High-yield glucose-secreting cyanobacteria and application thereof

Abstract

The invention relates to application of a cyanobacteria glucokinase inactivating agent in preparation of cyanobacteria with a glucose secretion function or high glucose yield. The invention further relates to cyanobacteria with a high glucose secretion function or high glucose yield and application of the cyanobacteria. Glucokinase in the cyanobacteria loses functions. According to the invention, glucokinase of cyanobacteria is knocked out, the mutant strain accidentally obtains the capacity of mass production of glucose and secretion of glucose to the outside of cells, the yield of glucose is increased by hundreds of times, the mutant strain provides possibility for subsequent industrial production of glucose by using cyanobacteria, and the mutant strain can be used for constructing a biological photovoltaic device, and has a wide application prospect. And glucose is continuously provided for the cathode cell and is used for being matched with a glucose oxidation electron transfer chain displayed extracellularly to generate reducing power and electrons, so that the light energy conversion efficiency is improved, and efficient transmembrane transfer of energy substances is realized.

Description

Technical Field

[0001] The invention belongs to the field of cyanobacteria metabolic engineering, and particularly relates to cyanobacteria capable of high-yielding and secreting glucose and applications thereof. Background Art

[0002] Glucose, one of the most common monosaccharides in nature, is an essential energy source for life. From human metabolism to industrial production, from food processing to biotechnology, glucose is used everywhere. It is not only a core substance for energy metabolism in organisms, but also a crucial raw material for modern science, technology, and industrial development.

[0003] Glucose is primarily produced by starch hydrolysis, with recent additions including microbial fermentation and chemical synthesis. However, starch hydrolysis is energy-intensive and environmentally polluting. Other methods, such as high production costs and numerous byproducts, have limited widespread adoption.

[0004] Cyanobacteria, also known as blue-green algae or blue-green algae, are among the oldest organisms on Earth and the only prokaryotic microorganisms capable of photosynthetic oxygen evolution. They utilize solar energy, CO₂, and H₂O to generate chemical energy and store it in biomass. Cyanobacteria boast high photosynthetic efficiency, converting nearly 10% of solar energy into biomass. In recent years, cyanobacteria have garnered significant attention for their potential applications in biotechnology. If cyanobacteria could synthesize and secrete glucose, this would significantly reduce glucose production costs and expand its application.

[0005] Synechococcus elongatus PCC 7002 is a single-celled marine algae that is resistant to high temperature, high light, and high salt. It can grow at a temperature of 38°C and can also withstand 500 μmol photons / m 2 / s light intensity, it typically divides by binary fission, with an average of 2.6 hours for a new generation under optimal conditions, making it the fastest dividing algae strain. Furthermore, Synechococcus PCC 7002 has a wide adaptability, is capable of facultative photoautotrophy and heterotrophy, and has a complete genome sequence and comprehensive genetic tools, making it a model strain for studying cyanobacterial genomic functions and biotechnology applications. Currently, no cyanobacteria can be directly used to produce glucose. Summary of the Invention

[0006] During our research on Synechococcus PCC 7002, we found that when the glucose kinase in its genome was knocked out, the mutant strain unexpectedly showed extremely high glucose production, and most of this glucose was secreted outside the cell, making the glucose content in the culture system as high as hundreds of milligrams per liter, while the growth of the algae was not affected.

[0007] Based on the above research, the present invention provides the use of a cyanobacterial glucokinase disabling agent in the preparation of cyanobacteria having glucose secretion function or high glucose production.

[0008] In the present invention, the glucokinase disabling agent refers to a reagent that can make the cyanobacteria lose glucokinase activity, for example, can completely lack the nucleic acid structure of the glucokinase gene in the cyanobacteria, or can make the glucokinase gene in the cyanobacteria partially lack or partially mutate and cause it to lose the nucleic acid structure of glucokinase function. Such nucleic acid structure can be DNA and / or RNA. The glucokinase disabling agent can also be a glucokinase function antagonist, for example, can be by adding a glucokinase function antagonist in the culture, or by expressing a gene with antagonism glucokinase function in the cyanobacteria and realizing.

[0009] Although the present invention does not specifically demonstrate experimental evidence other than knockout using homologous double crossover, those skilled in the art, after reading the contents disclosed herein and understanding the spirit of the present application, will fully be able to obtain such enlightenment: after disabling glucokinase, cyanobacteria can produce glucose in large quantities and transport it to the extracellular space. Those skilled in the art can achieve glucokinase disabling by any existing or future technology, but these should all be encompassed within the scope of protection of the present invention.

[0010] The present invention also provides a cyanobacterium with high glucose secretion function or high glucose yield, wherein the glucokinase in the cyanobacterium loses its function.

[0011] The loss of function of glucokinase means that glucokinase no longer has the function of phosphorylating glucose. We can disable glucokinase by methods such as deletion, mutation or expression of antagonists of this glucokinase. Cyanobacteria with disabled glucokinase will not be able to phosphorylate the glucose produced, and glucose will be produced in large quantities and transported outside the cell. Those skilled in the art can achieve the disablement of glucokinase by any existing or future technology, but these should all be encompassed within the scope of protection of the present invention.

[0012] The present invention also provides the use of the cyanobacteria in producing glucose.

[0013] The present invention also provides the use of the cyanobacteria in preparing photovoltaic biological devices.

[0014] The present invention also provides a method for making cyanobacteria secrete glucose or produce glucose, comprising the step of making the glucokinase in the cyanobacteria lose its function.

[0015] In a specific embodiment, the cyanobacteria is free of glucokinase by knocking out the glucokinase gene in the cyanobacteria.

[0016] In a specific embodiment, the cyanobacterium is Synechococcus, and the reference amino acid sequence of the glucokinase is shown in SEQ ID NO: 1.

[0017] The present invention knocks out the glucose kinase of cyanobacteria, and the mutant strain unexpectedly acquires the ability to produce large amounts of glucose and secrete it outside the cell, increasing the glucose yield by hundreds of times. This mutant strain not only provides the possibility of subsequent industrial production of glucose using cyanobacteria, but can also be used to construct a biophotovoltaic device to continuously provide glucose to the cathode pool, which is used to cooperate with the glucose oxidation electron transport chain displayed outside the cell to generate reducing power and electrons, thereby improving the efficiency of light energy conversion and realizing efficient transmembrane transfer of energy substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the knockout structure of the glucose kinase gene glk.

[0019] Figure 2 PCR was used to verify that the glucokinase gene in YD07 was completely knocked out.

[0020] Figure 3 Comparison of the growth curves of mutant strain YD07 and wild-type PCC7002.

[0021] Figure 4 The standard curve of extracellular glucose content.

[0022] Figure 5 The bar graph shows the changes in extracellular glucose content in the culture of mutant strain YD07 over time.

[0023] Figure 6 Schematic diagram of the biophotovoltaic device.

[0024] Figure 7 This is the culture status of the sodium alginate gel-solidified algae strain YD07 and the extracellular glucose content curve.

[0025] Figure 8 This is the current output curve of the biophotovoltaic device. DETAILED DESCRIPTION

[0026] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0027] 1. Construction of a glucokinase gene knockout mutant of Synechococcus PCC 7002

[0028] Synechococcus elongatus PCC 7002 was used as the chassis algae to construct a glucokinase gene glk knockout mutant.

[0029] The specific method is as follows: The glucokinase gene glk in the Synechococcus PCC 7002 genome is numbered SYNPCC7002_A2438, the amino acid sequence is shown in SEQ ID NO: 1, and the nucleic acid sequence is shown in SEQ ID NO: 2. With SYNPCC7002_A2438 as the center, approximately 500 bp of homology arms were amplified upstream and downstream, and a kanamycin resistance gene fragment was inserted between the two homology arms to obtain a knockout construct ( Figure 1 ), the knockout construct was cloned into a plasmid vector to obtain a glk gene knockout plasmid, which was used to transform wild-type Synechococcus PCC 7002, and knockout mutants were screened. Finally, a glucokinase knockout mutant YD07 was obtained, which was verified by PCR and showed that the glucokinase gene glk was knocked out ( Figure 2 ).

[0030] 2.YD07 secretes glucose

[0031] YD07 was inoculated into the culture medium and incubated at 30°C and 150 μmol photons·m -2 ·s -1 Culture was performed under the same light intensity and air containing 3% CO2 was introduced. Samples were taken every two days to measure OD 730 and glucose content.

[0032] The OD of YDO7 730 Draw a growth curve, such as Figure 3 As shown, the growth curve of YDO7 had no significant difference from that of the wild type, indicating that the knockout of glk did not affect the growth of the strain.

[0033] The glucose content was determined as follows: 1 mL of algal cells was collected and centrifuged at 13,000 rpm for 5 minutes. The supernatant was used as the sugar sample to be tested. The intracellular and extracellular glucose content was determined using the Sucrose / D-Glucose Assay Kit (Megazyme). To increase the number of uses of the kit, the original steps were optimized. The optimized experimental steps are as follows: 20 μL of the sample to be tested was placed in a 1.5 mL EP tube, 300 μL of GOPOD buffer was added, the mixture was thoroughly mixed, and the mixture was placed in a 50°C water bath for 20 minutes. 200 μL of the reaction mixture was placed in a 96-well plate and the A 510 And through the standard curve ( Figure 4 ) was converted to glucose content.

[0034] The results are as follows Figure 5 As shown, YD07 secreted a large amount of glucose into the culture environment during the culture process, and the extracellular glucose content reached a peak of nearly 400 mg / L on the 12th day of culture.

[0035] 3. YDO7 is used to construct a new biophotovoltaic device

[0036] The novel biophotovoltaic device of this embodiment includes an anode cell, a cathode cell, and a proton-permeable semipermeable membrane ( Figure 6 ).

[0037] The cathode cell is made of a light-transmitting material. It contains culture medium and a YD07 attachment sheet immersed in the culture medium. The YD07 attachment sheet is prepared as follows: an appropriate amount of algae solution is centrifuged at 6000 rpm for 15 minutes at room temperature. It is then mixed with a 0.5-2.5% w / w sodium alginate solution to create a sodium alginate-cyanobacteria mixture. This mixture is then solidified with calcium chloride (the sodium ions in sodium alginate are replaced by calcium ions in calcium chloride to form calcium alginate, which is insoluble in water, thus forming a cross-linked gel system). This forms a sodium alginate-YD07 hydrogel, whose structure supports normal YD07 growth and glucose synthesis.

[0038] The composition of the culture medium (1 L) is: 18 g / L NaCl, 0.6 g / L KCl, 1 g / L NaNO3, 5 g / L MgSO4·7H2O, 0.2775 g / L CaCl2, and 1 mL / L trace element stock solution. In a specific embodiment of the present invention, the trace element stock solution composition is: 2.86 g / L H3BO3, 1.81 g / L MnCl2·4H2O, 0.222 g / L ZnSO4·7H2O, 0.39 g / L Na2MoO4·2H2O, 0.08 g / L CuSO4·5H2O, 0.01 g / L CoCl2·6H2O, 16.8105 g / L EDTA-1Na, and 2.78 g / L FeSO4·7H2O.

[0039] Under conventional culture conditions, the cathode cell can produce 405 mg / L extracellular sugar ( Figure 7 ), while meeting the experimental requirements of the biophotovoltaic device, it simplifies the difficulty of replacing the algal culture medium. A fresh and abundant culture system will facilitate the long-term use of the biophotovoltaic device.

[0040] The extracellular display bacterial community, which supports the electron transport chain required for the complete oxidation of glucose, was immobilized on the cathode electrode. The upstream bacterial community ratio was PPGK:G6PDH:6PGDH:PGL = 8:1:3:3, and the downstream bacterial community ratio was RPI:Ru5PE:TK:TAL:PGI = 1:5:6:1:1, with an upstream-downstream ratio of 1:6. Carbon cloth was used as the cathode substrate. The extracellular display bacterial community was pretreated by centrifugation and then added dropwise to the cathode surface. After forming a stable biofilm at room temperature, a chitosan solution was added dropwise for biofilm fixation (modification method).

[0041] In the cathode cell, sodium alginate gel-encapsulated YD07 cells fixed light energy through photosynthesis, converted it into glucose, and excreted it into the external system. Glucose, as a high-energy intermediate metabolite, was utilized by the extracellular display bacteria and completely oxidized to generate electrons, thereby generating a photocurrent output for the experimental integrated biophotovoltaic device. After 29 hours of stable operation, the device achieved a maximum monitored photocurrent output density of 210 μA·cm -2 ( Figure 8 ).

[0042] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. Application of a cyanobacterial glucokinase disabling agent in the preparation of cyanobacteria with glucose secretion function or high glucose production.

2. The use according to claim 1, characterized in that The cyanobacterial glucokinase disabling agent is a reagent for knocking out the glucokinase gene in the cyanobacteria.

3. A cyanobacterium with high glucose production, characterized in that: The glucokinase in the cyanobacteria is nonfunctional.

4. Application of cyanobacteria according to claim 3 in producing glucose.

5. Use of the cyanobacteria according to claim 3 in the preparation of photovoltaic biodevices.

6. A method for making cyanobacteria produce high yield and secrete glucose, characterized in that: The method comprises the step of disabling the function of glucokinase in the cyanobacteria.

7. The method according to claim 6, characterized in that The glucokinase gene in the cyanobacteria is knocked out so that the cyanobacteria does not contain glucokinase.

8. The use according to claim 1, 2, 4 or 5, or the cyanobacteria according to claim 3, or the method according to claim 6 or 7, characterized in that: The cyanobacteria is Synechococcus, and the reference amino acid sequence of the glucokinase is shown in SEQ ID NO: 1.

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