CN115478024A

CN115478024A - Non-transgenic high-nucleic-acid saccharomyces cerevisiae strain and application thereof

Info

Publication number: CN115478024A
Application number: CN202211344795.9A
Authority: CN
Inventors: 徐丽丽; 鲍晓明; 夏天晴; 王赟; 李在禄; 曾杜文; 邱晨曦
Original assignee: Qilu University of Technology
Current assignee: Qilu University of Technology
Priority date: 2022-10-31
Filing date: 2022-10-31
Publication date: 2022-12-16
Anticipated expiration: 2042-10-31
Also published as: CN115478024B

Abstract

The invention relates to the field of bioengineering, in particular to non-transgenic saccharomyces cerevisiae with high nucleic acid content and application thereof. According to the application, a breeding technology is improved, a high-rRNA synthetic saccharomyces cerevisiae strain with good growth condition and high nucleic acid content is screened, a non-transgenic saccharomyces cerevisiae strain YM83 with high nucleic acid content is finally obtained through steps of multiple rounds of iterative mutagenesis and large-scale preliminary screening, growth, fluorescence value and RNA content determination and rescreening, expression system elimination and the like, the nucleic acid content of the strain is higher than that of most of saccharomyces cerevisiae used in the existing market, the strain can be stably inherited, is non-transgenic saccharomyces cerevisiae, can be applied to preparation and production of ribonucleic acid, nucleotide derivatives, yeast extracts and the like with high safety requirements, and the product can be widely applied to industries such as food, medicine, health products, agriculture, livestock breeding and the like and has a good market prospect.

Description

Non-transgenic high-nucleic-acid saccharomyces cerevisiae strain and application thereof

Technical Field

The invention relates to the field of bioengineering, in particular to non-transgenic saccharomyces cerevisiae with high nucleic acid content and application thereof.

Background

Ribonucleic acid (RNA for short) is used for performing important biological functions in cells, plays an extremely important role in the growth and health of human and animals, and has wide application and development prospects in various fields such as medicines, health-care products, foods, the breeding industry and the like. The degradation product nucleotide of RNA can be used as a medical intermediate, the derivative of the degradation product nucleotide is an effective component of various medicines, has the health-care effects of promoting the growth of animals, maintaining the health of intestinal tracts, improving the immunity of human and animals and the like, and can be prepared into health-care products or feed additives; 5 '-guanylic acid and 5' -inosinic acid (I + G) prepared by multi-enzyme accelerated directional conversion of RNA are very effective food flavoring agents, have a flavor development effect, can generate a synergistic effect when being mixed with monosodium glutamate, can improve the freshness by tens of times, and can be used as food flavoring agents; yeast is prepared into yeast extract by cell autolysis and directional enzymolysis technology, wherein the yeast extract is rich in various natural flavor nucleotides such as 5 '-inosinic acid (5' -IMP) and 5 '-guanylic acid (5' -GMP) (I + G, also called flavor nucleotide disodium), protein, polypeptide, amino acid and the like, has the characteristic of being rich in nutrition, can generate good flavor superposition effect, can reduce the addition of sodium salt, is an effective monosodium glutamate substitute seasoning, becomes a new generation of enhanced nutrition health food flavor enhancer, is widely applied to the field of food flavoring such as leisure food, meat products, instant noodles, soy sauce and the like, and is more and more favored by the market.

Most of the RNA production is obtained by microbial fermentation, so the key to the production of RNA related products is the strain, and in terms of the nucleic acid content of each strain and the nature of the strain, the current research on the increase of the nucleic acid content of the strain is mainly focused on Candida, and U.S. and Japanese patents disclose that variant Candida strains are used to obtain yeast cells with ribonucleic acid content of more than 12% and more than 20% of the dry cell weight, respectively, but Candida still has potential safety hazards to the health of human and animals. The saccharomyces cerevisiae does not produce any toxin, is a food-safe microorganism, has high growth and passage speed, is easy to culture, has high nutrition, contains rich nutritional components such as protein, nucleic acid, saccharides and the like, is a recognized optimal RNA source, can be used as high-quality protein to be added into feed, has the average nucleic acid content of 6-8 percent and is higher than most strains, and can meet the market demand only by further improvement.

Saccharomyces cerevisiae produces at least three major RNAs, ribosomal RNAs (rRNAs), messenger RNAs (mRNAs), and transfer RNAs (tRNAs), with the rRNAs being the most abundant. Therefore, increasing intracellular rRNA levels is a key to constructing high nucleic acid yeast strains. People usually improve the RNA content of yeast by optimizing culture conditions, adding glutamine and aspartic acid, or limiting strategies such as potassium sulfate and the like in the culture conditions, for example, chinese patent application CN102559522B discloses a high nucleic acid bread yeast and a preparation method thereof, glutamine and aspartic acid are added in the process of expanding culture of the bread yeast in the method, and the RNA content of the bread yeast is more than 12.0 percent; chinese patent application CN101760437A discloses a high nucleic acid baker's yeast and a preparation method thereof, and the method obtains baker's yeast with RNA reaching more than 9.5% by controlling the culture time of amplification culture and other conditions. These techniques are used for screening by measuring the RNA content of the strain one by one, and there is no mention of an increase in rRNA content. In recent years, some studies have been made to improve ribosome biosynthesis of strains by genetic engineering or the like, and further, some studies have been made to improve ribosome biosynthesisIncreasing the nucleic acid content of yeast, e.g. by knocking out the Upstream Activating Factor (UAF) of Saccharomyces cerevisiae in Harashima project group of JapanRRN10Gene, yeast growth deterioration, then using EMS mutagenesis treatmentRRN10Selecting the gene-deleted strain to obtain the inhibitor with better growth, and increasing the specific growth rate and RNA content to different degrees [ Chuwattanakul et al, construction of a Saccharomyces cerevisiae strain with a high level of RNA. J Biosci Bioeng. 2011, 112(1):1–7.]. This growth-based breeding strategy involves transgenic problems, requiring the use of yeast laboratory strains (haploids) with a simpler genetic background and selection markers as breeding starting strains; the Chinese Xiaodongguang professor topic group over-expresses the gene transcription function of ribosome in Saccharomyces cerevisiaeFHL1，IFH1And acting on ribosome biosynthesisSSF2Knock out of a negative regulator of cell growthHRP1The RNA content of the recombinant strain was Increased to 11.68% [ Guo et al, incorporated RNA production in%Saccharomyces cerevisiae by simultaneously overexpressing FHL1, IFH1, and SSF2 and deleting HRP1. Appl Microbiol Biotechnol . 2020, 104(18):7901-7913.](ii) a Chinese patent application CN108841737B discloses a 'nucleic acid-producing saccharomyces cerevisiae engineering bacterium containing Rpf1 gene and a construction method and application thereof', the method overexpresses saccharomyces cerevisiae nucleolar RNA binding protein gene Rpf1, and after shake flask fermentation, the nucleic acid content of a breeding strain reaches 11.39%; the Chinese patent application CN108660085B super-expression vacuole protein sorting receptor Pep1 gene, after flask-shaking fermentation, the nucleic acid content of the breeding strain can be up to 14.70%. Chinese patent application CN112175850A discloses 'an industrial saccharomyces cerevisiae with high nucleic acid yield and application thereof', the method overexpresses glucose transporter YHR094C, knocks out YOR185C and YGR088W genes, and the nucleic acid content of the saccharomyces cerevisiae obtained by breeding reaches 18.94%.

In general, the breeding of the industrial strains of the high-nucleic acid yeast is lack of a high-throughput screening system which directly reflects RNA synthesis change, transgenosis and the like. Through retrieval, the high-nucleic acid yeast strains which are bred by utilizing the high-throughput screening technology and have high rRNA synthesis and can be directly used for industrial production have no reports of related patents and related documents.

Disclosure of Invention

Aiming at the problems of insufficient nucleic acid production capacity, low nucleic acid yield, transgenosis and the like of the saccharomyces cerevisiae strain on the current market, a non-transgenic saccharomyces cerevisiae strain YM83 with higher nucleic acid content is finally obtained by improving a breeding technology, screening a high rRNA synthetic saccharomyces cerevisiae strain with good growth condition and increased nucleic acid content, performing multiple rounds of iterative mutagenesis and large-scale preliminary screening, re-screening the growth condition and fluorescence intensity, measuring the RNA content again, eliminating an expression system and the like, wherein the nucleic acid content of the strain is higher than that of most of saccharomyces cerevisiae strains used on the current market; the saccharomyces cerevisiae gene-modified saccharomyces cerevisiae gene can be stably inherited, can be applied to preparation and production of ribonucleic acid, nucleotide derivatives, yeast extracts and the like with high safety requirements, can be widely applied to industries such as food, medicine, health products, agriculture, livestock breeding and the like, and has good market prospect.

The technical scheme of the invention is as follows:

a non-transgenic high nucleic acid saccharomyces cerevisiae strain is provided, wherein the strain is saccharomyces cerevisiaeSaccharomycescerevisiaeIn 16.09.2022, china general microbiological culture Collection Center (CCM), address: no. 3 of Xilu No. 1 of Beijing, chaoyang, beijing, and the preservation number is CGMCC No. 25730.

Further, the strain is obtained by the following steps: transferring plasmids which can reflect rRNA synthesis change and contain a novel protein expression system into a starting strain, carrying out mutagenesis treatment on yeast cells containing the expression system by utilizing normal pressure room temperature plasma (ARTP), carrying out large-scale preliminary screening on the strains with remarkably improved fluorescence intensity and remarkably improved growth condition and fluorescence intensity based on a flow cytometry sorting technology, measuring the RNA content again, eliminating the plasmids with the novel protein expression system, and testing the stability of the strains.

Preferably, the strain is a saccharomyces cerevisiae industrial strain.

The application of the non-transgenic high-nucleic-acid saccharomyces cerevisiae strain is applied to the preparation of ribonucleic acid, nucleotide derivatives and yeast extracts.

The invention has the advantages of

The nucleic acid content of the strain is higher than that of most of saccharomyces cerevisiae used in the market at present, the strain is cultured in a YPD culture medium for 4 hours and 14 hours in a shaking way, the nucleic acid content can reach 0.252 g/g-DCW and 0.209 g/g-DCW respectively, and the nucleic acid content is respectively improved by 18.5 percent and 22 percent compared with the strain before mutagenesis; 18S rRNA of YM03 andyEGFP3the transcription level of the gene is respectively improved by 2.24 times and 6.14 times; the saccharomyces cerevisiae is a non-transgenic saccharomyces cerevisiae, can be applied to the preparation and production of ribonucleic acid, nucleotide derivatives, yeast extracts and the like with higher safety requirements, can be widely applied to the industries of food, medicine, health care products, agriculture, livestock breeding and the like, and has good market prospect. The strain and the screening strategy provided by the invention provide technical references for further breeding new strains of high-nucleic-acid yeast.

Drawings

FIG. 1 is a flow cytometer showing fluorescence distribution histogram of Saccharomyces cerevisiae industrial strain cells containing novel expression system

(a) Starting strain Y02; (b) strain Y03 containing Pol I & IRES mediated GFP expression; (c) cells of strain Y03 subjected to ARTP mutagenesis; boxed areas indicate fluorescence intensity > 1800 a.u; MFI represents the mean fluorescence intensity.

FIG. 2 is an ARTP mutagenesis lethality curve of the Y02 strain.

FIG. 3 shows the growth and fluorescence intensity of strains Y02 and YM 03. (a): a growth curve; (b): fluorescence intensity.

FIG. 4 shows the nucleic acid content of strains Y02 and YM03 grown in shake flasks for 4 h and 14 h.

FIG. 5 shows 18S rRNA and YM03 of strains Y02 and Y03yEGFP3The transcription level of (a).

FIG. 6 shows the growth of YM03 strain on a plate containing 400 mg/L G418 before and after the elimination of the plasmid pMIGK.

Fig. 7 is a measurement of growth and nucleic acid content before and after continuous passage of YM83, (a): a growth curve; (b): shake flask culture for 4 h and 14 h of nucleic acid content; YM83n: YM83 strain was continuously passaged for more than 10 generations.

Detailed Description

Example 1: high throughput screening fluorescence intensity threshold determination:

a novel protein expression system plasmid pMIGK which is used for high-throughput screening of high-nucleic acid yeast and suitable for industrial strains of saccharomyces cerevisiae and is mediated by RNA polymerase I and IRES is constructed, the plasmid is transferred into an initial strain (the strain is the industrial strain Y02 of saccharomyces cerevisiae stored in a laboratory of the applicant), 400 mg/L G418 is used for screening transformants, and a recombinant strain Y03 is obtained through verification.

Y03 was activated in a medium containing 400 mg/L YPD, and then cultured from an initial OD600 of about 0.2 to a mid-log phase (OD) ₆₀₀ About 1.0) and then utilizing MoFlo ^TM The XDP high-efficiency cell sorter detects the fluorescence intensity of Y02 and Y03 strains respectively, 50 ten thousand cells of the two strains are analyzed, the average fluorescence intensity (MFI) of the starting strain Y02 is 33.2 a.u. (figure 1 a), the MFI of the recombinant strain Y03 is increased to 54.8 a.u. (figure 1 b), compared with the Y02, the fluorescence distribution of the Y03 shifts to the right, the MFI is increased by 61.5%, the successful expression of the RNA polymerase I and IRES mediated novel protein expression system plasmid pMIGK in the saccharomyces cerevisiae industrial strain is shown, and the maximum fluorescence intensity 1800 a.u generated by the Y03 strain is used as a fluorescence intensity threshold value used in subsequent high-throughput screening.

Example 2: ARTP mutagenesis treatment

Inoculating the Y02 strain transformed with pMIGK into YPD liquid containing 400 mg/L G418, performing shake culture for 12-24 hours, activating for 2 times, transferring the bacterial liquid into fresh YPD containing 400 mg/L G418, and adjusting initial concentration OD ₆₀₀ To about 0.2, culturing at 30 deg.C with shaking to logarithmic phase (OD) ₆₀₀ About 1.0). Centrifuging 1mL bacterial solution at 8000 r/min, discarding supernatant, washing with normal saline for 2 times, diluting with normal saline containing 5% glycerol to obtain thallus concentration of 10 ⁶ ~10 ⁷ Uniformly coating 10 mu L of bacterial suspension on the surface of a sterile slide glass, and then coating the slide glassThe method is characterized by placing the sample on a carrying table of an ARTP mutation breeding system, placing the sample in an ARTP mutation breeding instrument (Techno-Siqing-sourced bioscience Co., ltd.) for mutation treatment, wherein the working gas of the mutation breeding instrument is 99.99% high-purity helium, the radio-frequency power is 90W, the helium flow is 10 SLM, the distance between the sample and a plasma emission source is fixed to be 3.5 mm, and a lethality curve is drawn by adjusting the treatment time, as shown in figure 2, when the treatment time is 20 s, the lethality of cells can reach 90%, and the mutation effect is optimal. Therefore, the Y03 is subjected to ARTP mutagenesis treatment under the treatment condition, after the sample treatment is finished, a slide is placed into an EP tube filled with 1mL of physiological saline by using tweezers, the vibration is continuously and fully performed, the thalli attached to the slide are fully eluted to prepare bacterial suspension, and the bacterial suspension is inoculated into YPD liquid culture medium containing 400 mg/L G418 to recover the growth of the bacterial suspension.

Example 3: scale sorting of high fluorescence intensity cells

The cells before and after mutagenesis in example 2 were cultured in YPD liquid medium containing 400 mg/L G418 for 12 hours to adjust the starting OD ₆₀₀ Culturing at 30 deg.C to middle logarithmic phase (OD) to about 0.2 ₆₀₀ About 1.0), centrifuging the culture solution at 8000 r/min for 2 min, discarding the supernatant, and then treating with 1

Washed 2 times with PBS buffer and then with 500. Mu.L of 1

Resuspending in PBS buffer to make cell suspension, using MoFlo ^TM Fluorescence intensity (fluorescence intensity unit is a.u.) of an FITC channel of an XDP high-efficiency cell sorter is measured under the conditions that excitation wavelength lambda ex =488 nm and emission wavelength lambda em = 507 nm, 50 ten thousand cells are analyzed respectively, compared with cells before mutagenesis, fluorescence distribution of the cells after mutagenesis is further shifted to the right, MFI is increased to 64.4 a.u., and compared with the cells before mutagenesis, the fluorescence distribution is increased by 17.5% (figure 1 c). Set the maximum fluorescence intensity of Y03 strain 1800 a.u. as the threshold for fluorescence intensity of cells after high throughput screening mutagenesis, cells above 1800 a.u. were sorted into 96-well plates containing liquid medium (care was taken to flow fineCell sorting, the Y03 strain cell suspension also required at least 3 cells to be sorted on a 96-well plate as a control).

Example 4: growth and fluorescence intensity rescreening:

the sorted 96-well plate was incubated at 30 ℃ for 12 hours, the 96-well plate was placed in a Biotek Synergy Neo2 multifunctional fluorescence microplate reader, the growth was measured at 600 nm, the green fluorescence intensity was measured under conditions of excitation wavelength λ ex =495 nm and emission wavelength λ em =510 nm, and as a result, as shown in FIG. 3, in the sorted cells, the OD of one well was present ₆₀₀ And fluorescence values were higher than for the control strain Y03 (FIGS. 3a and b).

Example 5: and (3) total RNA content determination and rescreening:

respectively inoculating the strain Y03 before mutagenesis and the strain YM03 with high fluorescence intensity and good growth condition into YPD liquid culture medium containing 400 mg/L G418, performing shake culture for 12-24 h, transferring the strain liquid into fresh YPD containing 400 mg/L G418, and adjusting initial OD ₆₀₀ About 0.2, shake-flask culture is carried out for 4 h and 14 h at 30 ℃, thalli are collected, one part of thalli is used for measuring the dry weight of cells, 1mL of bacterial liquid is collected, the RNA content is measured by using a perchloric acid method, the nucleic acid content of YM03 cultured for 4 h and 14 h can reach 0.252 g/g-DCW and 0.209 g/g-DCW, and is respectively improved by 18.5% and 22% compared with the strain Y03 before mutagenesis (figure 4), which indicates that the strain YM03 with improved fluorescence intensity screened by using the screening strategy is the strain with improved nucleic acid content.

Example 6: fluorescent quantitative PCR detection of 18S rRNA andyEGFP3level of transcription

Respectively inoculating strain Y03 before mutagenesis and strain YM03 with high fluorescence intensity and good growth condition into YPD liquid culture medium containing 400 mg/L G418, performing shake culture at 30 deg.C for 12-24 hr, transferring bacterial liquid into fresh YPD containing 400 mg/L G418, adjusting initial OD ₆₀₀ Shaking about 0.2 at 30 deg.C, culturing for 4 h, collecting thallus, extracting total RNA with UNIQ-10 column type Trizol total RNA extraction kit (Shanghai bioengineering Co., ltd.), reacting at 37 deg.C for 30 min with RNA free DNase I to remove genome DNA, taking RNA digested with DNase I as template, and reverse transcription kit (HiScript III RT Supermix for qP)CR (+ gDNA wiper), nykino Kinza Biotechnology Co., ltd.) and then using the cDNA as a template, 18S rRNA-F (5yEGFP3-F (5 'CCAGTTCCATGGCCAACCTTA-3') andyEGFP3-R (5ACT1-F (5ACT1-R (5 'AGTTTGGTCAATACCGGCAG-3') as primer, using ChamQ Universal SYBR qPCR Master Mix (Biotech Co., ltd. Of Nanjing NuoWei Zan) to perform real-time fluorescence quantitative PCR, selecting housekeeping geneACT1As an internal standard, the synthesis conditions of intracellular 18S rRNA of the strains Y03 and YM03 before and after mutagenesis are compared with the green fluorescent protein gene in a novel protein expression systemyEGFP3The transcription level of (a), PCR reaction conditions: 95 ℃,3min,95 ℃,15s,61 ℃,15s,72 ℃,30s, for 40 cycles; 5min at 72 ℃. As a result, as shown in FIG. 5, 18S rRNA and DNA of YM03 were compared with those of Y03 strain before mutagenesisyEGFP3The transcription level of the gene is respectively improved by 2.24 times and 6.14 times, which shows that the mutagenesis promotes the synthesis of the intracellular rRNA of the yeast and the expression of the green fluorescent protein of an expression system.

Example 7: elimination of high-throughput screening system YM03 obtained in example 5 was inoculated into YPD liquid medium, continuous culture at 30 ℃ and subcultured for more than 10 times, the bacterial liquid before and after subculture was spread on YPD plates containing 400 mg/L G418, and culture was carried out at 30 ℃ for 2 to 3 d, with the results shown in FIG. 6, in which YM03 before subculture had colonies growing, and in which colonies after subculture grew, indicating that plasmids containing a novel protein expression system were eliminated, and finally, a non-transgenic high-nucleic acid yeast strain YM83 was obtained.

Example 8: genetic stability testing of high nucleic acid yeast strains:

YM83 is inoculated into YPD liquid medium, continuous passage is carried out for more than 10 times, then the strains before and after the continuous passage are inoculated into the YPD liquid medium, shake flask culture is carried out at 30 ℃, the growth curve and the nucleic acid content of 4 h and 14 h are determined, and the genetic stability of the strains before and after the passage is tested, and the result is shown in figure 7, wherein: the growth conditions of the strain before and after passage are basically equivalent (figure 7 a), the RNA content of the strain is not greatly changed (figure 7 a) regardless of the culture for 4 h or 14 h before and after passage, and the strain keeps better stability.

Claims

1. A non-transgenic high-nucleic acid saccharomyces cerevisiae strain is characterized in that the strain is saccharomyces cerevisiaeSaccharomyces cerevisiaeIn 16 th of 2022, 09.month, the strain was deposited in "China general microbiological culture Collection center", with the accession number of CGMCC No. 25730.

2. The non-transgenic high nucleic acid s.cerevisiae strain according to claim 1, wherein said strain is obtained by the steps of: transferring plasmids which can reflect rRNA synthesis change and contain a novel protein expression system into a starting strain, carrying out mutagenesis treatment on yeast cells containing the expression system by utilizing normal-pressure room-temperature plasma ARTP, carrying out large-scale primary screening on the cells with remarkably improved fluorescence intensity based on a flow cytometry sorting technology, carrying out secondary screening on the strains with remarkably improved growth conditions and fluorescence intensity, measuring the RNA content again, eliminating the plasmids with the novel protein expression system, and carrying out strain stability test.

3. The non-transgenic high nucleic acid s.cerevisiae strain of claim 1, wherein said strain is an industrial strain of s.cerevisiae.

4. The use of a non-transgenic high nucleic acid s.cerevisiae strain according to claim 1 in the preparation of ribonucleic acids, nucleotides, nucleotide derivatives and yeast extracts.