CN115820440A - Tetraacetylphytosphingosine high-yield mutant strain and application thereof - Google Patents

Abstract

The invention discloses a mutation strain of saccharomyces quaternary Meng Maiye for producing tetraacetylphytosphingosine with high yield and application thereof. The mutant strain mSCU-JM06-8 of the yeast Meng Maiye produced in TAPS season is preserved in China general microbiological culture Collection center on 29 th 9 th 2021, with the preservation number of CGMCC No.23507. The strain is obtained by screening and ARTP mutagenesis technology breeding, and 5L fermentation tank amplification experiment is carried out on the strain. Experiments prove that the mSCU-JM06-8 strain can produce TAPS with high yield, the yield of TAPS produced by shake flask fermentation can reach 659.96 mg/L, and is improved by 400.33% compared with the original strain; the yield of the TAPS fermented in the 5L fermentation tank can reach 1070.86 mg/L, is improved by 495.97% compared with the original strain, and the strain is applied to biological fermentation of the TAPS to obtain higher yield of the TAPS, so that large-scale industrial production of the TAPS is realized, and a solid theoretical basis and data reference are provided for improving industrialization.

Description

Tetraacetylphytosphingosine high-yield mutant strain and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a quaternary Meng Maiye saccharomyces cerevisiae mutant strain capable of highly producing tetraacetylphytosphingosine and application thereof.

Background

Tetraacetylphytosphingosine (TAPS) is a sphingolipid substance widely present in cell membranes of animals, plants, fungi and prokaryotes. TAPS stimulates the synthesis of glucosylceramide in vitro, and glucosylceramide has protective function, and helps to strengthen skin barrier function, inhibit inflammation, inhibit angiogenesis, and lighten dark eye. The TAPS has good solubility, can be compounded with various cosmetic raw materials, and is suitable for anti-aging, repairing, eye cream and other products. In addition, phytosphingosine can be used as a starting material for synthesizing ceramide, so that TAPS can be converted into other products with higher commercial value, and can also be directly used as an effective component to participate in the preparation of cosmetics and skin care products.

The existing approaches for preparing the TAPS comprise chemical synthesis, separation and purification from natural sources and the like, but the chemical synthesis, the separation and purification of the TAPS are extremely expensive and have low yield, the synthesized TAPS cannot be used for food and cosmetics and is difficult to apply on a large scale, and is gradually replaced by a biological fermentation method, but the yield of the strain always limits the fermentation production of the TAPS, so that the obtaining of the strain with high yield of the TAPS is particularly important.

The normal pressure room temperature plasma (ARTP) mutagenesis system is capable of generating plasma jet with high active particle concentration at the temperature of 25-40 ℃ under atmospheric pressure, and changing the structure and permeability of biological cell wall and cell membrane by irradiation to further cause nucleic acid damage, and finally generating variant capable of stably inheriting. The ARTP can change the nucleotide structure of the microorganism through high-activity particles to further mutate and obtain a target cell product, so that the mutant strain has good stability, a vacuum device is not needed, and the operation is simple. Since it can obtain a large number of mutant strains in a short time, it has attracted more and more attention in recent years in the field of mutation breeding of microorganisms, and it has become an effective method for obtaining high-yield strains.

The invention screens a strain of quaternary Meng Maiye yeast for producing TAPS for the first time, and breeds and obtains a high-yield TAPS strain by ARTP mutagenesis technology, so that higher TAPS yield can be obtained, and no public report of the strain on the aspect of fermentation production of tetraacetyl phytosphingosine (TAPS) is seen at present.

Disclosure of Invention

The invention aims to provide a mutation strain of the quaternary Meng Maiye yeast for high yield of tetraacetyl phytosphingosine, the strain is obtained by screening and ARTP mutation breeding technology, and the strain is applied to biological fermentation of TAPS to obtain higher yield of TAPS, thereby realizing large-scale industrial production of TAPS.

The purpose of the invention is realized by the following technical scheme:

the invention provides a strain of Saccharomyces cerevisiae Meng Maiye (II)Meyerozyma guilliermondii) mSCU-JM06-8, which has been deposited at CGMCC (CGMCC, accession number: west road No. 1, north west of the republic of kyo, yang, institute of microbiology, academy of sciences of china, zip code: 100101 CGMCC No. 23507).

The invention is to screen a strain of seasonal Meng Maiye Saccharomyces cerevisiae SCU-JM06 producing TAPS from cellar mud collected from a certain winery in Sichuan as an initial strain, to mutate the strain by using Atmospheric and Room Temperature Plasma (ARTP), to obtain 1 strain of excellent mutagen with high TAPS yield through 3 rounds of screening, namely the seasonal Meng Maiye Saccharomyces cerevisiae (ARTP)Meyerozyma guilliermondii) mSCU-JM06-8, the TAPS yield of the strain fermented for 48h in a shake flask can reach 659.96 mg/L, and is improved by 400.33% compared with the original strain; the yield of TAPS fermented in a 5L fermentation tank can reach 1070.86 mg/L, and is improved by 495.97% compared with the original strain.

The invention also provides Meng Maiye yeast mSCU-JM06-8 which grows well on the agar culture medium, and the colony morphology is a milky white colony which is round and slightly convex, has smooth and viscous edge and is easy to pick up by wetting. The strain cells have typical yeast morphological characteristics, the cell morphology is round, and the reproduction mode is budding reproduction.

The selective breeding season Meng Maiye yeast mSCU-JM06-8 of the invention obtains a target gene SEQUENCE consisting of 572 base pairs (bp) by performing PCR amplification on 18S rDNA of the yeast mSCU-JM06-8 by taking 18S-ITS1 and 18S-ITS4 as primers, as shown in SEQUENCE LISTING. The 18S rDNA sequence of the strain mSCU-JM06-8 was compared to that of the phylogenetic tree of MEGA-X constructed strains by NCBI BLASTMeyerozyma guilliermondii strain KAML05The similarity of gene homology reaches 99 percent, and the strain can be identified as Pichia guilliermondii (Meyerozyma guilliermondii）。

Compared with the prior art, the invention has the following advantages:

1. the invention screens a strain of seasonal Meng Maiye saccharomyces cerevisiae for producing TAPS for the first time, obtains a high-yield TAPS strain capable of being stably inherited by breeding through an ARTP mutagenesis technology, and can obtain higher TAPS yield, thereby realizing the large-scale industrial production of the TAPS.

2. The yield of Tetraacetylphytosphingosine (TAPS) produced by shake flask fermentation of the selected strain of the season Meng Maiye yeast mSCU-JM06-8 can reach 659.96 mg/L, which is increased by 400.33% compared with the original strain; the yield of TAPS fermented in a 5L fermentation tank can reach 1070.86 mg/L, and is improved by 495.97% compared with the original strain. The strain is used for biological fermentation, and has wide application value and important commercial development significance for the industrial production of Tetraacetylphytosphingosine (TAPS).

Deposit description

The strain name is as follows: quaternary Meng Maiye yeast

Latin name:Meyerozyma guilliermondii

the strain number is as follows: mSCU-JM06-8

The preservation organization: china general microbiological culture Collection center

The preservation organization is abbreviated as: CGMCC

Address: west road No. 1, north west of the republic of kyo, yang, institute of microbiology, academy of sciences of china, zip code: 100101

The preservation date is as follows: 09 and 29 months in 2021

Registration number of the preservation center: CGMCC No.23507.

Drawings

FIG. 1 shows the cell microscopic morphology and colony morphology of TAPS producing starting strain SCU-JM 06.

FIG. 2 is a growth curve of the TAPS producing starting strain SCU-JM 06.

FIG. 3A is a plot of lethality of the strains at different ARTP mutagenesis times; b is a control group bacterial liquid flat plate coating pattern; c is a mutant 90s bacteria liquid flat plate coating pattern; d is a mutagenic 120s bacterium liquid flat plate coating pattern; e is the mutant 180s bacterial liquid plate coating pattern.

FIG. 4 is a graph comparing the TAPS yields of the starting strain SCU-JM06 and the mutagenized strain mSCU-JM06-8 by shake flask fermentation for 48h.

FIG. 5 is a graph of the genetic stability assay of the mutagenized strain mSCU-JM 06-8.

FIG. 6A is a strain molecular characterization electropherogram for the mutagenized strain mSCU-JM 06-8; b is an evolutionary tree constructed by the 18s rDNA-ITS sequence of the mutagenic strain mSCU-JM 06-8.

FIG. 7 is a graph showing the comparison of the TAPS production yields in the 5L fermentor between the starting strain SCU-JM06 and the mutant strain SCU-JM 06-8.

Detailed Description

The present invention is further illustrated with reference to the following examples, but the embodiments of the present invention are not limited thereto, and any technologies implemented based on the above contents of the present invention are within the scope of the present invention.

The media and formulations in the following examples are as follows:

YPD liquid medium: 20 g.L ^-1 Glucose, 10 g.L ^-1 Yeast extract, 20 g.L ^-1 Peptone

YPD solid Medium: 20 g.L ^-1 Glucose, 10 g.L ^-1 Yeast extract, 20 g.L ^-1 Peptone, 20 g.L ^-1 Agar.

TAPS fermentation medium: 20-30 g.L ^-1 Glucose, 1-2 g.L ^-1 Yeast extract, 5-10 g.L ^-1 Peptone, 0.67 g.L ^-1 Yeast nitrogen base, 5 g.L ^-1 L-serine, 0.2 g.L ^-1 Calcium chloride.

Example 1

The starting strain is mutagenized by an ARTP mutagenesis system, and the specific experimental steps are as follows:

(1) Measuring a strain growth curve: selecting single colony of SCU-JM06 strain, inoculating into 10mL YPD liquid culture medium, culturing at constant temperature of 180-200r/min at 28 deg.C for 12-16 hr to obtain seed solution, inoculating into a triangular flask (volume of 100 mL) containing 30mL YPD liquid culture medium at 1%, culturing at 28 deg.C at 180-200r/min, and detecting OD every 2 hr _600nm The blank was set to zero using sterile YPD liquid medium. The experiment was set up in 3 replicates, repeated 3 times, data recorded, and curves plotted. As shown in FIG. 2, the strain proliferated slowly in the first 4h and was in a lag phase; the thalli is rapidly proliferated within 4-12h, the growth rate is maximum, and the thalli is in a logarithmic phase; the number of the cells is in dynamic balance within 14-48h, and the stationary phase is the time. In order to meet the requirements of sufficient inoculation amount, vigorous strain growth activity and experimental uniformity, the late logarithmic phase bacterial liquid is finally selected as the seed liquid.

(2) Preparing a bacterial suspension: selecting single colony strain on the plate, placing in YPD liquid culture medium, shaking-culturing at 28 deg.C for 12 hr to logarithmic phase, washing with sterile physiological saline solution repeatedly, centrifuging for 3 times, and adding sterile physiological saline to obtain bacterial solution OD _600nm Diluting to about 0.6-0.8 to obtain bacterial suspension.

(3) The strains were mutagenized using an Atmospheric Room Temperature Plasma (ARTP) mutagenesis system:

a. placing a metal slide matched with the instrument in an ultraclean workbench, burning the metal slide for 20-30s by outer flame of an alcohol lamp, placing the metal slide on a sterile glass plate, cooling, and coating 10 mu L of prepared bacterial suspension on the slide to uniformly cover the surface of the slide with bacterial liquid;

b. transferring the flat plate with the slide glass to an operation cabin of the mutagenesis system which is disinfected by ultraviolet irradiation, placing the slide glass at a corresponding hole position by using sterile tweezers, clamping a slot card below each hole position of a carrying platform by using an EP (EP) tube containing 1mL of sterile physiological saline, adjusting a knob below the carrying platform to enable the slide glass and a working airflow port to be 2mm, and closing a cabin door;

c. the instrument power was set at 100W, the processing times were set at 0, 30, 60, 90, 120, 150, 180, 210, 240s, respectively, and the airflow parameter 10SLM. Clicking a 'start processing' button on an instrument screen, and automatically pressing settings to process a sample;

d. after the sample treatment is finished, the metal slide automatically falls into the corresponding EP tube in the clamping groove below, and the marking is carried out. And (3) oscillating the EP tube with the slide glass on a vortex oscillator for 1min to ensure that the thalli attached to the slide glass are fully eluted to obtain bacterial suspension. The bacterial suspension obtained above was diluted by 10 times for 3 gradients, and 100. Mu.L of each dilution was applied to YPD plates, and the different treatment times and dilution gradients were made 3 times in parallel. Placing in an incubator at 28 ℃ for 48-72h until colonies are formed, and counting. The number of colonies on YPD solid medium plates was recorded, and the lethality was calculated and plotted. The method comprises the steps of taking an SCU-JM06 strain as an initial strain to carry out ARTP mutation breeding, diluting bacterial suspension subjected to ARTP mutation treatment, coating the bacterial suspension on a YPD solid culture medium plate, culturing at 28 ℃, and counting after obvious bacteria grow on the plate. As shown in FIG. 3, the lethality of ARTP mutation is positively correlated with time, SCU-JM06 is not very sensitive to ARTP treatment, the lethality is only about 85% after 60s treatment, the lethality is about 90% after 90s treatment, the lethality can reach more than 97% after 120-180s treatment, and the lethality of the bacteria is not increased continuously after the continuous mutagenesis time. Because the mutation is random, positive and negative mutation and mutagenesis time have no definite rule and can be circulated, but because the longer the mutagenesis time is, the larger the degree of DNA chain and hydrogen bond breakage, the degree of thymine dimer formation and the error degree caused after host repair are increased, the larger the strain damage is, and the larger the change is compared with the original strain. Therefore, in order to improve the screening efficiency of the mutagenic strain, 180s is adopted as the mutagenesis time, and the lethality rate exceeds 97%.

Lethality (%) = [ (N-M)/N ]. Times.100%

N: number of colonies on control (mutagenesis time 0) bacterial liquid-coated solid plate

M: number of colonies on the plate coated with mutagenized cells

ARTP mutagenesis was performed for a treatment time of 180 s: placing the mutagenized slide glass in a 1mL centrifuge tube filled with 1mL sterile physiological saline, performing shaking elution by using a vortex oscillator to prepare a bacterial suspension, diluting, uniformly coating 100 mu L of the bacterial suspension on an YPD plate, and culturing at 28 ℃ for 48-72h.

Example 2

Screening of mutagenic strains, detection and identification of genetic stability:

(1) And (3) screening in a shaking flask: inoculating the obtained mutagenic strain into a YPD liquid culture medium, performing shake-flask culture to obtain a seed solution, inoculating into a triangular flask (with the volume of 100 mL) containing 30mL of liquid TAPS fermentation medium according to the inoculation amount of 1%, and fermenting at 28 ℃ for 48h at 180-200r/min to obtain a fermentation liquid. And (3) putting 2mL of shaking fermentation liquor into a 50mL centrifuge tube, adding 8mL of acetone, fully and uniformly mixing, sealing the centrifuge tube, performing ultrasonic treatment at room temperature for 30min to assist dissolution, centrifuging for 5min at 10000g, taking a 0.22 mu m microporous filter head of a supernatant, filtering, and detecting the content of TAPS in the fermentation liquor by HPLC. As shown in FIG. 4, the starting strain was selected from SCU-JM06, and 8 representative mutant strains were obtained by 3 rounds of mutagenesis and selection. Preliminary experiment results show that the forward mutation strains have 4 strains, wherein the two strains with the highest yield are mSCU-JM06-6 and mSCU-JM06-8, the TAPS yield of the shake flask fermentation liquor respectively reaches 312.27 mg/L and 659.96 mg/L, and the TAPS yields are respectively improved by 136.74% and 400.33% compared with the original strains.

(2) Testing the genetic stability of the strain: continuously passaging the mSCU-JM06-8 mutant strain with higher TAPS yield on a YPD plate for 6 times, performing shake flask fermentation in a TAPS fermentation medium, taking 48h fermentation liquor to detect the content of TAPS, and verifying the genetic stability of the TAPS production capacity of the mSCU-JM06-8 strain. The results show that the mutagenic strain mSCU-JM06-8 has better genetic stability within 6 generations, and the average yield is 654.75 mg/L (see FIG. 5).

(3) And (3) strain identification: and performing PCR amplification by using the extracted genome DNA of the mSCU-JM06-8 strain as a template and 18S-ITS1 and 18S-ITS4 as primers, and performing comparison analysis on the amplified product in NCBI Blast after sequencing. The extracted mSCU-JM06-8 strain DNA is subjected to PCR amplification, agarose gel electrophoresis detection shows that a specific amplification band appears at about 600bp, and the band is single and bright (see figure 6A), which indicates that the PCR amplification result meets the sequencing standard and can be used for sequencing. The 18S rDNA sequence of the strain mSCU-JM06-8 can be identified by NCBI BLAST comparisonThe yeast is also Meng Maiye, and the homology similarity of the genes reaches 99 percent. MEGA-X constructs a phylogenetic tree of strains (see FIG. 6B), which are related toMeyerozyma guilliermondii strain KAML05The relationship is closest.

Example 3

Production of TAPS by small-scale fermentation in 5L fermentation tank

The high producing strain mSCU-JM06-8 finally obtained in example 2 was subjected to small scale up cultivation in a 5L fermenter with the starting strain SCU-JM06 as a control. When a 5L fermentation tank is used for small-scale amplification fermentation, the fermentation temperature is 28 ℃, the stirring speed is 200r/min, the air flow is 0.8-2L/min, the pH is natural, and the total fermentation time is 48h. And after the fermentation is finished, detecting the content of the TAPS in the fermentation liquor by an HPLC method.

As can be seen from FIG. 7, in the 5L fermentor, the yield of TAPS produced by 48h fermentation of the strain mSCU-JM06-8 is 1070.86 mg/L, which is 495.97% higher than that of the starting strain.

SEQUENCE LISTING

<110> Chongqing Chuan Chong Tech & ltd

<120> tetraacetyl phytosphingosine high-yield mutagenesis strain and application thereof

　　<160> 1

　　<170> PatentIn version 3.3

　　<210> 1

　　<211> 572

　　<212> DNA

<213> season also Meng Maiye's yeast (Meyerozyma guilliermondii)

　　<400> 1

GAAGGATCAT TACAGTATTC TTTTGCCAGC GCTTAACTGC GCGGCGAAAA ACCTTACACA CAGTGTCTTT TTGATACAGA ACTCTTGCTT TGGTTTGGCC TAGAGATAGG TTGGGCCAGA GGTTTAACAA AACACAATTT AATTATTTTT ACAGTTAGTC AAATTTTGAA TTAATCTTCA AAACTTTCAA CAACGGATCT CTTGGTTCTC GCATCGATGA AGAACGCAGC GAAATGCGAT AAGTAATATG AATTGCAGAT TTTCGTGAAT CATCGAATCT TTGAACGCAC ATTGCGCCCT CTGGTATTCC AGAGGGCATG CCTGTTTGAG CGTCATTTCT CTCTCAAACC CCCGGGTTTG GTATTGAGTG ATACTCTTAG TCGGACTAGG CGTTTGCTTG AAAAGTATTG GCATGGGTAG TACTAGATAG TGCTGTCGAC CTCTCAATGT ATTAGGTTTA TCCAACTCGT TGAATGGTGT GGCGGGATAT TTCTGGTATT GTTGGCCCGG CCTTACAACA ACCAAACAAG TTTGACCTCA AATCAGGTAG GAATACCCGC TGAACTTAAG CA 572

Claims (5)

1. Yeast of China grass Meng Maiye for high yield of tetraacetyl phytosphingosine (S) ((S))Meyerozyma guilliermondii) mSCU-JM06-8, characterized in that: the yeast Meng Maiye is preserved in China general microbiological culture Collection center on 29 th 9 th 2021 with the preservation number of CGMCC No.23507.

2. The yeast Quaternary strain Meng Maiye of claim 1 (Meyerozyma guilliermondii) Application of mSCU-JM06-8 in fermentation production of tetraacetylphytosphingosine TAPS.

3. Use according to claim 2, characterized in that: said Quaternary yeast also Meng Maiye (yeast)Meyerozyma guilliermondii) mSCU-JM06-8 is collected from pit mud in a cellar and is obtained by 3 rounds of mutagenesis and screening, the strain is subjected to shake flask culture for 48h in a TAPS fermentation culture medium at 28 ℃ and 180-200r/min, and the yield of the TAPS is 659.96 mg/L.

4. Use according to claim 3, characterized in that: the TAPS fermentation medium comprises: 20-30 g. L ^-1 Glucose, 1-2 g.L ^-1 Yeast extract, 5-10 g. L ^-1 Peptone, 0.67 g. L ^-1 Yeast Nitrogen base, 5 g. L ^-1 L-serine, 0.2 g. L ^-1 Calcium chloride.

5. The use of claim 2, wherein: the season is also Meng Maiye Saccharomyces cerevisiae (Meyerozyma guilliermondii) mSCU-JM06-8 in a 5L fermentation tank, at 28 ℃, the stirring speed is 200r/min, the air flow is 0.8-2L/min, the pH is natural, and the TAPS yield after 48h fermentation is 1070.86 mg/L.

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