CN108893417B

CN108893417B - High-throughput screening system for high-nucleic-acid yeast breeding and application

Info

Publication number: CN108893417B
Application number: CN201810815659.0A
Authority: CN
Inventors: 鲍晓明; 徐丽丽; 曾杜文; 邱晨曦; 易勇; 张继祥; 吴倩; 杜显雨
Original assignee: Shandong Bio Sunkeen Co ltd; Qilu University of Technology
Current assignee: Shandong Bio Sunkeen Co ltd; Qilu University of Technology
Priority date: 2018-07-16
Filing date: 2018-07-16
Publication date: 2021-05-14
Anticipated expiration: 2038-07-16
Also published as: CN108893417A

Abstract

A high-throughput screening system for high-nucleic acid saccharomyces cerevisiae breeding is composed of a report plasmid YEp-Hyg B-yeGFP and a host cell containing YEp-Hyg B-yeGFP; the reporter plasmid YEp-Hyg B-yeGFP is annular and sequentially consists of a YEplac195 plasmid skeleton, a yeast enhanced green fluorescent protein gene yeGFP expression box and a hygromycin B resistance gene expression box from 5 'to 3'; the starting strain of the host cell is a saccharomyces cerevisiae industrial strain with nucleic acid content higher than a normal value through screening confirmation. The invention also discloses the application of the screening system in the breeding of the high nucleic acid yeast engineering bacteria. The screening system can make up the defect that the RNA content cannot be directly reflected in the traditional high-nucleic-acid yeast breeding, can be easily eliminated by continuous passage by virtue of the instability of the screening system in yeast, and has important significance for the application of the high-nucleic-acid yeast in the food industry.

Description

High-throughput screening system for high-nucleic-acid yeast breeding and application

Technical Field

The invention relates to the technical field of microorganisms, in particular to a high-throughput screening system for high-nucleic acid saccharomyces cerevisiae breeding and application thereof.

Background

Ribonucleic acid (RNA) not only performs biological functions in cells, but also is an important new food material, food additive, pharmaceutical intermediate, and the like. RNA and degradation products thereof, or corresponding derivatives thereof are effective components of a plurality of medicines [ Wang nan, Chua Xia, Li Yong, exogenous nucleotide and immune function research progress, food science, 2016,37(5):278 and 282 ], and have positive effects on health care efficacy, immunity improvement and the like of human and animals. In recent years, 5 '-inosinic acid (5' -IMP) and 5 '-guanylic acid (5' -GMP) prepared by multi-enzyme promoted directional transformation of RNA are called as flavor nucleotide disodium, have unique 'delicate flavor' taste and rich nutrition characteristics, become a new generation of enhanced nutrition and health care type nucleotide food flavor enhancer and have high economic value.

Ribonucleic acid is a material basis of life existence, almost all organisms contain ribonucleic acid, but all biological resources can not be used for extracting ribonucleic acid on a large scale, and the problems of low ribonucleic acid content, limited resources, industrial preparation incapability and the like exist. Although the direct fermentation of RNA by microorganisms such as Bacillus subtilis is industrially valuable, it is still technically difficult to extract RNA from the fermentation broth due to the high viscosity of the fermentation broth since RNA is a macromolecular substance, which limits its industrial production [ Jobingfu. microorganisms produce nucleosides and nucleic acids [ J ]. Industrial microorganisms, 1998, (28):22-27 ]. And As a food safety-grade (Generally managed As Safe, GRAS) microorganism, Saccharomyces cerevisiae (Saccharomyces cerevisiae) is a well-known optimal RNA source, not only has relatively high RNA content, but also the extracted mycoprotein still has high application value and is convenient for comprehensive utilization. At present, foreign yeast nucleic acid production is mainly concentrated in Brazil, and although some domestic beer manufacturers can change waste into valuable by extracting RNA from waste yeast generated in the beer production process, the recycling of resources is realized, but because the raw materials are poor in quality, the RNA content is low, the product quality is unstable, the relative conversion rate is low, and the influence on subsequent production and processing is large, the domestic pharmaceutical manufacturers can only import a large amount of high-content yeast RNA products from foreign countries such as Brazil, and the market is in a situation of short supply and short demand. Therefore, the breeding of high nucleic acid yeast is imperative and is the basis for solving the problem.

At present, the research on saccharomyces cerevisiae with high nucleic acid is one of the current research hotspots, and chinese patent application CN101760437A discloses a "bread yeast with high nucleic acid and a preparation method thereof", which prepares nucleic acid containing more than 20 wt% of the weight of the cell of the bread yeast by controlling the culture time of the amplification culture and other conditions, wherein the RNA reaches more than 9.5%. Chinese patent application CN102559522B discloses a high nucleic acid baker's yeast and its preparation method, in which glutamine and aspartic acid are added in the process of expanding culture of baker's yeast, nucleic acid containing more than 20 wt% relative to 100 wt% of baker's yeast thallus is prepared, wherein RNA is more than 12.0 wt%; chinese patent application CN106635851A discloses a method for breeding high-nucleic acid saccharomyces cerevisiae, which comprises the steps of primary screening and secondary screening, culture medium optimization, and amplification culture by small-scale test and pilot-scale fermentation process optimization to obtain the high-nucleic acid saccharomyces cerevisiae with the RNA content of 14.6-15.2%. Although the traditional breeding method avoids the problem of transgenosis, the RNA content detection operation is complicated, the error is large, and the large-scale screening is difficult to realize.

Other international research institutes have also made improvements in breeding methods, and the japanese Harashima group reported breeding measures based on growth, in which strains with severely deteriorated growth were constructed by knocking out the RRN10 gene in Upstream Activating Factor (UAF), and then selected to 7 growth-improved inhibitors by Ethyl Methanesulfonate (EMS) mutagenesis, which had different improvements in specific growth rate and RNA content [ Chuwattanakul V, Kim YH, Sugiyama M, Nishiuchi H, Miwa H, Kaneko Y, Harashima s.construction of a Saccharomyces cerevisiae aid with a high level of rna.j Biosci bioeng.2011,112(1): 1-7 ]. The breeding strategy based on growth realizes high-throughput screening, however, transgenic problems are involved in the breeding process, laboratory strains (haploid) which have simpler genetic background and screening markers are required to be used as breeding starting strains, and RNA content changes cannot be reflected. Therefore, the problems of high-throughput screening systems and transgenes which cannot reflect the change of RNA content at present become limiting factors for the breeding of high-nucleic-acid yeasts.

Saccharomyces cerevisiae, like other eukaryotes, produces at least three major RNAs, ribosomal RNA (rRNA), messenger RNA (mRNA), and transfer RNA (tRNA), with the highest content of rRNA. Therefore, increasing intracellular rRNA levels is a key to constructing high nucleic acid yeast strains. rDNA encoding rRNA is generally randomly located on chromosome XII in about 150-200 repeats, and RNA polymerase I forms a transcription initiation complex with other subunits in the rDNA promoter region to direct rDNA gene transcription. RNA polymerase I is the most active eukaryotic RNA polymerase, and its transcription initiation and extension efficiency for rDNA gene is significantly faster than polymerase II or III. But the search finds that: by utilizing the characteristic that RNA polymerase I has high-efficiency action in rDNA gene transcription, the expression of the reporter gene yeGFP is guided by an rDNA gene promoter, a high-pass gauge modeling screening system which can indirectly reflect rRNA content change through regulation disturbance of rRNA synthesis change caused by induction is constructed, and related articles or patents applied to screening of high-nucleic-acid yeast are not reported yet.

Disclosure of Invention

Aiming at the defects of the prior high nucleic acid yeast breeding, the invention provides a high-throughput screening system for breeding the high nucleic acid yeast and the application thereof in the breeding of the high nucleic acid yeast engineering bacteria, which can reflect the change of RNA content and avoid the transgenic problem, based on the characteristic that the RNA polymerase I plays a high-efficiency role in the rDNA gene transcription, and has important significance for the application of the high nucleic acid yeast in the food industry.

The invention relates to a high-throughput screening system for breeding high-nucleic-acid saccharomyces cerevisiae, which is characterized in that: the screening system consists of a reporter plasmid YEp-Hyg B-yeGFP and a host cell containing YEp-Hyg B-yeGFP; the reporter plasmid YEp-Hyg B-yeGFP is a shuttle plasmid constructed between saccharomyces cerevisiae and escherichia coli, is annular and sequentially consists of a YEplac195 plasmid framework, a yeast enhanced green fluorescent protein gene yeGFP expression cassette and a hygromycin B resistance gene expression cassette from 5 'to 3'; the starting strain of the host cell is a saccharomyces cerevisiae industrial strain with nucleic acid content higher than a normal value through screening confirmation.

In the high-throughput screening system for breeding the high-nucleic-acid saccharomyces cerevisiae, the following steps are carried out: the YEplac195 plasmid in the reporter plasmid YEp-Hyg B-yeGFP is a yeast episome plasmid, containing the ori of the yeast 2. mu. plasmid.

In the high-throughput screening system for breeding the high-nucleic-acid saccharomyces cerevisiae, the following steps are carried out: the expression cassette of the green fluorescent protein gene yeGFP in the reporter plasmid YEp-HygB-yeGFP consists of an rDNA promoter, an internal ribosome entry site IRES (internal ribosome entry site) sequence, a yeGFP gene expression cassette, a poly (T) sequence and an rDNA terminator from upstream to downstream in sequence; wherein, the sequence of the rDNA promoter is shown as SEQ ID No. 1, the sequence of IRES is shown as SEQ ID No. 2, the sequence of the yeGFP gene expression frame is shown as SEQ ID No. 3, the sequence of poly (T) is shown as SEQ ID No. 4, and the sequence of the rDNA terminator is shown as SEQ ID No. 5.

In the high-throughput screening system for breeding the high-nucleic-acid saccharomyces cerevisiae, the following steps are carried out: the hygromycin B resistance gene expression box in the reporter plasmid YEp-HygB-yeGFP consists of a TEF1 promoter, a hygromycin B (Hyg B) gene and a TEF1 terminator; wherein, the TEF1 promoter sequence is shown as SEQ ID NO. 6, the hygromycin B (Hyg B) gene sequence is shown as SEQ ID NO. 7, and the TEF1 terminator sequence is shown as SEQ ID NO. 8.

In the high-throughput screening system for breeding the high-nucleic-acid saccharomyces cerevisiae, the following steps are carried out: the starting strain of the host cell is preferably a yeast strain CGMCC No.9084 with relatively high nucleic acid content.

The invention relates to a construction method of a report plasmid YEp-HygB-yeGFP in a high-throughput screening system for high-nucleic acid saccharomyces cerevisiae breeding, which comprises the following steps:

(1) amplifying a hygromycin B resistance gene expression frame by adopting a PCR (polymerase chain reaction) method, then digesting the hygromycin B resistance gene expression frame and YEplac195 by using restriction enzymes, converting escherichia coli DH5 alpha by using a connecting solution, selecting a transformant, and obtaining a recombinant plasmid YEp-Hyg B by using PCR verification;

(2) amplifying rDNA promoter, IRES sequence, yeGFP gene, poly (T) and rDNA terminator by using a PCR method to obtain fusion PCR product rDNAP-IRES-yeGFP-poly (T) -rDNAT, then using restriction endonuclease to cut rDNAP-IRES-yeGFP-poly (T) -rDNAT and YEp-Hyg B, transforming Escherichia coli DH5 alpha by using a connecting solution, selecting a transformant, and using a PCR method to verify to obtain a recombinant plasmid YEp-Hyg B-yeGFP.

The invention relates to a method for constructing host cells containing a reporter plasmid YEp-HygB-yeGFP in a high-throughput screening system for breeding high-nucleic acid saccharomyces cerevisiae, which comprises the following steps:

(1) converting the reporter plasmid YEp-Hyg B-yeGFP into a saccharomyces cerevisiae industrial strain with the nucleic acid content higher than a normal value through screening by using a PEG-LiAc conversion method, an electric conversion method or a protoplast conversion method;

(2) screening positive transformants by using a screening culture medium containing hygromycin B, extracting yeast reverse-extracting plasmids from the recombinant yeast, and performing PCR verification to obtain host cells containing YEp-Hyg B-yeGFP;

(3) and (3) detecting the fluorescence intensity of the host cells by using a fluorescence spectrophotometer detection method, a multi-label analyzer detection method or a fluorescence microscope detection method, and determining the host cells of a high-throughput screening system for high-nucleic acid yeast breeding according to the GFP protein expression condition.

Among them, the preferred embodiment is: converting the yeast strain CGMCC No.9084 with relatively high nucleic acid content by the report plasmid YEp-Hyg B-yeGFP in the step (1) by using a PEG-LiAc conversion method; the method for detecting the fluorescence intensity in the step (3) selects a multi-label analyzer detection method, and comprises the following specific operation steps: taking 1mL of bacterial suspension to be detected in a 1.5mL EP tube, centrifuging at 12000rpm for 2min, discarding supernatant, and using ddH for bacteria₂O Wash twice to wash off residual media, add 1mL ddH₂Mixing by vortex oscillation, absorbing 200 μ L, transferring into black 96-hole enzyme standard tube, placing in multi-label analyzer, detecting with absorption light of 485nm and excitation light of 535nm,fluorescence/OD for Single cell fluorescence levels₆₀₀To indicate.

The invention discloses application of a high-throughput screening system for high-nucleic acid saccharomyces cerevisiae breeding in high-nucleic acid yeast engineering strain breeding, which is characterized in that the application method comprises the following steps:

(1) adopting ultraviolet mutagenesis, Ethyl Methane Sulfonate (EMS) mutagenesis or normal pressure Room Temperature Plasma (ARTP) mutagenesis method to mutate host cells containing YEp-Hyg B-yeGFP;

(2) adopting a flow cytometer to carry out high-throughput modeling to separate out yeast cells with improved fluorescence intensity;

(3) measuring the content of the cell nucleic acid of the strain with improved fluorescence intensity by adopting a perchloric acid method extraction or Trizol method extraction mode;

(4) the report plasmid YEp-Hyg B-yeGFP high-flux screening system contained in the bred high-nucleic acid yeast engineering bacteria is eliminated by adopting a mode of continuously culturing in a YPD liquid culture medium without screening pressure and transferring for more than 10 times, so that the high-nucleic acid yeast engineering bacteria without the high-flux screening system are obtained.

In the above application, a preferred embodiment is:

the step (1) adopts an ordinary pressure room temperature plasma (ARTP) mutagenesis method to mutate host cells containing YEp-Hyg B-yeGFP, and the method and the conditions are as follows: taking 1mL of bacterial liquid, suspending the bacterial liquid in a 1.5mL EP tube, centrifuging the bacterial liquid at 8000r/min for 2min, and removing supernatant; washing with physiological saline for 2 times, diluting with physiological saline containing 5% glycerol to obtain bacterial cell with concentration of 10⁶～10⁷Taking 10 mu L of bacterial suspension and uniformly coating the bacterial suspension on the surface of a sterile slide glass; then placing the slide glass on a carrying platform of an ARTP mutation breeding system, and placing the slide glass in an ARTP mutation breeding instrument for mutation treatment, wherein the working gas of the mutation instrument is 99.99% high-purity helium, the radio frequency power is 100W, the helium flow is 10SLM, the treatment time is set to be 120s, and the distance between the sample and a plasma emission source is 3 mm; after the sample is processed, putting the slide into an EP tube filled with 1mL of physiological saline by using a pair of tweezers, continuously and fully oscillating for 1min, and fully eluting thalli attached to the slide to form bacterial suspension; directly adding 10 mu L of the bacterial liquid into the control group1mL of physiological saline;

the method for separating the yeast cells with improved fluorescence intensity by adopting the flow cytometer to implement high-throughput modeling comprises the following steps: subjecting the non-mutagenized YEp-Hyg B-yeGFP-containing host cell suspension, i.e., the control cell suspension and the ARTP-mutagenized cell suspension of step (1), to centrifugation at 8000r/min for 2min, discarding the supernatant, then re-suspending with 500. mu.L of 1 XPBS solution, adding the corresponding fluorescein-labeled antibody, respectively, incubating at 25 ℃ for 1h, washing with 1 XPBS solution for 3 times, and re-suspending with 500. mu.L of 1 XPBS; then, after passing through a 40-micron filter screen, sorting by an ultra-rapid flow cytometry sorting system (MoFlo XDP), setting the fluorescence value of the control cell suspension as a threshold value, and sorting out cells with the fluorescence value larger than the threshold value, namely yeast cells with improved fluorescence intensity;

and (3) determining the content of the cell nucleic acid of the strain with improved fluorescence intensity by adopting a Trizol method, wherein the specific method comprises the following steps: culturing host cells containing the reporter plasmid and the cells with the fluorescence value larger than the threshold value in the step (2) in a YPD liquid culture medium containing 200mg/L hygromycin B for 12-24 hours, transferring the bacterial liquid into a fresh YPD liquid culture medium containing 200mg/L hygromycin B, and controlling the cell concentration to be OD₆₀₀About.0.1, shake-culturing at 30 ℃ to the final cell concentration OD₆₀₀1.0, centrifuging and collecting cells; every 5X 10⁷Adding 0.5ml of Trizol into the bacterial cells, and grinding by using liquid nitrogen; RNA extraction is carried out by adopting a UNIQ-10 column type Trizol total RNA extraction kit of Shanghai worker and the operation steps thereof, and the light absorption value of the extracted RNA is measured at 260 nm.

The high-throughput screening system for high-nucleic acid yeast breeding established by the invention can make up the defect that the traditional high-nucleic acid yeast breeding can not directly reflect the RNA content, and is bound to become a main method for high-nucleic acid yeast breeding. In addition, the high-throughput screening system for breeding the high-nucleic acid saccharomyces cerevisiae has the unique advantages that the screening system is constructed on high-copy episome plasmids suitable for industrial strains of the saccharomyces cerevisiae, and after the high-nucleic acid saccharomyces cerevisiae is obtained, the high-nucleic acid saccharomyces cerevisiae can be easily eliminated by continuous passage by virtue of the instability of the high-nucleic acid saccharomyces cerevisiae in the yeast, the operation is convenient, the transgenic problem is avoided, the high-nucleic acid saccharomyces cerevisiae has important significance for the application of the high-nucleic acid saccharomyces cerevisiae in the food industry, and the high-throughput screening system has huge economic value and wide market prospect.

Drawings

FIG. 1 is a PCR-verified image of the hygromycin B gene expression cassette constructed on YEplac195 in example 1

Wherein M: 1kb DNA marker; 1: hyg B-TEF1 terminator gene fragment amplified by colony PCR using primers Hyg B-F and pJ-TEF1-Nco I-R.

FIG. 2 is a PCR verification chart showing the construction of each element of the yeGFP gene expression cassette onto YEp-Hyg B in example 1

Wherein M: 1kb DNA marker; 1: a yeGFP-poly (T) -rDNA terminator gene fragment amplified using primers Asc I-yeGFP-F and rDNAT-Hind III-R.

FIG. 3 is a graph of fluorescence intensity measurements of industrial strains of Saccharomyces cerevisiae expressing the high throughput screening system of example 2.

FIG. 4 shows the results of measuring the nucleic acid content of the strain with increased fluorescence intensity screened by the high-throughput screening system in example 3.

Detailed Description

The technical solution of the present invention will be described in detail with reference to the following examples. These examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and substance of the invention and are intended to be within the scope of the invention.

Unless otherwise indicated, the examples follow conventional experimental conditions, such as those set forth in Sam brook et al Molecular cloning, A laboratory Manual (Sam brook J & Russell DW, Molecular cloning: a laboratory Manual,2001), or in accordance with the product specifications.

The starting strain used in this example, yeast strain CGMCC No.9084 was purchased from China general microbiological culture Collection center (CGMCC). The plasmid YEplac195 concerned is purchased from Biovector NTCC collection and is constructed as described in the literature [ Gietz RD, Sugino A. New year-Escherichia coli short vectors restricted with in vitro mutated genes puncturing six-base pair restriction sites, Gene.1988,74(2): 527. sup. 534 ].

Example 1: construction of reporter plasmid YEp-Hyg B-yeGFP

1. Plasmid YEp-Hyg B construction containing hygromycin B resistance gene

(1) Construction of hygromycin B resistance gene expression cassette

Plasmid YEp-CH is used as a template, and a primer Sal I-pJ-TEF1-F is used

(5'-CATTTCCCCGAAAAGTGCCACCTGACGTCGACATGGAGGCCCAGAATACC-3') and pJ-TEF1-Nco I-R (5'-CCTCCATGGCAGTATAGCGACCAGCATTC-3') amplifying about 1500bp hygromycin B gene expression frame Sal I-TEF1p-Hyg B-TEF1t-Nco I with Sal I and Nco I enzyme cutting sites, wherein PCR amplification conditions comprise pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 45s, annealing at 52 ℃ for 15s, extension at 72 ℃ for 1.5min, 30 cycles and final extension at 72 ℃ for 5 min.

(2) Plasmid YEp-Hyg B construction containing hygromycin B resistance gene

Digesting the plasmid YEplac195 and the hygromycin B gene expression frame Sal I-TEF1p-HygB-TEF1t-Nco I by using Sal I and Nco I, then connecting, transforming escherichia coli DH5 alpha, selecting a transformant, extracting a plasmid, and using a primer

Hyg B-F (5'-ATGCCTGAACTCACCGCG-3') and

the colony PCR verification is carried out on pJ-TEF1-Nco I-R (5'-CCTCCATGGCAGTATAGCGACCAGCATTC-3'), and the PCR amplification conditions are pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 45s, annealing at 56 ℃ for 15s, extension at 72 ℃ for 2min, 30 cycles and final extension at 72 ℃ for 5 min. The amplification reaches a band of about 1300bp (shown in figure 1), which indicates that the hygromycin B expression cassette is successfully connected to YEplac195, and the recombinant plasmid YEp-HygB containing the hygromycin B gene expression cassette is obtained.

2. Construction of reporter plasmid YEp-Hyg B-yeGFP

(1) Amplification of elements of the expression cassette for the reporter gene yeGFP

Amplification of rDNA promoter: takes the genome DNA of the saccharomyces cerevisiae Y08 as a template and utilizes a primer SacI-rDNAP-F

(5'-CATTTCCCCGAAAAGTGCCACCTGACGTCGACATGGAGGCCCAGAATACC-3') and rDNap-IRES-R

(5'-CTTTAGCGGCTTAACTGTGCCCTCCATGGCAGTATAGCGACCAGCATTCAC-3') performing PCR amplification to obtain about 600bp rDNA promoter fragment with IRES element homology arm, wherein the PCR amplification conditions comprise pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 45s, annealing at 52 ℃ for 15s, extension at 72 ℃ for 40s, 30 cycles and final extension at 72 ℃ for 5 min.

Amplification of IRES fragment: the sequence of CrPV gene spacer (IGR) IRES was consulted in Genome of NCBI (national Center for Biotechnology information), the IRES sequence was obtained by whole gene synthesis, and then PCR amplification was performed using plasmid pUC57-IRES containing the IRES sequence as a template, using primers rDNap-IRES-F (5'-GAAAGCAGTTGAAGACAAGTTCGAAAAGAGAAAGCAAAAATGTGATCTTGC-3') and Asc I-IRES-R (5'-TTGGCGCGCCTTGAAATGTAGCAGGTAAATTTC-3') to obtain about 250bp of IRES element fragments with homologous arms of the rDNA promoter element, under conditions of 95 ℃ pre-denaturation for 3min, 95 ℃ denaturation for 45s, 52 ℃ annealing for 15s, 72 ℃ for 20s, 30 cycles, and 72 ℃ final elongation for 5 min.

Amplification of rDNA promoter fragment fused to IRES fragment: respectively taking an rDNA promoter fragment with an IRES element homologous arm and an IRES element fragment with an rDNA promoter element homologous arm as templates, carrying out fusion amplification on the Sac I-rDNAP-IRES-Asc I fragment with Sac I enzyme cutting sites and Asc I enzyme cutting sites of about 850bp by using primers Sac I-rDNAP-F and Asc I-IRES-R, wherein the PCR amplification conditions comprise pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 45s, annealing at 52 ℃ for 15s, extension at 72 ℃ for 50s, 30 cycles and final extension at 72 ℃ for 5 min.

Fourthly, amplification of the yeGFP gene open reading expression frame: the method is characterized in that a laboratory-preserved plasmid pJFE3-yeGFP plasmid is used as a template, primers Asc I-yeGFP-F (5'-TTGGCGCGCCATGTCTAAAGGTGAAGAATTA-3') and yeGFP-Xho I-R (5'-CCGCTCGAGTTATTTGTACAATTCATCCATACC-3') are used for PCR amplification to obtain a 714bp yeast enhanced green fluorescent protein yeGFP gene open reading frame, and amplification conditions comprise pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 45s, annealing at 52 ℃ for 15s, extension at 72 ℃ for 50s, 30 cycles and final extension at 72 ℃ for 5 min.

Obtaining poly (T) sequence: because it is difficult to obtain poly (T) sequence by means of PCR, said invention utilizes artificial synthesis mode to construct on plasmid pUC57-poly (T), then utilizes Xho I and Xba I double enzyme digestion to obtain poly (T) containing enzyme cutting site.

Amplification of rDNA terminator fragment: the genome DNA of saccharomyces cerevisiae Y08 is used as a template, primers rDNat-Xba I-F (5'-CTAGTCTAGATTTTTATTTCTTTCTAAGTGGGTAC-3') and rDNat-Hind III-R (5'-GATGCTAGCTTGTGAAAGCCCTTCTCTTTC-3') are utilized to carry out PCR amplification to obtain about 300bp of rDNA terminator fragments containing Xba I and Hind III enzyme cutting sites, and the amplification conditions are pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 45s, annealing at 50 ℃ for 15s, extension at 72 ℃ for 25s, 30 cycles and final extension at 72 ℃ for 5 min.

Wherein:

the rDNA promoter sequence is shown in SEQ ID No. 1;

the IRES sequence is shown as SEQ ID No. 2;

the sequence of the yeGFP gene expression frame is shown as SEQ ID No. 3;

the poly (T) sequence is shown in SEQ ID No. 4.

The rDNA terminator sequence is shown in SEQ ID No. 5.

(2) Construction of reporter plasmid YEp-Hyg B-yeGFP

The elements in the recombinant plasmid YEp-Hyg B and the reporter gene expression frame are respectively cut by corresponding restriction enzymes, then the restriction enzymes are connected, escherichia coli is transformed, then corresponding verification is carried out, escherichia coli DH5 alpha is transformed after 4 times of connection, transformants are selected to extract plasmids, finally the transformants are verified by PCR (shown in figure 2) by primers Asc I-yeGFP-F and rDNat-HindIII-R, the PCR amplification conditions are that the transformants are pre-denatured at 94 ℃ for 10min, denatured at 94 ℃ for 30s, annealed at 52 ℃ for 30s, extended at 72 ℃ for 5min, circulated for 30 min and finally extended at 72 ℃ for 10 min.

And amplifying a band of about 1530bp by PCR (polymerase chain reaction), and indicating that an open reading frame, poly (T) and rDNA terminator of the yeGFP gene are successfully connected to YEp-Hyg B to finally obtain a recombinant plasmid YEp-Hyg B-yeGFP of a novel expression vector, wherein the expression vector contains an expression frame of a hygromycin B resistance gene and an expression frame of a reporter gene yeGFP, and the yeGFP gene is transcribed and translated by adding an IRES sequence and a poly (T) sequence under the control of the rDNA promoter and the terminator.

Example 2: construction of a Saccharomyces cerevisiae host cell containing a reporter plasmid

1. Reporter plasmid transformed Saccharomyces cerevisiae host cell

The yeast strain CGMCC No.9084 (named as Y08) with relatively high nucleic acid content is confirmed by primary screening at the early stage of conversion of the control plasmid YEp-Hyg B and the report plasmid YEp-Hyg B-yeGFP, the used conversion method is a PEG-LiAc mediated saccharomyces cerevisiae conversion method, a YPD plate containing 200mg/L hygromycin B is used for screening transformants, the transformants are selected, the plasmids are extracted from the yeast, then primers Sac I-rDNAP-F and URA3-Xho I-R are used for PCR amplification, the PCR amplification conditions are pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 45s, annealing at 52 ℃ for 15s, extension at 72 ℃ for 1.5min, 30 cycles and final extension at 72 ℃ for 5 min. A band of about 1400bp is amplified by PCR, which indicates that the expression vector is successfully transformed into the saccharomyces cerevisiae.

2. Fluorescence intensity detection

Selecting control group strains separated by plate streaking, and naming Y081 (namely empty plasmid YEp-Hyg B without yeGFP gene expression frame) and experimental group strains, naming Y082 (namely expressing yeGFP gene and containing reporter plasmid YEp-Hyg B-yeGFP) and inoculating single colony of saccharomyces cerevisiae into YPD containing 200mg/L hygromycin B, carrying out shake activation culture twice at 30 ℃, carrying out overnight culture for about 10 hours, taking 1mL of to-be-detected bacterial suspension into 1.5mL of EP tube, centrifuging at 12000rpm for 2min, discarding supernatant, and using ddH for bacteria₂O washing twice to wash out residual culture medium, adding 1mL ddH₂Mixing by vortex oscillation, absorbing 200 mu L of the mixture, transferring the mixture into a black 96-hole enzyme standard tube, detecting the intensity of Green Fluorescent Protein (GFP) by using a multi-label analyzer (Pekinelmer), setting the absorption light to be 485nm, detecting the excitation light to be 535nm, and detecting the level of single-cell fluorescence by using the fluorescence value/OD₆₀₀To indicate. Through detection (as shown in figure 3), the fluorescence intensity of the control group strain is lower, and the fluorescence intensity of the experimental group strain expressing the yeGFP gene is higher. The yeGFP gene is shown to realize transcription under the action of rDNA promoter, terminator and poly (T), translation is realized under the action of translation initiation function of IRES, and a high-throughput screening system for high-nucleic acid yeast breeding is successfully established in industrial strains of saccharomyces cerevisiae.

Example 3: screening of high nucleic acid yeast engineering bacteria by using novel high-throughput screening system

1. Mutagenesis of reporter plasmid-containing host cells using ARTP

(1) Strain activation: inoculating a saccharomyces cerevisiae industrial strain Y082 expressing a high-throughput screening system to a YPD plate containing 200mg/L hygromycin B, carrying out inverted culture at 30 ℃ for 2-3 days, then inoculating the strain to YPD liquid containing 200mg/L hygromycin B, carrying out shake culture for 12-24 hours, transferring a bacterial liquid to fresh YPD containing 200mg/L hygromycin B, and controlling the bacterial concentration OD₆₀₀Approximately equal to 0.1, culturing at 30 ℃ for several hours with shaking culture, and controlling the final concentration OD of the thalli₆₀₀About 1.0 or less, as an ARTP mutagenesis starting strain.

(2) ARTP mutagenesis: taking 1mL of bacterial liquid, suspending the bacterial liquid in a 1.5mL EP tube at 8000r/min, centrifuging for 2min, and discarding the supernatant. Washing with physiological saline for 2 times, diluting with physiological saline containing 5% glycerol to obtain bacterial cell with concentration of 10⁶～10⁷And (3) uniformly coating 10 mu L of the bacterial suspension on the surface of a sterile slide. Then, the slide glass is placed on a carrying table of an ARTP mutation breeding system and is placed in an ARTP mutation breeding instrument (Wuxi Si Qingyuan Biotech Co., Ltd.) for mutation treatment, wherein the working gas of the mutation instrument is 99.99% high-purity helium, the radio frequency power is 100W, the helium flow is 10SLM, the treatment time is set to be 120s, and the distance between the sample and a plasma emission source is 3 mm. After the sample is processed, the slide glass is put into an EP tube filled with 1mL of physiological saline by using a pair of tweezers, the continuous and sufficient oscillation is carried out for 1min, the thalli attached to the slide glass are sufficiently eluted to form bacterial suspension, and 10 mu L of bacterial liquid is directly added into a control group to be added into 1mL of physiological saline.

2. High-throughput modeling of mutagenized bacteria to sort yeast cells with improved fluorescence intensity

The non-mutagenized suspension of YEp-Hyg B-yeGFP-containing host cells, i.e., the control cell suspension and the ARTP-mutagenized cell suspension of step (1), were centrifuged at 8000r/min for 2min, the supernatant was discarded, and then resuspended in 500. mu.L of 1 XPBS solution, the corresponding fluorescein-labeled antibody was added, and incubated at 25 ℃ for 1h, washed 3 times with 1 XPBS solution, and resuspended in 500. mu.L of 1 XPBS. Then, the cells were screened through a 40 μm screen, sorted by an ultra-rapid flow cytometry sorting system (MoFloXDP), the fluorescence value of the control cell suspension was set as a threshold, and the sorted cells having a fluorescence value larger than the threshold were cultured in YPD. Finally, a saccharomyces cerevisiae strain with improved fluorescence intensity is selected and named as saccharomyces cerevisiae strain Y083.

3. Determination of nucleic acid content of strain with improved fluorescence intensity

Inoculating the starting strain Y082 and the strain Y083 with improved fluorescence intensity into a YPD liquid culture medium containing 200mg/L hygromycin B, carrying out shake culture for 12-24 hours, transferring the bacterial liquid into a fresh YPD containing 200mg/L hygromycin B, and controlling the cell concentration to be OD₆₀₀About 0.1, culturing at 30 deg.C for several hours with shaking, and when the final concentration of cells is OD₆₀₀And (2) about 1.0, collecting 1mL of bacterial liquid, extracting RNA by using a UNIQ-10 column type Trizol total RNA extraction kit (Shanghai's worker Co., Ltd.), specifically referring to a product specification, measuring an absorbance value of the extracted RNA at 260nm, and determining that the nucleic acid content of Y083 is higher than that of an original strain Y082 (shown in figure 4), which indicates that the fluorescence intensity-improved strain Y083 screened by the high-throughput screening system is a nucleic acid content-improved strain.

Example 4: elimination of high throughput screening systems

And connecting the saccharomyces cerevisiae strain Y083 with improved fluorescence intensity and nucleic acid content proved to be improved to a YPD liquid culture medium without hygromycin B, continuously culturing and transferring for more than 10 times, eliminating a high-throughput screening system of a reporter plasmid YEp-Hyg B-yeGFP contained in the bred high-nucleic acid yeast engineering bacteria, and finally obtaining the high-nucleic acid yeast strain without the high-throughput screening system.

Sequence listing

<110> university of Qilu Industrial science, san Qi Bio Inc. of Shandong

<120> high-throughput screening system for high-nucleic-acid yeast breeding and application

<141> 2018-7-10

<160>8

<210> 1

<211> 600

<212> DNA

<213> Artificial sequence

<221> rDNA promoter sequence

<222>（1）…（600）

<400> 1

agaaaacata gaatagttac cgttattggt aggagtgtgg tggggtggta tagtccgcat 60

tgggatgtta ctttcctgtt atggcatgga tttcccttta gggtctctga agcgtatttc 120

cgtcaccgaa aaaggcagaa aaagggaaac tgaagggagg atagtagtaa agtttgaatg 180

gtggtagtgt aatgtatgat atccgttggt tttggtttcg gttgtgaaaa gttttttggt 240

atgatatttt gcaagtagca tatatttctt gtgtgagaaa ggtatatttt gtatgttttg 300

tatgttcccg cgcgtttccg tattttccgc ttccgcttcc gcagtaaaaa atagtgagga 360

actgggttac ccggggcacc tgtcactttg gaaaaaaaat atacgctaag atttttggag 420

aatagcttaa attgaagttt ttctcggcga gaaatacgta gttaaggcag agcgacagag 480

agggcaaaag aaaataaaag taagatttta gtttgtaatg ggaggggggg tttagtcatg 540

gagtacaagt gtgaggaaaa gtagttggga ggtacttcat gcgaaagcag ttgaagacaa 600

<210> 2

<211> 180

<212> DNA

<213> Artificial sequence

<221> IRES sequence

<222>（1）…（180）

<400> 2

aaagcaaaaa tgtgatcttg cttgtaaata caattttgag aggttaataa attacaagta 60

gtgctatttt tgtatttagg ttagctattt agctttacgt tccaggatgc ctagtggcag 120

ccccacaata tccaggaagc cctctctgcg gtttttcaga ttaggtagtc gaaaaaccta 180

<210> 3

<211> 717

<212> DNA

<213> Artificial sequence

<221> yeGFP gene expression cassette sequence

<222>（1）…（717）

<400> 3

atgtctaaag gtgaagaatt attcactggt gttgtcccaa ttttggttga attagatggt 60

gatgttaatg gtcacaaatt ttctgtctcc ggtgaaggtg aaggtgatgc tacttacggt 120

aaattgacct taaaatttat ttgtactact ggtaaattgc cagttccatg gccaacctta 180

gtcactactt tcggttatgg tgttcaatgt tttgctagat acccagatca tatgaaacaa 240

catgactttt tcaagtctgc catgccagaa ggttatgttc aagaaagaac tatttttttc 300

aaagatgacg gtaactacaa gaccagagct gaagtcaagt ttgaaggtga taccttagtt 360

aatagaatcg aattaaaagg tattgatttt aaagaagatg gtaacatttt aggtcacaaa 420

ttggaataca actataactc tcacaatgtt tacatcatgg ctgacaaaca aaagaatggt 480

atcaaagtta acttcaaaat tagacacaac attgaagatg gttctgttca attagctgac 540

cattatcaac aaaatactcc aattggtgat ggtccagtct tgttaccaga caaccattac 600

ttatccactc aatctgcctt atccaaagat ccaaacgaaa agagagacca catggtcttg 660

ttagaatttg ttactgctgc tggtattacc catggtatgg atgaattgta caaataa 717

<210> 4

<211> 50

<212> DNA

<213> Artificial sequence

<221> poly (T) sequence

<222>（1）…（50）

<400> 4

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 50

<210> 5

<211> 284

<212> DNA

<213> Artificial sequence

<221> rDNA terminator sequence

<222>（1）…（284）

<400> 5

tttttatttc tttctaagtg ggtactggca ggagccgggg cctagtttag agagaagtag 60

actgaacaag tctctataaa ttttatttgt cttaagaatt ctatgatccg ggtaaaaaca 120

tgtattgtat atatctatta taatatacga tgaggatgat agtgtgtaag agtgtaccat 180

ttactaatgt atgtaagtta ctatttacta tttggtcttt ttatttttta tttttttttt 240

ttttttcgtt gcaaagatgg gttgaaagag aagggctttc acaa 284

<210> 6

<211> 344

<212> DNA

<213> Artificial sequence

<221> TEF1 promoter sequence

<222>（1）…（344）

<400> 6

gacatggagg cccagaatac cctccttgac agtcttgacg tgcgcagctc aggggcatga 60

tgtgactgtc gcccgtacat ttagcccata catccccatg tataatcatt tgcatccata 120

cattttgatg gccgcacggc gcgaagcaaa aattacggct cctcgctgca gacctgcgag 180

cagggaaacg ctcccctcac agacgcgttg aattgtcccc acgccgcgcc cctgtagaga 240

aatataaaag gttaggattt gccactgagg ttcttctttc atatacttcc ttttaaaatc 300

ttgctaggat acagttctca catcacatcc gaacataaac aacc 344

<210> 7

<211> 804

<212> DNA

<213> Artificial sequence

<221> hygromycin B (Hyg B) gene sequence

<222>（1）…（804）

<400> 7

atgtcgaaag ctacatataa ggaacgtgct gctactcatc ctagtcctgt tgctgccaag 60

ctatttaata tcatgcacga aaagcaaaca aacttgtgtg cttcattgga tgttcgtacc 120

accaaggaat tactggagtt agttgaagca ttaggtccca aaatttgttt actaaaaaca 180

catgtggata tcttgactga tttttccatg gagggcacag ttaagccgct aaaggcatta 240

tccgccaagt acaatttttt actcttcgaa gacagaaaat ttgctgacat tggtaataca 300

gtcaaattgc agtactctgc gggtgtatac agaatagcag aatgggcaga cattacgaat 360

gcacacggtg tggtgggccc aggtattgtt agcggtttga agcaggcggc agaagaagta 420

acaaaggaac ctagaggcct tttgatgtta gcagaattgt catgcaaggg ctccctatct 480

actggagaat atactaaggg tactgttgac attgcgaaga gcgacaaaga ttttgttatc 540

ggctttattg ctcaaagaga catgggtgga agagatgaag gttacgattg gttgattatg 600

acacccggtg tgggtttaga tgacaaggga gacgcattgg gtcaacagta tagaaccgtg 660

gatgatgtgg tctctacagg atctgacatt attattgttg gaagaggact atttgcaaag 720

ggaagggatg ctaaggtaga gggtgaacgt tacagaaaag caggctggga agcatatttg 780

agaagatgcg gccagcaaaa ctaa 804

<210> 8

<211> 198

<212> DNA

<213> Artificial sequence

<221> TEF1 terminator sequence

<222>（1）…（198）

<400> 8

actgacaata aaaagattct tgttttcaag aacttgtcat ttgtatagtt tttttatatt 60

gtagttgttc tattttaatc aaatgttagc gtgatttata ttttttttcg cctcgacatc 120

atctgcccag atgcgaagtt aagtgcgcag aaagtaatat catgcgtcaa tcgtatgtga 180

atgctggtcg ctatactg 198

Claims

1. A method for breeding high nucleic acid yeast engineering bacteria by applying a high-throughput screening system comprises the following steps:

(1) transforming the saccharomyces cerevisiae cells by a high-throughput screening system; wherein the high-throughput screening system is characterized in that an episome plasmid YEplac195 shuttled between saccharomyces cerevisiae and escherichia coli is taken as a skeleton, and yeast enhanced green fluorescent protein genes are connected in sequence from 5' to 3yeGFPExpression cassettes and selection marker gene expression cassettes; wherein, the yeast enhanced green fluorescent protein geneyeGFPThe expression cassette comprises rDNA promoter, internal ribosome entry site IRES sequence, internal ribosome entry site,yeGFPGene expression frame, poly (T) sequence, rDNA terminator, rDNA promoter sequence shown in SEQ ID No. 1, IRES sequence shown in SEQ ID No. 2,yeGFPthe gene expression frame sequence is shown as SEQ ID No. 3, the poly (T) sequence is shown as SEQ ID No. 4, and the rDNA terminator sequence is shown as SEQ ID No. 5; the screening marker gene expression cassette is hygromycin B resistance gene expression cassette consisting ofTEF1Promoter, hygromycin B: (Hyg B) Gene, gene,TEF1The composition of the terminator,TEF1the promoter sequence is shown as SEQ ID NO. 6, hygromycin B (A)Hyg B) The gene sequence is shown as SEQ ID NO. 7,TEF1the terminator sequence is shown as SEQ ID NO. 8;

(2) mutagenizing the host cells containing the high-throughput screening system in the step (1) by adopting an ultraviolet mutagenesis method, an Ethyl Methane Sulfonate (EMS) mutagenesis method or an Atmospheric and Room Temperature Plasma (ARTP) mutagenesis method;

(3) adopting a flow cytometer to carry out high-throughput modeling to separate out yeast cells with improved fluorescence intensity;

(4) measuring the content of the cell nucleic acid of the strain with improved fluorescence intensity by adopting a perchloric acid method extraction or Trizol method extraction mode;

(5) the high-throughput screening system contained in the bred high-nucleic acid yeast engineering bacteria is eliminated by adopting a mode of continuously culturing in a YPD liquid culture medium without screening pressure and transferring for more than 10 times, so that the non-transgenic high-nucleic acid yeast engineering bacteria without the high-throughput screening system are obtained.

2. The method for breeding the high-nucleic acid yeast engineering bacteria by using the high-throughput screening system according to claim 1, wherein the method for transforming the saccharomyces cerevisiae cells by using the high-throughput screening system in the step (1) comprises the following steps: 1) transforming a high-throughput screening system into a saccharomyces cerevisiae industrial strain with nucleic acid content higher than a normal value through screening confirmation by using a PEG-LiAc transformation method, an electric transformation method or a protoplast transformation method; 2) screening positive transformants by using a screening culture medium containing hygromycin B, reversely extracting yeast plasmids from the recombinant yeast, and carrying out PCR verification to obtain host cells containing a high-throughput screening system; wherein the industrial strain of Saccharomyces cerevisiae with nucleic acid content higher than normal value is yeast strain CGMCC number 9084.

3. The method for breeding the high-nucleic-acid yeast engineering bacteria by using the high-throughput screening system according to claim 1, wherein the construction method of the high-throughput screening system in the step (1) is as follows: 1) amplifying the hygromycin B resistance gene expression frame by adopting a PCR method, then digesting the hygromycin B resistance gene expression frame and YEplac195 by using restriction endonucleases, transforming escherichia coli DH5 alpha by using a connecting solution, selecting transformants, and obtaining a recombinant plasmid YEp-Hyg B(ii) a 2) Amplification of rDNA promoter, IRES sequence by PCR,yeGFPGene, poly (T) and rDNA terminator to obtain fusion PCR product rDNap-IRES-yeGFPPoly (T) -rDNat, followed by restriction endonuclease cleavage of rDNap-IRES-yeGFPPoly (T) -rDNat and YEp-Hyg BThe ligation solution is transformed into Escherichia coli DH5 alpha, transformants are selected and verified by a PCR method to obtain a recombinant plasmid YEp-Hyg B-yeGFP。

4. The method for breeding the high nucleic acid yeast engineering bacteria by using the high-throughput screening system according to claim 1, wherein the method and conditions for mutagenizing the host cells containing the high-throughput screening system by using the normal pressure room temperature plasma (ARTP) mutagenesis method in the step (2) are as follows: taking 1mL of bacterial liquid, suspending in 1.5mL of EP tube, centrifuging at 8000r/min for 2min, discardingSupernatant fluid; washing with physiological saline for 2 times, diluting with physiological saline containing 5% glycerol to obtain bacterial cell with concentration of 10⁶~10⁷Taking 10 mu L of bacterial suspension and uniformly coating the bacterial suspension on the surface of a sterile slide glass; then placing the slide glass on a carrying platform of an ARTP mutation breeding system, and placing the slide glass in an ARTP mutation breeding instrument for mutation treatment, wherein the working gas of the mutation instrument is 99.99% high-purity helium, the radio frequency power is 100W, the helium flow is 10SLM, the treatment time is set to be 120s, and the distance between the sample and a plasma emission source is 3 mm; after the sample is processed, putting the slide into an EP tube filled with 1mL of physiological saline by using a pair of tweezers, continuously and fully oscillating for 1min, and fully eluting thalli attached to the slide to form bacterial suspension; control group was added 10. mu.L of bacterial suspension directly to 1mL of physiological saline.

5. The method for selectively breeding the yeast engineering bacteria with high nucleic acid content by using the high-throughput screening system as claimed in claim 1, wherein the method for performing high-throughput modeling separation to select the yeast cells with improved fluorescence intensity by using a flow cytometer in the step (3) comprises the following steps: respectively 8000r/min of host cell suspension without mutagenesis, namely control cell suspension containing a high-throughput screening system and cell suspension subjected to ARTP mutagenesis in the step (2), centrifuging for 2min, discarding supernatant, then re-suspending with 500 mu L of 1 XPBS solution, respectively adding corresponding fluorescein labeled antibodies, incubating for 1h at 25 ℃, washing for 3 times with 1 XPBS solution, and re-suspending in 500 mu L of 1 XPBS; and then, after passing through a 40-micron filter screen, sorting by an ultra-rapid flow cytometry sorting system MoFlo XDP, and setting the fluorescence value of the control cell suspension as a threshold value, so that cells with the fluorescence value larger than the threshold value can be sorted, namely the yeast cells with improved fluorescence intensity.

6. The method for selectively breeding the high-nucleic-acid yeast engineering bacteria by using the high-throughput screening system according to claim 1, wherein the step (4) of determining the content of the cell nucleic acid of the strain with the improved fluorescence intensity by using a Trizol method comprises the following specific steps: the host cells containing the high-throughput screening system and the cells with the increased fluorescence intensity in the step (3) are put in YPD liquid culture medium containing 200mg/L of hygromycin BCulturing for 12-24 hours, transferring the bacterial liquid into a fresh YPD liquid culture medium containing 200mg/L hygromycin B, and controlling the cell concentration to be OD₆₀₀About.0.1, shake-culturing at 30 ℃ to the final cell concentration OD₆₀₀1.0, centrifuging and collecting cells; every 5X 10⁷Adding 0.5ml of Trizol into the bacterial cells, and grinding by using liquid nitrogen; RNA extraction is carried out by adopting a UNIQ-10 column type Trizol total RNA extraction kit of Shanghai worker and the operation steps thereof, and the light absorption value of the extracted RNA is measured at 260 nm.