CN114774316B - Streptococcus equi subsp zooepidemicus mutant strain and application thereof - Google Patents
Streptococcus equi subsp zooepidemicus mutant strain and application thereof Download PDFInfo
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Abstract
The Streptococcus equi zooepidemicus mutant strain is characterized in that Streptococcus equi zooepidemicus SFPE-A17 is preserved in China general microbiological culture Collection center at 11 months and 12 days of 2021, the preservation address is No. 3 of North Cheng West Lu No. 1 of the sunward area in Beijing, and the preservation number is CGMCC No.23795. By utilizing the Streptococcus equi subsp zooepidemicus SFPE-A17, the growth speed of the strain is obviously accelerated in the process of fermenting hyaluronic acid, the strain begins to accumulate hyaluronic acid earlier, the yield of the hyaluronic acid is improved by 42.9 percent compared with the original strain, the production cost can be obviously reduced, the molecular weight of the hyaluronic acid is reduced by one time, and the average molecular weight distribution is 0.54 multiplied by 10 6 Da can improve the industrial efficiency in the medium and low molecular weight hyaluronic acid market, and has important industrial application prospect.
Description
Technical Field
The invention relates to an streptococcus equi subsp zooepidemicus mutant strain and application thereof, belonging to the technical field of biological fermentation.
Background
Hyaluronic acid is a high molecular polysaccharide substance formed by connecting D-glucuronic acid and N-acetylglucosamine through beta-1, 3 and beta-1, 4 glycosidic bonds, and is also called hyaluronic acid. Hyaluronic acid has a very strong moisturizing effect and is called an ideal natural moisturizing factor. It is the substance with the best moisturizing performance for cosmetics found in nature at present. Hyaluronic acid has different molecular weights, ranging from thousands to millions of daltons, and its properties and applications vary depending on the molecular weight. Hyaluronic acid with small molecular weight (molecular weight 10kDa-100 kDa) can permeate into dermis and be easily absorbed by human body, so that the hyaluronic acid can be used in the fields of health food, beauty food and drug carriers. The market for hyaluronic acid worldwide has currently exceeded $ 100 billion.
The production method of hyaluronic acid mainly comprises two methods of animal tissue extraction and microbial fermentation. The former has high production cost and is easy to generate immune reaction due to factors such as limited source, low yield and the like. Therefore, hyaluronic acid is currently produced mainly by fermentation. At present, the industrial hyaluronic acid production strains mainly comprise streptococcus zooepidemicus, streptococcus equi and the like.
Meanwhile, chemical mutagenesis is mostly adopted for streptococcus zooepidemicus strain mutagenesis, such as alkylating agent, diethyl sulfate, nitrogen mustard hydrochloride, 5-bromouracil and the like, the chemical mutagens have specificity to the chemical mutagens, and one mutagen acts on a certain part of a gene; meanwhile, the application is limited due to the teratogenesis and carcinogenesis effects on human bodies.
The application of hyaluronic acid in various fields is increasingly wide, but the application and the modification of wild bacteria are limited to a certain extent by limited gene manipulation technology. At present, the genetic engineering bacteria meeting the industrial production requirements do not reach the level of wild bacteria in terms of yield, and the improvement and the process optimization of the wild bacteria have practical value and significance.
Disclosure of Invention
In order to solve the technical problems, the invention provides streptococcus equi subsp zooepidemicus with high hyaluronic acid yield and application thereof. The invention separates and purifies Streptococcus zooepidemicus producing hyaluronidase from hyaluronic acid fermentation liquor, carries out mutation breeding by an Atmospheric pressure room temperature plasma (ARTP) mutation system, carries out comparative transcriptomics sequencing on mutant strains, and verifies differentially expressed genes on a molecular level, and finally obtains a Streptococcus equi zooepidemicus subsp. Meanwhile, the hyaluronic acid prepared by the strain through liquid fermentation has high yield and short production period. The production is easy to control and is suitable for industrial large-scale production.
The invention aims to provide Streptococcus equi subsp subsp.zooepidemicus (Streptococcus equi) SFPE-A17, which is preserved in China general microbiological culture Collection center (CGMCC) at 11, 12 and 11 months in 2021, with the preservation address of No. 3 Hospital No. 1 of Xingyang district, beijing, and the preservation number of CGMCC No.23795.
The streptococcus equi subsp zooepidemicus strain is characterized in that: spherical or oval, chain-shaped, capsular, spore-free, flagellar, gram-positive.
The expression level of glucosamine-6-phosphate deaminase (nagB) of the streptococcus equi subsp zooepidemicus is 2-20% of that of a wild type, and the expression level of glucosamine-1-phosphate-acetyltransferase (glmU) of the streptococcus equi subsp zooepidemicus is 2-4 times of that of the wild type.
The second purpose of the invention is to provide a hyaluronic acid production method, which adopts Streptococcus equi subsp.
Further, the method is to culture Streptococcus equi subsp.
Further, the fermentation temperature is 30-37 ℃ in the fermentation process.
Further, the rotating speed is 150-250 rpm in the fermentation process.
Further, the fermentation medium comprises the following components: 8-12 g/L of glucose, 4-6 g/L of yeast powder, 14-16 g/L of tryptone and K 2 HPO 4 ·3H 2 O 1.6～1.8g/L，MgSO 4 ·7H 2 0.6 to 0.7g/L of O and 2.5 to 3.0g/L of sodium glutamate.
Further, the method comprises a step of treating the fermentation broth, comprising adding 0.1% -1% SDS for 10-30min; and diluting the fermentation liquor, centrifuging to remove thalli, adding ethanol with the volume of 3-5 times that of the fermentation liquor, treating for 2-4 hours, centrifuging to obtain an alcohol precipitation product, and drying to obtain a crude hyaluronic acid product.
The third purpose of the invention is to provide a microbial agent, which comprises the Streptococcus equi subsp.
The fourth purpose of the invention is to provide a mutagenesis method of Streptococcus equi subsp. Zooepidemicus SFPE-A17, which adopts plasma with normal pressure and room temperature to carry out four rounds of mutagenesis and adopts Bovine Serum Albumin (BSA) combined with hexadecyl trimethyl ammonium bromide (CTAB) to carry out screening.
Further, the method comprises the following steps:
(1) Carrying out mutation breeding on the plasma at normal pressure and room temperature: putting the starting strain into an ARTP mutagenesis system for mutagenesis;
(2) Screening of a mutant strain: diluting the mutagenized bacterial suspension, coating, culturing and screening, screening the bacterial strain by adopting 30-60g/L BSA solution under an acidic condition, and performing rescreening by complexing the supernatant of the fermentation liquor with CTAB solution at 300-500nm for absorbance detection; the strain with the highest absorption value after four rounds of mutagenesis is the Streptococcus equi subsp.
The invention has the beneficial effects that:
when the Streptococcus equi subsp zooepidemicus SFPE-A17 is used for fermenting hyaluronic acid, the growth speed of a strain is obviously accelerated, the strain begins to accumulate hyaluronic acid earlier, the yield of the hyaluronic acid is improved by 42.9 percent compared with the original strain, the production cost can be obviously reduced, the molecular weight of the obtained hyaluronic acid is reduced by one time, and the average molecular weight distribution is 0.54 multiplied by 10 6 Da can improve the industrial efficiency in the medium and low molecular weight hyaluronic acid market, and has important industrial application prospect.
Biological material preservation:
streptococcus equi subsp zooepidemicus SFPE-A17, the strain is preserved in China general microbiological culture Collection center at 11 months and 12 days in 2021, the preservation address is No. 3 of West Lu No. 1 Hospital in the sunward district of Beijing, and the preservation number is CGMCC No.23795.
Description of the drawings:
FIG. 1: compared with the growth curve of wild Streptococcus zooepidemicus, the growth speed of the strain is obviously accelerated and hyaluronic acid begins to accumulate earlier.
FIG. 2: compared with the yield and the molecular weight of hyaluronic acid fermented by Streptococcus equi subsp. Zooepidemicus) SFPE-A17 and wild Streptococcus zooepidemicus, the yield of the hyaluronic acid is improved by 42.9 percent compared with the original strain, the production cost can be obviously reduced, the molecular weight of the hyaluronic acid is reduced by one time, and the molecular weight average distribution is 0.54 multiplied by 10 6 Da, the industrial efficiency can be improved in the middle-low molecular weight hyaluronic acid market.
FIG. 3: streptococcus equi subsp. zooepidemicus SFPE-A17 and wild Streptococcus equi differential gene volcano graph, X axis represents difference multiple value after log2 conversion, Y axis represents-log 10 The transformed significance values, gray, represent up-regulated differential genes (344), and black, down-regulated differential genes (374).
FIG. 4: the Streptococcus equi subsp zooepidemicus SFPE-A17 and wild Streptococcus zooepidemicus partially significantly differentially express gene real-time fluorescence quantitative PCR (RT-qPCR) verification, and the up-regulation and down-regulation trend of the gene expression is consistent with the transcriptomic sequencing result.
Detailed Description
The present invention is further described below with reference to specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.
Example 1: mutagenesis of Streptococcus equi subsp.zooepidemicus SFPE-A17 original Strain
(1) Preparing a bacterial suspension: streptococcus equi subsp.zooepidemicus SFPE-A17 is obtained by mutagenesis based on wild Streptococcus zooepidemicus, inoculating the wild Streptococcus zooepidemicus into TSA culture medium (tryptone 15.0g/L, phytone 5.0g/L, sodium chloride 5.0 g/L), culturing at 37 deg.C and 220rpm for 10-12 hr to obtain OD 600 0.5-0.7, so that the thalli are in logarithmic growth phase. Centrifuging 10mL of the above seed culture solution at 12000rpm for 2min, collecting thallus, washing with sterile normal saline for 3 times, and suspending thallus in sterile normal saline to obtain OD 600 In the bacterial suspension of 0.6-0.8.
(2) ARTP mutagenesis: accurately sucking 10 mu L of bacterial suspension on the surfaces of a plurality of sterilized slides, and uniformly coating. And (4) starting the ARTP mutagen apparatus correctly according to the steps, and carrying out ultraviolet sterilization for 15-20min. The slides of the inoculum were transferred into the ARTP chamber using sterile forceps and placed in the corresponding grooves. Setting relevant parameters of a mutagen: the power was set to 120W, the gas flow was set to 10SLM, and the sample processing time was 30s, 60s, 90s, 120s, 150s, 180s, 210s, and 240s, respectively. After all samples were mutagenized, the sterilized slides were dropped into an EP tube containing 1mL of sterile water and removed. Shaking and eluting an EP tube for 2min by using an oscillator, adding sterile water into bacterial suspensions with different mutagenesis time gradients for gradient dilution, and coating 0.2mL of diluted mutagenesis bacterial liquid on the surface of a solid culture medium for culture, wherein the components of the culture medium comprise 10g/L of glucose, 5g/L of yeast powder, 15g/L of tryptone and K 2 HPO 4 ·3H 2 O 1.7g/L，MgSO 4 ·7H 2 0.65g/L of O, 2.8g/L of sodium glutamate and 2 percent of agar powder, and culturing at the constant temperature of 37 ℃ for about 24-48h. Counting and comparing the colony number in the plate, calculating the mutagenesis lethality, and selecting the mutagenesis time with 90% lethality as the time parameter of subsequent multiple mutagenesis.
(3) Screening of a mutant strain: the single colonies which are well grown by separation are numbered and picked and transferred to the same solid culture medium, and the primary screening of the mutant strains is carried out on a flat plate. Since the hyaluronic acid can generate white precipitate under the acidic condition after being combined with Bovine Serum Albumin (BSA), 5mL of 50g/L BSA solution is paved on a solid culture mediumAfter the reaction for 10 minutes, 12.5% (v/v) glacial acetic acid was added to the reaction mixture to react for 20 minutes. Selecting strains with obviously increased white sedimentation circle, and inoculating the strains in a liquid culture medium (10 g/L glucose, 5g/L yeast powder, 15g/L tryptone, K) 2 HPO 4 ·3H 2 O 1.7g/L，MgSO 4 ·7H 2 O0.65 g/L, sodium glutamate 2.8 g/L) in a 96-deep well plate, and culturing at 37 deg.C and 220rpm for 24h. The plates were centrifuged at 5000rpm for 30min to give a supernatant containing HA. 50 μ L of the supernatant was transferred to a new 96-well microplate containing 100 μ L of cetyltrimethylammonium bromide (CTAB) solution (CTAB solution was prepared by dissolving 25mg CTAB in 1mL of 0.05mM NaCl solution). The reaction mixture was shaken well to disperse it, and the absorbance of the sample at 400nm was measured using a microplate spectrophotometer microplate reader, and finally, it was inoculated into a 250mL flask containing 50mL of fermentation medium to verify the HA production behavior. The determination number A17 with the highest absorption value after four rounds of mutagenesis is the screened bacterium with the best hyaluronic acid production capacity. The screened Streptococcus equi subsp zooepidemicus (Streptococcus equi subsp. Zooepidemicus) SFPE-A17 mutant is entrusted to China general microbiological culture Collection center for preservation and is named as follows: streptococcus zooepidemicus SFPE-A17 with the preservation number of CGMCC No.23795.
Example 2: comparing transcriptomics to verify the gene expression difference between Streptococcus equi subsp
The mutant strain samples selected for comparative transcriptomic sequencing are subjected to RNA extraction by a Trizol method, sent to the Huada Gene company and subjected to subsequent operation through the company process. And detecting the original data obtained by sequencing, and comparing clean reads obtained after data filtration to a wild type reference genome of streptococcus zooepidemicus after quality control is qualified. And (3) detecting clean reads, and performing gene quantification and various analyses based on gene level after secondary quality control, including principal component analysis, sample correlation, differential gene screening and the like. It was found that the up-regulation gene was 344 and the down-regulation gene was 374, compared to the original strain. Reverse transcription and real-time fluorescent quantitative PCR are carried out on part of genes with larger expression difference multiples(RT-qPCR) validation. The reverse transcription and RT-qPCR methods are described in the kit instructions of Shanghai Czeri qPCR premix (SYBR Green I). Taking streptococcus zooepidemicus 16s rDNA as housekeeping gene, taking cDNA obtained after reverse transcription as a template, and taking the Ct value obtained after reaction as 2 -ΔΔCt The calculation method quantifies the transcription level and the relative expression amount. Part of the significantly differentially expressed genes are shown in table 1, and the verification of the gene expression level is shown in fig. 4. The up-regulation trend and the down-regulation trend of the gene expression level are consistent with the sequencing result of transcriptomics.
Table 1 partially significantly different genes
Example 3: production of hyaluronic acid Using Streptococcus equi subsp. zooepidemicus SFPE-A17
Inoculating the activated SFPE-A17 strain of the Streptococcus equi subsp zooepidemicus into a Streptococcus seed liquid culture medium, wherein the liquid loading amount is 10mL, and then placing the Streptococcus equi subsp zooepidemicus SFPE-A17 strain into a constant-temperature shaking table for shaking culture for about 12-14 h, the temperature of the shaking table is set to be 37 ℃, and the rotating speed is 220rpm. After the culture is finished, inoculating the streptococcus into a streptococcus fermentation medium in an ultra-clean bench in an inoculation amount of 4%, wherein the liquid loading amount is 50mL, the streptococcus is subjected to shake culture in a constant-temperature shaking table with the temperature set to 37 ℃ and the rotating speed set to 220rpm, and the shaking flask fermentation period is about 24 hours.
The fermentation broth was treated with 1% SDS for 10min to release the capsular hyaluronic acid. Diluting the fermentation liquor, centrifuging, removing thalli, adding ethanol with four times of volume, treating for 2-4 hours, centrifuging to obtain an ethanol precipitation product, and drying to obtain a hyaluronic acid crude product. The fermentation result shows that the growth rate of Streptococcus equi subsp. Zooepidemicus SFPE-A17 is improved, and the average molecular weight distribution of hyaluronic acid in a shake flask is 0.54 multiplied by 10 6 Da, the yield can reach 0.813g/L, which is higher than that of the original strainThe improvement is 42.9 percent.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitutions or changes made by the person skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the invention is subject to the claims.
Claims (8)
1. Streptococcus equi subsp zooepidemicus (Streptococcus equi subsp.zooepidemicus) SFPE-A17 is characterized in that the strain is preserved in China general microbiological culture Collection center on 12 months at 11 months in 2021, the preservation address is No. 3 of Xilu No. 1 of Beijing province in the sunny region, and the preservation number is CGMCC No.23795.
2. A method for producing hyaluronic acid, which comprises using the Streptococcus equi subsp. Zooepidemicus of claim 1: (A)Streptococcus equi subsp.zooepidemicus) SFPE-A17 is used as a production bacterium to produce hyaluronic acid by fermentation.
3. The method for producing hyaluronic acid according to claim 2, wherein said method is performed by using Streptococcus equi subspStreptococcus equi subsp.zooepidemicus) The SFPE-A17 strain is cultured in a fermentation medium for 10 to 24h.
4. The method for producing hyaluronic acid according to claim 3, wherein the fermentation temperature is 37 ℃ during fermentation.
5. The method for producing hyaluronic acid according to claim 3, wherein the rotation speed is 150 to 250rpm during the fermentation process.
6. The method of producing hyaluronic acid according to claim 3, wherein the composition of the fermentation medium is: 8-12 g/L of glucose, 4-6 g/L of yeast powder, 14-16 g/L of tryptone and K 2 HPO 4 ·3H 2 O 1.6~1.8 g/L，MgSO 4 ·7H 2 0.6 to 0.7g/L of O and 2.5 to 3.0g/L of sodium glutamate.
7. The method for producing hyaluronic acid according to claim 2, wherein said method further comprises a treatment step of the fermentation broth, comprising adding 0.1% -1% SDS to the fermentation broth for 10-30min; and after treatment, diluting the fermentation liquor, centrifuging to remove thalli, adding ethanol with the volume of 3-5 times of the fermentation liquor, treating for 2-4 hours, centrifuging to obtain an ethanol precipitation product, and drying to obtain a crude hyaluronic acid product.
8. A microbial agent comprising the Streptococcus equi subsp zooepidemicus of claim 1 (A), (B), (C), and (C)Streptococcus equi subsp.zooepidemicus）SFPE-A17。
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