Polysialic acid high-producing strain as well as rapid screening method and application thereof

Abstract

The invention relates to the field of microorganisms, and provides a polysialic acid high-producing strain as well as a rapid screening method and application thereof. The screening method comprises the following steps: firstly carrying out primary screening on Escherichia coli mutant colonies through propidium iodide fluorescence color development, then carrying out negative screening by using a rifampicin-containing flat plate, inoculating the obtained target colonies to a high-flux porous plate, carrying out micro-fermentation secondary screening, and finally carrying out performance verification and confirmation by adopting shake flask fermentation. According to the screening method, the cell membrane permeability increasing strains are accurately and directionally screened, so that the screening efficiency of the polysialic acid high-yield mutant strains is remarkably improved. Through the implementation of the method, a polysialic acid high-yield strain is successfully screened, the strain is Escherichia coli, the strain is preserved in the China General Microbiological Culture Collection Center on May 19, 2025, and the preservation number is CGMCC NO.34585. The polysialic acid high-yield strain can be used for producing polysialic acid. The yield of polysialic acid of the strain in a 50L fermentation tank can reach 27.13 g / L.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to a high-yield polysialic acid strain, a rapid screening method, and its application. Background Technology

[0002] Polysialic acid (PSA), as a valuable biopolymer, shows broad application prospects in pharmaceuticals, food, and cosmetics. Its innovative applications in drug delivery systems and infant nutrition products are driving market demand growth. PSA production is undergoing a technological transformation from traditional extraction to modern bio-fermentation synthesis, with the acquisition of high-yield strains being the core engine of this transformation. Shi et al. (2022) used ARTP mutagenesis to achieve a PSA yield of 0.95 g / L in shake-flask fermentation, and after process optimization, the yield reached 16.1 g / L. CN 112553120 screened a PSA-producing strain with a maximum yield of 19.3 g / L; US 8722365 achieved a PSA yield of 1.5 g / L through genetic modification. While traditional mutation and molecular modification can alter strain characteristics, the rapid and effective screening of target strains depends on the establishment of targeted and efficient screening methods. Polysialic acid (PSA) is an extracellular polysaccharide synthesized by *Escherichia coli*. Both the monomer and the product require transmembrane transport, and transmembrane resistance is one of the key factors limiting the efficient synthesis of PSA. Targeting transmembrane transport efficiency and improving the cell membrane permeability of bacterial strains holds promise for establishing a targeted and efficient screening method for high-yield PSA strains, thereby significantly improving strain screening efficiency and helping to further reduce the industrialization cost of PSA. Summary of the Invention

[0003] One of the objectives of this invention is to address the problem of high workload and low efficiency in screening target strains after traditional mutagenesis of polysialic acid-producing microorganisms, and to provide a method for targeted and efficient screening of high-yield polysialic acid strains and its practical application.

[0004] The second objective of this invention is to further reduce the industrialization cost of polysialic acid and to provide a high-yield polysialic acid production strain and its application in the fermentation preparation of polysialic acid.

[0005] One objective of this invention is achieved through the following technical solution: a highly efficient screening method for high-yielding polysialic acid strains, comprising the following steps: (1) Propidine iodide fluorescence colorimetric screening: Escherichia coli mutant colonies were activated and cultured at 37°C for 8-24 h. A sterile filter membrane was laid on the colony plate, and propidine iodide was sprayed evenly on the surface of the filter membrane. After incubation for 30 min, red fluorescent colonies were marked under a gel imaging system at a wavelength of 535 nm. These colonies were the initial screening candidate colonies. (2) Hydrophobic antibiotic plate negative screening: The candidate colonies in the initial screening are numbered and inoculated onto conventional plates and plates containing hydrophobic antibiotics respectively. Colonies that grow on conventional plates but cannot grow or are severely inhibited on plates containing hydrophobic antibiotics are selected to obtain the target strain with increased cell membrane permeability. (3) High-throughput secondary screening in deep well plate: The target colony was inoculated into a high-throughput multi-well plate containing 200 μL of culture medium for fermentation. The PSA yield was rapidly detected by an enzyme-linked immunosorbent assay (ELISA) using the resorcinol-hydrochloric acid method. The top 30% of high-yielding strains were selected for secondary screening. (4) Shake flask fermentation performance confirmation: The top 30% of high-yielding strains in PSA production were screened and subjected to shake flask fermentation for final performance confirmation, thus obtaining high-yielding PSA strains.

[0006] Based on the above screening method, the primary screening chromogenic agent for candidate colonies is propidium iodide, with a working concentration of 5~20 μg / mL.

[0007] Based on the above screening method, the hydrophobic antibiotics used in the negative screening plates include, but are not limited to, neomycin, erythromycin, and rifampin, with rifampin being preferred.

[0008] Furthermore, the concentration of hydrophobic antibiotics added to the negative screening plates was 10~100 μg / mL.

[0009] The second objective of this invention is achieved through the following technical solution: An *Escherichia coli* strain was deposited on May 19, 2025, at the China General Microbiological Culture Collection Center, Beijing, with accession number CGMCC NO.34585. The 16S rRNA of this *Escherichia coli* strain has the sequence shown in SEQ ID NO.1.

[0010] An application of the aforementioned Escherichia coli in the fermentation preparation of polysialic acid, the application comprising the following steps: (1) After culturing Escherichia coli at 37 °C for 16 h, inoculate it into 100 mL of LB liquid medium and place it in a shaker at 37 °C and 250 rpm until the OD600 is 2~6; (2) The Escherichia coli culture after step (1) was transferred to a 6L seed tank LB medium at a volume concentration of 0.1% and cultured until the OD600 was 2~6. The temperature was controlled at 37±1 ℃, the tank pressure was 0.05 MPa, the air flow rate was 0.8 vvm, and the stirring was 150~300 rpm to obtain the seed culture. (3) The seed liquid obtained in step (2) was inoculated into a 30L fermenter culture medium at a volume concentration of 2% and fermented for 48h. The temperature was controlled at 37±1℃, the tank pressure at 0.05 MPa, the air flow rate at 0.8~1.1 vvm, and the dissolved oxygen was controlled at 20% by stirring and dissolved oxygen linkage to prepare polysialic acid.

[0011] In step (3), the fermentation tank culture medium consists of: 200 g / L glucose, 15 g / L yeast extract powder, 5 g / L ammonium sulfate, 0.6 g / L magnesium sulfate heptahydrate, 40 mmol / L dipotassium hydrogen phosphate trihydrate, 1 g / L defoamer, and pH 8.0 before defoaming.

[0012] In step (3): (a) The potassium ion concentration in the fermenter culture medium is less than or equal to 50 mmol / L; (b) When the dissolved oxygen in the fermenter medium drops to 0-1% and OD600 is greater than 20, when OD600 is greater than 40, and when OD600 is greater than 60, xylose with a working concentration of 5 g / L should be added once, and glucose with a working concentration of 25 g / L should be added again after 3 hours.

[0013] The beneficial effects of this invention are that by combining propidium iodide fluorescence detection and negative screening with hydrophobic antibiotics, colonies with increased cell membrane permeability can be rapidly and accurately screened, reducing transmembrane resistance of sialic acid monomers and polysialic acid, thereby obtaining strains with high polysialic acid biosynthesis efficiency. Ultimately, this provides a targeted and efficient screening method for high-yielding polysialic acid strains. Through practical application of this method, a high-yielding polysialic acid-producing *Escherichia coli* strain was successfully screened, achieving a polysialic acid yield of 27.13 g / L in a 50 L fermenter.

[0014] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0015] Figure 1 The yield distribution of high-throughput screening strains under different experimental conditions; Figure 2 The molecular weight determination results of PSA prepared in a 50 L container for strain CGMCC NO.34585. Detailed Implementation

[0016] Example 1: Escherichia coli mutation The working strain of *Escherichia coli* obtained from natural isolation was used. A small amount of the thawed bacterial suspension was dipped into an inoculation loop and evenly spread onto the slant of an LB tube. The tube was then incubated at 37 °C for 10 h. The slant was rinsed with 10 mL of phosphate buffer, vortexed for 5 min, centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the tube was resuspended in 10 mL of fresh phosphate buffer. The suspension was vortexed for 5 min to obtain the suspension to be mutated. The suspension containing the mutant was serially diluted and inoculated onto LB plates at a rate of 0.1 mL / plate. The plates were incubated at 37 °C for 10 h, and the bacteria were counted. 50 μL of the suspension to be mutated was dropped onto a slide and irradiated with arterial plasma (ARTP) for different durations (5 s, 10 s, 15 s, 20 s). The slide was then transferred to sterile physiological saline to wash off the irradiated bacteria. After serial dilution, the slide was inoculated onto LB plates at a rate of 0.1 mL / plate. The plates were incubated at 37 °C for 10 h, and the bacteria were counted. Based on the counting results, the lethality rate was calculated, and the plate colonies irradiated for 10 s (lethality rate 81.5%) were finally selected as the mutant initiation screening strains.

[0017] Example 2: Propidium iodide fluorescence colorimetric primary screening Forty agar plates irradiated with ARTP for 10 s were used. Two plates were randomly selected as the control group, and the remaining 38 plates were used as the experimental group. A sterile filter membrane was placed on all agar plates. The control group was left untreated, while the experimental group was sprayed evenly with a sterile propidium iodide aqueous solution of 10 μg / mL. All plates were incubated at 37°C for 30 min. After incubation, the filter membrane was removed from the agar plates, and the agar plates were observed at 535 nm using a gel imaging system. All 49 colonies in the control group showed no fluorescence. Of the 1016 colonies in the experimental group, 57 showed varying degrees of red fluorescence. The red fluorescent colonies were the candidate mutants with increased cell membrane permeability.

[0018] Example 3: Negative screening of hydrophobic antibiotics using a plate The colonies from Example 2 were used. The control group was numbered A1-A49, and the experimental group's red fluorescent colonies were numbered B1-B57. Each colony was individually transferred to a standard LB agar plate and an LB agar plate supplemented with 20 μg / mL rifampicin (a hydrophobic antibiotic) using a toothpick. All plates were incubated at 37°C for approximately 24 h, and the diameter of each colony was measured. The results are shown in Table 1 below. In the control group, except for A17 and A42, there was no significant difference in growth diameter between the antibiotic-treated plates and the LB agar plates. In the experimental group, 26.3% of the colonies that could not grow on antibiotic-treated plates but grew normally on LB agar plates, and 59.6% of the colonies whose growth was significantly inhibited on antibiotic-treated plates compared to LB agar plates, were identified as having increased cell membrane permeability. This indicates that the method of using propidium iodide fluorescence to screen for strains with increased cell membrane permeability is accurate and effective.

[0019] Table 1. Summary of negative selection results of mutant and propidium iodide fluorescent screening strains on plates. .

[0020] Example 4: High-throughput fermentation and secondary screening of target colonies Forty-nine non-mutated colonies, forty-nine mutant colonies (A1~A49), and forty-nine colonies selected for propidium iodide fluorescence screening followed by rifampicin negative screening (growth inhibition colonies) were inoculated one loop at a time into 96-well deep-well plates containing 200 μL of fermentation medium and incubated at 37°C with shaking for 30 h. After incubation, PSA yield was rapidly detected using the resorcinol-hydrochloric acid method with a microplate reader, and the yield distribution of each experimental group was statistically analyzed. Figure 1 As shown in the figure. The results showed that the yield of the non-mutant strain (control) was mainly distributed in the range of 0.6-0.8 g / L, the yield of the mutant strain was mainly distributed in the range of 0.4-0.6 g / L, and the yield of the strains subjected to propidium iodide fluorescence colorimetric screening followed by rifampicin negative screening was mainly distributed in the range of 1.0-1.2 g / L. This indicates that the combined screening method of propidium iodide fluorescence colorimetric screening followed by rifampicin negative screening can quickly and efficiently screen out positive mutant strains from mutant strains.

[0021] Example 5: Confirmation of the shake-flask fermentation performance of high-yield strains The top 30% yield strains under each of the three experimental conditions in Example 4 were selected and inoculated one loop into liquid LB seed medium (50 mL / 250 mL), and cultured at 37°C and 250 rpm for 12 h. Then, they were inoculated into shake flask fermentation medium (30 mL / 250 mL) at a 4% (v / v) inoculation rate and cultured at 37°C and 250 rpm for 48 h. After fermentation, the PSA yield was determined using the resorcinol-hydrochloric acid method. The yield of the non-mutated strain (control) was 1.52-1.84 g / L, the yield of the mutant strain was 1.40-1.71 g / L, and the yield of the strains screened by propidium iodide fluorescence colorimetric development and rifampicin negative screening was 2.11-2.66 g / L. The strain with the highest yield was B26# (2.66 g / L). After isolation and purification, this strain was deposited by our company's strain preservation center (HDCC000121), and then on May 19, 2025, it was deposited at the China General Microbiological Culture Collection Center, with the accession number CGMCC NO.34585.

[0022] The shake-flask fermentation medium formula is as follows: glucose 30 g / L, yeast extract powder 2.5 g / L, ammonium sulfate 6 g / L, magnesium sulfate heptahydrate 0.6 g / L, dipotassium hydrogen phosphate trihydrate 40 mmol / L, calcium carbonate 10 g / L, and pH 8.0 before digestion.

[0023] Example 6: Preparation of PSA by fermentation of CGMCC strain NO.34585 in a 50 L tank Take a working glycerol tube of CGMCC strain NO.34585, dip a small amount of thawed bacterial culture into it with an inoculation loop, and evenly spread it on the LB slant. Incubate at 37 ℃ overnight (approximately 16 h). Take a loopful of bacterial growth from the slant and inoculate it into liquid LB shake flask seed medium (100 mL / 500 mL). Incubate at 37 ℃ and 250 rpm on a shaker for 8 h, controlling the OD. 600 Between 2 and 6; the cultured shake-flask seed culture was inoculated into LB medium (6 L / 10 L) in a seed tank at a ratio of 0.1% (v / v), the temperature was controlled at 37±1 ℃, the tank pressure at 0.05 MPa, the air flow rate at 0.8 vvm, the stirring was 150~300 rpm, and the culture was carried out for 5 h, controlling the OD. 600 Between 2 and 6; the cultured seed culture was inoculated into the fermenter medium (30 L / 50 L) at a ratio of 2% (v / v). The temperature was controlled at 37±1 ℃, the pressure at 0.05 MPa, and the air flow rate at 0.8~1.1 vvm. The dissolved oxygen was controlled at 20% by agitation and dissolved oxygen control. After inoculation, the dissolved oxygen dropped rapidly, reaching zero in about 10 hours, and the OD... 600 Once the OD600 reaches 20 or higher, administer xylose for the first time at 5 g / L (based on total volume), followed by glucose at 25 g / L 3 hours later. When the OD600 reaches 40 or higher, administer xylose for the second time at 5 g / L, followed by glucose at 25 g / L 3 hours later. 600 When the concentration reached above 60%, xylose (5 g / L) was added for the third time, followed by glucose (25 g / L) 3 hours later. The pH was automatically controlled to 7.0 using ammonia water throughout the fermentation process, with a fermentation cycle of 48 hours. The PSA yield (resorcinol-hydrochloric acid method) was 27.13 g / L after tank discharge. After preliminary purification, the PSA molecular weight was determined to be 34211 Da. Figure 2 As shown.

[0024] The basic fermentation formula consists of: 200 g / L glucose, 15 g / L yeast extract, 5 g / L ammonium sulfate, 0.6 g / L magnesium sulfate heptahydrate, 40 mmol / L dipotassium hydrogen phosphate trihydrate, and 1 g / L defoamer, with a pre-fermentation pH of 8.0. Feeding includes a carbon source mixture: 400 g / L glucose, 80 g / L xylose; ammonia; and defoamer.

[0025] In Example 2, 57 out of 1016 ARTP mutant activated colonies were successfully stained with propidium iodide, indicating that ARTP mutagenesis can effectively enhance the activity of Escherichia coli (Escherichia coli). Escherichia coli Cell membrane permeability was assessed, and the initial screening elimination rate using propidium iodide fluorescence detection was 94.39%.

[0026] In Example 3, there was no significant difference in the growth diameter of the control group colonies on rifampicin and LB plates, while 85.9% of the propidium iodide fluorescent screening strains showed significantly inhibited or no growth on rifampicin plates, further confirming that the cell membrane permeability of most propidium iodide fluorescent screening strains was improved.

[0027] In Example 4, the yield distribution range of strains selected by propidium iodide fluorescence screening followed by rifampicin negative screening was significantly higher than that of the non-mutated and mutated unscreened groups, indicating that the propidium iodide fluorescence screening followed by rifampicin negative screening combined screening method can quickly and efficiently screen out positive mutant strains from mutant strains.

[0028] In Example 5, during the shake-flask rescreening confirmation, strain B26#, which yielded the highest output among the candidate strains selected through propidium iodide fluorescence staining primary screening and rifampicin negative screening, was successfully obtained. It was deposited on June 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.34585. Through the implementation of a potassium concentration control and glucose / xylose dual carbon source control strategy, this strain ultimately achieved a PSA constant of 27.13 g / L in a 50 L fermenter, reaching an advanced level. In summary, the combined screening method of ARTP mutation-propidium iodide fluorescence staining primary screening-hydrophobic antibiotic negative screening-high-throughput plate rescreening is feasible and effective in the rapid screening of high-yielding PSA strains.

Claims

1. A rapid screening method for high-yielding polysialic acid strains, characterized in that, Includes the following steps: (1) Propidine iodide fluorescence colorimetric screening: Escherichia coli mutant colonies were activated and cultured at 37°C for 8-24 h. A sterile filter membrane was laid on the colony plate, and propidine iodide was sprayed evenly on the surface of the filter membrane. After incubation for 30 min, red fluorescent colonies were marked under a gel imaging system at a wavelength of 535 nm. These colonies were the initial screening candidate colonies. (2) Hydrophobic antibiotic plate negative screening: The candidate colonies in the initial screening are numbered and inoculated onto conventional plates and plates containing hydrophobic antibiotics respectively. Colonies that grow on conventional plates but cannot grow or are severely inhibited on plates containing hydrophobic antibiotics are selected to obtain the target strain with increased cell membrane permeability. (3) High-throughput secondary screening in deep well plates: The target colonies were inoculated into high-throughput multi-well plates containing 200 μL of culture medium for fermentation culture. The PSA yield was quickly detected by an enzyme-linked immunosorbent assay (ELISA) reader using the resorcinol-hydrochloric acid method. The top 30% of the strains with the highest yield were selected for secondary screening. (4) Shake flask fermentation performance confirmation: The top 30% of high-yielding strains in PSA production were screened and subjected to shake flask fermentation for final performance confirmation, thus obtaining high-yielding PSA strains.

2. The rapid screening method according to claim 1, characterized in that, In step (1), propidium iodide is used as the primary screening chromogenic agent for candidate colonies, with a working concentration of 5~20 μg / mL.

3. The rapid screening method according to claim 1, characterized in that, In step (2), the hydrophobic antibiotic is one of neomycin, erythromycin, or rifampin, and the concentration of the hydrophobic antibiotic added to the plate containing the hydrophobic antibiotic is 10~100μg / mL.

4. The rapid screening method according to claim 1, characterized in that, In step (2), the hydrophobic antibiotic is preferably rifampicin.

5. An Escherichia coli strain obtained by screening using the rapid screening method described in claim 1, characterized in that, The Escherichia coli was deposited at the China General Microbiological Culture Collection Center on June 9, 2025, with accession number CGMCC NO.34585.

6. The *Escherichia coli* according to claim 5, characterized in that, The 16S rRNA sequence of this strain is shown in SEQ ID NO.

1.

7. The use of the Escherichia coli of claim 5 in the fermentation preparation of polysialic acid.

8. The application according to claim 7, characterized in that, The application includes the following steps: (1) After culturing Escherichia coli at 37 °C for 16 h, inoculate it into 100 mL of LB liquid medium and incubate it in a shaker at 37 °C and 250 rpm until OD. 600 It ranges from 2 to 6; (2) Transfer the Escherichia coli culture from step (1) to a 6L seed tank LB medium at a volume concentration of 0.1% and culture until OD. 600 Set the temperature to 37±1 ℃, control the pressure in the tank to 0.05 MPa, the air flow rate to 0.8 vvm, and stir at 150~300 rpm to obtain the seed liquid in the tank. (3) The seed liquid obtained in step (2) was inoculated into a 30L fermenter culture medium at a volume concentration of 2% and fermented for 48h. The temperature was controlled at 37±1℃, the tank pressure at 0.05 MPa, the air flow rate at 0.8~1.1 vvm, and the dissolved oxygen was controlled at 20% by stirring and dissolved oxygen linkage to prepare polysialic acid.

9. The application according to claim 7, characterized in that, In step (3), the fermentation tank culture medium consists of: 200 g / L glucose, 15 g / L yeast extract powder, 5 g / L ammonium sulfate, 0.6 g / L magnesium sulfate heptahydrate, 40 mmol / L dipotassium hydrogen phosphate trihydrate, 1 g / L defoamer, and pH 8.0 before defoaming.

10. The application according to claim 7, characterized in that, In step (3): (a) The potassium ion concentration in the fermenter culture medium is less than or equal to 50 mmol / L; (b) When the dissolved oxygen content of the fermenter culture medium drops to 0-1% and OD 600 When OD is greater than 20, 600 When OD is greater than 40, 600 When the concentration is greater than 60, add xylose at a working concentration of 5 g / L once, and add glucose at a working concentration of 25 g / L 3 hours later.

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