Application of sll0528 gene in improving the yield of exopolysaccharide in cyanobacterium synechocystis sp. pcc6803

Abstract

The application discloses application of an sll0528 gene in improving production of exocellular polysaccharide of Synechococcus sp. PCC6803, and belongs to the field of industrial microorganisms. The sll0528 gene in Synechococcus sp. PCC6803 is knocked out by a method of homologous recombination to obtain a Synechococcus sp. PCC6803 strain asll0528 with improved production of exocellular polysaccharide. The sll0528 gene is knocked out in combination with a salt stress culture mode, so that the salt resistance protection mechanism of Synechococcus sp. PCC6803 can be better utilized to produce more exocellular polysaccharide. The method is simple, easy to operate, low in cost, and can be used for improving the production of exocellular polysaccharide of Synechococcus sp. PCC6803. The method has important theoretical and practical significance for further construction of a high-yield exocellular polysaccharide genetically engineered bacterium, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of industrial microbiology, specifically relating to the application of the sll0528 gene in increasing the extracellular polysaccharide yield of Synechocystis PCC6803. Background Technology

[0002] Cyanobacteria, also known as blue-green algae, are a large class of prokaryotic microorganisms capable of photosynthesis. During their growth, cyanobacteria produce abundant extracellular polysaccharides, which can be used as industrial adhesives, bioflocculators, thickeners, emulsifiers, soil conditioners, and biosorbents. Furthermore, due to their bioactivities, including antitumor, hypoglycemic, antioxidant, antiradiation, immunomodulatory, and freeze-drying protective effects, cyanobacterial polysaccharides are gradually becoming important raw materials for developing high-value-added products such as medical drugs and health supplements.

[0003] Microbial extracellular polysaccharides possess characteristics such as renewability, excellent biosafety, biodegradability, and biocompatibility; furthermore, cyanobacteria exhibit advantages such as autotrophic growth, rapid reproduction, strong adaptability, and large-scale cultivation. Currently, most commercial production of microbial extracellular polysaccharides is based on heterotrophic bacterial fermentation, while photosynthetically autotrophic cyanobacteria represent a novel source of natural polymers, and research on the production of extracellular polysaccharides from cyanobacteria has received increasing attention in recent years.

[0004] However, the low yield of extracellular polysaccharides (ECPs) in cyanobacteria is a major factor restricting their large-scale production. ECP yield is easily affected by various factors, including cyanobacterial species, genetic characteristics, and culture conditions, and is regulated by relevant genes. Synechocystis PCC6803 has a simple genetic background and complete genetic information, making it a model organism for studying cyanobacterial genetic engineering, new energy development, and new raw material production. This invention uses it as the research object, combining gene knockout with changes in culture conditions to increase the ECP yield of Synechocystis PCC6803, thereby promoting the industrial development and application of cyanobacterial ECPs. Summary of the Invention

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of this invention is to provide an application of the sll0528 gene in increasing the extracellular polysaccharide production of Synechocystis PCC6803.

[0006] This invention also provides an application of the sll0528 gene in increasing the extracellular polysaccharide production of Synechocystis PCC6803 under salt stress.

[0007] This invention utilizes the salt-resistant protection mechanism of Synechocystis PCC6803 to produce more extracellular polysaccharides by knocking out the sll0528 gene and combining it with salt stress culture. The method of this invention is simple, easy to operate, and low in cost, and can be used to increase the yield of extracellular polysaccharides from Synechocystis PCC6803.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] This invention provides an application of the sll0528 gene in increasing the extracellular polysaccharide production of Synechocystis PCC6803.

[0010] Knocking out the sll0528 gene increases the extracellular polysaccharide production of Synechocystis PCC6803.

[0011] Furthermore, the application of the sll0528 gene in constructing the Synechocystis PCC6803 algal strain with increased extracellular polysaccharide production.

[0012] Furthermore, the Synechocystis PCC6803 algal strain with increased extracellular polysaccharide production was obtained by knocking out the sll0528 gene in Synechocystis PCC6803 through homologous recombination.

[0013] Among them, the *Syntrophus cytogenes* strain PCC6803, which has increased extracellular polysaccharide production, can be used to construct genetically engineered bacteria that produce extracellular polysaccharides.

[0014] Furthermore, the application of the sll0528 gene in increasing the extracellular polysaccharide production of Synechocystis PCC6803 under salt stress;

[0015] Furthermore, the salt stress is NaCl salt stress; the concentration used is 0.5–1.2 M, preferably 0.9 M;

[0016] Specifically, the steps include the following:

[0017] After knocking out the sll0528 gene in Synechocystis PCC6803, a gene knockout strain △sll0528 was obtained, which is a Synechocystis PCC6803 strain with increased extracellular polysaccharide production. Then, the gene knockout strain △sll0528 was subjected to salt stress treatment to achieve the goal of increasing the extracellular polysaccharide production of Synechocystis.

[0018] Furthermore, the salt stress is NaCl salt stress; the concentration used is 0.5-1.2M, preferably 0.9M;

[0019] Furthermore, the salt stress treatment time is 0.5 to 24 hours; preferably 0.5 to 4 hours.

[0020] The nucleotide sequence or gene sequence of the sll0528 gene is shown in (a), (b), or (c):

[0021] (a) The cDNA sequence of the sll0528 gene, as shown in SEQ ID NO.1;

[0022] (b) Genomic DNA sequence of the sll0528 gene;

[0023] (c) DNA sequences that are more than 90% homologous to (a) or (b) and encode the same functional protein.

[0024] The present invention has the following advantages and effects compared with the prior art:

[0025] (1) In this invention, the sll0528 gene in Synechocystis PCC6803 is knocked out by homologous recombination to obtain a Synechocystis PCC6803 knockout strain △sll0528 with increased extracellular polysaccharide production; its extracellular polysaccharide production is relatively higher than that of wild-type algae strains.

[0026] (2) In this invention, the knockout strain △sll0528 of Synechocystis PCC6803 was cultured in BG11 medium containing 0.5-1.2M (preferably 0.9M) NaCl to induce an adaptive protective mechanism, and its extracellular polysaccharide production was significantly increased compared with that under normal conditions; that is, the extracellular polysaccharide production of the knockout strain △sll0528 in BG11 medium containing 0.5-1.2M (preferably 0.9M) was significantly better than that of the wild-type algal strain.

[0027] (3) The method of the present invention is simple, reliable, easy to operate, and suitable for large-scale promotion and application. It has important theoretical and practical significance for the further construction of high-yield extracellular polysaccharide genetically engineered bacteria and has broad application prospects. Attached Figure Description

[0028] Figure 1 The relative extracellular polysaccharide yields of wild-type Synechocystis PCC6803 and knockout strain △sll0528 under salt culture conditions are given; where WT: wild-type Synechocystis PCC6803; KO: knockout strain △sll0528. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0030] The wild-type strain of Synechocystis PCC6803 used in this embodiment of the invention was isolated and purified from ATCC27184 (ATCC is the abbreviation for the American Culture Collection Center, and 27184 is the strain number).

[0031] The gene knockout strain △sll0528 is disclosed in the patent "201811591708.3, Application of sll0528 gene in improving the ethanol tolerance of Synechocystis PCC6803".

[0032] Example 1

[0033] Culture of Synechocystis and preparation and yield determination of extracellular polysaccharides.

[0034] (1) Wild-type Synechocystis PCC6803 and gene knockout strain △sll0528 with an initial OD730 of about 0.5 were inoculated in BG11 medium containing 0.9M NaCl and cultured for 0.5-24h. After centrifugation at 8000rpm for 10min, the supernatant of the culture medium was collected.

[0035] (2) The supernatant obtained in step (1) was concentrated by rotary evaporation at 45°C for 5 times, and 3 times the volume of anhydrous ethanol was added. The polysaccharide was precipitated overnight at 4°C and centrifuged at 8000 rpm for 10 min.

[0036] (3) Add 3% trichloroacetic acid solution to the precipitate obtained in step (2), stir thoroughly until the precipitate no longer dissolves, and centrifuge at 4°C to collect the supernatant.

[0037] (4) The supernatant obtained in step (3) is filtered through a 0.45 μm microporous filter membrane, and the supernatant is precipitated again with 3 times the volume of anhydrous ethanol. The precipitate is obtained by centrifugation and freeze-drying to obtain crude polysaccharide, i.e. extracellular polysaccharide.

[0038] (5) Standard Curve: Accurately weigh glucose standard to prepare a 1 mg / mL glucose standard solution, and accurately dilute it to the required concentration with distilled water. Add 1 mL of 5% (mass fraction) phenol solution to each test tube, shake well, add 5 mL of concentrated H2SO4, shake well, and then boil in a water bath for 30 min. After cooling, take samples into a 96-well plate and measure the absorbance at 490 nm. Plot a glucose standard curve with the standard glucose concentration as the abscissa and OD490 as the ordinate to obtain the linear regression equation.

[0039] (6) The extracellular polysaccharide yield of Synechocystis PCC6803 was determined by the phenol-sulfuric acid method: The obtained extracellular polysaccharides were fully dissolved in 1 mL of distilled water, and 1 mL of 5% (mass fraction) phenol solution was added to each test tube. After shaking, 5 mL of concentrated H2SO4 was added, and the mixture was shaken well and then boiled in a water bath for 30 min. After cooling, samples were taken into a 96-well plate, and the OD490 of the supernatant was measured. The extracellular polysaccharide content was calculated according to the linear regression equation in step (5).

[0040] Polysaccharides are first hydrolyzed into monosaccharides under the action of sulfuric acid, and then rapidly dehydrated to form uronic acid derivatives. These derivatives then react with phenol to form an orange-yellow compound, which is then determined by colorimetry. The relative yield of extracellular polysaccharides is as follows: Figure 1As shown, compared with the wild-type *Synostemma pentaphyllum* strain PCC6803 under normal culture conditions, the extracellular polysaccharide production of the gene knockout strain △sll0528 increased by approximately 15%. Salt stress treatment significantly increased extracellular polysaccharide production. Treatment with 0.9M NaCl for 0.5 hours resulted in an extracellular polysaccharide increase of over 60% compared to the untreated strain, while the increase in the wild-type strain was approximately 40%. Compared to the wild-type strain under the same 0.5-hour salt stress treatment, the extracellular polysaccharide production of the gene knockout strain △sll0528 increased by 30%.

[0041] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. sll0528 The application of genes in increasing the extracellular polysaccharide production of Synechocystis PCC6803 is characterized by: Knockout sll0528 Genes increase the extracellular polysaccharide production of Synechocystis PCC6803; sll0528 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that: The aforementioned sll0528 Application of genes in constructing Synechocystis PCC6803 algal strain with increased extracellular polysaccharide production.

3. The application according to claim 2, characterized in that: The *Syntrophus synergists* strain PCC6803, which exhibits increased extracellular polysaccharide production, utilizes homologous recombination to enhance the production of extracellular polysaccharides in *Syntrophus synergists* PCC6803. sll0528 Obtained by knocking out the gene.

4. The application according to claim 3, characterized in that: The *Syntrophus synergists* strain PCC6803, which exhibits increased extracellular polysaccharide production, is used to construct genetically engineered bacteria that produce extracellular polysaccharides.

5. The application according to any one of claims 1 to 4, characterized in that: sll0528 Application of genes in increasing the extracellular polysaccharide production of Synechocystis PCC6803 under salt stress.

6. The application according to claim 5, characterized in that: The application specifically includes the following steps: Knockout of Synechocystis PCC6803 sll0528 Gene knockout strains were obtained after gene sequencing △ sll0528 The strain PCC6803 of Synechocystis, which exhibits increased extracellular polysaccharide production, was then subjected to gene knockout. sll0528 Salt stress treatment was applied to increase the production of extracellular polysaccharides in Synechocystis.

7. The application according to claim 6, characterized in that: The salt stress described is NaCl salt stress; the concentration used is 0.5–1.2 M.

8. The application according to claim 6 or 7, characterized in that: The salt stress treatment time is 0.5 to 24 hours.

9. The application according to claim 8, characterized in that: The salt stress treatment time is 0.5 to 4 hours.

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