A method for transforming Escherichia coli to produce phycocyanin

Abstract

The present invention discloses a method for modifying Escherichia coli to produce phycocyanin, belonging to the fields of genetic engineering and bioengineering. The method heterologously expresses the ho1 and pcyA genes from Synechocystis sp. PCC6803, converting protoheme into biliverdin, an intermediate in phycocyanin synthesis, while reducing the accumulation of biliverdin, an intermediate in phycocyanin synthesis. Furthermore, the E. coli genome is modified to overexpress genes involved in the phycocyanin metabolic pathway, resulting in a recombinant E. coli strain with a high phycocyanin production. When the recombinant strain is cultured for 36 hours in a system using inexpensive glycerol as a substrate, the phycocyanin yield reaches 147 mg / L.

Description

Technical Field

[0001] The invention relates to a method for transforming Escherichia coli to produce phycocyanin, belonging to the technical field of genetic engineering and bioengineering. Background Art

[0002] Phycocyanin is a natural pigment unique to certain algae. Due to its unique spectroscopic properties, it is widely used as an excellent fluorescent probe molecule in fields such as immune disease diagnosis and photodynamic therapy. At the same time, because it also has anti-inflammatory, antioxidant, and free radical scavenging functions, it also has significant potential application value in the fields of food, cosmetics, and pharmaceutical health products. As an important industrial microorganism, Escherichia coli has many advantages such as fast reproduction speed, clear genetic background, and mature gene editing technology. It is an important host strain for heterologous protein expression, so E. coli is considered as a host strain for the synthesis of phycocyanin. The biosynthesis of phycocyanin starts with pentaminolevulinic acid (ALA). The synthesis pathway of ALA in E. coli is the C5 pathway, and glutamate is converted to ALA through the C5 pathway. Two ALA molecules are catalyzed by porphobilinogen synthase to produce one molecule of porphobilinogen (PBG). Four molecules of PBG, under the action of porphobilinogen deaminase, form 1-hydroxymethylbichrome (HMB). Uroporphyrinogen synthase cyclizes 1-hydroxymethylbichrome to form uroporphyrinogen III. Subsequently, porphyrinogen III is converted to protoporphyrin IX through decarboxylation and oxidation catalyzed by porphyrinogen III decarboxylase, coporphyrinogen III oxidase, and protoporphyrinogen oxidase. Finally, ferrochelatase incorporates iron into protoporphyrin to form heme. After heme formation, heme oxygenase (Ho1) uses oxygen to degrade heme through a redox reaction to form biliverdin IX, releasing one molecule of CO. Biliverdin is then catalyzed by ferredoxin oxidoreductase (PcyA) to form free phycocyanin (PCB).

[0003] Currently, phycocyanin is primarily extracted from spirulina, using methanol to pyrolyze the spirulina to obtain crude phycocyanin, which is then separated and purified to obtain purer phycocyanin. However, the long methanol pyrolysis process not only requires large quantities of spirulina as raw material but also consumes significant amounts of energy to provide heat. Therefore, there is a need for a superior genetic engineering technology that could extract a single, pure form of PCB through fermentation with recombinant Escherichia coli, thereby avoiding the complex phycocyanin extraction process from spirulina and reducing the cost of pyrolysis. Summary of the Invention

[0004] The present invention aims to construct a recombinant strain that is conducive to the accumulation of phycocyanin, reduce the production cost of phycocyanin, lay a foundation for industrial production, and promote its potential guiding value and significance for the development of synthetic biology.

[0005] The invention provides a recombinant Escherichia coli for synthesizing phycocyanin. The BL21 (DE3) is used as a starting strain and the ho1 gene and the pcyA gene derived from Synechocystis sp. PCC6803 are heterologously expressed.

[0006] In one embodiment, pRSFDuet-1 is used as an expression vector to express the ho1 gene and the pcyA gene.

[0007] In one embodiment, the ho1 gene and the pcyA gene are expressed by fusion with the short peptide tags RIDD and RIAD.

[0008] In one embodiment, the recombinant Escherichia coli also enhances the synthesis of heme.

[0009] In one embodiment, the recombinant E. coli further undergoes at least one of the following improvements:

[0010] (1) Overexpression of endogenous hemB (Gene ID: 945017) in E. coli BL21(DE3), overexpression of endogenous hemC (Gene ID: 947759) in E. coli BL21(DE3), and overexpression of endogenous hemD (Gene ID: 948587) in E. coli BL21(DE3);

[0011] (2) Overexpression of hemE (Gene ID: 948497) endogenous to Escherichia coli BL21 (DE3), overexpression of hemF (Gene ID: 946908) endogenous to Escherichia coli BL21 (DE3), overexpression of hemG (Gene ID: 948331) endogenous to Escherichia coli BL21 (DE3), and overexpression of hemH (Gene ID: 947532) endogenous to Escherichia coli BL21 (DE3).

[0012] In one embodiment, the hemB gene from Escherichia coli is integrated and expressed to achieve asymmetric condensation of ALA to the monopyrrole compound porphobilinogen.

[0013] In one embodiment, the hemC gene from Escherichia coli is integrated and expressed to convert porphobilinogen into the destabilized linear tetrapyrrole, hydroxymethylcholane.

[0014] In one embodiment, the hemD gene from Escherichia coli is integrated and expressed, and hydroxymethylcholane is cyclized to the tetrapyrrole ring of uroporphyrinogen III.

[0015] In one embodiment, the hemE gene from Escherichia coli is integrated and expressed to decarboxylate the four acetate chains of uroporphyrinogen III to form coproporphyrinogen III.

[0016] In one embodiment, the hemF gene from Escherichia coli is integrated and expressed to decarboxylate coproporphyrinogen III to produce protoporphyrinogen IX.

[0017] In one embodiment, the hemG gene from Escherichia coli is integrated and expressed to mediate the six-electron oxidation of protoporphyrinogen IX to protoporphyrin IX.

[0018] In one embodiment, the hemH gene from Escherichia coli is integrated and expressed, so that ferrous ions are chelated into the protoporphyrin IX macrocycle to form proheme.

[0019] In one embodiment, short peptide tags RIDD and RIAD are fused to the C-terminus of Hol and the N-terminus of PcyA to construct a multi-enzyme complex to enhance the synthesis of phycocyanin.

[0020] In one embodiment, the hemEFGH gene is integrated at the location of the heme degradation gene yfeX.

[0021] In one embodiment, the hemBCD gene is integrated at the locus of the arsenate transporter gene arsB.

[0022] In one embodiment, the nucleotide sequences of the heme oxygenase ho1 encoding gene and the ferredoxin oxidoreductase pcyA encoding gene are shown as SEQ ID NO.1 and SEQ ID NO.2.

[0023] In one embodiment, the nucleotide sequences of the short peptide tags RIDD and RIAD are shown as SEQ ID NO. 3 and SEQ ID NO. 4, respectively.

[0024] In one embodiment, the nucleotide sequence of the gene hemBCD encoding the hemB gene, hemC gene, and hemD gene is shown in SEQ ID NO.5.

[0025] In one embodiment, the nucleotide sequence of the gene hemEFGH encoding the hemE gene, hemF gene, hemG gene, and hemH gene is shown in SEQ ID NO.6.

[0026] The present invention provides a method for producing phycocyanin, which utilizes the recombinant Escherichia coli to ferment and produce phycocyanin.

[0027] In one embodiment, the recombinant Escherichia coli is inoculated into a fermentation system and cultured at 35-37° C. for 2-3 hours. 0.5 mM IPTG is added to induce and synthesize phycocyanin at 25° C. and 200-220 rpm, and fermented for 24-48 hours.

[0028] The present invention provides the use of the recombinant Escherichia coli in producing products containing phycocyanin.

[0029] Beneficial effects:

[0030] The present invention heterologously expresses the ho1 and pcyA genes from Synechocystis sp. PCC6803, converting protoheme into biliverdin, an intermediate in phycocyanin synthesis, while reducing the accumulation of biliverdin. Furthermore, the Escherichia coli genome was modified to overexpress genes involved in the phycocyanin metabolic pathway, resulting in a high-phycocyanin-producing recombinant E. coli strain. When cultured for 36 hours in a system using inexpensive glycerol as a substrate, the recombinant strain achieved a phycocyanin yield of 147 mg / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the plasmid map of the recombinant vector pRSFDuet-ho1-pcyA.

[0032] Figure 2 This is the plasmid map of the recombinant vector pRSFDuet-ho1-RIDD-RIAD-pcyA.

[0033] Figure 3 This is the synthetic pathway diagram of phycocyanin synthesized by Escherichia coli.

[0034] Figure 4 is the amount of phycocyanin synthesized by recombinant Escherichia coli shake flask fermentation.

[0035] Figure 5 LC-MS images of recombinant E. coli, control and standard.

[0036] Figure 6 This is the amount of phycocyanin synthesized in a 5L fermentation tank. DETAILED DESCRIPTION

[0037] (1) Culture medium

[0038] LB medium: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride. Add 15 g / L agar when preparing LB solid medium.

[0039] Fermentation medium (per L): KH2PO4 6 g, K2HPO4·3H2O 16.4 g, (NH4)2SO4 5 g, anhydrous citric acid 1 g, MgSO4·7H2O 1 g, yeast powder 10 g, glycerol 30 g, maltodextrin 10 g, vitamin B1 0.1 g and trace element solution 1 mL.

[0040] Trace element solution: Fe(III) citrate 100 g / L, ZnCl3 18 g / L, MnSO4·H2O 14.64 g / L, CuSO4·5H2O 0.75 g / L, Na2MoO4·2H2O 2 g / L, CaCl2.2H2O 2 g / L, H3BO3 3.0 g / L, CoCl2·6H2O 2.5 g / L, NiSO4·6H2O 2.5 g / L and HCl 100 mL.

[0041] (II) PCR reaction system and amplification conditions: 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), 10–50 ng of template DNA, 25 μL of 2× Phanta Max Master Mix, and double-distilled water to 50 μL. Amplification conditions: 95°C pre-incubation for 3 min, followed by 30 cycles (95°C for 15 s, 55°C for 15 s, and 72°C for 15 s), followed by extension at 72°C for 5 min.

[0042] The seamless cloning reaction system is as follows: 40 ng of target gene, 100 ng of vector, 5 μL of enzyme mixture, and 10 μL of sterile ultrapure water. The reaction conditions are as follows: 50°C for 60 min. Immediately place on ice after the reaction. A 10 μL aliquot is transformed into competent E. coli JM109.

[0043] (III) Preparation of competent E. coli cells: Use TaKaRa's Competent Cell Preparation Kit. Refer to the instructions for the specific procedure. Prepared competent cells are stored at -80°C and can be subsequently transformed with plasmids or fragments.

[0044] (IV) Chemical transformation of E. coli: Streak E. coli JM109 on a solid LB plate and incubate at 37°C for 12 h. Select a single colony and inoculate it into liquid LB medium and grow it at 37°C, 220 rpm for 10 h. Transfer the inoculum to fresh 25 mL of liquid LB medium at a rate of 1% and incubate it at 37°C for 1.5-2 h. 600 When the conjugate is grown to 0.6-1, the bacteria are harvested to prepare competent cells.

[0045] (V) HPLC Assay of Phycocyanin: Phycocyanin concentration was determined using a high-performance liquid chromatography system (Waters Corporation, USA) using a 250 × 4.6 mm, 5 μm column (Thermo-Fisher, MA, USA) and a UV detector (Waters 2487) at 380 nm. The initial mobile phase composition was 40% solvent A (acetonitrile containing 0.1% trifluoroacetic acid) and 60% solvent B (0.1% trifluoroacetic acid in water). The solvent gradient was 0.8 ml / min, using the following gradient: 40% B for 0 min, 55% B for 30 min. The column temperature was 25°C.

[0046] (VI) Strain information is shown in Table 1:

[0047] Table 1 Strains and genes involved in the present invention

[0048]

[0049] Example 1: Synthesis of key genes for phycocyanin biosynthesis

[0050] Suzhou Jinweizhi Co., Ltd. was commissioned to carry out whole-gene chemical synthesis of the heme oxygenase Ho1 gene (KEGG accession number sll1184) from Synechocystis sp. PCC 6803, the nucleotide sequence of which is shown in SEQ ID NO.1; synthesize the phycocyanin:ferredoxin oxidoreductase gene PcyA (KEGG accession number slr0116) from Synechocystis sp. PCC 6803, the nucleotide sequence of which is shown in SEQ ID NO.2; and synthesize the short peptide tag RIDD shown in SEQ ID NO.3 and the short peptide tag RIAD shown in SEQ ID NO.4.

[0051] Example 2: Construction of gene expression cassettes related to the phycocyanin biosynthesis pathway

[0052] Escherichia coli BL21(DE3) was used as the starting strain for phycocyanin synthesis. First, to construct the phycocyanin synthesis pathway, PCR amplification was performed using the synthetic sequence of ho1 (SEQ ID NO. 1) as a template with primer pair F1 / R1 and PhantaMasterMix (Vazyme) high-fidelity pfu enzyme. Conditions included initial denaturation at 95°C for 3 minutes, 30 cycles of amplification at 95°C for 15 seconds, 55°C for 15 seconds, and 72°C for 30 seconds, followed by extension at 72°C for 5 minutes. The PCR product was purified to obtain fragment ho1. PCR amplification was performed using the vector pRSFDuet-1 as a template with primer pair F2 / R2 and PhantaMasterMix (Vazyme) high-fidelity pfu enzyme. Conditions included initial denaturation at 95°C for 3 minutes, 30 cycles of amplification at 95°C for 15 seconds, 55°C for 15 seconds, and 72°C for 2 minutes, followed by extension at 72°C for 5 minutes. The PCR product was purified. The fragment ho1 and the vector pRSFDuet-1 were recombined into the vector pRSFDuet-ho1 by seamless cloning and assembly. The vector was then transformed into E. coli JM109. The resulting vector was sent to Shanghai Bioengineering for sequencing. After correct alignment, the recombinant vector pRSFDuet-ho1 was obtained.

[0053] Using the synthetic sequence of pcyA shown in SEQ ID NO. 2 as a template, PCR amplification was performed using primer pair F3 / R3 and Phanta MasterMix (Vazyme) high-fidelity pfu enzyme. Conditions included initial denaturation at 95°C for 3 minutes, 30 cycles of amplification at 95°C for 15 seconds, 58°C for 15 seconds, and 72°C for 30 seconds, followed by extension at 72°C for 5 minutes. The PCR product was purified to obtain the pcyA fragment. Using the recombinant vector pRSFDuet-ho1 as a template, PCR amplification was performed using primer pair F4 / R4 and Phanta MasterMix (Vazyme) high-fidelity pfu enzyme. Conditions included initial denaturation at 95°C for 3 minutes, 30 cycles of amplification at 95°C for 15 seconds, 58°C for 15 seconds, and 72°C for 3 minutes, followed by extension at 72°C for 5 minutes. The PCR product was purified to obtain the vector fragment pRSFDuet-ho1. The fragment pcyA and the vector fragment pRSFDuet-ho1 were recombined into the vector pRSFDuet-ho1-pcyA by seamless cloning and assembly. Escherichia coli JM109 was transformed. The obtained vector was sent to Shanghai Bioengineering for sequencing. After correct alignment, the correct recombinant vector pRSFDuet-ho1-pcyA was obtained. The recombinant vector pRSFDuet-ho1-pcyA was transformed into Escherichia coli BL21 (DE3) to obtain the engineered strain St07. The engineered strain was cultured in seed culture medium at 37°C and 220r / min for 12 hours to obtain the seed solution (OD 600 3 ± 0.2), and then inoculated into a fermentation medium containing kanamycin at a final concentration of 50 μg / mL at a 2% inoculum. After culturing at 37°C and 220 rpm for 3 hours, IPTG was added to a final concentration of 0.5 mM. Phycocyanin synthesis was induced and continued at 25°C and 220 rpm for 48 hours. Figure 4 The yield of phycocyanin in the fermentation broth of St07 was 9.1 mg / L.

[0054] Table 2 Primer sequences

[0055]

[0056]

[0057] Example 3: Enhancement of endogenous heme synthesis pathway

[0058] As a direct precursor for phycocyanin synthesis, in order to promote the supply of protoheme, the endogenous gene hemBCD (nucleotide sequence such as SEQ ID NO.5) of Escherichia coli was overexpressed and integrated into the arsenate transporter gene arsB site.

[0059] The gene fragment PT7-lacO-RBS-hemB-PT7-lacO-RBS-hemCD was amplified from the plasmid pRSF-hemBCD (published in the paper "Metabolic engineering of Escherichia coli for secretory production of free haem") using primer pair F5 / R5. Using the Escherichia coli BL21 (DE3) genome as a template, the upstream and downstream homology arms U1 and D1 of the arsB site were amplified using primer pairs F6 / R6 and F7 / R7, respectively, and the fragments were purified. Using the purified fragments U1 and DI as templates, the primer pairs F8 / R8 and F9 / R9 were used to amplify and gradually obtain the integration cassette U-PT7-lacO-RBS-hemB-PT7-lacO-RBS-hemCD-D1, and the fragment was purified. In order to obtain pTarget-arsB for the integration site arsB, pTarget was used as a template and amplified with primers F10 / R10, and the fragment was purified; the purified fragment was transformed into Escherichia coli JM109, the plasmid was extracted and sequenced to obtain the correct recombinant vector pTarget-arsB.

[0060] To generate electroporation-competent E. coli BL21(DE3) containing pCas, the pCas plasmid was transformed into competent E. coli BL21(DE3). A single colony obtained from the transformation was transferred to 4 mL of LB medium, and kanamycin was added to a final concentration of 50 μg / mL. The culture was incubated at 30°C for 12 hours. The bacterial suspension was then inoculated into 50 mL of LB medium at a 2% inoculum, and kanamycin was added to a final concentration of 50 μg / mL and a 10 mM arabinose solution. After incubation at 30°C and 220 rpm for 4-6 hours, when the OD reached 0.6, the suspension was transferred to a 50 mL centrifuge tube and incubated on ice for 15 minutes. The suspension was centrifuged at 4000 rpm for 10 minutes at 4°C, the supernatant removed, and the suspension resuspended in 10 mL of 10% glycerol. This process was repeated twice, and the suspension was aliquoted into 100 μL portions and stored at -80°C. To competent E. coli BL21(DE3) cells, 400 ng of the recombinant vector pTarget-arsB and 1200 ng of the integration cassette U-PT7-lacO-RBS-hemB-PT7-lacO-RBS-hemCD-D1 were added. The cells were incubated on ice for 10 minutes, then transferred to a 1 mm electroporation cuvette that had been pre-chilled for 10 minutes and electroporated at 1.8 kV. After electroporation, 1 ml of LB liquid medium was added and incubated at 30°C for 1.5 hours. Colony PCR was performed using primer pair F11 / R11. Once a single colony was confirmed, pTarget-arsB and pCas9 were removed according to the protocol described in the literature to obtain the engineered strain E. coli BL21(DE3)ΔarsB::hemBCD. The recombinant vector pRSFDuet-ho1-pcyA was then transformed into E. coli BL21(DE3)ΔarsB::hemBCD to obtain the engineered strain St18. The engineered strain St18 was cultured in a seed culture medium at 37°C and 220 rpm for 12 hours to obtain a seed solution (OD 600 3 ± 0.2), and then inoculated into a fermentation medium containing kanamycin at a final concentration of 50 μg / mL at a 2% inoculum. After culturing at 37°C and 220 rpm for 3 hours, IPTG was added at a concentration of 0.5 mM. Phycocyanin synthesis was induced and continued at 25°C and 220 rpm for 48 hours. Figure 4 The yield of phycocyanin in the fermentation broth of St18 was 15.0 mg / L.

[0061] To further improve the supply of protohemoglobin, the endogenous gene hemEFGH of Escherichia coli (corresponding nucleotide sequence is shown in sequence SEQ ID NO.6) was overexpressed and integrated into the yfeX site. The gene fragment PT7-lacO-RBS-hemEFG-PT7-lacO-RBS-hemH was amplified from the plasmid pET-hemEFGH (disclosed in the paper "Metabolic engineering of Escherichia coli for secretory production of freehaem") using primer pair F12 / R12. Using the Escherichia coli BL21 (DE3) genome as a template, the upstream and downstream homology arms U1 and D1 of the yfeX site were amplified, respectively, using the primer pairs F13 / R13 and F14 / R14, and the fragments were purified. Using the purified fragments U2 and D2 as templates, the primer pairs F15 / R15 and F16 / R16 were amplified to gradually obtain the integration cassette U2-PT7-lacO-RBS-hemEFG-PT7-lacO-RBS-hemH-D2, and the fragments were purified. In order to obtain pTarget-yfeX for the integration site yfeX, plasmid pTarget was used as a template, amplified with primers F17 / R17, and the fragment was purified; the purified fragment was transformed into Escherichia coli JM109, the plasmid was extracted and sequenced to obtain the correct recombinant vector pTarget-yfeX.

[0062] Table 3 Primer sequences

[0063]

[0064]

[0065] Example 4: Construction of gene expression cassettes related to the phycocyanin synthase multienzyme complex

[0066] Using the synthetic RIDD sequence shown in SEQ ID NO. 3 as a template, primer pair F19 / R19 was used for amplification. The amplified fragment was purified to obtain the RIDD fragment. Using the recombinant vector pRSFDuet-ho1-pcyA as a template, primer pair F20 / R20 was used for amplification using Phanta MasterMix (Vazyme) high-fidelity pfu enzyme. The conditions were pre-denaturation at 95°C for 3 minutes, 30 cycles of amplification at 95°C for 15 seconds, 58°C for 15 seconds, and 72°C for 3 minutes, followed by extension at 72°C for 5 minutes. The PCR product was purified to obtain the recombinant vector backbone pRSFDuet-ho1-pcyA. The RIDD fragment and the pRSFDuet-ho1-pcyA backbone were recombined by seamless cloning to obtain the recombinant vector. The recombinant vector was transformed into Escherichia coli JM109, and the plasmid was extracted and sequenced to confirm the correct recombinant vector pRSFDuet-ho1-GGGGS-RIDD-pcyA.

[0067] Then, using the RIAD synthetic sequence as a template, the primer pair F21 / R21 was designed, and amplification was performed with this pair of primers to amplify the RIAD fragment, which was then purified. Using the recombinant vector pRSFDuet-ho1-GGGGS-RIDD-pcyA as a template, the primer pair F22 / R22 was designed, and amplification was performed with this pair of primers. Phanta MasterMix (Vazyme) high-fidelity pfu enzyme was selected, and the conditions were pre-denaturation at 95°C for 3 minutes; 30 cycles of amplification were performed at 95°C for 15 seconds, 58°C for 15 seconds, and 72°C for 3 minutes; and extension was performed at 72°C for 5 minutes. The PCR product was purified to obtain the recombinant vector backbone pRSFDuet-ho1-pcyA. The fragment RIAD and the vector pRSFDuet-ho1-GGGGS-RIDD-pcyA backbone were recombined by seamless cloning to obtain a recombinant vector, which was transformed into Escherichia coli JM109. The plasmid was extracted and sequenced to verify the correct recombinant vector pRSFDuet-ho1-GGGGS-RIDD-RIAD-GGGGS-pcyA. The recombinant vector pRSFDuet-ho1-GGGGS-RIDD-RIAD-GGGGS-pcyA was transformed into Escherichia coli BL21 (DE3) ΔarsB::hemBCDΔyfeX::hemEFGH to obtain the engineered strain St26. The engineered strain St26 was cultured in a seed medium at 37°C and 220 rpm for 12 hours to obtain a seed solution (OD 6003 ± 0.2), and then inoculated into a fermentation medium containing kanamycin at a final concentration of 50 μg / mL at a 2% inoculum. After culturing at 37°C and 220 rpm for 3 hours, IPTG was added at a concentration of 0.5 mM. Phycocyanin synthesis was induced and continued at 25°C and 220 rpm for 48 hours. Figure 4 The yield of phycocyanin in the fermentation broth of St26 was 23.4 mg / L.

[0068] Table 4 Primer sequences

[0069]

[0070] Example 5: 5L fermenter culture

[0071] The strain St26 constructed in Example 3 was fermented in a 5 L fermenter system.

[0072] Pick large and round colonies and place them in a 250 ml shake flask with 25 ml LB liquid, culture at 37 ° C and 220 rpm for 12 hours, then transfer them to a 250 ml shake flask with 25 ml LB liquid at a 1% inoculum size, culture at 37 ° C and 220 rpm for 12 hours, transfer them to a 5 L fermentor with 2.5 L liquid at a 4% inoculum size, culture at 37 ° C until the dissolved oxygen rebounds, then cool to 25 ° C, add a final concentration of 0.5 mM IPTG for induction and start feeding. During the fermentation process, the dissolved oxygen is controlled at 40%, the feeding flow rate is 20 ml / h, and the pH is controlled at 7.0 ± 0.03 by automatically adding 50% ammonia water.

[0073] Feed medium: 50% (v / v) glycerol, 0.1 g / L FeCl3, 6.25 g / L (NH4)2SO4, 15 g / L MgSO4·7H2O, feeding rate is 20 ml / L.

[0074] like Figure 6 As shown, after 36 hours of fermentation, the phycocyanin production of the engineered strain can reach 147 mg / L, which is 51.2% higher than that of the initial strain.

[0075] Comparative Example 1:

[0076] The specific implementation method is the same as Example 2, except that pRSFDuet-1 is replaced by pETDuet-1 plasmid. The results show that the phycocyanin yield of the constructed recombinant bacteria after 48 hours of fermentation is 6.0 mg / L.

[0077] Comparative Example 2:

[0078] The specific implementation method is the same as Example 2, except that pRSFDuet-1 is replaced by pCDFDuet-1 plasmid. The results show that the phycocyanin yield of the constructed recombinant bacteria after 48 hours of fermentation is 5.9 mg / L.

[0079] Comparative Example 3:

[0080] The specific implementation method is the same as Example 2, except that pRSFDuet-1 is replaced by pACYCDuet-1 plasmid. The results show that the phycocyanin yield of the constructed recombinant bacteria after 48 hours of fermentation is 5.2 mg / L.

[0081] Comparative Example 4:

[0082] The specific implementation method is the same as that of Example 5, except that the fermentation conditions are controlled to be inoculated for 6-7h, OD 600 The temperature was lowered and induced when the temperature was about 10. The results showed that the phycocyanin production after 52 hours of fermentation was 74 mg / L.

[0083] Comparative Example 5:

[0084] The specific implementation method is the same as that of Example 5, except that the feed medium does not contain FeCl3. The results show that the phycocyanin yield after 52 hours of fermentation is 117 mg / L.

[0085] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A recombinant Escherichia coli for synthesizing phycocyanin, characterized in that: Using BL21 (DE3) as the starting strain and pRSFDuet-1 as the expression vector, the heme oxygenase gene was expressed by fusing the short peptide tags RIDD and RIAD. ho1 and ferredoxin oxidoreductase genes pcyA , and integrate expression hemBCD Genes and hemEFGH Gene; The heme oxygenase gene ho1 and ferredoxin oxidoreductase genes pcyA The nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively; described hemBCD In genes hemB The nucleotide sequence is shown in Gene ID: 945017; hemC The nucleotide sequence is shown in Gene ID: 947759; hemD The nucleotide sequence is shown in Gene ID: 948587; hemEFGH In genes hemE The nucleotide sequence is shown in Gene ID: 948497; hemF The nucleotide sequence is shown in Gene ID: 946908; hemG The nucleotide sequence is shown in Gene ID: 948331; hemH The nucleotide sequence is shown in Gene ID: 947532.

2. The recombinant Escherichia coli according to claim 1, characterized in that Arsenate transporter gene arsB Site integration hemBCD Gene.

3. The recombinant Escherichia coli according to claim 1 or 2, characterized in that Heme degradation genes yfeX Location Integration hemEFGH Gene.

4. A method for producing phycocyanin, characterized in that: Phycocyanin is produced by fermentation using the recombinant Escherichia coli according to any one of claims 1 to 3.

5. The method according to claim 4, characterized in that The recombinant E. coli was inoculated into a fermentation system, cultured at 35-37° C. for 2-3 hours, induced with IPTG, and fermented for 24-48 hours.

6. Use of the recombinant Escherichia coli according to any one of claims 1 to 3 in the production of a product containing phycocyanin.

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