Nucleic acid molecule, expression vector and recombinant engineering strain for preparing indigoidine

Abstract

The invention relates to a nucleic acid molecule for preparing indigoidine, an expression vector and a recombinant engineering strain. The nucleic acid molecule comprises a sequence as shown in SEQ ID NO.2 or a nucleotide sequence which has at least 98% sequence identity with the SEQ ID NO.2. According to the present invention, the Wlpl protein encoded by the nucleic acid molecule is derived from Williamsiapylosphaerae bacteria, the amino acid sequence of the Wlpl protein is represented by SEQ ID NO.1, the protein has excellent 4 '-phosphoric acid pantetheine transferase catalytic activity, and when the Wlpl protein is used for catalyzing the synthesis process of glutamine to synthesize the indigoidine, the yield of the indigoidine can achieve 14.26 g / L, such that the indigoidine yield is substantially improved, and the good industrialization prospect is provided;

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to a nucleic acid molecule, an expression vector, and a recombinant engineering strain for preparing indigoidine. Background Art

[0002] The structural formula of indigoidine is shown as follows. It is a natural blue pigment derived from microorganisms. Two molecules of L-glutamine are condensed to form indigoidine under the catalysis of nonribosomal peptide synthetase (NRPS) and phosphopantetheinyl transferase (PPTase). As a metabolite of microorganisms, i.e., a biological pigment, indigoidine not only has a unique chemical structure, including a cyclic structure, a dicarboximide, and an imine group, but is also closely related to L-glutamine in terms of function. It has bright colors, strong stability, and remarkable antioxidant and antibacterial properties. It is a new type of natural blue pigment with wide potential applications in multiple fields such as textiles, food, and medicine.

[0003]

[0004] Currently, the production methods of indigoidine mainly include wild strain fermentation and engineering microorganism fermentation. The wild strain fermentation requires a long period and has a low yield, and does not meet the conditions for large-scale production. In contrast, the engineering microorganism fermentation method has a controllable reaction and a feasible prospect for industrialization.

[0005] One of the cores of the engineering microorganism fermentation method for producing indigoidine is to screen 4'-phosphopantetheinyl transferase with high activity to efficiently activate the process of indigoidine synthase catalyzing the synthesis of indigoidine. Currently, the publicly reported 4'-phosphopantetheinyl transferase for indigoidine production mainly comes from the genus Bacillus or the genus Streptomyces. However, the reported 4'-phosphopantetheinyl transferase has problems such as insufficient catalytic activity and low indigoidine yield, and there is still room for further improvement. Therefore, developing a new type of 4'-phosphopantetheinyl transferase is of great significance for promoting the yield of indigoidine. Summary of the Invention

[0006] The object of the present invention is to overcome the defects in the prior art, and provide a nucleic acid molecule, an expression vector and a recombinant engineering strain for preparing indigoidine, which can efficiently convert glutamine to produce indigoidine, and the yield of indigoidine is significantly improved.

[0007] To achieve the above object, the technical solutions adopted by the present invention are as follows:

[0008] A nucleic acid molecule for preparing indigoidine, the nucleic acid molecule comprising a nucleotide sequence as shown in SEQ ID NO.2 or having at least 85% identity with SEQ ID NO.2.

[0009] As a further technical solution, the nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO.2 or having at least 98% identity with SEQ ID NO.2.

[0010] As a further technical solution, the nucleic acid molecule is obtained by codon optimization of the 4'-phosphopantetheine transferase gene Wlpl, and the nucleic acid molecule is used to encode the Wlpl protein.

[0011] As a further technical solution, the 4'-phosphopantetheine transferase gene Wlpl is derived from Williamsia phyllosphaerae.

[0012] An expression vector containing the nucleic acid molecule.

[0013] A recombinant engineering strain containing the nucleic acid molecule or the expression vector.

[0014] As a further technical solution, the host bacteria of the recombinant engineering strain include one or more of Corynebacterium glutamicum, Escherichia coli, Bacillus subtilis (preferably Corynebacterium glutamicum).

[0015] A whole cell catalyst comprising the recombinant engineering strain.

[0016] Use of the nucleic acid molecule, the expression vector, the recombinant engineering strain or the whole cell catalyst as described above in the preparation of indigoidine.

[0017] A method for producing indigoidine, using the nucleic acid molecule, the expression vector, the recombinant engineering strain or the whole cell catalyst as described in any one of the above to produce indigoidine.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] 1. The present invention obtained the gene Wlpl encoding 4'-phosphopantetheine transferase with high activity through a large number of screenings. Through gene alignment, the protein encoded by it is a new 4'-phosphopantetheine transferase. After optimizing this gene, a recombinant engineering strain was constructed, which can produce indigoidine in high yield.

[0020] 2. The present invention uses Corynebacterium glutamicum ATCC 13032 as the starting strain, and utilizes the gene Wlpl encoding 4'-phosphopantetheine transferase with high activity after codon optimization to construct a recombinant Corynebacterium glutamicum with high yield of indigoidine. At the shake-flask fermentation level, the yield of indigoidine reaches 14.26 g / L, showing great potential for industrial application. Description of the Drawings

[0021] Figure 1 is the metabolic pathway diagram of indigoidine;

[0022] Figure 2 is the gene sequence alignment result of 4'-phosphopantetheine transferase Wlpl from Williamsia phyllosphaerae and 4'-phosphopantetheine transferases from other different sources;

[0023] Figure 3 is the high-performance liquid chromatography diagram of indigoidine produced by the engineering strain C9 in Example 3;

[0024] Figure 4 is the mass spectrometry diagram of indigoidine produced by the engineering strain C9 in Example 3. Detailed Embodiments

[0025] The technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0026] In addition, it should be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0027] In the present invention,

[0028] 1. BHIS liquid medium (seed medium): Brain heart infusion broth 38.5 g / L, sorbitol 91 g / L.

[0029] 2. CGXII medium (fermentation medium): Glucose 80 g / L, magnesium sulfate heptahydrate 0.25 g / L, ammonium sulfate 20 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, urea 5 g / L, MOPS 42 g / L, and metal ions 10 mL / L.

[0030] 3. Strain shake flask fermentation method: Pick a single colony and inoculate it into 4 mL of liquid BHIS medium containing 1‰ chloramphenicol, place it in a shaker at 30 °C overnight. Then, pipette 2 mL of the culture broth and inoculate it into 50 mL of CGXII fermentation medium containing 1‰ chloramphenicol antibiotic. Shake it in a shaker at 37 °C until the OD600 reaches 0.6 - 0.8. Then, induce the addition of 0.05 - 0.5 mM IPTG for induced recombinant expression for indigoidine production, and lower the temperature to 25 °C. Continue to culture for at least 72 - 96 h and then perform HPLC to detect the yield of indigoidine.

[0031] 4. High-performance liquid chromatography (HPLC) detection of indigoidine, the chromatographic conditions are as follows:

[0032] Use a ZORBAX Eclipse XDB-C18, 150 mm × 4.6 mm × 3.5 μm chromatographic column;

[0033] Mobile phase A: Pure water, B: Methanol, gradient elution; The elution gradient is as follows in the table:

[0034] Time (min) Phase A (volume percentage) % Phase B (volume percentage) % 0 80 20 9 50 50 13 80 20 18 80 20

[0035] Mobile phase flow rate: 0.8 mL / min;

[0036] Detection wavelength: 610 nm;

[0037] Chromatographic column temperature: 30 °C;

[0038] Injection volume: 10 μL;

[0039] Detector: Agilent 1260 Infinity II VWD detector;

[0040] Ion chromatography detection parameters:

[0041] The elution time of indigoidine was determined by separation using a CarboPac PA10 chromatographic column (4 mm × 250 mm). Gradient elution was performed using three solutions: eluent A: ultrapure water; eluent B: 1 M sodium acetate; eluent C: 250 mM NaOH; linear gradient elution was carried out at a flow rate of 1 mL / min.

[0042] Mass spectrometry detection parameters:

[0043] Mass spectrometry analysis was performed using a MALDI SYNAPT Q-TOF mass spectrometer (Waters, Milford, MA, USA). Capillary: 3.5 kV; cone, 20 V; source block temperature, 100 °C; desolvation temperature, 400 °C; desolvation gas flow rate: 700 L / h; cone gas flow rate: 50 L / h; collision energy: 6 eV; mass range (m / z): 50 - 1000; detector voltage: 2000 V.

[0044] Example 1: Construction of recombinant plasmid

[0045] 1. Construction of recombinant plasmid pXMJ19-bpsA(RBS)Wlpl

[0046] The sequence of gene Wlpl was codon-optimized to obtain codon-optimized Wlpl, whose optimized nucleotide sequence is shown in SEQ ID NO.2, that is, an indigoidine nucleic acid molecule was prepared. The sequences of Wlpl encoded by it were aligned with the sequences of other enzymes (Kspn, Tpsp, SspN, Spep, Rsap, Cdpc, NspN, Nmis), and their similarities were 41.53%, 43.75%, 52.66%, 50.00%, 47.39%, 43.60%, 48.58% and 52.83% respectively. Therefore, Wlpl does not have a very high similarity with other phosphopantetheinyl transferases, and its highly conserved sequence region is shown in Figure 2 (boxed, drawn, bolded and blackened), and Wlpl has unique high catalytic activity.

[0047] Using plasmids pTac-bpsA(RBS)sfp2 and pXMJ19 as templates, codon-optimized Wlpl was cloned onto the pXMJ19 vector to construct the recombinant plasmid pXMJ19-bpsA(RBS)Wlpl. The construction of the recombinant plasmid pXMJ19-bpsA(RBS)Wlpl was completed by Suzhou Anshengda Biotechnology Co., Ltd.

[0048] Among them, the construction method of plasmid pTac-bpsA(RBS)sfp2 is described in the patent document with the publication number CN118086163A. Specifically, the gene fragment bpsA(RBS)sfp2 controlled by the tac promoter was codon-optimized and then ligated between the restriction enzyme sites EcoRI and XbaI of the MCS of pTrc99a to obtain plasmid pTac-bpsA(RBS)sfp2.

[0049] Plasmid pXMJ19 was commercially purchased.

[0050] 2. Construction of pXMJ19-bpsA(RBS)Cdpc: The sequence of gene Cdpc was codon-optimized to obtain codon-optimized Cdpc, and its optimized nucleotide sequence is shown in SEQ ID NO.3. Using plasmids pTac-bpsA(RBS)sfp2 and pXMJ19 as templates, the codon-optimized Cdpc was cloned onto the pXMJ19 vector to construct the recombinant plasmid pXMJ19-bpsA(RBS)Cdpc. The construction of the recombinant plasmid pXMJ19-bpsA(RBS)Cdpc was completed by Suzhou Anshengda Biotechnology Co., Ltd.

[0051] 3. Construction of pXMJ19-bpsA(RBS)Rsap: The sequence of gene Rsap was codon-optimized to obtain codon-optimized Rsap, and its optimized nucleotide sequence is shown in SEQ ID NO.4. Using plasmids pTac-bpsA(RBS)sfp2 and pXMJ19 as templates, the codon-optimized Rsap was cloned onto the pXMJ19 vector to construct the recombinant plasmid pXMJ19-bpsA(RBS)Rsap. The construction of the recombinant plasmid pXMJ19-bpsA(RBS)Rsap was completed by Suzhou Anshengda Biotechnology Co., Ltd.

[0052] 4. Construction of pXMJ19-bpsA(RBS)SspN: The sequence of gene SspN was codon-optimized to obtain codon-optimized SspN, and its optimized nucleotide sequence is shown in SEQ ID NO.5. Using plasmids pTac-bpsA(RBS)sfp2 and pXMJ19 as templates, the codon-optimized SspN was cloned onto the pXMJ19 vector to construct the recombinant plasmid pXMJ19-bpsA(RBS)SspN. The construction of the recombinant plasmid pXMJ19-bpsA(RBS)SspN was completed by Suzhou Anshengda Biotechnology Co., Ltd.

[0053] 5. Construction of pXMJ19-bpsA(RBS)Nmis: The sequence of gene Nmis was codon-optimized to obtain codon-optimized Nmis, and its optimized nucleotide sequence is shown in SEQ ID NO.6. Using plasmids pTac-bpsA(RBS)sfp2 and pXMJ19 as templates, the codon-optimized Nmis was cloned onto the pXMJ19 vector to construct the recombinant plasmid pXMJ19-bpsA(RBS)Nmis. The construction of the recombinant plasmid pXMJ19-bpsA(RBS)Nmis was completed by Suzhou AnShengDa Biotechnology Co., Ltd.

[0054] 6. Construction of pXMJ19-bpsA(RBS)Kspn: The sequence of gene Kspn was codon-optimized to obtain codon-optimized Kspn, and its optimized nucleotide sequence is shown in SEQ ID NO.7. Using plasmids pTac-bpsA(RBS)sfp2 and pXMJ19 as templates, the codon-optimized Kspn was cloned onto the pXMJ19 vector to construct the recombinant plasmid pXMJ19-bpsA(RBS)Kspn. The construction of the recombinant plasmid pXMJ19-bpsA(RBS)Kspn was completed by Suzhou AnShengDa Biotechnology Co., Ltd.

[0055] 7. Construction of pXMJ19-bpsA(RBS)Tpsp: The sequence of gene Tpsp was codon-optimized to obtain codon-optimized Tpsp, and its optimized nucleotide sequence is shown in SEQ ID NO.8. Using plasmids pTac-bpsA(RBS)sfp2 and pXMJ19 as templates, the codon-optimized Tpsp was cloned onto the pXMJ19 vector to construct the recombinant plasmid pXMJ19-bpsA(RBS)Tpsp. The construction of the recombinant plasmid pXMJ19-bpsA(RBS)Tpsp was completed by Suzhou AnShengDa Biotechnology Co., Ltd.

[0056] 8. Construction of pXMJ19-bpsA(RBS)Spep: The sequence of gene Spep was codon-optimized to obtain codon-optimized Spep, and its optimized nucleotide sequence is shown in SEQ ID NO.9. Using plasmids pTac-bpsA(RBS)sfp2 and pXMJ19 as templates, the codon-optimized Spep was cloned onto the pXMJ19 vector to construct the recombinant plasmid pXMJ19-bpsA(RBS)Spep. The construction of the recombinant plasmid pXMJ19-bpsA(RBS)Spep was completed by Suzhou AnShengDa Biotechnology Co., Ltd.

[0057] 9. Construction of pXMJ19-bpsA(RBS)NspN: The sequence of gene NspN was codon-optimized to obtain codon-optimized NspN, and its optimized nucleotide sequence is shown in SEQ ID NO.10. Using plasmids pTac-bpsA(RBS)sfp2 and pXMJ19 as templates, the codon-optimized NspN was cloned onto the pXMJ19 vector to construct the recombinant plasmid pXMJ19-bpsA(RBS)NspN. The construction of the recombinant plasmid pXMJ19-bpsA(RBS)NspN was completed by Suzhou Anshengda Biotechnology Co., Ltd.

[0058] Example 2

[0059] The recombinant plasmids in Example 1 were respectively transformed into the competent cells of Corynebacterium glutamicum ATCC 13032, placed on ice for 20 minutes, then electrotransformed twice at 1800V by an electroporator, and pre-warmed BHIS liquid medium was added. Subsequently, heat shock was performed in a 46°C water bath for 8 minutes, and then it was placed in a 30°C incubator for shaking culture for 2 hours. After the culture was completed, low-speed centrifugation was performed for 3 minutes to remove part of the supernatant, and the remaining bacterial solution was resuspended and spread on the BHIS solid plate with corresponding resistance, and cultured overnight at 30°C. Then, single colonies were picked for shake flask fermentation culture, and the yield of indigoidine was measured by HPLC. The fermentation results of each strain are shown in Table 1.

[0060] Table 1 Information of engineering strains for indigoidine production

[0061]

[0062] As shown in Table 1, the yield of the screened and codon-optimized Wlpl strain C9 increased most significantly, demonstrating the high efficiency of its enzymatic catalytic activity and higher production potential.

[0063] Example 3

[0064] To further verify that the fermentation product of strain C9 is indigoidine, the fermentation product of strain C9 in Example 2 was detected by HPLC and LC-MS. The results showed that compared with the indigoidine standard product ( Figure 3 and Figure 4 ), the fermentation product of strain C9 contained indigoidine, the liquid phase peak time remained consistent, and its mass spectrometry molecular weight was the same as that of the standard product.

[0065] The above-described embodiments are only preferred embodiments of the present invention and not an exhaustive list of the feasible embodiments of the present invention. Any obvious modifications made by those of ordinary skill in the art without departing from the principle and spirit of the present invention shall be considered to be included within the scope of the claims of the present invention.

Claims

1. A nucleic acid molecule for preparing indigoidine, characterized in that: The nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO.2 or having at least 85% identity with SEQ ID NO.

2.

2. A nucleic acid molecule for preparing indigoidine according to claim 1, characterized in that: The nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO.2 or having at least 98% identity with SEQ ID NO.

2.

3. A nucleic acid molecule for preparing indigoidine according to claim 1, characterized in that: The nucleic acid molecule is obtained by codon optimization of the 4'-phosphopantetheine transferase gene Wlpl, and the nucleic acid molecule is used to encode the Wlpl protein.

4. A nucleic acid molecule for preparing indigoidine according to claim 3, characterized in that: The 4'-phosphopantetheine transferase gene Wlpl is derived from Williamsiaphyllosphaerae.

5. An expression vector containing the nucleic acid molecule of claim 1.

6. A recombinant engineering strain, characterized in that: Containing the nucleic acid molecule according to claim 1 or the expression vector according to claim 2.

7. The recombinant engineering strain according to claim 6, characterized in that The host bacteria of the recombinant engineering strain include one or more of Corynebacterium glutamicum, Escherichia coli, and Bacillus subtilis.

8. A whole cell catalyst, characterized in that The invention comprises a recombinant engineering strain as described in any one of claims 6 to 7.

9. Use of the nucleic acid molecule according to any one of claims 1 to 4, the expression vector according to claim 5, the recombinant engineering strain according to any one of claims 6 to 7 or the whole cell catalyst according to claim 8 in the preparation of indigoidine.

10. A method for producing indigoidine, characterized in that: Indigoidine is produced using the nucleic acid molecule according to any one of claims 1 to 4, the expression vector according to claim 5, the recombinant engineered strain according to any one of claims 6 to 7, or the whole-cell catalyst according to claim 8.

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