Light-driven synthetic flora and application thereof

Abstract

The invention discloses a light-driven synthetic flora and application thereof. The light-driven synthetic flora comprises phototrophic bacteria and heterotrophic bacteria, the phototrophic bacteria are genetically modified synechococcus slender, and the gene modification comprises construction and expression of heterologous sucrose permease, glyceraldehyde dehydrogenase and sucrose synthase in the synechococcus slender; and the heterotrophic bacteria are the P.adami. According to the light-driven synthetic flora disclosed by the invention, carbon dioxide is converted into cane sugar through photosynthesis of phototrophic bacteria, and the cane sugar is secreted out of cells to provide a carbon source for heterotrophic bacteria; carrying out cell growth and polyhydroxyalkanoate synthesis on the heterotrophic bacteria by using cane sugar; therefore, the light-driven synthetic flora provided by the invention can realize direct conversion of carbon dioxide into polyhydroxyalkanoate without additional carbon sources, has the advantage of environmental friendliness, and provides a new economical and feasible scheme and approach for production of polyhydroxyalkanoate.

Description

Technical Field

[0001] The present application relates to the technical field of light-driven synthetic bacterial communities, and in particular to a light-driven synthetic bacterial community and its application. Background Art

[0002] Polyhydroxyalkanoates (PHA) are a class of naturally occurring intracellular polyesters typically synthesized by microorganisms. Due to their excellent biodegradability, they are considered an ideal alternative to traditional petroleum-based plastics. In recent years, demand for PHAs in packaging, agriculture, medicine, and other fields has grown rapidly. However, the industrial production cost of PHAs remains high, primarily due to the high demand for carbon sources during the synthesis process and the high cost of carbon sources. Therefore, it is necessary to develop a cost-effective, efficient, and sustainable production technology for PHAs.

[0003] The carbon sources used by microorganisms to synthesize polyhydroxyalkanoates (PHA) have evolved over many generations. From first-generation carbon sources such as glucose and vegetable oils, to second-generation carbon sources such as cellulose and wastewater, or industrial and agricultural waste, to third-generation carbon sources represented by carbon dioxide, the choice of carbon sources is gradually shifting towards non-food and non-land-competing sources. However, the production of PHAs based on third-generation carbon sources still faces numerous technical challenges, such as insufficient genetic modification tools for photoautotrophic organisms, limited carbon conversion efficiency, and long culture cycles. Summary of the Invention

[0004] The purpose of this application is to provide a new light-driven synthetic bacterial community and its application.

[0005] This application adopts the following technical solutions:

[0006] The first aspect of the present application discloses a light-driven synthetic bacterial community, which includes phototrophic bacteria and heterotrophic bacteria; the phototrophic bacteria of the present application are genetically modified Synechococcus elongatus, and the genetic modification includes constructing and expressing heterologous sucrose permease, glyceraldehyde dehydrogenase and sucrose synthase in Synechococcus elongatus; the heterotrophic bacteria of the present application are Priesteria aeruginosa.

[0007] It should be noted that the light-driven synthetic bacterial community of the present application converts carbon dioxide into sucrose through photosynthesis by phototrophic bacteria, and secretes sucrose outside the cell to provide a carbon source for heterotrophic bacteria; heterotrophic bacteria use sucrose for cell growth and polyhydroxyalkanoate synthesis; therefore, the light-driven synthetic bacterial community of the present application can realize the direct conversion of carbon dioxide into polyhydroxyalkanoates without the need for an external carbon source, and has high environmental friendliness. In one implementation of the present application, after culturing the light-driven synthetic bacterial community of the present application for 2 days, the polyhydroxyalkanoate content can reach 0.319 g / L; as the culture time is extended to the 4th day, the polyhydroxyalkanoate content further increases to 0.362 g / L. In addition, the light-driven synthetic bacterial community of the present application can determine the biomass of the phototrophic bacteria and heterotrophic bacteria by chlorophyll content and total cell dry weight.

[0008] It should also be noted that compared with the wild-type Synechococcus elongatus that has not been genetically modified, the genetically modified Synechococcus elongatus used in this application has a significantly improved sucrose yield. At the fourth week of culture, the sucrose concentration can reach 4.66 g / L; and more importantly, the genetically modified Synechococcus elongatus can synergize with Priesteria aeruginosa to form a light-driven synthetic bacterial community. The two grow interactively and can achieve the conversion of carbon dioxide to polyhydroxyalkanoates without adding an exogenous carbon source.

[0009] It should also be noted that in one implementation of the present application, only genetically modified Synechococcus elongatus is used as a phototrophic bacterium and Priesteria aeruginosa is used as a heterotrophic bacterium to form a light-driven synthetic bacterial community for the preparation of polyhydroxyalkanoates; it can be understood that on this basis, it is not ruled out that other algae and / or bacterial species can be added to the light-driven synthetic bacterial community to form a more stable or more functional microecosystem.

[0010] In one implementation of the present application, the nucleic acid sequence of sucrose permease is the sequence shown in SEQ ID NO.1.

[0011] In one implementation of the present application, the sucrose permease is derived from Escherichia coli.

[0012] In one implementation of the present application, the glyceraldehyde dehydrogenase is the sequence shown in SEQ ID NO.2.

[0013] In one implementation of the present application, the glyceraldehyde dehydrogenase is derived from Synechococcus.

[0014] In one implementation of the present application, the sucrose synthase is the sequence shown in SEQ ID NO.3.

[0015] In one implementation of the present application, the sucrose synthase is derived from Synechocystis sp.

[0016] The second aspect of the present application discloses the application of the light-driven synthetic bacterial consortium in the preparation of polyhydroxyalkanoates.

[0017] The third aspect of the present application discloses a method for preparing polyhydroxyalkanoate, comprising co-culturing the light-driven synthetic bacterial community of the present application and collecting the polyhydroxyalkanoate from the co-culture product.

[0018] In one implementation of the present application, the culture medium used for co-culture contains 50-150 mmol / L of sodium chloride.

[0019] In one implementation of the present application, the culture medium used for co-cultivation is BG11 culture medium.

[0020] In one implementation of the present application, the BG11 culture medium contains 50-150 mmol / L sodium chloride, 1-6 mmol / L ammonium chloride, 0.2-0.3 g / L yeast powder, and 2-4 g / L 4-hydroxyethylpiperazineethanesulfonic acid.

[0021] In one implementation of the present application, the co-cultivation conditions are 15-38° C., 60-220 rpm, and continuous light conditions for 2-4 days.

[0022] In one implementation of the present application, the preparation method of the present application further includes inoculating and culturing the phototrophic bacteria of the light-driven synthetic bacterial community for 7-14 days, then inoculating heterotrophic bacteria, and then co-culturing.

[0023] The beneficial effects of this application are:

[0024] The light-driven synthetic bacterial community of the present application converts carbon dioxide into sucrose through photosynthesis by phototrophic bacteria, and secretes sucrose outside the cells to provide a carbon source for heterotrophic bacteria; the heterotrophic bacteria use sucrose for cell growth and polyhydroxyalkanoate synthesis; therefore, the light-driven synthetic bacterial community of the present application can directly convert carbon dioxide into polyhydroxyalkanoates without the need for an external carbon source, has the advantage of being environmentally friendly, and provides a new economically feasible solution and approach for the production of polyhydroxyalkanoates. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The growth and sucrose secretion test results of the phototrophic bacteria in the examples of this application are as follows;

[0026] Figure 2 This is the result of the co-growth test of phototrophic bacteria and heterotrophic bacteria in the embodiment of the present application;

[0027] Figure 3 The results of the test of heterotrophic bacteria and their polyhydroxyalkanoate synthesis ability under different nitrogen sources in the examples of this application are as follows;

[0028] Figure 4 This is the growth detection result of the phototrophic bacteria in the light-driven synthetic bacterial community in the embodiment of the present application;

[0029] Figure 5 This is the growth detection result of heterotrophic bacteria in the light-driven synthetic bacterial community in the embodiment of the present application;

[0030] Figure 6 This is a fluorescence microscope photo of the light-driven synthetic bacterial colony in the examples of this application;

[0031] Figure 7 is the standard curve of chlorophyll content and dry weight of phototrophic bacteria in the examples of this application;

[0032] Figure 8 This is the biomass detection result of phototrophic bacteria and heterotrophic bacteria in the light-driven synthetic bacterial community in the embodiment of the present application;

[0033] Figure 9 This is the result of detecting the polyhydroxyalkanoate content of the light-driven synthetic bacterial community in the examples of the present application. DETAILED DESCRIPTION

[0034] Light-driven synthetic bacterial consortia strategies are gradually demonstrating their potential in addressing the many technical challenges facing the production of polyhydroxyalkanoates (PHA) based on third-generation carbon sources. By constructing synthetic bacterial consortia that synergize with photoautotrophs and heterotrophs, the photoautotrophs can fix carbon dioxide through photosynthesis and convert it into an organic carbon source, providing key substances for the growth and production of heterotrophs. Furthermore, the introduction of heterotrophic PHA-synthesizing strains has further enhanced the production performance of light-driven synthetic bacterial consortia. These strains not only excel in carbon source utilization efficiency and PHA synthesis capacity, but also possess strong environmental adaptability and tolerance. By metabolically collaborating with photoautotrophs, they are able to synthesize PHA from photosynthesis products. Compared with traditional PHA production processes, this light-driven synthetic bacterial consortium-based strategy not only eliminates dependence on sugar-based feedstocks and reduces production costs, but also significantly reduces the carbon footprint of industrialization, providing a new technical path towards achieving carbon neutrality.

[0035] Based on the above research and understanding, the present application creatively pioneered the development of a new light-driven synthetic bacterial community, which includes phototrophic bacteria and heterotrophic bacteria; among them, the phototrophic bacteria is a genetically modified Synechococcus elongatus, and the genetic modification includes constructing and expressing heterologous sucrose permease, glyceraldehyde dehydrogenase and sucrose synthase in Synechococcus elongatus; the heterotrophic bacteria is Priesteria aeruginosa.

[0036] The artificial photosynthetic community of the present application reduces dependence on external traditional substrates, reduces production costs, improves the economic feasibility of polyhydroxyalkanoate production, and provides a new solution for the sustainable production of green chemicals and bioplastics.

[0037] The present invention is further described in detail below through specific examples. The following examples are only used to further illustrate the present invention and should not be construed as limiting the present invention.

[0038] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0039] Example 1 Determination of the constituent species of the light-driven synthetic bacterial community

[0040] 1. Construction of phototrophic bacteria

[0041] (1) Construction of pCyano-cscB recombinant plasmid

[0042] The construction of the sucrose permease recombinant plasmid (pCyano-cscB) refers to patent application CN118956708A. The nucleic acid sequence of the sucrose permease in this example is the sequence shown in SEQ ID NO. 1, which is derived from the genomic DNA of Escherichia coli ATCC700927.

[0043] SEQ ID NO.1:

[0044]

[0045] (2) Construction of pHN1-gapA recombinant plasmid

[0046] The glyceraldehyde dehydrogenase recombinant plasmid (pHN1-gapA) in this example uses the glyceraldehyde dehydrogenase gapA gene derived from Synechococcus PCC 7002, and the amino acid sequence encoded by the gene is shown in SEQ ID No. 2. The theophylline riboswitch sequence was inserted before the gapA gene sequence and sent to Jinweizhi Company for synthesis to obtain the Riboswitch-gapA gene fragment. The pHN1_lacUV5 plasmid was double-digested with NEB restriction endonucleases, and the digested plasmid was recovered using AxyPrepDNAGel Extraction Kit from Axygen. The Riboswitch-gapA gene fragment and the recovered pHN1_lacUV5 plasmid were seamlessly cloned using ClonExpress Ultra One Step Cloning Kit from Novozymes to obtain the recombinant plasmid pHN1-gapA.

[0047] SEQ ID No. 2:

[0048] MIRVAINGFGRIGRNFLRCWAGRENSQLQVVGINATTDTKSNAHMLRYDTMLGKFDGEIDYDANSLTVNGNVIKCCSDRNPLNLPWKEWGVDLVIESTGVFNTEEGSSKHITAGAQKVLITAPGKGGHIGTYVVGVNADQYGHDKQNVISNASCTTNCLAPIVKVLNDR FGIVKGTMTTVHSYTGDQRILDNSHRDLRRARAAAENIVPTSTGAAKAVALVIPEMKGKLNGIAMRVPTPNVSVVDLVAQVAKPTIAEEVNQVLKEASETYMKGILAYTEEPLVSCDFRGTDVSSTIDGSLTLAMDGDLIKVVAWYDNEWGYSQRVVDLAEIVAQNWQG*

[0049] (3) Construction of pAM-sps recombinant plasmid

[0050] The sucrose synthase recombinant plasmid (pAM-sps) uses the sucrose synthase sps gene derived from Synechocystis PCC6803, and the amino acid sequence encoded by the gene is shown in SEQ ID No. 3. The gene was synthesized by Jinweizhi Company to obtain the sps gene fragment. The pAM plasmid was double-digested with NEB restriction endonucleases, and the digested plasmid was recovered using AxyPrep DNAGel Extraction Kit from Axygen Company. The sps gene fragment and the recovered pAM plasmid were seamlessly cloned using ClonExpress Ultra One Step Cloning Kit from Novozymes Company to obtain the recombinant plasmid pAM-sps.

[0051] SEQ ID No.3:

[0052] *

[0053] (4) Construction

[0054] Take a Synechococcus elongatus suspension in the exponential growth phase, collect the algae by centrifugation, wash three times with BG11 medium, and resuspend in BG11 medium. Then, add the recombinant plasmid, mix thoroughly, and incubate at 30°C in the dark for one day. After incubation, spread a nitrocellulose filter on a BG11 plate. Transfer the mixture of algal cells and recombinant plasmid DNA to the filter and incubate at 30°C in low light for one day. After incubation, transfer the filter to a BG11 plate containing the corresponding antibiotic and incubate at 30°C in low light until a transformed algae strain is obtained. The transformed algae strain is streaked onto a BG11 plate containing the corresponding antibiotic and incubated at 30°C in low light until a monoclonal strain is obtained that can be subcultured. The monoclonal strain selected from the resistance plate is inoculated into BG11 medium containing the corresponding antibiotic and incubated at 30°C in low light for 3 to 5 days until the culture turns green. PCR amplification is performed using the engineered algae genome as a template and primers for the exogenous gene transferred to the recombinant plasmid. When introducing the recombinant plasmid pCyano-cscB, use 10 μg / mL spectinomycin for resistance selection. When introducing the recombinant plasmid pHN1-gapA, use 10 μg / mL chloramphenicol for resistance selection. When introducing the recombinant plasmid pAM-sps, use 20 μg / mL kanamycin for resistance selection.

[0055] In this example, the heterologous sucrose permease, glyceraldehyde dehydrogenase, and sucrose synthase genes on the recombinant plasmids pCyano-cscB, pHN1-gapA, and pAM-sps were integrated into the genome of Synechococcus elongatus through homologous recombination, and the resulting engineered algae was the phototrophic bacterium of this example.

[0056] (5) Verification of sucrose secretion ability of phototrophic bacteria

[0057] The phototrophic bacteria introduced with the above recombinant plasmid were inoculated into BG11 culture medium (initial biomass OD730 was 0.1) and cultured under continuous illumination for 3 days. Then 150mmol / L sodium chloride solution, 1mmol / L IPTG and 1mmol / L theophylline solution were added and cultured under continuous illumination for 4 weeks. Samples were taken every week and OD730 was detected by microplate reader to characterize the concentration of phototrophic bacteria. At the same time, the phototrophic bacteria liquid was taken, the supernatant was collected by centrifugation and filtered through a 0.22μm filter membrane, and the sucrose content was characterized by a sucrose detection kit. Three parallels were set for each group. The results are shown in the figure below. Figure 1 shown.

[0058] Figure 1The growth of the phototrophic bacteria and changes in sucrose secretion are shown. With increasing culture time, the OD730 gradually increases, indicating that the phototrophic bacteria have entered the proliferation phase. The OD730 increase is highest at the third week of culture, indicating that the cell growth rate has reached its peak and entered the logarithmic phase. Meanwhile, sucrose secretion is low in the early stages of culture. As the cyanobacterial growth progresses, sucrose secretion increases rapidly, reaching a sucrose concentration of 4.66 g / L by the fourth week of culture.

[0059] 2. Heterotrophic bacteria that can grow together with phototrophic bacteria

[0060] The Priestia aryabhattai MIBE00003 used in this example refers to patent application CN115975861A.

[0061] The phototrophic bacteria were inoculated into BG11 medium containing the corresponding antibiotics and cultured under continuous illumination for 3 days. The phototrophic bacteria solution was spread on the BG11 plate and cultured under illumination until dense green appeared. Activate Priesteria aeruginosa overnight and dilute the bacterial solution to OD600 = 0.8. Use sterile tweezers to spread 4 pieces of sterile filter paper on the BG11 plate, add 10 μL of the diluted Priesteria aeruginosa solution, repeat 3 times, and use sterile water as a control. Culture the plate under illumination at 30°C overnight and observe the growth. The results are as follows Figure 2 shown.

[0062] Figure 2 Shows the co-growth of phototrophic bacteria and Priesteria aeruginosa, where the green color shows the growth of the phototrophic bacteria. Figure 2 The results showed that as the control group with sterile water, no microorganisms grew near the bottom filter paper, while Priesteria africana grew around other filter papers, indicating that there was no antagonism between the phototrophic bacteria and the heterotrophic bacteria, and the two could grow together.

[0063] Example 2 Optimization of nitrogen source types for light-driven synthetic bacterial communities

[0064] The heterotrophic bacteria were inoculated into 2216E medium and cultured at 30°C and 180 rpm for 1 day, then transferred to a fermentation medium and cultured at 30°C and 180 rpm for 2 days to obtain a fermentation broth. The fermentation medium composition consisted of 30 g / L sucrose, 9 g / L disodium hydrogen phosphate, 1.5 g / L potassium dihydrogen phosphate, 30 g / L sodium chloride, 0.2 g / L magnesium sulfate, 0.02 g / L calcium chloride, 0.0012 g / L ferric ammonium citrate, and 100 μL of a trace element solution (1 g / L zinc sulfate, 0.3 g / L manganese chloride, 3 g / L boric acid, 2 g / L cobalt chloride, 0.1 g / L copper chloride, 0.2 g / L nickel chloride, and 0.3 g / L sodium molybdate). In this example, inorganic nitrogen sources and organic nitrogen sources were used for testing. The types of inorganic nitrogen sources tested included ammonium sulfate, ammonium chloride, and urea, and the organic nitrogen sources included soybean meal powder, soybean cake powder, peanut cake powder, corn steep liquor, and yeast powder. The obtained fermentation broth was transferred to a centrifuge tube, the bacterial precipitate was collected by centrifugation, and freeze-dried. A certain amount of freeze-dried sample of the strain was weighed and placed in a 10mL esterification tube for methylation reaction. After the reaction was completed, ice bath was placed for 10 minutes, and 1mL of deionized water was added to the esterification tube to obtain a methylated product sample in the lower layer. The above-mentioned methylated product sample was used to determine the content of polyhydroxyalkanoate by gas chromatography. A DB-WAX model chromatographic column was selected as the stationary phase, helium was used as the mobile phase, the injection volume was 1μL, the injection temperature was 250°C, and the flow rate was 0.7mL / min. Three parallels were set up for each group. The results are as follows Figure 3 shown.

[0065] Figure 3 The effects of different nitrogen sources on bacterial growth and polyhydroxyalkanoates (PHA) were demonstrated. It can be seen that the content of PHA synthesized by heterotrophic strains using organic nitrogen sources is significantly higher than that synthesized using inorganic nitrogen sources. When using inorganic nitrogen sources, ammonium chloride is most suitable for heterotrophic bacteria to synthesize PHA, with a PHA content of 1.94 g / L. When using organic nitrogen sources, yeast extract is the most effective, with the cell dry weight of heterotrophic bacteria reaching 9.57 g / L, the cumulative amount of PHA to cell dry weight being 63.62%, and the content being 6.09 g / L. Therefore, ammonium chloride and yeast extract were selected as nitrogen sources for the construction and application of light-driven synthetic bacterial communities.

[0066] Example 3 Growth Interactions of Light-Driven Synthetic Bacteria

[0067] Phototrophic bacteria were inoculated into BG11 medium containing the corresponding antibiotics and activated for 3 days to produce seeds. The resulting seed solution was inoculated into fresh BG11 medium, with the initial biomass OD730 controlled at 0.1, and cultured under continuous illumination for 3 days. Then, 150 mmol / L sodium chloride solution, 1 mmol / L IPTG, and 1 mmol / L theophylline were added, and cultured under continuous illumination for 7 days. 4 mmol / L ammonium chloride solution, 0.214 g / L yeast powder solution, and 3 g / L HEPES solution (pH = 7.5) were added, along with an overnight activated A. aegypti culture (OD approximately 8). The initial OD730 and OD600 values ​​were measured, and cultured under continuous illumination for 4 days. Starting from the second day, samples were taken every other day to measure the OD730 and OD600 values. Three replicates were set up, and a control without the addition of heterotrophic bacteria was used.

[0068] The growth of phototrophic and heterotrophic bacteria in the light-driven synthetic bacterial consortium is as follows: Figure 4 and Figure 5 As shown. Figure 4 It can be seen that compared with the pure culture of phototrophic bacteria, the addition of heterotrophic bacteria significantly increased the biomass of phototrophic bacteria, indicating that in the light-driven synthetic bacterial community, heterotrophic bacteria have a positive promoting effect on the growth of phototrophic bacteria. Figure 5 The results showed that the biomass of heterotrophic bacteria continued to increase on the fourth day of culture, indicating that the phototrophic bacteria were able to stably secrete sucrose, providing an adequate carbon source for the growth of heterotrophic bacteria. Comprehensive analysis shows that this light-driven synthetic bacterial community can be stably maintained, forming a synergistic symbiotic relationship: the phototrophic bacteria fix carbon dioxide, convert it into sucrose, and secrete it extracellularly, providing the required carbon source for the heterotrophic bacteria; at the same time, the presence of the heterotrophic bacteria further promotes the growth of the phototrophic bacteria.

[0069] The light-driven synthetic bacterial colony was observed using a fluorescence microscope. Figure 6 Under white light conditions, the distribution of phototrophic and heterotrophic bacteria can be clearly observed simultaneously; while under green light conditions, only phototrophic bacteria showing red fluorescence can be observed.

[0070] Example 4 Biomass Characterization of Light-Driven Synthetic Bacteria

[0071] The biomass of the phototrophic bacteria in the light-driven synthetic bacterial consortium was characterized by plotting a chlorophyll-biomass standard curve for the phototrophic bacteria. Combined with the total dry weight data, the biomass of the heterotrophic bacteria in the light-driven synthetic bacterial consortium was calculated.

[0072] Inoculate the phototrophic bacteria into BG11 culture medium and culture under continuous illumination. Take an appropriate amount of algae culture solution for dilution, and the dilution multiples are 1, 2, 4, 8 and 16 respectively. Take 1 mL of diluted algae solution, centrifuge at 12000 rpm for 7 minutes, and remove the supernatant. Add 1 mL of methanol to the precipitate and vortex to mix. Wrap the sample with aluminum foil and place it at 4°C for 20 minutes to extract the pigment in the cells. Centrifuge at 12000 rpm and 4°C for 7 minutes. Then use methanol as a blank and measure the absorbance of the sample at 665 nm and 720 nm. At the same time, take an appropriate amount of diluted algae solution and centrifuge at 10000 rpm for 5 minutes to collect the precipitate. Freeze at -80°C overnight, vacuum dry for 24 hours, and weigh to calculate the dry weight. Set up 3 parallels.

[0073] Figure 7 The standard curve of chlorophyll content and dry weight of phototrophic bacteria was displayed. The results showed that the standard curve had a high degree of fit and the determination coefficient R 2 The value reached 0.9991, indicating that chlorophyll content can be used as a basis for accurately estimating the dry weight of phototrophic bacteria cells.

[0074] Phototrophic bacteria were activated by inoculating them into BG11 medium containing the appropriate antibiotics and incubated under continuous illumination for 3 days to produce seeds. The seed solution was inoculated into fresh BG11 medium to an initial biomass OD730 of 0.1 and incubated under continuous illumination for 3 days. 150 mmol / L sodium chloride solution, 1 mmol / L IPTG, and 1 mmol / L theophylline were then added, and the culture was incubated under continuous illumination for 7 days. After the incubation period, 4 mmol / L ammonium chloride solution, 0.214 g / L yeast extract solution, and 3 g / L HEPES solution (pH = 7.5) were added, along with 125 μL of Priesteria aeruginosa culture (OD approximately 8), and the culture was incubated under continuous illumination for 4 days. Starting from the second day, samples were taken every other day to measure chlorophyll content and total dry weight. The chlorophyll content was applied to the chlorophyll-biomass standard curve for the phototrophic bacteria to obtain the dry weight of the phototrophic bacteria. The dry weight of the heterotrophic bacteria was then subtracted from the total dry weight to obtain the dry weight of the heterotrophic bacteria. Three replicates were set up.

[0075] Figure 8 The biomass of the phototrophic and heterotrophic bacteria in the light-driven synthetic microbial consortium was analyzed over time. The results showed that the biomass of the phototrophic bacteria increased significantly with increasing culture time, indicating that the heterotrophic bacteria promoted their growth. Simultaneously, the biomass of the heterotrophic bacteria also continued to increase, further demonstrating that the sucrose secreted by the phototrophic bacteria supports the continued growth of the heterotrophic bacteria. These results demonstrate that the phototrophic and heterotrophic bacteria in the light-driven synthetic microbial consortium have established a healthy symbiotic relationship, achieving coordinated coexistence and stable growth.

[0076] Example 5 Characterization of polyhydroxyalkanoates from light-driven synthetic bacterial colonies

[0077] The phototrophic bacteria were inoculated into BG11 medium containing the corresponding antibiotics for activation and cultured under continuous light for 3 days to obtain seed liquid. The seed liquid was inoculated into new BG11 medium, the initial biomass OD730 was controlled to be 0.1, and cultured under continuous light for 3 days. Then 150mmol / L sodium chloride solution, 1mmol / L IPTG and 1mmol / L theophylline solution were added, and cultured under continuous light for 7 days. After the culture was completed, 4mmol / L ammonium chloride solution, 0.214g / L yeast powder solution and 3g / L HEPES (pH=7.5) were added, and 125μL heterotrophic bacteria liquid (OD of about 8) was added, and cultured under continuous light for 4 days. Samples were taken every 2 days of culture, and the content of polyhydroxyalkanoates was detected by gas chromatography-mass spectrometry. The injection volume was 1 μL, the split ratio was 15:1, the carrier gas was helium, the flow rate was 1 mL / min, the injection port temperature was 280°C, and the column temperature program was: 60°C for 2 min, then 10°C / min to 110°C, and then 20°C / min to 200°C. Three replicates were set up.

[0078] Figure 9 The results show that the changes in polyhydroxyalkanoate content in the light-driven synthetic bacterial consortium at different culture times were demonstrated. After 2 days of culture, the polyhydroxyalkanoate content reached 0.319 g / L. As the culture time was extended to the fourth day, the polyhydroxyalkanoate content further increased to 0.362 g / L. These results show that without the addition of an exogenous carbon source, the light-driven synthetic bacterial consortium can effectively convert carbon dioxide into polyhydroxyalkanoates, achieving efficient carbon fixation and biopolymer synthesis.

[0079] The above content is a further detailed description of the present application in conjunction with specific implementation methods, and the specific implementation of the present application cannot be considered to be limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application.

Claims

1. A light-driven synthetic bacterial community, characterized in that: Includes phototrophic and heterotrophic bacteria; The phototrophic bacteria is genetically modified Synechococcus elongatus, and the genetic modification includes constructing and expressing heterologous sucrose permease, glyceraldehyde dehydrogenase and sucrose synthase in Synechococcus elongatus; The heterotrophic bacteria is Priesteria africana.

2. The light-driven synthetic bacterial community according to claim 1, characterized in that: The nucleic acid sequence of the sucrose permease is the sequence shown in SEQ ID NO.1; Preferably, the sucrose permease is derived from Escherichia coli.

3. The light-driven synthetic bacterial community according to claim 1, characterized in that: The glyceraldehyde dehydrogenase is the sequence shown in SEQ ID NO.2; Preferably, the glyceraldehyde dehydrogenase is derived from Synechococcus.

4. The light-driven synthetic bacterial community according to any one of claims 1 to 3, characterized in that: The sucrose synthase is the sequence shown in SEQ ID NO.3; Preferably, the sucrose synthase is derived from Synechocystis.

5. Use of the light-driven synthetic bacterial consortium according to any one of claims 1 to 4 in the preparation of polyhydroxyalkanoates.

6. A method for preparing polyhydroxyalkanoate, characterized in that: The method comprises co-culturing the light-driven synthetic bacterial community according to any one of claims 1 to 4, and collecting polyhydroxyalkanoates from the co-cultured products.

7. The preparation method according to claim 6, characterized in that: The culture medium used in the co-culture contains 50-150 mmol / L of sodium chloride.

8. The preparation method according to claim 7, characterized in that: The culture medium used in the co-cultivation is BG11 culture medium; Preferably, the BG11 culture medium contains 50-150 mmol / L sodium chloride, 1-6 mmol / L ammonium chloride, 0.2-0.3 g / L yeast powder, and 2-4 g / L 4-hydroxyethylpiperazineethanesulfonic acid.

9. The preparation method according to claim 6, characterized in that: The co-cultivation conditions are 15-38° C., 60-220 rpm, and co-cultivation for 2-4 days under continuous light conditions.

10. The preparation method according to any one of claims 6 to 9, characterized in that: The invention also comprises the step of first inoculating and culturing the phototrophic bacteria in the light-driven synthetic bacterial community according to any one of claims 1 to 4 for 7 to 14 days, then inoculating the heterotrophic bacteria, and then co-culturing.

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