATMGel Extraction Kit (Omega, USA).

[0038] Table 1. Used for constructing carriers DsBCI Primer sequence of the target fragment

[0039] 3. Ligation and transformation of the target fragment with the linearized vector The linearized pETduet-1 vector obtained above was then used in combination with the pETduet-1 vector amplified by PCR. DsBCI -1, pETduet- DsBCI -2 and pETduet- DsBCI -3 The target fragment was ligated using the XK pTOPO-TA / Blunt SimpleCloning Kit (Xinkailai, Guangzhou). The reaction mixture consisted of: 1 μL XKpTOPO-TA / Blunt Simple Vector, 1 μL 10×Toposmart, 4 μL target fragment, and 5 μL linearized vector, with sterile water added to a final volume of 10 μL. The ligation reaction was performed at 25°C for 15 min. The ligation yielded pETduet- DsBCI -1, pETduet- DsBCI -2 and pETduet- DsBCI -3 Three types of plasmids. Figure 1 pETduet- DsBCI -1 is the construction graph. Figure 2 pETduet- DsBCI -2's construction graph. Figure 3 pETduet- DsBCI -3 Construction diagram. The three plasmids contain the following components: T7 promoter, lac operator gene, DsBCI -1 / DsBCI -2 / DsBCI -3 gene, S-Tag, T7 terminator, f1 origin of replication, ampicillin resistance gene (AmpR), AmpR promoter, ori origin of replication, bom site, rop gene, lacI gene, and lacI promoter.

[0040] Five minutes beforehand, thaw the DH5α competent cells on ice. Place the ligation reaction solution (the ligated plasmid) on ice. Add 5 μL of the ligation reaction solution to 50 μL of DH5α competent cells, mix well, and incubate on ice for 30 minutes. Then, heat-shock the plasmid in a preheated water bath at 42°C for 1 minute, followed by immediate incubation on ice for 2 minutes. Add 400 μL of LB lysate and incubate at 37°C for 200 rpm for 1 hour. Spread 200 μL of the plasmid onto a plate containing ampicillin and incubate at 37°C for 12 hours.

[0041] 4. Bacterial culture PCR identification and sequencing Bacterial PCR identification was performed under aseptic conditions. Ten single colonies were picked from each plate using a 2.5 μL pipette tip and thoroughly mixed in 10 μL of sterile water for PCR identification. The PCR identification system consisted of: 5 μL of 2×Taq PCR mix (Colony), 2 μL of bacterial culture, 1 μL each of the verification primers and the target fragment amplification primers, and sterile water to a final volume of 10 μL. Positive strains from the plates were selected for sequencing and stored.

[0042] Example 3 Based on Dunaliella salina β-carotene isomerase DsBCI Construction of engineered bacteria 1. Plasmid extraction Plasmids were extracted using the OMEGA EZNATM Plasmid Mini Kit, following the kit's instructions. The recombinant plasmid pETduet- was extracted. DsBCI -1, pETduet- DsBCI -2, pETduet- DsBCI -3 and W07 plasmids. Among them, the W07 plasmid was successfully constructed in the laboratory in the past and can be used as a vector for carotene production in Escherichia coli. The main components of this plasmid are: p15A origin of replication, chloramphenicol resistance gene (CmR), cat promoter, tet promoter, crtE gene (GGPP synthase), crtY gene (lycopene β-cyclase), crtI gene (phytopene dehydrogenase), and crtB gene (phytopene synthase). Figure 4 The spectrum of plasmid W07 is shown.

[0043] 2. Transformation of Escherichia coli BL21(DE3) The recombinant plasmid pETduet- DsBCI -1, pETduet- DsBCI -2, pETduet- DsBCI -3 was co-transformed with W07 plasmid (laboratory-preserved). The transformation method was as follows: BL21(DE3) competent cells were thawed on ice 5 min in advance, and the reaction solution (extracted plasmid) was placed on ice. 5 μL of the reaction solution was added to 50 μL of BL21(DE3) competent cells, mixed well, and placed on ice for 30 min. Then, it was heat-shocked in a preheated water bath at 42℃ for 1 min, and immediately placed on ice for 2 min. 400 μL of LB lysate was added, and the cells were incubated at 37℃ for 200 rpm for 1 hour. 200 μL was plated on an ampicillin-containing plate and incubated overnight at 37℃ for 12-16 h. Positive bacteria on the plate were selected for colony PCR to screen for the target strain, which is based on Dunaliella salina β-carotene isomerase. DsBCI Engineered bacteria.

[0044] 3. Transformant expansion culture and carotenoid extraction Expanded culture: Transformants (engineered bacteria based on Dunaliella salina β-carotene isomerase DsBCI) were placed in 50 ml of LB medium supplemented with ampicillin and cultured at 37°C with shaking at 200 rpm. When the OD of the bacterial culture reached 0.6-0.8, IPTG was added for induction, and the culture was continued for 48 h.

[0045] Carotenoid extraction: The bacterial culture was centrifuged at 8000 rpm for 2 minutes at 4°C, and the supernatant was discarded to collect the bacterial cells. Then, the *E. coli* cells were resuspended in 20 ml of deionized water and centrifuged for 2 minutes under the same conditions, after which the supernatant was discarded. Next, 0.5 ml of sterile water was added to fully resuspend the cells, and 4.5 mL of acetone was added with gentle shaking. The mixture was then vortexed to fully disperse the cells, and the mixture was sonicated for 15 minutes in the dark. After sonication, the mixture was centrifuged at 10000 rpm for 10 minutes at 4°C, and thoroughly shaken and vortexed. 1 ml of the supernatant was then added to the sample and stored at -20°C.

[0046] β-carotene content in transformants was determined by liquid chromatography: Phase A consisted of 97% (v / v) methanol, 1 g / L butylated hydroxytoluene, and 30 mL / L deionized water. Phase B consisted of 1 g / L butylated hydroxytoluene dissolved in 100% tert-butyl methyl ether.

[0047] To prepare a standard curve for β-carotene: Dilute a 100 μg / mL stock solution of β-carotene with acetone to prepare the following concentrations: 0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, and 10 μg / mL. Filter each concentration using a 0.22 μm pore size filter. The standard curve is given by y = 34.791x + 59.973 (where x is the peak area and y is the β-carotene content). Use a C30 column (C30YMC carotenoid column, 5 μm, 250 x 4.6 mm).

[0048] The column was washed with 100% methanol for 60 min. The liquid chromatography method was as follows: detection at 473 nm for 40 min, followed by gradient elution: 0 min, 90% (v / v) A phase and 10% (v / v) B phase; 10 min, 60% (v / v) A phase and 40% (v / v) B phase; 20 min, 50% (v / v) A phase and 50% (v / v) B phase; 25 min, 10% (v / v) A phase and 90% (v / v) B phase; 29.5 min, 90% (v / v) A phase and 10% (v / v) B phase. The column was washed at a flow rate of 1 mL / min until equilibration.

[0049] Based on the previously obtained standard curve for β-carotene, y = 34.791x + 59.973 (where x is the peak area and y is the β-carotene content), control strains (containing only the W07 plasmid) and three strains based on Dunaliella salina β-carotene isomerases were obtained. DsBCI Liquid phase diagram of engineered bacteria, such as Figure 5 As shown; the results indicate that, compared with the control strain (containing only the W07 plasmid), the W07 plasmid and the three β-carotene isomerases described in this invention are co-expressed. DsBCI The engineered bacteria showed a significant increase in the production of 9-cis-β-carotene. Among them, the co-expressing... DsBCI-1 The engineered bacteria produced 1.40 μg / mL of 9-cis-β-carotene. DsBCI-2 The yield of the engineered bacteria was 3.00 μg / mL, and the co-expression... DsBCI-3 The engineered strain produced 3.92 μg / mL of 9-cis-β-carotene, while the control strain produced only 0.65 μg / mL. Regarding all-trans-β-carotene production, the co-expressed strain... DsBCI-1 and DsBCI-2 The yields of the engineered strains were 5.61 μg / mL and 8.91 μg / mL, respectively, both lower than the control strain (12.11 μg / mL); while the co-expressed strains... DsBCI-3 The engineered strain achieved a 9-trans β-carotene yield of 14.41 μg / mL, significantly higher than the control strain. In summary, all three engineered strains constructed in this invention can effectively synthesize 9-cis β-carotene, with the co-expressing strain being the most effective. DsBCI-3 The engineered bacteria showed the best results, with the highest yield of 9-cis-β-carotene and also better yield of all-trans-β-carotene than the control.

[0050] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Dunaliella salina ( Dunaliellasalina β-Carotene isomerase DsBCI -1, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

1.

2. Dunaliella salina β-carotene isomerase DsBCI -2, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

3.

3. Dunaliella salina β-carotene isomerase DsBCI -3, characterized in that Its amino acid sequence is shown in SEQ ID NO:

5.

4. Encoding the Dunaliella salina β-carotene isomerase as described in claim 1 DsBCI The gene with -1 is characterized by, Its nucleotide sequence is shown in SEQ ID NO:

2.

5. Encoding the Dunaliella salina β-carotene isomerase as described in claim 2 DsBCI The -2 gene is characterized by, Its nucleotide sequence is shown in SEQ ID NO:

4.

6. Encoding the Dunaliella salina β-carotene isomerase as described in claim 3 DsBCI The -3 gene is characterized by, Its nucleotide sequence is shown in SEQ ID NO:

6.

7. Based on Dunaliella salina β-carotene isomerase DsBCI The method for constructing engineered bacteria is characterized by, Includes the following steps: (1) Dunaliella salina ( Dunaliellasalina Using cDNA as a template, PCR amplification was performed using specific primer pairs, and the nucleotide sequence of the gene as described in claim 4, 5 or 6 was cloned. The specific primer pair for cloning the gene described in claim 4 is as follows: Upstream primer: as shown in SEQ ID NO: 7, Downstream primer: as shown in SEQ ID NO: 8; The specific primer pair for cloning the gene described in claim 5 is as follows: Upstream primer: as shown in SEQ ID NO: 9, Downstream primer: as shown in SEQ ID NO: 10; The specific primer pair for cloning the gene described in claim 5 is as follows: Upstream primer: as shown in SEQ ID NO: 11, Downstream primer: as shown in SEQ ID NO: 12; (2) The gene is ligated into an expression vector to obtain a recombinant expression vector; (3) Transform the recombinant vector into host cells to obtain a product based on Dunaliella salina β-carotene isomerase. DsBCI Engineered bacteria.

8. The construction method according to claim 7, characterized in that, The expression vector is pETduet-1; the host cell is Escherichia coli BL21(DE3).

9. The Dunaliella salina β-carotene isomerase constructed by the method described in claim 7. DsBCI Engineered bacteria.

10. The use of the engineered bacteria according to claim 9 in the production of 9-cis-β-carotene.

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