Construction method of cyanobacteria-corynebacterium glutamicum artificial photosynthetic mixed bacteria system

Abstract

The invention discloses a construction method of a cyanobacteria-corynebacterium glutamicum artificial photosynthetic mixed bacteria system. The construction method comprises the following steps: step S1, amplifying by taking plasmid pUC-tho as a template to obtain a theophylline inducible promoter Ptho; step S2, carrying out amplification by taking a Synechocystis sp. PCC 6803 genome as a template so as to obtain a strong promoter Pcpc560, and carrying out amplification on the strong promoter Pcpc560 so as to obtain a strong promoter Pcpc560; step S3, carrying out amplification by taking the plasmid pUC-PAL as a template to obtain an arabidopsis thaliana phenylalanine lyase gene pal; step S4, carrying out amplification by taking the plasmid pUC-cscB as a template to obtain an escherichia coli W sucrose transporter gene cscB; step S5, carrying out amplification by taking the integrated plasmid Pcp3031 as a template, so as to obtain a terminator Trbcl; and step S6, fusing the strong promoter Pcpc560, the arabidopsis phenylalanine lyase gene pal and the terminator Trbcl, and respectively connecting with an expression vector pSI-SPE to obtain the expression vector containing the gene pal. According to the invention, a'cyanobacteria-corynebacterium glutamicum 'artificial photosynthetic mixed bacteria system is constructed by simulating a mutualistic symbiotic relationship ubiquitous in nature, and negative carbon biosynthesis of cis-muconic acid is realized.

Description

Technical Field

[0001] The present invention relates to a method for constructing a cyanobacterium-Corynebacterium glutamicum artificial photosynthetic mixed bacterium system. Background Art

[0002] Cis,cis-Muconic acid (ccMA) is an unsaturated dicarboxylic acid with the molecular formula C6H6O4 and a molecular weight of 142.11 g / mol. Its molecular structure contains two carboxyl groups (-COOH) and a conjugated double bond, presenting a cis configuration and being prone to addition, polymerization and other reactions. Cis,cis-Muconic acid is a platform compound with various application values and is commonly used as a starting material for the synthesis of various value-added compounds, such as fatty acids, malonamides and benzoic acids. The biosynthetic pathway of cis,cis-Muconic acid has been successfully applied to Escherichia coli, Corynebacterium glutamicum, Pseudomonas and Saccharomyces cerevisiae, and is mainly synthesized through related metabolites in the shikimate pathway using carbon sources such as glucose. Although the biosynthesis of cis,cis-Muconic acid has been successful in various microorganisms, it mostly focuses on the transformation of single engineered strains. Moreover, cis,cis-Muconic acid is mostly synthesized by microorganisms using organic carbon sources, and the research on negative carbon biosynthesis has not been reported.

[0003] Photosynthetic cyanobacteria have excellent nitrogen fixation and carbon fixation abilities, with a clear genetic background, simple genetic manipulation and fast growth rate, and have excellent application prospects in the field of biomanufacturing. Synechococcus elongatus PCC 7942 belongs to the Cyanophyta and the genus Synechococcus, and is a typical photosynthetic autotroph in the freshwater environment. As the first cyanobacterium to achieve stable transformation, it has rich genetic tools and is commonly used in the research of biosynthetic applications and environmental stress responses. Synechococcus elongatus PCC 7942 can accumulate sucrose intracellularly under the stress of moderate salt ions. For example, under the stress of 200 mM NaCl, it accumulates nearly 300 mM of sucrose intracellularly (calculated based on the culture volume). CscB is a sucrose / proton symporter. Under the stress of salt ions such as NaCl, by expressing the sucrose transporter gene cscB in Synechococcus elongatus PCC 7942, the extracellular secretion of cyanobacterial sucrose can be achieved, avoiding the economic costs brought by cell lysis and recovery.

[0004] Corynebacterium glutamicum is a facultative aerobe that can regulate metabolic flux through redox balance under low-oxygen conditions and preferentially synthesize products such as glutamic acid, which is commonly used in the industrial production of amino acids. Corynebacterium glutamicum can maintain a high carbon source consumption rate under both aerobic and anaerobic conditions and has high tolerance to osmotic pressure and various chemicals, making these characteristics make it a commonly used strain in fermentation production. As an intermediate in the β-ketoadipic acid pathway of Corynebacterium glutamicum, cis-muconic acid provides an inherent advantage for its synthesis in Corynebacterium glutamicum. In previous studies, by enhancing the expression of core enzymes and key genes in Corynebacterium glutamicum and increasing the metabolic flux of precursors, the production of 10.35 g / L cis-muconic acid was achieved with 0.29 mol / mol glucose as the carbon source in a 50 mL shake flask, which is also the highest titer reported for the synthesis of cis-muconic acid using glucose as the carbon source.

[0005] Considering the dual advantages of cyanobacteria and Corynebacterium glutamicum, namely that cyanobacteria can use photosynthetic carbon fixation to produce organic carbon sources, and Corynebacterium glutamicum can use organic carbon sources to produce cis-muconic acid. The present invention proposes a method for constructing a cyanobacteria-Corynebacterium glutamicum artificial photosynthetic mixed culture system. Summary of the Invention

[0006] The object of the present invention is to provide a method for constructing a cyanobacteria-Corynebacterium glutamicum artificial photosynthetic mixed culture system to overcome the existing defects. By simulating the mutually beneficial symbiotic relationship commonly existing in nature, a "cyanobacteria-Corynebacterium glutamicum" artificial photosynthetic mixed culture system is constructed, realizing the negative carbon biosynthesis of cis-muconic acid.

[0007] The technical solution to achieve the above object is: A method for constructing a cyanobacteria-Corynebacterium glutamicum artificial photosynthetic mixed culture system, comprising: Step S1, amplifying the theophylline-inducible promoter Ptho using plasmid pUC-tho as a template; Step S2, amplifying the strong promoter Pcpc560 using the genome of Synechocystis sp. PCC 6803 as a template; Step S3, amplifying the Arabidopsis thaliana phenylalanine lyase gene pal using plasmid pUC-PAL as a template; Step S4, amplifying the Escherichia coli W sucrose transporter gene cscB using plasmid pUC-cscB as a template; Step S5, amplifying the terminator Trbcl using the integrative plasmid Pcp3031 as a template; Step S6: Fuse the strong promoter Pcpc560, Arabidopsis thaliana phenylalanine lyase gene pal, and terminator Trbcl, and ligate them to the expression vector pSI-SPE respectively to obtain an expression vector containing the gene pal. Step S7: Fuse the theophylline-inducible promoter Ptho, Escherichia coli W sucrose transporter gene cscB, and terminator Trbcl, and ligate them to the expression vector pSII-CM to obtain an expression vector containing the gene cscB. Step S8: Transfer the expression vector containing the gene pal into Synechococcus elongatus PCC 7942, and screen to obtain the strain 7942-PAL that secretes trans-cinnamic acid. Then transfer the expression vector containing the gene cscB into the strain 7942-PAL, and screen to obtain the strain 7942-PAL-cscB that secretes both tCA and sucrose. Step S9: Amplify the upstream and downstream homologous arm fragments of the gene catB using the Corynebacterium glutamicum ATCC 13032 genome as a template, and amplify the upstream and downstream homologous arm fragments of the gene phdR using the Corynebacterium glutamicum 13032 genome as a template. Step S10: Fuse the upstream and downstream homologous arm fragments of the gene catB, and fuse the upstream and downstream homologous arm fragments of the gene phdR, and ligate them to the expression vector pk18mobrpsl respectively to obtain two recombinant expression vectors. Step S11: Transfer the two obtained recombinant expression vectors into Corynebacterium glutamicum 13032 in sequence to obtain the cis-muconic acid-producing strain Cgb-CP. Step S12: Use the obtained 7942-PAL-cscB and Cgb-CP to form an artificial photosynthetic mixed-bacteria system for negative-carbon synthesis of cis-muconic acid.

[0008] Preferably, in step S1, the plasmid pUC-tho is synthesized from the nucleotide sequence of the promoter Ptho. In step S3, the plasmid pUC-PAL is synthesized from the nucleotide sequence of the Arabidopsis thaliana phenylalanine lyase gene pal. In step S4, the plasmid pUC-cscB is synthesized from the Escherichia coli W sucrose transporter gene cscB. In step S6, the expression vector pSI-SPE is an integrative plasmid for Synechococcus PCC 7942, and the integration site is the neutral site NSI. In step S7, the expression vector pSII-CM is an integrative plasmid for Synechococcus elongatus PCC 7942, and the integration site is the neutral site NSII.

[0009] Preferably, the culture medium used in the artificial photosynthetic mixed-bacteria system is the 1 / 2M4 culture medium derived from the NBG and CG12 culture media used for the separate culture of the genetically engineered bacteria Synechococcus elongatus PCC7942 and Corynebacterium glutamicum ATCC 13032.

[0010] Preferably, the composition of 1 L of the NBG culture medium is as follows: Sodium nitrate 1.5 g, sodium carbonate 0.02 g, magnesium sulfate heptahydrate 0.075 g, dipotassium hydrogen phosphate 0.04 g, ethylenediaminetetraacetic acid 0.001 g, boric acid 2.86 g, ammonium ferric citrate 0.006 g, calcium chloride dihydrate 0.036 g, cobalt nitrate hexahydrate 0.0494 g, copper sulfate pentahydrate 0.079 g, sodium molybdate dihydrate 0.39 g, zinc sulfate heptahydrate 0.222 g, manganese chloride tetrahydrate 1.81 g, theophylline 0.36 g, TES 2.29 g, sodium chloride 8.766 g.

[0011] Preferably, the composition of 1 L of the CG12 culture medium is as follows: Ammonium sulfate 10 g, urea 2 g, potassium dihydrogen phosphate 1 g, dipotassium hydrogen phosphate 1 g, magnesium sulfate heptahydrate 0.25 g, MOPS 42 g, biotin 0.2 mg, calcium chloride 0.01 g, protocatechuic acid 0.033 g, ferrous sulfate heptahydrate 0.011 g, manganese sulfate monohydrate 0.011 g, zinc sulfate heptahydrate 0.001 g, copper sulfate 0.2 mg, nickel chloride hexahydrate 0.02 mg.

[0012] Preferably, the M4 culture medium is composed by supplementing different components of the CG12 culture medium on the basis of NBG, and among them, 1 / 2M4 is obtained by halving the concentration of the effective components of CG12 added on the basis of M4.

[0013] Preferably, the culture temperature of the artificial photosynthetic mixed-bacteria system is 30 °C.

[0014] Preferably, at 30 °C, the genetically engineered bacteria Synechococcus elongatus PCC 7942 and Corynebacterium glutamicum ATCC 13032 obtained are inoculated at an inoculation ratio of 4:1 using the 1 / 2M4 culture medium.

[0015] The beneficial effects of the present invention are as follows: By using synthetic biology methods, the present invention synthesizes cis-muconic acid around an artificial photosynthetic mixed culture system, obtains the culture medium, culture temperature and inoculation ratio for establishing the mixed culture system, and obtains a two-strain artificial photosynthetic mixed culture system for the efficient biosynthesis of cis-muconic acid, named 2S-MA. Experiments prove that the present invention can achieve the direct biosynthesis of high-value chemicals from CO2, which has important theoretical and practical significance for constructing mixed culture systems using photosynthetic microorganisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a flow chart of a method for constructing a cyanobacterium-corynebacterium glutamicum artificial photosynthetic mixed culture system of the present invention; Figure 2 is a sucrose production curve of strain 7942-PAL-cscB in Example 1 of the present invention at 37 °C using M1-4 and 1 / 2M1-1 / 2M4 culture media; Figure 3 is a trans-cinnamic acid production curve of strain 7942-PAL-cscB in Example 1 of the present invention at 37 °C using M1-4 and 1 / 2M1-1 / 2M4 culture media; Figure 4 is a sucrose production curve of strain 7942-PAL-cscB in Example 1 of the present invention at 30 °C using M1-4 and 1 / 2M1-1 / 2M4 culture media; Figure 5 is a trans-cinnamic acid production curve of strain 7942-PAL-cscB in Example 1 of the present invention at 30 °C using M1-4 and 1 / 2M1-1 / 2M4 culture media; Figure 6 is a cis-muconic acid production curve of strain Cgb-CP in Example 1 of the present invention at 37 °C using CG12 and M1-4, 1 / 2M1-1 / 2M4 culture media; Figure 7 is a cis-muconic acid production curve of strain Cgb-CP in Example 1 of the present invention at 30 °C using CG12 and M1-4, 1 / 2M1-1 / 2M4 culture media; Figure 8 is a production curve of sucrose, trans-cinnamic acid and cis-muconic acid of the mixed culture system in Example 2 of the present invention at 30 °C in 1 / 2M4 culture medium using a 1:1 inoculation ratio; Figure 9 is a production curve of sucrose, trans-cinnamic acid and cis-muconic acid of the mixed culture system in Example 2 of the present invention at 30 °C in 1 / 2M4 culture medium using a 4:1 inoculation ratio; Figure 10It is the production curve graph of sucrose, trans-cinnamic acid and cis-muconic acid of the mixed bacteria system in Example 2 of the present invention at 30°C in 1 / 2 M4 medium with an inoculation ratio of 8:1. Figure 11 It is the production curve graph of sucrose, trans-cinnamic acid and cis-muconic acid of the mixed bacteria system in Example 2 of the present invention at 30°C in 1 / 2 M4 medium with an inoculation ratio of 16:1. Detailed implementation manners

[0017] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the accompanying drawings. In the description of the present invention, it should be noted that the orientation or positional relationship indicated by the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. is based on the orientation or positional relationship shown in the accompanying drawings. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation to the present invention. In addition, the terms "first", "second", and "third" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance.

[0018] Next, the present invention will be further described in conjunction with the accompanying drawings.

[0019] As Figure 1 shown, a method for constructing a cyanobacteria-Corynebacterium glutamicum artificial photosynthetic mixed bacteria system includes: Step S1, amplifying the theophylline-inducible promoter Ptho using plasmid pUC-tho as a template.

[0020] In the embodiment, plasmid pUC-tho is synthesized from the nucleotide sequence of promoter Ptho.

[0021] Step S2, amplifying the strong promoter Pcpc560 using the genome of Synechocystis sp. PCC 6803 as a template.

[0022] Step S3, amplifying the Arabidopsis phenylalanine lyase gene pal using plasmid pUC-PAL as a template.

[0023] In the embodiment, plasmid pUC-PAL is synthesized from the nucleotide sequence of the Arabidopsis phenylalanine lyase gene pal.

[0024] Step S4, amplifying the Escherichia coli W sucrose transporter gene cscB using plasmid pUC-cscB as a template; In the embodiment, plasmid pUC-cscB is synthesized from the Escherichia coli W sucrose transporter gene cscB.

[0025] Step S5: Amplify the terminator Trbcl using the integrative plasmid Pcp3031 as a template.

[0026] Step S6: Fuse the strong promoter Pcpc560, the Arabidopsis phenylalanine lyase gene pal, and the terminator Trbcl, and ligate them respectively with the expression vector pSI-SPE to obtain an expression vector containing the gene pal.

[0027] In the example, the expression vector pSI-SPE is an integrative plasmid for Synechococcus PCC 7942, with the integration site being the neutral site NSI and carrying spectinomycin resistance.

[0028] Step S7: Fuse the theophylline-inducible promoter Ptho, the Escherichia coli W sucrose transporter gene cscB, and the terminator Trbcl, and ligate them with the expression vector pSII-CM to obtain an expression vector containing the gene cscB.

[0029] In the example, the expression vector pSII-CM is an integrative plasmid for Synechococcus elongatus PCC 7942, with the integration site being the neutral site NSII and carrying chloramphenicol resistance.

[0030] Step S8: Transfer the expression vector containing the gene pal into Synechococcus elongatus PCC 7942, and screen to obtain the strain 7942-PAL that secretes trans-cinnamic acid. Then transfer the expression vector containing the gene cscB into the strain 7942-PAL, and screen to obtain the strain 7942-PAL-cscB that secretes both tCA and sucrose.

[0031] Step S9: Amplify the upstream and downstream homologous arm fragments of the gene catB using the Corynebacterium glutamicum ATCC 13032 genome as a template, and amplify the upstream and downstream homologous arm fragments of the gene phdR using the Corynebacterium glutamicum 13032 genome as a template.

[0032] Step S10: Fuse the upstream and downstream homologous arm fragments of the gene catB, and fuse the upstream and downstream homologous arm fragments of the gene phdR. Then ligate them respectively with the expression vector pk18mobrpsl to obtain two recombinant expression vectors.

[0033] Step S11: Transfer the two obtained recombinant expression vectors into Corynebacterium glutamicum 13032 in sequence to obtain the cis-muconic acid producing strain Cgb-CP. Step S12: Combine the obtained 7942-PAL-cscB and Cgb-CP to form an artificial photosynthetic mixed bacteria system for negative carbon synthesis of cis-muconic acid.

[0034] In the examples, the culture media used in the artificial photosynthetic mixed bacteria system are eight culture media derived from the culture media NBG and CG12 used when the genetically engineered bacteria Synechococcus elongatus PCC7942 and Corynebacterium glutamicum ATCC 13032 are cultured separately, namely M1, M2, M3, M4, and 1 / 2M1, 1 / 2M2, 1 / 2M3, and 1 / 2M4 culture media. The M1, M2, M3, M4, and 1 / 2M1, 1 / 2M2, 1 / 2M3, and 1 / 2M4 culture media are composed by supplementing different components of the CG12 culture media to the NBG. Among them, 1 / 2M1 is obtained by halving the concentration of the effective components of CG12 added on the basis of M1, 1 / 2M2 is obtained by halving the concentration of the effective components of CG12 added on the basis of M2, 1 / 2M3 is obtained by halving the concentration of the effective components of CG12 added on the basis of M3, and 1 / 2M4 is obtained by halving the concentration of the effective components of CG12 added on the basis of M4, as shown in Table 1 and Table 2 specifically.

[0035] In the examples, the composition of 1 L of the NBG culture medium: Sodium nitrate 1.5 g, sodium carbonate 0.02 g, magnesium sulfate heptahydrate 0.075 g, dipotassium hydrogen phosphate 0.04 g, ethylenediaminetetraacetic acid 0.001 g, boric acid 2.86 g, ammonium ferric citrate 0.006 g, calcium chloride dihydrate 0.036 g, cobalt nitrate hexahydrate 0.0494 g, copper sulfate pentahydrate 0.079 g, sodium molybdate dihydrate 0.39 g, zinc sulfate heptahydrate 0.222 g, manganese chloride tetrahydrate 1.81 g, theophylline 0.36 g, TES 2.29 g, sodium chloride 8.766 g.

[0036] In the examples, the composition of 1 L of the CG12 culture medium: Ammonium sulfate 10 g, urea 2 g, potassium dihydrogen phosphate 1 g, dipotassium hydrogen phosphate 1 g, magnesium sulfate heptahydrate 0.25 g, MOPS 42 g, biotin 0.2 mg, calcium chloride 0.01 g, protocatechuic acid 0.033 g, ferrous sulfate heptahydrate 0.011 g, manganese sulfate monohydrate 0.011 g, zinc sulfate heptahydrate 0.001 g, copper sulfate 0.2 mg, nickel chloride hexahydrate 0.02 mg.

[0037] Table 1 Other components contained in the M1-4 and 1 / 2M1-4 culture media Table 2 Other components contained in the M1-4 and 1 / 2M1-4 culture media In the examples, the growth and production of the genetically engineered bacteria of Synechococcus elongatus PCC 7942 and Corynebacterium glutamicum ATCC 13032 were investigated under eight culture media and two temperatures. Finally, the culture medium for the artificial photosynthetic mixed-bacteria system was determined to be 1 / 2M4, and the culture temperature was 30°C.

[0038] In the examples, at 30°C, the obtained genetically engineered bacteria of Synechococcus elongatus PCC 7942 and Corynebacterium glutamicum ATCC 13032 were cultured in the 1 / 2M4 culture medium at inoculation ratios of 1:1, 4:1, 8:1, and 16:1. The production of sucrose, trans-cinnamic acid, and cis-muconic acid in the co-culture system was detected. Finally, the inoculation ratio of the genetically engineered bacteria of Synechococcus elongatus PCC 7942 and Corynebacterium glutamicum ATCC 13032 was determined to be 4:1.

[0039] The present invention will be further described below with specific examples: Example 1: Determination of the optimal culture medium 1 / 2M4 and the optimal temperature 30°C.

[0040] Under the conditions of a light intensity of 65 μmol photons m -2 s -1 , a rotation speed of 160 rpm, and a temperature of 37°C, the strain 7942-PAL-cscB was inoculated into 20 mL of M1-4 and 1 / 2M1-4 culture media at an initial OD 750 = 0.04. A total of 3 biological replicates were used in each group. After culturing for 5 days, 40 μL of the culture supernatant was taken as the test sample. The sucrose was quantified using a sucrose detection kit and a sucrose production curve was plotted, as shown in Figure 2 . Then, 500 μL of the culture supernatant was taken as the test sample, and the trans-cinnamic acid was quantified using HPLC and a production curve was plotted, as shown in Figure 3 .

[0041] Under the conditions of a light intensity of 65 μmol photons m -2 s -1 , a rotation speed of 160 rpm, and a temperature of 30°C, at an initial OD 750= 0.04. Inoculate strain 7942-PAL-cscB into 20 mL of M1-4 and 1 / 2 M1-4 media respectively. A total of 3 biological replicates are used in each group. Cultivate for 5 days, take 40 μL of the culture supernatant as the sample to be tested, use a sucrose detection kit to quantify sucrose and plot the sucrose production curve, as Figure 4 shown. Then take 500 μL of the culture supernatant as the sample to be tested, use HPLC to quantify trans-cinnamic acid and plot the production curve, as Figure 5 shown.

[0042] Under the conditions of light intensity of 65 μmol photons m -2 s -1 , rotation speed of 160 rpm, and temperature of 37 °C, inoculate strain Cgb-CP into 20 mL of CG12, M1-4 and 1 / 2 M1-4 media respectively with an initial OD 630 = 0.04. Add sucrose with a final concentration of 2 g / L and trans-cinnamic acid standard with a final concentration of 15 mg / L. A total of 3 biological replicates are used in each group. Cultivate for 48 h, add 1 g / L of sucrose at 24 h. Take 500 μL of the culture supernatant as the sample to be tested, use HPLC to quantify cis-muconic acid and plot the production curve, as Figure 6 shown.

[0043] Under the conditions of light intensity of 65 μmol photons m -2 s -1 , rotation speed of 160 rpm, and temperature of 30 °C, inoculate strain Cgb-CP into 20 mL of CG12, M1-4 and 1 / 2 M1-4 media respectively with an initial OD 630 = 0.04. Add sucrose with a final concentration of 2 g / L and trans-cinnamic acid standard with a final concentration of 15 mg / L. A total of 3 biological replicates are used in each group. Cultivate for 48 h, add 1 g / L of sucrose at 24 h. Take 500 μL of the culture supernatant as the sample to be tested, use HPLC to quantify cis-muconic acid and plot the production curve, as Figure 7 shown.

[0044] It can be seen from Figure 2-5 that at 37 °C is more favorable for the sucrose production of 7942-PAL-cscB than at 30 °C in different media, and the result is similar for the production of trans-cinnamic acid. From Figure 6 , 7It can be seen that a temperature of 30°C is more conducive to the synthesis of cis-muconic acid by Cgb-CP, and the product is synthesized in M4, 1 / 2M4, and CG12 media. At 37°C, the product is only synthesized in CG12 medium, while 7942-PAL-cscB cannot secrete sucrose in CG12 medium. Therefore, 30°C was determined as the final temperature for the mixed culture system. On the basis of establishing 30°C, by comparing Figure 4 and Figure 5 the production of 7942-PAL-cscB in M4 and 1 / 2M4 media in

[0045] Example 2: Determination of the optimal mixed culture inoculation ratio.

[0046] The 1 / 2M4 medium obtained in Example 1 was used to screen the inoculation ratio of the artificial photosynthetic mixed culture system of the two strains at a culture temperature of 30°C.

[0047] At a light intensity of 65 μmol photons m -2 s -1 , a rotation speed of 160 rpm, and a temperature of 30°C, the strain 7942-PAL-cscB was inoculated into the NBG medium with an initial OD 750 = 0.1. A total of 3 biological replicates were used in each group and cultured for 3 days.

[0048] After culturing 7942-PAL-cscB alone for 3 days, the medium was supplemented with 1 / 2M4 medium and mixed at a ratio of 1:1, 4:1, 8:1, and 16:1 of 7942-PAL-cscB and Cgb-CP. The mixed culture system was cultured at a light intensity of 65 μmol photons m -2 s -1 , a rotation speed of 160 rpm, and a temperature of 30°C for 48 h. Six time points were selected during the 48 h. 40 μL of the culture supernatant was taken as the test sample, and a sucrose detection kit was used to quantify sucrose and plot the sucrose production curve. 500 μL of the culture supernatant was taken as the test sample, and HPLC was used to quantify trans-cinnamic acid and plot the production curve, as Figures 8-11 shown.

[0049] In Figures 8-11It can be seen that when the inoculation ratios of 7942-PAL-cscB and Cgb-CP are 1:1, 4:1, 8:1, and 16:1, 4.98, 6.77, 4.68, and 4.64 mg / L of cis-muconic acid are synthesized respectively in 48 hours. Therefore, the inoculation ratio of 7942-PAL-cscB and Cgb-CP at 4:1 is determined as the inoculation ratio of the artificial photosynthetic mixed bacteria system of the two strains.

[0050] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing a cyanobacteria-Corynebacterium glutamicum artificial photosynthetic mixed bacteria system, characterized in that, Comprising: Step S1: Amplify the theophylline-inducible promoter Ptho using plasmid pUC-tho as a template; Step S2: Amplify the strong promoter Pcpc560 using the genome of Synechocystis sp. PCC 6803 as a template; Step S3: Amplify the Arabidopsis phenylalanine lyase gene pal using plasmid pUC-PAL as a template; Step S4: Amplify the Escherichia coli W sucrose transporter gene cscB using plasmid pUC-cscB as a template; Step S5: Amplify the terminator Trbcl using the integrative plasmid Pcp3031 as a template; Step S6: Fuse the strong promoter Pcpc560, the Arabidopsis phenylalanine lyase gene pal, and the terminator Trbcl, and ligate them respectively with the expression vector pSI-SPE to obtain an expression vector containing the gene pal; Step S7: Fuse the theophylline-inducible promoter Ptho, the Escherichia coli W sucrose transporter gene cscB, and the terminator Trbcl, and ligate them with the expression vector pSII-CM to obtain an expression vector containing the gene cscB; Step S8: Transfer the expression vector containing the gene pal into Synechococcus elongatus PCC 7942, and screen to obtain the strain 7942-PAL that secretes trans-cinnamic acid. Then transfer the expression vector containing the gene cscB into the strain 7942-PAL, and screen to obtain the strain 7942-PAL-cscB that secretes both tCA and sucrose; Step S9: Amplify the upstream and downstream homologous arm fragments of the gene catB using the genome of Corynebacterium glutamicum ATCC 13032 as a template, and amplify the upstream and downstream homologous arm fragments of the gene phdR using the genome of Corynebacterium glutamicum 13032 as a template; Step S10: Fuse the upstream and downstream homologous arm fragments of the gene catB, and fuse the upstream and downstream homologous arm fragments of the gene phdR. Then ligate them respectively with the expression vector pk18mobrpsl to obtain two recombinant expression vectors; Step S11: Transfer the two obtained recombinant expression vectors into Corynebacterium glutamicum 13032 in sequence to obtain the cis-muconic acid-producing strain Cgb-CP; Step S12: Combine the obtained 7942-PAL-cscB and Cgb-CP to form an artificial photosynthetic mixed-bacteria system for negative-carbon synthesis of cis-muconic acid.

2. The construction method of a cyanobacteria-corynebacterium glutamicum artificial photosynthetic mixed bacteria system according to claim 1, characterized in that In the said Step S1, the plasmid pUC-tho is synthesized from the nucleotide sequence of the promoter Ptho; In the said Step S3, the plasmid pUC-PAL is synthesized from the nucleotide sequence of the Arabidopsis phenylalanine lyase gene pal; In the said Step S4, the plasmid pUC-cscB is synthesized from the Escherichia coli W sucrose transporter gene cscB; In the said Step S6, the expression vector pSI-SPE is an integrative plasmid for Synechococcus PCC 7942, and the integration site is the neutral site NSI; In the step S7, the expression vector pSII-CM is an integrative plasmid for Synechococcus elongatus PCC 7942, and the integration site is the neutral site NSII.

3. The construction method of a cyanobacteria-corynebacterium glutamicum artificial photosynthetic mixed bacteria system according to claim 1, characterized in that, The culture medium used in the artificial photosynthetic mixed bacteria system is the 1 / 2M4 medium derived from the NBG and CG12 media used for the separate culture of the genetically engineered bacteria Synechococcus elongatus PCC 7942 and Corynebacterium glutamicum ATCC 13032.

4. The construction method of a cyanobacteria-corynebacterium glutamicum artificial photosynthetic mixed bacteria system according to claim 3, characterized in that Composition of 1 L of NBG medium: Sodium nitrate 1.5 g, sodium carbonate 0.02 g, magnesium sulfate heptahydrate 0.075 g, dipotassium hydrogen phosphate 0.04 g, ethylenediaminetetraacetic acid 0.001 g, boric acid 2.86 g, ammonium ferric citrate 0.006 g, calcium chloride dihydrate 0.036 g, cobalt nitrate hexahydrate 0.0494 g, copper sulfate pentahydrate 0.079 g, sodium molybdate dihydrate 0.39 g, zinc sulfate heptahydrate 0.222 g, manganese chloride tetrahydrate 1.81 g, theophylline 0.36 g, TES 2.29 g, sodium chloride 8.766 g.

5. The construction method of a cyanobacteria-corynebacterium glutamicum artificial photosynthetic mixed bacteria system according to claim 3, characterized in that, Composition of 1 L of CG12 medium: Ammonium sulfate 10 g, urea 2 g, potassium dihydrogen phosphate 1 g, dipotassium hydrogen phosphate 1 g, magnesium sulfate heptahydrate 0.25 g, MOPS 42 g, biotin 0.2 mg, calcium chloride 0.01 g, protocatechuic acid 0.033 g, ferrous sulfate heptahydrate 0.011 g, manganese sulfate monohydrate 0.011 g, zinc sulfate heptahydrate 0.001 g, copper sulfate 0.2 mg, nickel chloride hexahydrate 0.02 mg.

6. The construction method of a cyanobacteria-corynebacterium glutamicum artificial photosynthetic mixed bacteria system according to claim 3, characterized in that, The M4 medium is composed by supplementing different components of the CG12 medium on the basis of NBG. Among them, 1 / 2M4 is obtained by halving the concentration of the effective components of CG12 added on the basis of M4.

7. The construction method of a cyanobacteria-corynebacterium glutamicum artificial photosynthetic mixed bacteria system according to claim 3, characterized in that The culture temperature of the artificial photosynthetic mixed bacteria system is 30 °C.

8. The construction method of a cyanobacteria-corynebacterium glutamicum artificial photosynthetic mixed bacteria system according to claim 7, characterized in that, At 30 °C, the genetically engineered bacteria Synechococcus elongatus PCC 7942 and Corynebacterium glutamicum ATCC 13032 obtained are inoculated at an inoculation ratio of 4:1 using the 1 / 2M4 medium.

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