CN106119274B - High expression or low expression site of saccharomyces cerevisiae - Google Patents

Abstract

The invention belongs to the technical field of genes, and particularly relates to determination of a site effect of saccharomyces cerevisiae and screening of high-expression or low-expression sites of genes, in particular to a series of vectors for inserting genes into high-expression or low-expression sites and high-expression and low-expression sites of the genes in the saccharomyces cerevisiae. The invention researches the site effect of the saccharomyces cerevisiae on the whole genome scale, reveals the distribution rule of the site effect on the saccharomyces cerevisiae genome, screens and obtains the high expression sites of the saccharomyces cerevisiae such as YBR128C, YDR448W, YGR240C, YHR142W, YML059C, YPL014W and YPR028W sites, and the low expression sites of the saccharomyces cerevisiae such as YBR001C, YGR038W, YIL092W, YLR371W and YPL241C sites, and provides rational design basis for expressing exogenous metabolites and byproducts by using saccharomyces cerevisiae cells as chassis cells.

Description

High expression or low expression site of saccharomyces cerevisiae

Technical Field

The invention belongs to the technical field of genes, and particularly relates to determination of a site effect of saccharomyces cerevisiae and screening of high-expression or low-expression sites of genes, in particular to a series of high-expression or low-expression sites of genes in the saccharomyces cerevisiae and vectors for inserting the genes into the high-expression sites and the low-expression sites.

Background

Saccharomyces cerevisiae is one of the major model organisms in microbial research and is often used to express foreign proteins. Researchers often use biotechnology means to introduce exogenous genes and even a series of metabolic pathways into saccharomyces cerevisiae cells, thereby synthesizing some natural products, including important pharmaceutical and chemical products such as terpenes, polyunsaturated fatty acids, and the like.

Foreign genes and metabolic pathways are generally introduced by episomal vectors that are present in the Saccharomyces cerevisiae cells and are otherwise integrated into the Saccharomyces cerevisiae genome. Compared with the former, the latter not only ensures the stability of the exogenous gene, but also does not need to add antibiotics or special chemical substances in the culture medium to maintain the screening pressure, thereby simplifying the culture medium and reducing the production cost. In industrial production, researchers therefore often integrate foreign genes or metabolic pathways into the genome of s.cerevisiae.

Generally, methods for increasing the expression of a foreign gene include increasing the copy number of the foreign gene or selecting a stronger promoter. Prior studies have shown that the expression level of a gene varies when the gene is inserted at different positions on a chromosome, a phenomenon known as site effect. This phenomenon has been demonstrated in the chromosomes of many organisms, such as E.coli, Salmonella typhimurium, lactococcus lactis, Saccharomyces cerevisiae, Drosophila and humans. However, the related research aiming at the site effect of the saccharomyces cerevisiae is less at present. In 1998, Yamane et al used lacZ as a reporter gene and integrated it at different positions on Saccharomyces cerevisiae chromosome III, characterized the site effect of Saccharomyces cerevisiae genome by the expression amount of beta-galactosidase, and found that there are two high-expression hot spot regions and seven low-expression inhibition regions on chromosome III. In 2001, Thompson et al used a similar characterization method to study the site effects of 24 randomly selected sites on the yeast genome. In 2009, Bai et al also used lacZ as a reporter gene and selected 20 sites for relevant studies. However, the above studies are limited to individual sites or a certain chromosome, and the distribution rule and action mechanism of site effects in the Saccharomyces cerevisiae genome cannot be revealed from a global perspective.

Disclosure of Invention

The invention aims to disclose the distribution rule of the site effect on the saccharomyces cerevisiae genome and the sites with high expression and low expression of genes from the global perspective, thereby providing theoretical basis and guidance for expressing exogenous genes and metabolic pathways by using the saccharomyces cerevisiae.

The invention selects proper promoter and reporter gene, and uses the homologous recombination method and reporter gene fragment (RFP) to replace the KanMX expression frame of the selected strain of the saccharomyces cerevisiae single gene knockout library respectively. The relative fluorescence intensity of each recombinant strain in the logarithmic growth phase is respectively detected by a fluorescence microplate reader, so as to represent the difference of gene expression intensity at different positions in the chromosome. The results showed that there were 162 loci in the total of the high and low groups in 16 chromosomes of s.cerevisiae. The site with the highest expression is located at YBR128C, where the relative fluorescence intensity of RFP is the greatest, reaching 12.98. In addition, the higher expression sites include YDR448W, YGR240C, YHR142W, YML059C, YPL014W and YPR 028W. The lower expression sites are more concentrated near telomeres and centromeres, such as YBR001C, YGR038W, YIL092W, YLR371W and YPL241C sites.

Therefore, the invention provides the application of the loci YBR128C, YDR448W, YGR240C, YHR142W, YML059C, YPL014W and YPR028W of saccharomyces cerevisiae in high expression of exogenous metabolites by taking saccharomyces cerevisiae as a chassis cell.

The invention also provides the application of the YBR001C, YGR038W, YIL092W, YLR371W and YPL241C loci of saccharomyces cerevisiae in low-expression byproducts by taking saccharomyces cerevisiae as underplate cells.

Further, the present invention provides a vector having a gene inserted into at least one site of Saccharomyces cerevisiae YBR128C, YDR448W, YGR240C, YHR142W, YML059C, YPL014W, YPR028W, YBR001C, YGR038W, YIL092W, YLR371W or YPL 241C.

Wherein, the promoter in the vector is TEF1P or URA 3P.

Preferably, the reporter gene in the vector of the present invention is RFP, GFP or LacZ.

The invention researches the site effect of the saccharomyces cerevisiae on the whole genome scale, reveals the distribution rule of the site effect on the saccharomyces cerevisiae genome, screens and obtains the high expression sites of the saccharomyces cerevisiae such as YBR128C, YDR448W, YGR240C, YHR142W, YML059C, YPL014W and YPR028W sites, and the low expression sites of the saccharomyces cerevisiae such as YBR001C, YGR038W, YIL092W, YLR371W and YPL241C sites, and provides rational design basis for expressing exogenous metabolites and byproducts by using saccharomyces cerevisiae cells as chassis cells.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows a schematic representation of the plasmid PUC 19;

FIG. 2 shows electrophoretic verification of promoter and reporter gene, wherein lane 1 is URA3P, lane 2 is ADH1P, lane 3 is PGI1P, lane 4 is PGK1P, lane 5 is TEF1P, lane 6 is TEF2P, lane 7 is THDH3P, lane 8 is Mark DL2000, lane 9 is GFP, lane 10 is RFP, lane 11 is Mark 2K Plus;

FIG. 3 shows selection plates for correct transformants, where a is a selection plate with SD-LEU auxotrophic medium as positive control and b is a selection plate with YPD + G418 medium as negative control;

FIG. 4 shows an electrophoretic picture of the verification fragment, wherein lane 1 is URA3P + GFP, lane 2 is ADH1P + GFP, lane 3 is PGI1P + GFP, lane 4 is PGK1P + GFP, lane 5 is TEF1P + GFP, lane 6 is TEF2P + GFP, lane 7 is THDH3P + GFP, lane 8Mark DL 2000;

FIG. 5 shows a comparison of fluorescence intensities for different promoters and different reporter genes, wherein a, b are YCR001C, c, d are YOL001W, e, f are YPR 015C; a. c, e report gene is GFP, b, d, f report gene is RFP;

FIG. 6 shows a schematic diagram of an alternative procedure for RFP substitution of the KanMX sequence;

FIG. 7 shows a library fluorescence intensity distribution diagram of RFP-expressing strains;

FIG. 8 is a graph showing fluorescence relative expression intensity at each site on chromosome 2;

FIG. 9 shows a map of high expression sites and low expression sites at 162 sites in 16 chromosomes of yeast;

FIG. 10 is a graph showing a comparison of the difference in RFP copy number between 10 strains;

FIG. 11 shows the transcription of the RFP gene at 10 sites, wherein,show Fluoresence Intensity，mRNA is shown;

FIG. 12 shows a comparison of fluorescence intensities of 10 different promoters in position, whereThe URA3P + RFP is shown,TEF1P + RFP;

FIG. 13 is a graph showing the relative fluorescence intensity and galactosidase activity of 10 different reporter genes at sites, wherein,shown are TEF1P + RFP,shown are TEF1P + GFP,TEF1P + LacZ is shown.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

For a further understanding of the present invention, reference will now be made in detail to the following examples.

Examples 1,

Materials and methods

1. Strains and culture media

The single-gene knockout library strain culture medium is YPD (glucose 20G/L, yeast extract 10G/L, peptone 20G/L) culture medium, and 200mg/mL G418 is added.

The recombinant strain replacing the reported gene has LEU2 gene, and the culture medium is SD-LEU (6.7g/L yeast nitrogen source without amino acid, 2g/L amino acid mixture, 20g/L glucose, 20mg/L histidine, 20mg/L tryptophan, 20mg/L uracil).

2. Construction of expression cassette

The expression plasmid is constructed on the basis of the plasmid PUC19 (figure 1), and comprises KANMX-L, a terminator CYC1T, a promoter, a reporter gene (RFP, GFP) and a terminator, auxotrophy screening markers LEU2 and KANMX-R, and enzyme cutting sites on both sides of an expression frame are SACI and BAMHI. Wherein KANMX-L and KANMX-R are homology arms integrated into the chromosome of the yeast single gene knockout strain. CYC1T was designed to eliminate the effect of the promoter prior to the integration site. LEU2 is a selection marker for auxotrophy. Different promoters and reporter genes are respectively ligated into this expression cassette. The plasmid was treated with both SacI and BamHI restriction enzymes and the reporter fragment was recovered from the gel for subsequent transformation experiments.

3. Transformation method

High-throughput yeast transformation was performed using 96-well plates, and strains in single gene knockouts were transferred to 96-well plates containing 150. mu.L of YPD + G418 per well, cultured overnight at 30 ℃ and transferred the next day. Transferred to a 96-well plate containing 150. mu.L LYPD + G418 per well, and incubated at 30 ℃ for 6 h. Centrifuging at 4000g for 2min, and removing supernatant. Resuspend with 200. mu.L water, centrifuge at 4000g for 2min, remove supernatant. The cells were resuspended in 200 μ L0.1M LiAc (Lithium acetate). Centrifuge at 4000g for 2min and discard the supernatant. Then, the cells were resuspended in 40. mu.L of 0.1M LiAc, and 160. mu.L of a transformation mix (transformation mix) containing PEG 50% 124. mu.L, 1M LiAc 16. mu.L, SSDNA 10. mu.L (10mg/mL), and 10. mu.L of the desired gene fragment (containing 200ng of the desired gene fragment) were added to each well. Incubating at 30 deg.C for 30min, adding 10 μ L dimethyl sulfoxide, heat-shocking at 42 deg.C for 30min, centrifuging at 4000g for 5min, discarding supernatant, adding 2000 μ L5mM CaCl2 for resuspension, centrifuging at 4000g for 2min, discarding supernatant, and re-suspending in water. Spread on SD-LEU auxotrophic plates and cultured at 30 ℃. Positive and negative screens (SD-Leu and YPD + G418) and PCR were used to verify the insertion of the target gene into the genome.

4. Fluorescence measurement

The transformants which were confirmed to be correct were transferred into 96-well plates containing 150. mu.L of SD-Leu medium per well, and cultured overnight at 30 ℃. The next day, 10. mu.L of the inoculum was transferred to a 96-well plate containing 150. mu.L of LSD-Leu medium per well and cultured for 5 hours. Centrifuged at 4000g for 2min and resuspended in PBS (phosphate-buffered saline). OD600 and fluorescence measurements were performed. The emission light and excitation light of red fluorescent protein (Mcherry RFP) are 587nm/610nm, respectively, and the emission light and excitation light of green fluorescent protein are 485nm/510nm, respectively. The site effect of the insertion site is characterized by the relative fluorescence intensity of the sample obtained from the measured fluorescence intensity of the sample/OD 600 of the sample.

Second, result in

1. Construction of the plasmids involved

The method comprises the steps of taking plasmids with promoters URA3P, ADH1P, PGI1P, PGK1P, TEF1P, TEF2P and TDH3P as templates, designing primers, introducing enzyme cutting sites Bgl II at the 5 'end and Xbal I at the 3' end of the promoters respectively, and amplifying to obtain seven promoters URA3P, ADH1P, PGI1P, PGK1P, TEF1P, TEF2P and TDH3P (figure 2). And then, using plasmids with GFP and RFP genes as templates, designing primers, introducing a restriction enzyme site Xbal I restriction enzyme site at the 5 'end of the reporter gene, introducing a Sal I restriction enzyme site at the 3' end of the reporter gene, and respectively amplifying to obtain the reporter genes GFP and RFP (figure 2). The substitution of the promoter or reporter gene, respectively, on the basis of the plasmid PUC19M1, resulted in the combination of 14 plasmids, which are listed in Table 1.

TABLE 1 related plasmids

Name of plasmid	Promoters	Reporter gene	Screening markers
				PUC19M1	URA3P	RFP	Leu
PUC19M2	ADH1P	RFP	Leu
				PUC19M3	PGI1P	RFP	Leu
PUC19M4	PGK1P	RFP	Leu
				PUC19M5	TEF1P	RFP	Leu
PUC19M6	TEF2P	RFP	Leu
				PUC19M7	TDH3P	RFP	Leu
PUC19M8	URA3P	GFP	Leu
				PUC19M9	ADH1P	GFP	Leu
PUC19M10	PGI1P	GFP	Leu
				PUC19M11	PGK1P	GFP	Leu
PUC19M12	TEF1P	GFP	Leu
				PUC19M13	TEF2P	GFP	Leu
PUC19M14	TDH3P	GFP	Leu

2. Homologous recombination and validation of reporter genes

The 14 plasmids were treated with SacI and BamHI restriction enzymes, respectively, and reporter fragments were recovered and purified by gel extraction and transformed into single gene deletion strains YCR011, YOL001W and YPR015C, respectively, to obtain 42 recombinant strains. Correct transformants were selected using SD-LEU auxotrophic medium as a positive control and YPD + G418 medium as a negative control, and the results are shown in fig. 3. The genome of the screened strain was extracted as a template, and the insertion of the objective gene was verified using the corresponding verification primer, and the results are shown in fig. 4. And the deletion of the KANMX fragment was verified using the corresponding primers, resulting in the correct transformants.

The fluorescence intensities of GFP and RFP at different insertion sites under the action of different promoters were measured using a luciferase reader (FIG. 5). Excitation light 485nm and emission light 510nm for GFP assay. RFP measurement of 485nm excitation light, emitted light 510 nm.

The results of the fluorescence measurements show that the autofluorescence of yeast itself has a large effect on the weaker promoter when GFP is used as a reporter gene. The background of RFP is much lower relative to GFP, and has less effect on the assay results. Compared with a strong promoter, a weak promoter is easier to amplify the signal of the reporter gene, so that the difference between sites is easier to distinguish, and in conclusion, the experiment finally selects URA3P as the promoter and RFP as the reporter gene for subsequent experiments.

3. Establishment of high-throughput homologous recombination method and replacement of single-gene knockdown bacterium

The Saccharomyces cerevisiae single gene knockout library (MAT alpha library) uses Saccharomyces cerevisiae strain BY4742 as a background strain, each recombinant strain has an Open Reading Frame (ORF) replaced BY a KanMX expression cassette, and has G418 resistance. The invention selects 1044 loci from a saccharomyces cerevisiae single gene knockout library, the selection principle is that one locus is selected every 12KB in 16 chromosomes, and the selection of each chromosome locus is shown in table 2. And then, selecting red fluorescent protein as a reporter gene, carrying out homologous integration on the reporter gene into a selected single-gene knockout library through yeast transformation to replace a KanMX sequence, thereby constructing a single-site fluorescent marker library of the saccharomyces cerevisiae, and representing the gene expression level of the site by using the relative fluorescence intensity of each site. The replacement process is shown in fig. 6.

TABLE 2 selection of individual chromosomal sites

	Length KB	Percentage of total length%	Number of selecting points
				Chromosome 1	230.2	1.91	19
Chromosome 2	812.3	6.73	70
				Chromosome 3	316.1	2.62	29
Chromosome 4	1531.4	12.67	129
				Chromosome 5	576.8	4.78	49
Chromosome 6	270.1	2.24	24
				Chromosome 7	1090.9	9.04	94
Chromosome 8	562.6	4.66	47
				Chromosome 9	439.8	3.64	39
Chromosome 10	745.7	6.18	62
				Chromosome 11	666.8	5.53	59
Chromosome 12	1078.1	8.93	88
				Chromosome 13	924.2	7.66	79
Chromosome 14	784.3	6.51	68
				Chromosome 15	1091.2	9.04	92
Chromosome 16	948.1	7.86	81

And replacing the KANMX fragment in the selected strain with the reporter gene fragment in sequence, on one hand, carrying out forward screening by using an SD-LEU auxotroph culture medium and carrying out reverse screening by using an YPD + G418 culture medium, on the other hand, carrying out colony PCR verification by using corresponding primers, and carrying out genotype verification on the selected strain again. Finally, the recombinant strain containing the reporter gene is obtained and stored for subsequent fluorescence measurement experiments.

4. Fluorescence measurement results of the replacement Strain

Based on the relative expression intensity of RFP gene, the RFP expression strain library is divided into 5 groups, high, medium, low and low, which are respectively used(FIG. 7). The number of strains in the 5 groups was 91, 161, 410, 311 and 71, respectively, accounting for 8.7%, 15.4%, 39.3%, 19.8 and 6.8% of the total. Among the 1044 sites selected, the higher group and the middle groupThe group and the lower group occupied the majority, and only a few genes appeared in the upper and lower groups. The highest relative fluorescence intensity in the high group reached 12.98, the lowest relative fluorescence intensity in the low group was only 0.98, and the maximum difference reached a 13.21-fold difference.

TABLE 3 results of fluorescence measurements of the replacement strains

Relative intensity of expression of fluorescence	Percentage of the strain in the genome	TEL/CEN
			Height of	8.7％	0
Is higher than	15.4％	6.0％
			In	39.3	25.6％
Is lower than	29.8	51.3％
			Is low in	6.8％	17.1％

In order to further study the distribution rule of the site effect on the chromosome, chromosome 2 was used as a study sample to perform the fluorescence relative expression intensity analysis of each site, and the results are shown in fig. 8.

From FIG. 8, it can be seen that relative fluorescence expression of RFP was low at the sites adjacent to the telomeres and adjacent to the centromere. Further statistics were made on the distribution of strains with replacement sites adjacent to telomeres and centromeres in the pool of RFP-expressing strains in each group. As can be seen from comparison of the results in Table 3, the distribution of the 1044 loci in the whole genome was most concentrated in the middle group, but the strains adjacent to telomeres and centromeres were mainly concentrated in the lower group, occupying 51.3%, and the strains adjacent to telomeres and centromeres were not present in the high group. It is therefore believed that the lower effect of sites in the region adjacent to the centromere and telomere is probably due to the closer binding of these proteins to the DNA at these sites, which results in lower expression levels of the genes at these sites.

5. High expression site and low expression site on chromosome

In 16 chromosomes, there were 162 loci in the sum of the high and low groups, as shown in FIG. 9. The site with the highest expression is located at YBR128C, where the relative fluorescence intensity of RFP is the greatest, reaching 12.98. In addition, the higher expression sites include YDR448W, YGR240C, YHR142W, YML059C, YPL014W and YPR 028W. The lower expression sites are more concentrated near telomeres and centromeres.

6. Factors affecting the site Effect

The expression of a gene can be affected in many ways, such as copy number, promoter strength, etc. To demonstrate that the site effect is really due to the effect of the positional difference, the effect of the gene copy number must be removed. 5 strains with strong relative fluorescence intensity of RFP, namely YBR128C, YDR448W, YGR240C, YML059C and YPL014W, are selected from the high group, and 5 strains with weak relative fluorescence intensity of RFP, namely YBR001C, YGR038W, YIL092W, YLR371W and YPL241C are selected from the low group. The results of comparing the difference in RFP copy number between 10 strains by extracting the genome of each of 10 strains and using ALG9 as a reference gene and Q-PCR technique are shown in FIG. 10.

The maximum difference between the copy numbers of RFPs of 10 strains is only 1.39 times, so that the maximum difference between the copy numbers and the relative fluorescence intensities of RFPs is 1.39 times. However, the difference in relative fluorescence intensity of RFPs in the high and low groups is much larger than this value, so that the difference due to the site effect is not due to the influence of copy number.

Since the relative intensity of the site effect is estimated from the fluorescence measurement value after transcription and translation of the RFP gene used, it is necessary to confirm whether the transcription level of the RFP gene corresponds to the relative intensity. The applicants determined the transcription of RFP gene at 10 sites using ALG9 gene as reference gene, and the results are shown in fig. 11, wherein the transcription level of RFP gene in the high group is still significantly higher than that in the low group in the comparison of the strains in the high and low groups, but the difference of RFP gene transcription is reduced relative to the difference of relative fluorescence intensity of RFP, presumably due to mRNA instability or translational regulation.

To investigate whether the effect of the site effect is influenced by the different promoter strengths at the same insertion sites when the promoters are changed, the following experiment was designed. Firstly, a new expression frame of replacing URA3P promoter by TEF1P promoter is constructed, RFP is selected as reporter genes, and then the new expression frame is introduced into the 10 sites by a homologous recombination method. As shown in FIG. 12, after the promoter was replaced, the trend of difference among 10 sites was not changed, but the fold difference was changed little. The maximum difference between the sites was 13.2 fold when URA3P + RFP was used as the expression cassette, but the maximum difference between the sites was only 3.9 fold when the promoter was changed to the strong promoter TEF1P, which shows that the difference between the sites was reduced when the strong promoter was changed.

The strength of the promoter has an effect on the difference between sites, and applicants further confirmed whether the site effect is also affected by the replacement of the reporter gene. Further constructing a gene expression cassette which has the same promoter (TEF1P) but replaces a reporter gene, respectively replacing the RFP gene with expression cassettes of GFP and LacZ genes, introducing the RFP gene into the 10 high-low difference sites by utilizing homologous recombination again, obtaining a new recombinant strain, and respectively measuring the relative fluorescence intensity and the galactosidase activity of GFP at each site to represent the relative expression intensity of the site. As shown in FIG. 13, the replacement of the reporter gene by the strong promoter TEF1P had little effect on the site effect.

Claims (1)

1. The application of the YBR128C locus of saccharomyces cerevisiae in high expression of exogenous metabolites by taking saccharomyces cerevisiae as a chassis cell.