CN108660085B - Nucleic acid-producing saccharomyces cerevisiae engineering bacterium and construction method and application thereof - Google Patents

Abstract

The invention provides a saccharomyces cerevisiae strain for producing nucleic acid and a construction method and application thereof, which comprise gene overexpression and fermentation verification of breeding strains. The selected yeast strain is the saccharomyces cerevisiae(s) by taking the Pep1 gene as the target geneSaccharomyces cerevisiae) The vacuole protein sorting receptor Pep1 is over-expressed, and compared with the parent strain, the constructed saccharomyces cerevisiae (A)Saccharomyces cerevisiae) The basic fermentation performance of the strain is not affected, after the shake flask fermentation, the nucleic acid content of the bred strain reaches 14.70 percent, the nucleic acid content of the parent strain is 9.32 percent, and the bred strain is improved by 57.72 percent compared with the parent strain. The bred saccharomyces cerevisiae strain obviously improves the content of nucleic acid and is widely applied to yeast and nucleic acid industries.

Description

Nucleic acid-producing saccharomyces cerevisiae engineering bacterium and construction method and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, relates to breeding of industrial microorganisms, and particularly relates to a saccharomyces cerevisiae engineering bacterium for producing nucleic acid.

Background

The nucleic acid yeast industry is a new industry in China. Ribonucleic acid (RNA) is widely used in food industry, pharmaceutical industry and agricultural production. In the food industry, nucleic acids are an essential raw material for the development of novel food seasonings. The novel seasonings such as the second generation monosodium glutamate (strong monosodium glutamate), the third generation monosodium glutamate (flavor monosodium glutamate), the specially fresh soy sauce and the like in the world are all the results of the application of nucleic acid and derivatives thereof. In the pharmaceutical industry, nucleic acid is a raw material for preparing medicines for treating coronary heart disease, tumor, myocardial infarction and other diseases. In agriculture, nucleic acids and derivatives thereof are widely used as growth promoting substances for crops (such as rice, melons, fruits, beans, etc.).

The intracellular RNA of the saccharomyces cerevisiae mainly comprises: messenger RNA, ribosomal RNA, transfer RNA, and non-coding RNA. The ribosomal RNA is the RNA with the highest content, accounts for about 82% of the total RNA amount, and is the RNA with the largest relative molecular mass. Therefore, the method for effectively breeding the high-nucleic-acid saccharomyces cerevisiae is used for increasing the yield or the generation rate of the intracellular ribosome RNA.

In recent years, with the consideration of food safety, more and more research is focused on the work of breeding high-nucleic-acid saccharomyces cerevisiae, but not on strains such as candida which do not belong to gras (general cloning as safe).

Yangxin, Dingyue, Caojing, etc. research on RNA preparation by saccharomyces cerevisiae fermentation [ J ] pharmaceutical biotechnology, 2013(4) 310-.

The articles Chuwattakanakul V, Kim Y H, Sugiyama M, et al, Construction of a Saccharomyces cerevisiae strain with a high level of RNA [ J ]. Journal of bioscience & Bioengineering, 2011, 112(1) 1. A yeast strain with high nucleic acid yield is obtained by knocking out RRN10 gene in Saccharomyces cerevisiae, then selecting a reverted mutant strain by EMS mutagenesis, and finally integrating and expressing RRN10 gene in the mutant strain, so that the nucleic acid content is increased by 30%.

The article Peterson M R, Emr S D, The class C Vps complex functions as multiple stages of The vacuolar transport path [ J ] Traffic, 2001, 2(7):476 + 486, has disclosed

The function of the vacuolar protein sorting receptor PEP1 (also known as VPS10 or VPT 1) indicates that it plays a very critical role in the protein secretion process. It is primarily involved in the transport of soluble CPY proteins from pre-vacuolar organelles (e.g., endosomes) to the vacuole. The article Marcusson, Eric G, Horazdovsky, et al, The lubricating receiver for yeast vacuum boiler Y is encoded by The VPS10 gene [ J ]. Cell,1994, 77(4):579 indicates: the subcellular fractionation method localizes the PEP1 (also known as VPS10 or VPT 1) gene to the late golgi stage for protein sorting from endosomes to golgi. The PEP1 protein is in the cytoplasmic tail domain in saccharomyces cerevisiae, and multiple rounds of sorting are performed by repeated cycles from golgi to endosome: the sorting signal PEP1 protein of protein fragment CPY interacts with p2PCY to form a receptor-ligand complex, which is packaged into vesicles, PEP1 protein releases p2PCY, then receptor PEP1 returns to golgi to continue a new round of sorting, and CPY precursors continue to be transported to vacuoles until their activity is formed. When PEP1 is deleted, although the process of transporting CPY protein from endoplasmic reticulum to golgi is continuous, CPY precursor substance is secreted to endoplasmic reticulum and cannot be transported to vacuole due to the absence of cytoplasmic tail structure receptor, and thus cannot activate its activity, thereby affecting the physiological activity of cells.

The current research in the art on the vacuolar protein sorting receptor is primarily focused on the effect of the protein's functionality on the physiological activity of cells.

Disclosure of Invention

The invention aims to solve the problems of low strain performance and low nucleic acid yield of a nucleic acid-producing yeast strain and provide a saccharomyces cerevisiae with high nucleic acid yield.

In order to solve the technical problems, the technical scheme of the invention is as follows:

a high-nucleic acid-yield saccharomyces cerevisiae engineering strain is obtained by taking saccharomyces cerevisiae as an initial strain and performing overexpression of vacuolar protein sorting receptor gene PEP1 (also called VPS10 or VPT 1) by a genetic engineering means, wherein the gene sequence of the PEP1 is shown as SEQID No. 1.

The Gene ID of the PEP1 Gene is: 852264.

furthermore, the high-yield nucleic acid saccharomyces cerevisiae engineering bacteria are obtained by constructing a strong promoter, a vacuole protein sorting receptor gene PEP1 and a connecting fragment of a screening marker gene, and integrating the connecting fragment into a gene of host bacteria saccharomyces cerevisiae by using rDNA non-transcription spacer region 1 (NTS 1) as an insertion site through a homologous recombination method.

Furthermore, the rDNA is a sequence of Saccharomyces cerevisiae 25 s rDNA (Gene ID: 9164935), 5.8 s rDNA (Gene ID: 9164934) and 18 s rDNA (Gene ID: 9164923), and the rDNA nucleic acid sequence is shown in a nucleic acid sequence table SEQ ID NO. 2.

Preferably, the starting strain is saccharomyces cerevisiae (A)Saccharomyces cerevisiae)W303a。

Preferably, the strong promoter is the strong promoter PGK 1.

Preferably, the selectable marker gene is a Kan gene fragment.

Preferably, the host bacterium is Saccharomyces cerevisiae W303 a.

The invention also aims to provide a construction method of the high-yield nucleic acid saccharomyces cerevisiae engineering bacteria, which comprises the following specific steps:

(1) connecting a strong promoter PGK1 to a vector plasmid Yep352 to obtain a recombinant plasmid Yep352-PGK 1;

(2) taking the total DNA genome of the saccharomyces cerevisiae W303a as a template, carrying out PCR amplification to obtain a PEP1 gene fragment, and connecting the PEP1 gene fragment to a recombinant plasmid Yep352-PGK1 to obtain a recombinant plasmid Yep352-PGK1-PEP 1;

(3) carrying out PCR amplification by using the recombinant plasmid Yep352-PGK1-PEP1 as a template to obtain a strong promoter PGK1 and PEP1 connecting fragment;

(4) performing PCR by taking a genome of a starting saccharomyces cerevisiae strain W303a as a template to obtain an upstream homology arm and a downstream homology arm of an insertion site rDNA non-transcription interval region 1;

(5) and (3) converting the fragments obtained in the step (3) and the step (4) and the screening marker Kan fragment into the starting saccharomyces cerevisiae strain by a lithium acetate conversion method, wherein the upstream homology arm, the Kan fragment, the PGK1 and PEP1 connecting fragment and the downstream homology arm are sequentially connected, and screening to obtain the engineering bacteria for expressing the PEP1 gene in multiple copies.

Further, in the step (1), the upstream homology arm is rDNA-A fragment, and the downstream homology arm is rDNA-B fragment.

Preferably, the Kan fragment with the screening marker is obtained by the following method: PCR was carried out using the plasmid PUC6 as a template to obtain a fragment of the selectable marker gene Kan.

The invention also aims to provide the application of the high-yield nucleic acid saccharomyces cerevisiae engineering bacteria in nucleic acid production.

Preferably, the fermentation method of the high-yield nucleic acid saccharomyces cerevisiae engineering bacteria is as follows:

seed liquid culture: picking a ring of bacterial sludge from the inclined plane, inoculating the saccharomyces cerevisiae engineering bacteria into a YPD culture medium test tube, and culturing for 10-12 h at the temperature of 28-30 ℃ and under the condition of 180-190 rpm to obtain seed liquid;

fermentation culture: inoculating the seed liquid into YPD liquid culture medium according to the inoculation amount of 3-5% (volume percentage), and fermenting and culturing at 28-30 ℃ and 180-190 rpm for 4 h.

Above, the integration sites are as follows: sun H, Zang X, Liu Y, et al, expression of a polymeric human/salmon cellulose gene integrated into the yeast cells, gene using rDNA sequences as recombination sites [ J ]. Applied Microbiology & Biotechnology, 2015, 99(23): 10097-.

Has the advantages that:

1. however, the prior publications do not relate to the high yield relationship between the protein sorting receptor PEP1 and nucleic acid, the relevance between the two, and other problems. On the premise of keeping good fermentation performance, the high-yield nucleic acid saccharomyces cerevisiae engineering bacteria provided by the invention can copy over-expressed vacuolar protein sorting receptor gene PEP1, and unexpectedly obtain the technical effect of remarkably improving the yield of nucleic acid. The problem of low performance of the nucleic acid-producing yeast strain is solved, the growth performance and the fermentation activity of the nucleic acid-producing yeast strain are obviously improved, and the yield of nucleic acid is obviously improved.

2. The yield of the saccharomyces cerevisiae nucleic acid obtained by breeding is obviously improved. After the verification of shake flask fermentation, the content of the nucleic acid of the over-expressed PEP1 strain reaches 14.70%, and the content of the nucleic acid of the starting strain W303a is 9.32%, which is increased by 57.72%.

Drawings

FIG. 1 is a diagram of the construction and validation electrophoresis of the target fragment and the recombinant plasmid; wherein, M in the figure (a) is marker, and 1 is a PGK promoter electrophoresis band; in the diagram (b), M is marker, and 1 is a PCR verification band of the plasmid Yep 352-PGK; in the diagram (c), M is marker, and 1 is PEP1 gene obtained by PCR amplification; (d) in the figure, M is marker, 1 is a PCR verification electrophoretogram connecting a fragment PEP1 to Yep352-PGK1 (recombinant plasmid Yep352-PGK1-PEP 1);

FIG. 2 is an electrophoretogram of a target fragment, wherein lane M is a 10000 bp DNA marker, lane 1 is an electrophoretic band of rDNA-B fragment, 1084 bp; lane 2 is the Kan fragment electrophoresis band, 1613 bp; lane 3 is an electrophoresis band of a fragment connecting the promoter and PEP1, 6512 bp; lane 4 is the rDNA-A fragment electrophoresis band, 1462 bp;

FIG. 3 is a schematic diagram of homologous recombination of a fragment of interest with a yeast genome, wherein (1) shows a haploid genome of a starting s.cerevisiae strain;

FIG. 4 is a verification electrophoresis diagram of successfully constructed engineered Saccharomyces cerevisiae, in which lane M is 5000 bp DNA maker, lane 1 is the PCR product of verification primers M1-U/M1-D, and lane 2 is the PCR product of verification primers M2-U/M2-D;

FIG. 5 is a growth curve of the over-expressed PEP1 strain versus the starting strain.

Detailed Description

The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.

The saccharomyces cerevisiae used in the invention can adopt any source of saccharomyces cerevisiae engineering bacteria.

Example 1 construction of Yep352-PGK-PEP1 plasmid (Strong promoter PGK1, i.e., including PGK1 promoter (PGK 1 p) and PGK1 terminator (PGK 1 t) fragment, described below)

PCR amplification was carried out using a plasmid containing PGK1 as a template (for example, plasmid pUC-PGK1 disclosed in the patent application No. 201410277435.0 as a template) and primers PGK1-U (sequence shown by SEQ ID NO. 17) and PGK1-D (sequence shown by SEQ ID NO. 18) to obtain a PGK1 promoter and terminator ligated fragment (1771 bp), and plasmid Yep352 was digested with restriction enzyme KpnI, and then ligated using a recombinase, 15-20 bp homology arms designed at both ends of the primers, to obtain a Yep352-PGK1 recombinant plasmid. The genome of the saccharomyces cerevisiae W303a is taken as a template, and PCR amplification is carried out by using a primer P-U (a sequence shown by SEQ ID NO. 19) and a primer P-D (a sequence shown by SEQ ID NO. 20) to obtain a PEP1 (a sequence shown by SEQ ID NO. 1) fragment of 4740 bp. The Yep352-PGK1 recombinant plasmid is cut by restriction enzyme Xho I, and connected by using recombinase and 15-20 bp homology arms designed at two ends of a primer to obtain the Yep352-PGK1-PEP1 recombinant plasmid (wherein, the cutting site of the Xho I enzyme is between PGK1p and PGK1 t).

Verifying a strong promoter PGK1 by using verification primers PGKY-U (a sequence shown in SEQ ID NO. 21) and PGKY-D (a sequence shown in SEQ ID NO. 22); the PEP1 fragment is verified by verification primers PY-U (shown as a sequence in SEQ ID NO. 23) and PY-D (shown as a sequence in SEQ ID NO. 24).

As shown in FIG. 1, the target fragment and recombinant plasmid were constructed to verify the electrophoretogram, wherein lane M is 5000 bp DNAmaker. Lane 1 in FIG. (a) is the electrophoretic band of the strong promoter PGK1, 1771 bp, correct in size; FIG. 1 shows the PCR-verified band of the plasmid Yep352-PGK1, 2823 bp, correct size of the band, and correct ligation of the strong promoter PGK 1; in the figure (c), lane 1 is the PEP1 gene obtained by PCR amplification, the length is 4740bp, and the figure shows that the size of the band is correct and the band is single; (d) the figure shows that the fragment PEP1 is connected to Yep352-PGK1, the size of the band is 1309 bp, and the PEP1 is connected correctly.

The primers used for the whole procedure are shown in Table 1.

TABLE 1 PCR primers in the construction of vectors

Example 2 construction of high-yield nucleic acid Saccharomyces cerevisiae engineering bacteria

(the strong promoter PGK1 described below, i.e., including the PGK1 promoter (PGK 1 p) and the PGK1 terminator (PGK 1 t) fragment)

The main construction process of the strain is as follows (as attached figures 2 and 3):

1) obtaining the fragment of interest

Firstly, taking a saccharomyces cerevisiae W303a genome as a template, and using primers rDNA-AU (a sequence shown in SEQ ID NO. 5) and rDNA-AD (a sequence shown in SEQ ID NO. 6) to perform PCR amplification to obtain an upstream homologous arm rDNA-A fragment (a sequence shown in SEQ ID NO. 3) of 1462 bp; taking a saccharomyces cerevisiae BY23 genome as a template, and using primers rDNA-BU (a sequence shown in SEQ ID NO. 11) and rDNA-BD (a sequence shown in SEQ ID NO. 12), carrying out PCR amplification to obtain a downstream homologous arm rDNA-B fragment (a sequence shown in SEQ ID NO. 4) of 1084 bp; using plasmid PUC6 as a template, and using primers Kan-U (shown as a sequence in SEQ ID NO. 7) and Kan-D (shown as a sequence in SEQ ID NO. 8) to perform PCR to obtain a Kan fragment of 1613 bp; using the recombinant plasmid YEP352-PGK1-PEP1 prepared in example 1 as a template and primers PEP1-U (sequence shown in SEQ ID NO. 9) and PEP1-D (sequence shown in SEQ ID NO. 10), a strong promoter PGK1 and PEP1 junction fragment of 6512 bp was obtained by PCR.

The primers used for the whole procedure are shown in Table 2.

TABLE 2 PCR primers in engineering bacteria construction

FIG. 2 shows an electrophoretogram of a target fragment, wherein lane M is 10000 bp DNA marker, lane 1 is rDNA-B fragment electrophoresis band, 1084 bp, correct size; lane 2 is the Kan fragment electrophoresis band, 1613 bp, correct size; lane 3 is an electrophoresis band of a fragment connecting the promoter and PEP1, and the electrophoresis band is 6512 bp and has correct size; lane 4 is the rDNA-A fragment electrophoresis band, 1462bp, correct size;

2) construction of multi-copy over-expressed PEP1 saccharomyces cerevisiae engineering bacteria

And (2) transforming the 4 fragments obtained in the step (1) into a saccharomyces cerevisiae engineering bacterium W303a by using a lithium acetate transformation method, wherein an upstream homology arm rDNA-A, Kan fragment, a PGK1p-PEP1-PGK1t connecting fragment and a downstream homology arm rDNA-B are sequentially connected, and the multi-copy over-expression PEP1 saccharomyces cerevisiae engineering bacterium is obtained (as shown in figure 3).

Verification of the engineering bacteria of saccharomyces cerevisiae with multi-copy over-expression of PEP1 (shown in fig. 4):

two groups of primers are respectively designed, namely: M1-U (shown as a sequence in SEQ ID NO. 13), M1-D (shown as a sequence in SEQ ID NO. 14), M2-U (shown as a sequence in SEQ ID NO. 15) and M2-D (shown as a sequence in SEQ ID NO. 16) by taking plasmids in haploid transformants with better growth as templates, carrying out PCR amplification and verifying recombinants. The PCR products were subjected to 0.8% agarose gel electrophoresis, respectively. A3930 bp band and a 3000 bp band were obtained, respectively, indicating that the fragments had been successfully recombined into the haploid genome of s.cerevisiae and that the recombination positions were correct.

FIG. 4 shows a successfully constructed engineered Saccharomyces cerevisiae validation electrophoresis, in which lane M is 5000 bp DNAmaker, lane 1 is a PCR product of validation primers M1-U/M1-D, and is a specific band of 3930 bp in size, which is consistent with the expected size; lane 2 is the PCR product of the verification primer M2-U/M2-D, and a specific band with a size of 3000 bp can be seen by 0.8% agarose gel electrophoresis, and the size is consistent with the expectation, which indicates that the fragment has been successfully recombined into the haploid genome of Saccharomyces cerevisiae and the recombination position is also correct;

EXAMPLE 3 Shake flask fermentation experiment with overexpression of PEP1 Strain

1. Comparison of growth Performance of the over-expressed PEP1 Strain with the original Strain

The experimental group is Saccharomyces cerevisiae W303a overexpressing PEP1, and the control group is Saccharomyces cerevisiae W303a original strain.

1) And (3) measuring a growth curve:

seed liquid culture: using inoculating loop, picking out a loop from the slant, inoculating into YPD medium test tube, and culturing at 30 deg.C and 180 rpm for 12 h.

Fermentation culture: the seed solution was inoculated into 100 ml of Yepd broth, and 2 ml was sampled every one or two hours (depending on growth). Taking out 1 ml of bacterial liquid, diluting the bacterial liquid by a certain multiple to OD₆₀₀The value is in the range of 0.4-0.6, and the absorbance is measured at a wavelength of 600 nm. And drawing a growth curve according to the corresponding relation between the absorbance value and the sampling time.

As shown in FIG. 5, the growth performance of the over-expressed PEP1 strain was not affected at all compared with the starting strain W303a, and the fermentation termination time (to OD) of the over-expressed strain was determined at the same initial OD value₆₀₀Time of = 1) was 4 hours, and fermentation termination time (to OD) of starting strain₆₀₀Time of = 1) was 6 hours, and the overexpression strain was about 2 hours earlier than the starting strain, thereby illustrating that the fermentation activity of the engineered bacteria of the present invention was significantly improved.

2. Extraction and determination of intracellular total RNA:

(the following methods for determining nucleic acids are from the documents Chuwattakanakul V, Kim Y H, Sugiyama M, et. restriction of a Saccharomyces cerevisiae strain with a high level of the RNA. [ J ]. Journal of Bioscience & Bioengineering, 2011, 112(1): 1.)

The experimental group is Saccharomyces cerevisiae W303a overexpressing PEP1, and the control group is Saccharomyces cerevisiae W303a original strain.

1) Dry weight curve: using inoculating loop, picking out a loop from the slant, inoculating into YPD medium test tube, and culturing at 30 deg.C and 180 rpm for 12 h. Inoculating the primary seed solution into 100 ml Yepd liquid culture medium, sampling every 2 hours to determine its absorbance value at 600 nm and dry weight, and determining dry weight (y) versus OD₆₀₀(x) Dry weight curve y =0.382x +0.832 was made.

2) Fermentation culture of strain

First-stage seed liquid: respectively picking a ring from the inclined planes by utilizing an inoculating ring, inoculating the ring into a YPD culture medium test tube, and culturing for 12 h at 30 ℃ and 180 rpm;

inoculating the first seed solution to 50 ml YEPD liquid medium to make initial OD₆₀₀The same, shake-flask culture at 30 ℃ and 180 rpm to OD₂₆₀=1.0 (mid-exponential growth), divided into three tubes, and the OD per tube was measured₆₀₀Centrifuging, washing and collecting thalli; the thalli is resuspended by 0.5 mol/L perchloric acid, water bath is carried out at 70 ℃ for 20 min, supernatant fluid is collected by centrifugation and diluted by 50 times, OD value of the diluted solution at 260 nm is measured, and the intracellular total RNA content (%) is calculated according to the following formula.

(OD₂₆₀*2500)/(12.224*OD₆₀₀+26.624)

The results are shown in Table 3, Table 3 shows: in shake flask fermentation experiments, control group: the original strain W303a has a nucleic acid content of 9.32% (mass percent) of the dry weight of the strain, and the experimental group: the nucleic acid content of the over-expression strain obtained by the invention reaches 14.70% of the dry weight of the thallus, and is increased by 57.72% compared with the parent strain. The invention can improve the intracellular nucleic acid quantity of the yeast strain to a certain extent, and provides a theoretical basis for breeding the high-yield nucleic acid yeast strain.

TABLE 3 nucleic acid content of the strains

Bacterial strains	RNA％
		Control group	9.32
Experimental group	14.70

Note: the data shown are the average of the results of three replicates per group

While some embodiments of the invention have been presented herein, it will be appreciated by those skilled in the art that changes may be made to the embodiments of the invention without departing from the spirit thereof. The above-described embodiments are merely exemplary and should not be taken as limiting the scope of the invention.

Sequence listing

<110> Tianjin science and technology university

<120> high-nucleic acid-yield saccharomyces cerevisiae engineering bacterium and construction method and application thereof

<130>2018, 3 and 2 months

<141>2018-05-30

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cgtaaaaccg atgaaaattg ttttattggg aatattccac tttctgaatt ttcaagaaat 3900

atcaaaaact gttcttgtac aagacaagat ttcgagtgtg attacaactt ttacaaagct 3960

aacgatggta cttgtaaatt agtcaaagga ctaagcccag caaatgctgc agacgtttgt 4020

aaaaaagagc cagatttaat cgaatatttt gaatcgtcag gctacagaaa gatccctcta 4080

tcaacctgtg agggtggcct gaaattggat gctccctcat caccacatgc ttgcccagga 4140

aaagaaaaag aattcaagga aaagtactca gtaagtgccg gtccctttgc atttattttc 4200

atttcaattc ttttaataat tttctttgcc gcatggtttg tatatgacag aggtatcaga 4260

agaaatgggg gatttgcaag gtttggagaa attaggctag gtgacgatgg tttaatagaa 4320

aacaataata ctgacagagt tgtcaataac attgtgaaat caggatttta cgttttctca 4380

aatatcgggt ctcttttaca gcacacaaaa actaatatag cgcatgctat ctccaaaatt 4440

agaggaaggt ttggaaacag aacaggtcca agctactcat ccctgatcca tgatcaattt 4500

ttggatgaag cagatgacct gcttgctggc cacgatgaag acgccaatga cttatccagt 4560

ttcatggatc agggtagtaa ttttgaaatc gaagaagatg atgttccaac acttgaagaa 4620

gagcatacat catatacaga tcaacctacg accaccgatg ttccagatac attaccagaa 4680

ggaaatgagg aaaacatcga caggcctgat tctacagcgc catctaacga aaaccagtag 4740

<210>2

<211>2250

<212>DNA

<213>Saccharomyces cerevisiae rDNA

<400>2

ggaacctcta atcattggct ttacctcata aaactgatac gagcttctgc tatcctgagg 60

gaaacttcgg caggaaccag ctactagatg gttcgattag tctttcgccc ctatacccaa 120

attcgacgat cgatttgcac gtcagaaccg ctacgagcct ccaccagagt ttcctctggc 180

ttcaccctat tcaggcatag ttcaccatct ttcgggtccc aacagctatg ctcttactca 240

aatccatccg aagacatcag gatcggtcga ttgtgcacct cttgcgaggc cccaacctac 300

gttcactttc attacgcgta tgggttttac acccaaacac tcgcatagac gttagactcc 360

ttggtccgtg tttcaagacg ggcggcatat aaccattatg ccagcatcct tgacttacgt 420

cgcagtcctc agtcccagct ggcagtattc ccacaggcta taatacttac cgaggcaagc 480

tacattccta tggatttatc ctgccaccaa aactgatgct ggcccagtga aatgcgagat 540

tcccctaccc acaaggagca gagggcacaa aacaccatgt ctgatcaaat gcccttccct 600

ttcaacaatt tcacgtactt tttcactctc ttttcaaagt tcttttcatc tttccatcac 660

tgtacttgtt cgctatcggt ctctcgccaa tatttagctt tagatggaat ttaccaccca 720

cttagagctg cattcccaaa caactcgact cttcgaaggc actttacaaa gaaccgcact 780

cctcgccaca cgggattctc accctctatg acgtcctgtt ccaaggaaca tagacaagga 840

acggccccaa agttgccctc tccaaattac aactcgggca ccgaaggtac cagatttcaa 900

atttgagctt ttgccgcttc actcgccgtt actaaggcaa tcccggttgg tttcttttcc 960

tccgcttatt gatatgctta agttcagcgg gtactcctac ctgatttgag gtcaaacttt 1020

aagaacattg ttcgcctaga cgctctcttc ttatcgataa cgttccaata cgctcagtat 1080

aaaaaagatt agccgcagtt ggtaaaacct aaaacgaccg tacttgcatt atacctcaag 1140

cacgcagaga aacctctctt tggaaaaaaa aaacatccaa tgaaaaggcc agcaatttca 1200

agttaactcc aaagagtatc actcactacc aaacagaatg tttgagaagg aaatgacgct 1260

caaacaggca tgccccctgg aataccaagg ggcgcaatgt gcgttcaaag attcgatgat 1320

tcacggaatt ctgcaattca cattacgtat cgcatttcgc tgcgttcttc atcgatgcga 1380

gaaccaagag atccgttgtt gaaagttttt aatattttaa aattcccagt tacgaaaatt 1440

cttgtttttg acaaaaattt aatgaataaa taaaattgtt tgtgtttgtt acctctgggc 1500

cccgattgct cgaatgccca aagaaaaagt tgcaaagata tgaaaactcc acagtgtgtt 1560

gtattgaaac ggttttaatt gtcctataac aaaagcacag aaatctctca ccgtttggaa 1620

tagcaagaaa gaaacttaca agcctagcaa gaccgcgcac ttaagcgcag gcccggctgg 1680

actctccatc tcttgtcttc ttgcccagta aaagctctca tgctcttgcc aaaacaaaaa 1740

aaatccattt tcaaaattat taaatttctt taatgatcct tccgcaggtt cacctacgga 1800

aaccttgtta cgacttttag ttcctctaaa tgaccaagtt tgtccaaatt ctccgctctg 1860

agatggagtt gcccccttct ctaagcagat cctgaggcct cactaagcca ttcaatcggt 1920

actagcgacg ggcggtgtgt acaaagggca gggacgtaat caacgcaagc tgatgacttg 1980

cgcttactag gaattcctcg ttgaagagca ataattacaa tgctctatcc ccagcacgac 2040

ggagtttcac aagattacca agacctctcg gccaaggtta gactcgctgg ctccgtcagt 2100

gtagcgcgcg tgcggcccag aacgtctaag ggcatcacag acctgttatt gcctcaaact 2160

tccatcggct tgaaaccgat agtccctcta agaagtggat aaccagcaaa tgctagcacc 2220

actatttagt aggttaaggt ctcgttcgtt 2250

<210>3

<211>1462

<212>DNA

<213> Saccharomyces cerevisiae upstream homology arm rDNA-A ()

<400>3

gttaactata ggaaatgagc ttttctcaat tctctaaact tatacaagca ctcatgtttg 60

ccgctctgat ggtgcggaaa aaactgctcc atgaagcaaa ctgtccgggc aaatcctttc 120

acgctcggga agctttgtga aagcccttct ctttcaaccc atctttgcaa cgaaaaaaaa 180

aaaaaaaata aaaaataaaa agaccaaata gtaaatagta acttacatac attagtaaat 240

ggtacactct tacacactat catcctcatc gtatattata atagatatat acaatacatg 300

tttttacccg gatcatagaa ttcttaagac aaataaaatt tatagagact tgttcagtct 360

acttctctct aaactaggcc ccggctcctg ccagtaccca cttagaaaga aataaaaaac 420

aaatcagaca acaaaggctt aatctcagca gatcgtaaca acaaggctac tctactgctt 480

acaatacccc gttgtacatc taagtcgtat acaaatgatt tatccccacg caaaatgaca 540

ttgcaattcg ccagcaagca cccaaggcct ttccgccaag tgcaccgttg ctagcctgct 600

atggttcagc gacgccacaa ggacgcctta ttcgtatcca tctatattgt gtggagcaaa 660

gaaatcaccg cgttctagca tggattctga cttagaggcg ttcagccata atccagcgga 720

tggtagcttc gcggcaatgc ctgatcagac agccgcaaaa accaattatc cgaatgaact 780

gttcctctcg tactaagttc aattactatt gcggtaacat tcatcagtag ggtaaaacta 840

acctgtctca cgacggtcta aacccagctc acgttcccta ttagtgggtg aacaatccaa 900

cgcttaccga attctgcttc ggtatgatag gaagagccga catcgaagaa tcaaaaagca 960

atgtcgctat gaacgcttga ctgccacaag ccagttatcc ctgtggtaac ttttctggca 1020

cctctagcct caaattccga gggactaaag gatcgatagg ccacactttc atggtttgta 1080

ttcacactga aaatcaaaat caagggggct tttacccttt tgttctactg gagatttctg 1140

ttctccatga gcccccctta ggacatctgc gttatcgttt aacagatgtg ccgccccagc 1200

caaactcccc acctgacaat gtcttcaacc cggatcagcc ccgaatggga ccttgaatgc 1260

tagaacgtgg aaaatgaatt ccagctccgc ttcattgaat aagtaaagaa actataaagg 1320

tagtggtatt tcactggcgc cgaagctccc acttattcta caccctctat gtctcttcac 1380

aatgtcaaac tagagtcaag ctcaacaggg tcttctttcc ccgctgattc tgccaagccc 1440

gttcccttgg ctgtggtttc gc 1462

<210>4

<211>1084

<212>DNA

<213> Saccharomyces cerevisiae downstream homologous arm rDNA-B ()

<400>4

acctaccgac caactttcat gttctgtttc gacctacctc ttgtaaatga caaatcacct 60

ttttcatcgt atgcacctta ttctccacat cacaatgcac tattgctttt gctttttcac 120

ctgtcatatc ctattgctat tagatgaaat ataataaaaa ttgtcctcca cccataacac 180

ctctcactcc cacctactga acatgtctgg accctgccct catatcacct gcgtttccgt 240

taaactatcg gttgcggcca tatctaccag aaagcaccgt ttcccgtccg atcaactgta 300

gttaagctgg taagagcctg accgagtagt gtagtgggtg accatacgcg aaactcaggt 360

gctgcaatct ttatttcttt tttttttttt tttttttttt ttttttctag tttcttggct 420

tcctatgcta aatcccataa ctaacctacc attcgattca gaaaaattcg cactatccag 480

ctgcactctt cttctgaaga gttaagcact ccattatgct cattgggttg ctactacttg 540

atatgtacaa acaatattct cctccgatat tcctacaaaa aaaaaaaaaa aaacactccg 600

gttttgttct cttccctcca tttccctctc ttctacggtt aatactttcc tcttcgtctt 660

tttctacacc ctcgtttagt tgcttcttat tccttcccgc tttcctgcac taacattttg 720

ccgcattaca ctatatgatc gtagtacatc ttacaactcc gcataccgcg tcgccgcgtc 780

gccgcgtcgc caaaaattta cttcgccaac cattccatat ctgttaagta tacatgtata 840

tattgcactg gctattcatc ttgcactttt cctctttctt cttcccagta gcctcatcct 900

tttacgctgc ctctctggaa cttgccatca tcattcccta gaaactgcca tttacttaaa 960

aaaaaaaaaa aaaaaaaaat gtccccactg ttcactgttc actgttcact tgtctcttac 1020

atctttcttg gtaaaatcgt agttcgtagt attttttttc atatcaaagg catgtcctgt 1080

taac 1084

<210>5

<211>26

<212>DNA

<213> Artificial sequence ()

<400>5

gttaactata ggaaatgagc ttttct 26

<210>6

<211>44

<212>DNA

<213> Artificial sequence ()

<400>6

ctgcagcgta cgaagcttca gctggcgaaa ccacagccaa ggga 44

<210>7

<211>44

<212>DNA

<213> Artificial sequence ()

<400>7

tcccttggct gtggtttcgc cagctgaagc ttcgtacgct gcag 44

<210>8

<211>45

<212>DNA

<213> Artificial sequence ()

<400>8

gttttggata gatcagttag agcataggcc actagtggat ctgat 45

<210>9

<211>45

<212>DNA

<213> Artificial sequence ()

<400>9

atcagatcca ctagtggcct atgctctaac tgatctatcc aaaac 45

<210>10

<211>45

<212>DNA

<213> Artificial sequence ()

<400>10

catgaaagtt ggtcggtagg ttaacgaacg cagaattttc gagtt 45

<210>11

<211>46

<212>DNA

<213> Artificial sequence ()

<400>11

aactcgaaaa ttctgcgttc gttaacctac cgaccaactt tcatgt 46

<210>12

<211>18

<212>DNA

<213> Artificial sequence ()

<400>12

accccaccac actcctac 18

<210>13

<211>18

<212>DNA

<213> Artificial sequence ()

<400>13

accccaccac actcctac 18

<210>14

<211>18

<212>DNA

<213> Artificial sequence ()

<400>14

acgctcgtca tcaaaatc 18

<210>15

<211>18

<212>DNA

<213> Artificial sequence ()

<400>15

tcacctgcgt ttccgtta 18

<210>16

<211>18

<212>DNA

<213> Artificial sequence ()

<400>16

ccctgggagg agttatct 18

<210>17

<211>44

<212>DNA

<213> Artificial sequence ()

<400>17

acgaattcga gctcggtacc tctaactgat ctatccaaaa ctga 44

<210>18

<211>39

<212>DNA

<213> Artificial sequence ()

<400>18

tcgacggatc cccgggtacc taacgaacgc agaattttc 39

<210>19

<211>40

<212>DNA

<213> Artificial sequence ()

<400>19

ggaattccag atctcctcga gatgatatta cttcattttg 40

<210>20

<211>39

<212>DNA

<213> Artificial sequence ()

<400>20

atctatcgca gatccctcga gctactggtt ttcgttaga 39

<210>21

<211>19

<212>DNA

<213> Artificial sequence ()

<400>21

tctaactgat ctatccaaa 19

<210>22

<211>18

<212>DNA

<213> Artificial sequence ()

<400>22

aggaacgtgc tgctactc 18

<210>23

<211>18

<212>DNA

<213> Artificial sequence ()

<400>23

tttgtgctct tatgggac 18

<210>24

<211>18

<212>DNA

<213> Artificial sequence ()

<400>24

attcttcggc attagtta 18

Claims (5)

1. The application of the saccharomyces cerevisiae engineering bacteria in nucleic acid production is characterized in that: the engineering bacteria are obtained by over-expressing vacuolar protein sorting receptor gene PEP1 by a genetic engineering means, and the sequence of the PEP1 gene is shown as SEQ ID NO. 1; the engineering bacteria are obtained by constructing a strong promoter, a vacuole protein sorting receptor gene PEP1 and a connecting fragment of a screening marker gene, and integrating the connecting fragment into a gene of host bacteria saccharomyces cerevisiae by using an rDNA non-transcription interval region 1 as an insertion site through a homologous recombination method; the strong promoter is PGK 1.

2. The use of the engineered saccharomyces cerevisiae strain for producing nucleic acid as claimed in claim 1, wherein the selectable marker gene is Kan gene.

3. The use of the engineered saccharomyces cerevisiae strain of claim 1 for producing nucleic acid, wherein the host strain is saccharomyces cerevisiae W303 a.

4. The use of the engineered saccharomyces cerevisiae strain of claim 1 in nucleic acid production, wherein the engineered saccharomyces cerevisiae strain comprises: the construction method of the engineering bacteria comprises the following steps:

(1) connecting a strong promoter PGK1 to a vector plasmid Yep352 to obtain a recombinant plasmid Yep352-PGK 1;

(2) taking the total DNA genome of the saccharomyces cerevisiae W303a as a template, carrying out PCR amplification to obtain a PEP1 gene fragment, and connecting the PEP1 gene fragment to a recombinant plasmid Yep352-PGK1 to obtain a recombinant plasmid Yep352-PGK1-PEP 1;

(3) carrying out PCR amplification by using the recombinant plasmid Yep352-PGK1-PEP1 as a template to obtain a PGK1-PEP1 connecting fragment;

(4) PCR is carried out by taking a saccharomyces cerevisiae strain W303a genome as a template to obtain an upstream homology arm and a downstream homology arm of an insertion site rDNA non-transcription interval region 1;

(5) PCR is carried out by taking the plasmid PUC6 as a template to obtain a screening marker gene Kan fragment;

(6) and (3) transforming the upstream homology arm, the Kan fragment, the PGK1-PEP1 connecting fragment and the downstream homology arm fragment obtained in the steps (3), (4) and (5) into a host saccharomyces cerevisiae strain W303a by a lithium acetate transformation method, and screening to obtain the engineering bacteria for expressing the PEP1 gene in multiple copies.

5. The use of the engineered saccharomyces cerevisiae strain of claim 1 in nucleic acid production, wherein the engineered saccharomyces cerevisiae strain comprises: the culture method of the engineering bacteria comprises the following steps:

seed liquid culture: picking a ring of bacterial sludge from the inclined plane, inoculating the saccharomyces cerevisiae engineering bacteria into a YPD culture medium test tube, and culturing for 10-12 h at the temperature of 28-30 ℃ and under the condition of 180-190 rpm to obtain seed liquid;

fermentation culture: inoculating the seed liquid into YPD liquid culture medium according to the inoculation amount of 3-5%, and fermenting and culturing at 28-30 ℃ and 180-190 rpm for 4 h.

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