CN114958838A - High-strength saccharomyces cerevisiae artificial small promoter - Google Patents
High-strength saccharomyces cerevisiae artificial small promoter Download PDFInfo
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Abstract
The invention discloses a high-strength saccharomyces cerevisiae artificial small promoter. The invention provides a saccharomyces cerevisiae artificial promoter mutant library which is obtained by randomly mutating all or part of 1 st to 6 th positions from the 3' end of a nucleotide sequence of a saccharomyces cerevisiae artificial promoter UASF-E-C-core 1. The saccharomyces cerevisiae artificial promoter mutant provided by the invention is specifically any one of the following: 14 promoter mutants with the nucleotide sequence shown in SEQ ID No. 10-23. The invention provides a group of high-strength saccharomyces cerevisiae artificial small promoters, wherein the strength of the small promoter with the strongest strength is about 3.3 times of that of a natural strong promoter TDH3 and 7 times of that of a starting promoter. The strength range of the artificial small promoter is remarkably widened, and the promoter engineering is better applied to gene expression regulation and control of a saccharomyces cerevisiae metabolic pathway.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a group of high-strength saccharomyces cerevisiae artificial small promoters.
Background
Saccharomyces cerevisiae, as a eukaryotic model organism, has the advantages of rapid growth, easy culture, high safety, clear genetic background and the like, and is widely applied to synthetic biology. Saccharomyces cerevisiae cell factories have been developed for the production of recombinant proteins, biofuels and fine chemicals, among others. The promoter is an important transcription regulation element and plays an important role in the regulation of gene expression of a saccharomyces cerevisiae gene pathway and a metabolic pathway. The construction of metabolic pathways often involves the expression of multiple genes, the expression levels of which span several orders of magnitude. The fine regulation of these genes makes the selection of suitable promoters challenging.
At present, a series of constitutive promoters endogenous to yeast, such as pPGK1, pTEF1, pFBA11, pGPD/pTDH3, inducible strong promoter pGAL1 and the like, are commonly used for expressing exogenous genes in a saccharomyces cerevisiae cell factory. However, it is worth noting that these yeast endogenous promoters have some limitations, such as insufficient number of well-characterized promoters, poor dynamic range, and susceptibility to interference from endogenous substances, which hinder their application in metabolic engineering. In recent years, research on synthetic promoters of yeasts has been rapidly developed, especially in the model microorganism Saccharomyces cerevisiae. However, the problem is that the promoter frameworks for promoter modification at present are all from yeast endogenous promoters, and the obtained new promoters are generally large and have poor sequence diversity, so that the stability after multiple recombinations is poor. The promoter obtained by hybridization significantly improves the problem of poor sequence diversity, but has the defects that the obtained promoter is too large and depends on endogenous sequences such as UAS (upstream activation sequence) to play a role. To solve these problems, Alper et al obtained a series of small promoters of 125bp size independent of the yeast endogenous promoter by total synthesis. The length of the promoter is shortened by 80 percent compared with the integrated promoter. However, it reports that the strength of the strongest small promoter is about 70% of that of the yeast endogenous strong promoter TDH3, and this strength range is still difficult to satisfy the needs of researchers.
Disclosure of Invention
The invention aims to provide a group of high-strength saccharomyces cerevisiae artificial small promoters.
In a first aspect, the invention claims a saccharomyces cerevisiae artificial promoter mutant library.
The saccharomyces cerevisiae artificial promoter mutant library claimed by the invention is obtained by randomly mutating all or part of 1 st to 6 th positions from the 3' end of a nucleotide sequence of a saccharomyces cerevisiae artificial promoter UASF-E-C-core 1.
Wherein the nucleotide sequence of the Saccharomyces cerevisiae artificial promoter UASF-E-C-core1 is shown as 29-153 th site of SEQ ID No. 4.
In a specific embodiment of the invention, all the 1 st to 6 th positions from the 3 'end of the nucleotide sequence of the Saccharomyces cerevisiae artificial promoter UASF-E-C-core1 (the 29 th to 153 th positions of SEQ ID number 4) are randomly mutated by means of PCR, the 1 st to 6 th positions from the 3' end of the nucleotide sequence of the primer corresponding to the Saccharomyces cerevisiae artificial promoter UASF-E-C-core1 (the 29 th to 153 th positions of SEQ ID No. 4) are NNNNNNNN, and N is A or T or C or G.
In a second aspect, the invention claims an artificial promoter mutant of Saccharomyces cerevisiae.
The saccharomyces cerevisiae artificial promoter mutant claimed by the invention can be any one of the following mutants:
(A1) the nucleotide sequence is shown as SEQ ID No.19 (corresponding to 528 in FIG. 1);
(A2) the nucleotide sequence is shown as SEQ ID No.13 (corresponding to 510 in FIG. 1);
(A3) the nucleotide sequence is shown as SEQ ID No.11 (corresponding to 503 in FIG. 1);
(A4) the nucleotide sequence is shown as SEQ ID No.10 (corresponding to 501 in FIG. 1);
(A5) the nucleotide sequence is shown as SEQ ID No.12 (corresponding to 507 in FIG. 1);
(A6) the nucleotide sequence is shown as SEQ ID No.14 (corresponding to 512 in FIG. 1);
(A7) the nucleotide sequence is shown as SEQ ID No.20 (corresponding to 532 in FIG. 1);
(A8) the nucleotide sequence is shown as SEQ ID No.21 (corresponding to 536 in FIG. 1);
(A9) the nucleotide sequence is shown as SEQ ID No.16 (corresponding to 517 in FIG. 1);
(A10) the nucleotide sequence is shown as SEQ ID No.18 (corresponding to 525 in FIG. 1);
(A11) the nucleotide sequence is shown as SEQ ID No.15 (corresponding to 514 in FIG. 1);
(A12) the nucleotide sequence is shown as SEQ ID No.17 (corresponding to 523 in FIG. 1);
(A13) the nucleotide sequence is shown as SEQ ID No.22 (corresponding to 540 in FIG. 1);
(A14) the nucleotide sequence is shown as SEQ ID No.23 (corresponding to 545 in FIG. 1).
In a third aspect, the invention claims the application of the saccharomyces cerevisiae artificial promoter mutant in promoting the expression of a target gene in saccharomyces cerevisiae.
In a fourth aspect, the invention claims the application of the saccharomyces cerevisiae artificial promoter mutant in improving the expression level of the target gene in the saccharomyces cerevisiae.
In a fifth aspect, the present invention claims an expression cassette comprising an artificial promoter mutant of Saccharomyces cerevisiae as described in the previous second aspect.
The expression cassette consists of the saccharomyces cerevisiae artificial promoter mutant, a target gene and a terminator which are connected in sequence.
In a specific embodiment of the present invention, the terminator is specifically an SPG5 terminator.
Further, the nucleotide sequence of the SPG5 terminator is shown in 23 rd to 213 th of SEQ ID No. 2.
In a sixth aspect, the invention claims a recombinant vector comprising an expression cassette as described in the fifth aspect above.
In a specific embodiment of the invention, the backbone vector of the recombinant vector is saccharomyces cerevisiae universal expression vector pRS 313.
In a seventh aspect, the present invention claims a recombinant s.cerevisiae strain comprising the recombinant vector described above in the sixth aspect.
In a particular embodiment of the invention, the recipient saccharomyces cerevisiae is in particular BY 4742. Of course, other s.cerevisiae strains are theoretically possible.
In an eighth aspect, the present invention claims the application of the Saccharomyces cerevisiae artificial promoter mutant library of the first aspect or the Saccharomyces cerevisiae artificial promoter mutant of the second aspect or the expression cassette of the fifth aspect or the recombinant vector of the sixth aspect or the recombinant Saccharomyces cerevisiae of the seventh aspect in gene expression regulation of Saccharomyces cerevisiae metabolic pathway.
The invention provides a group of high-strength saccharomyces cerevisiae artificial small promoters, wherein the strength of the small promoter with the strongest strength is about 3.3 times of that of a natural strong promoter TDH3 and 7 times of that of a starting promoter. The strength range of the artificial small promoter is remarkably widened, and the promoter engineering is better applied to gene expression regulation and control of a saccharomyces cerevisiae metabolic pathway.
Drawings
FIG. 1 is a comparison of the strength of a high strength artificial mini-promoter with a yeast endogenous strong promoter.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 construction of YPL001 plasmid as blank control
1. Amplification of GFP Gene and SPG5 terminator
GFP gene was synthesized by Kinsley, and PCR was performed using a synthetic plasmid (obtained by inserting the GFP gene shown at positions 25 to 741 of SEQ ID No.1 into pUC 57) as a template, using primers GFPzeo-up2/GFPzeo-down2 (see Table 1) as an amplification system for TAKARADNA polymerase 5 Xbuffer 10. mu.l, DnMu.l of tp mix, 1. mu.l each of primers (see Table 1), cDNA, template 0.5. mu.l, 0.5. mu.l of PrimerSTAR HS polymerase (2.5U/. mu.L), and distilled water to a total volume of 50. mu.l. The amplification conditions were: pre-denaturation at 98 ℃ for 2 min (1 cycle); denaturation at 98 ℃ for 10 seconds, annealing at 56 ℃ for 15 seconds, and extension at 72 ℃ for 1 minute (30 cycles); extension at 72 ℃ for 8 min (1 cycle). The resulting amplification product was designated GFP (SEQ ID No.1, the fragment containing the 24bp plasmid pRS313 homologous region, the 20bp SPG5 homologous region, and the GFP gene sequence). And purifying the obtained PCR amplification product by using a PCR product purification kit of Shanghai biological engineering Co., Ltd, and obtaining a purified product for later use. The terminator SPG5 (as described above) was amplified using the Saccharomyces cerevisiae BY4742 genome as a template and the primers SPG5-up2/SPG5-down2 (see Table 1), and the amplification product was named SPG5(SEQ ID No.2, this fragment contains a 22bp GFP homologous region, a 22bp plasmid pRS313 homologous region, and a SPG5 terminator sequence). And purifying the obtained PCR amplification product by using a PCR product purification kit of Shanghai biological engineering Co., Ltd, and keeping the purified product for later use.
2. PCR amplification of expression vectors
The target expression vector was amplified with the primer 313-up2/313-down2 (see Table 1) using Saccharomyces cerevisiae universal expression vector pRS313 (from Addgene, His3 marker) as template. The amplification system and conditions were the same as in step 1 of example 1. The resulting amplified product was named pRS313-His-vector (SEQ ID No.3, this fragment is the pRS313 vector backbone with a 24bp GFP homology region and a 22bp SPG5 homology region). And (3) recovering and purifying the obtained PCR amplification product by using a PCR glue recovery kit of Shanghai biological engineering Co.
3. Construction of YPL001 plasmid by CPEC method
CPEC system: phusion High-Fidelity DNA polymerase 5 x Buffer 5. mu.l, Dntp mix 1, DMSO 0.75. mu.l, two purified fragments (the GFP gene and SPG5 terminator amplified purified fragments obtained in step 1) and pRS313-His-vector obtained in step 2 were added equimolar, Phusion High-Fidelity DNA polymerase (2U/. mu.L) 0.5. mu.l, and distilled water was added to a total volume of 25. mu.l. The amplification conditions were: 30 seconds at 98 ℃ (1 cycle); 10 seconds at 98 ℃, 30 seconds at 55 ℃ and 15 seconds at 72 ℃ (15 cycles); extension for 10 min at 72 ℃ (1 cycle). Transfer 5. mu.l of PCR product into Trans1-T1 competent cells in ice bath for 30 min, heat shock at 42 ℃ for 30 sec, and immediately ice for 2 min. After adding 800. mu.l of LB medium and incubating at 250rpm and 37 ℃ for 1 hour, the bacterial solution was spread on LB plate containing ampicillin and cultured overnight, PCR screening was carried out using YpL001-YZ-up/YpL001-YZ-down (see Table 1) with a positive clone band size of 1504 bp. And performing liquid culture on the positive clone, extracting a positive clone plasmid, and performing sequencing verification, wherein a sequencing result shows that a target fragment is inserted into the vector pRS313-His to obtain the plasmid YPL 001. A plasmid extraction kit of Aisijin is used to extract the target plasmid YPL001 for later use.
TABLE 1 construction of YPL001 and YPL002 plasmid primers
Example 2 construction of YPL002 plasmid as Positive control
The strongest artificial small promoter reported by Alper group, UASF-E-C-core1 (positions 29-153 of SEQ ID No. 4), was synthesized by Kinsley. The PCR product obtained by amplification with the primers Core11-up/Core11-down (see Table 1) was named UASF-E-C-Core1(SEQ ID No.4, which contains a 20bp plasmid pRS313 homologous region, a 24bp GFP homologous region, and UASF-E-C-Core1) in the same manner as in step 1 of example 1. The YPL001 plasmid constructed in example 1 was used as a template, and PCR amplification was carried out using primers YpL001-up/YpL001-down (see Table 1), and the resulting amplification product was designated pRS313-His-GFP-SPG5(SEQ ID No.5, which contains pRS313 vector backbone, GFP gene and SPG5 terminator). And (3) recovering and purifying the obtained PCR amplification product by using a PCR glue recovery kit of Shanghai biological engineering Co. The two fragments were ligated by the CPEC method (same procedure as in example 1, step 3). Transfer 5. mu.l of PCR product into Trans1-T1 competent cells in ice bath for 30 min, heat shock at 42 ℃ for 30 sec, and immediately ice for 2 min. After adding 800. mu.l of LB medium, incubating at 250rpm and 37 ℃ for 1 hour, plating the broth on LB plates containing ampicillin overnight, and performing PCR screening using primer Core11-up/GFPzeo-down2 (see Table 1), the band size of the positive clone was 890 bp. And performing liquid culture on the positive clone, extracting a positive clone plasmid, and performing sequencing verification, wherein a sequencing result shows that a target fragment is inserted into the vector pRS313 to obtain the plasmid YPL 002. A plasmid extraction kit of Aisijin is used to extract a target plasmid YPL002 for later use.
Example 3 construction of strains Ypl001, Ypl002, Ypl007, Ypl008
Construction of YPL007 and YPL008 plasmids
1. Amplification of pGPD/pTDH3, pTEF1 promoters
PCR amplification was carried out using Saccharomyces cerevisiae BY4742 genome as template and primers GPD-Dai-up/GPD-Dai-down, TEF1-Dai-up/TEF1-Dai-down (see Table 2), respectively. The amplification system is TAKARADNA polymerase 5 Xbuffer 10. mu.l, Dntp mix 4. mu.l, primers 1. mu.l each, cDNA, template 0.5. mu.l, PrimerSTAR HS polymerase (2.5U/. mu.L) 0.5. mu.l, and distilled water was added to a total volume of 50. mu.l. The amplification conditions were: pre-denaturation at 98 ℃ for 2 min (1 cycle); denaturation at 98 ℃ for 10 seconds, annealing at 56 ℃ for 15 seconds, and extension at 72 ℃ for 1 minute (30 cycles); extension at 72 ℃ for 8 min (1 cycle). The amplified products were designated pTDH3(SEQ ID No.6, which contains the 20bp plasmid pRS313 homologous region, the 24bp GFP homologous region and the TDH3 promoter sequence), pTEF1(SEQ ID No.7, which contains the 20bp plasmid pRS313 homologous region, the 24bp GFP homologous region and the TEF1 promoter), respectively. And purifying the obtained PCR amplification product by using a PCR product purification kit of Shanghai biological engineering Co., Ltd, and obtaining a purified product for later use.
TABLE 2 construction of YPL007 and YPL008 plasmid primers
Primer name | Sequence (5 '-3') |
GPD-Dai-up | GGAGAAAATACCGCATCAGGATACTAGCGTTGAATGTTAGCG |
GPD-Dai-down | GAAAAGTTCTTCTCCTTTACTCATTTTGTTTGTTTATGTGTGTTTATTC |
TEF1-Dai-up | GGAGAAAATACCGCATCAGGAGTGATCCCCCACACACCATAG |
TEF1-Dai-down | GAAAAGTTCTTCTCCTTTACTCATTTTGTAATTAAAACTTAGATTAG |
YpL001-YZ-up | CCAAAGGTGTTCTTATGTAGTG |
YpL001-YZ-down | CTTTAGGGTTCCGATTTAGTGC |
2. Amplification expression vector
Using YPL001 constructed in example 1 as a template, PCR amplification was carried out using primers YpL001-YZ-up/YpL001-YZ-down (see Table 1), and the resulting amplification product was designated pRS313-His-GFP-SPG5(SEQ ID No.5, which contains pRS313 vector backbone, GFP gene and SPG5 terminator). And (3) recovering and purifying the obtained PCR amplification product by using a PCR glue recovery kit of Shanghai biological engineering Co., Ltd, and obtaining a purified product for later use.
3. Construction of YPL007 and YPL008 plasmids by CPEC method
CPEC system: phusion High-Fidelity DNA polymerase 5 x Buffer 5. mu.l, Dntp mix 1, DMSO 0.75. mu.l, purified fragment (pTDH 3 or pTEF1 obtained in step 1) and pRS313-His-GFP-SPG5 were added equimolar, Phusion High-Fidelity DNA polymerase (2U/. mu.L) 0.5. mu.l, and distilled water was added to a total volume of 25. mu.l. The amplification conditions were: 30 seconds at 98 ℃ (1 cycle); 10 seconds at 98 ℃, 30 seconds at 55 ℃ and 15 seconds at 72 ℃ (15 cycles); extension for 10 min at 72 ℃ (1 cycle). Transfer 5. mu.l of PCR product into Trans1-T1 competent cells in ice bath for 30 min, heat shock at 42 ℃ for 30 sec, and immediately ice for 2 min. Adding 800 ul LB culture medium, incubating at 250rpm and 37 ℃ for 1 hour, coating the bacterial liquid on LB plate containing ampicillin, culturing overnight, and PCR screening with primer GPD-Dai-up or TEF1-Dai-up/GFPzeo-down2 (see Table 1 and Table 2), the positive clone band size is 1557bp or 1187 bp. And (3) performing liquid culture on the positive clone, extracting a positive clone plasmid, and performing sequencing verification, wherein the sequencing result shows that the target fragment is inserted into the vector pRS313-His-GFP-SPG5 to obtain plasmids YPL007 (corresponding to pTDH3) and YPL008 (corresponding to pTEF 1). The plasmid extraction kit of Aisijin company is used to extract the target plasmid for later use.
Secondly, Ypl001, Ypl002, Ypl007 and Ypl008 strain are constructed
Starting with Saccharomyces cerevisiae BY4742(Saccharomyces cerevisiae BY4742, described in Carrier baker brochmann et al, 1998, Yeast,14: 115-. 1mL (OD. about.0.6-1.0) was dispensed into 1.5mL EP tubes, centrifuged at 4 ℃ at 10000g for 1min, the supernatant was discarded, the precipitate was washed with sterile water (4 ℃), centrifuged under the same conditions, and the supernatant was discarded. The cells were incubated at 25 ℃ for 20min with 1mL of a treatment solution (10mM LiAc; 10mM DTT; 0.6M sorbitol; 10mM Tris-HCl (pH7.5), DTT being added when the treatment solution was used). After centrifugation, the supernatant was discarded, 1mL of 1M sorbitol (0.22 μ M aqueous membrane filtration sterilization) was added to the cells for resuspension, and the cells were centrifuged to discard the supernatant (resuspended twice with 1M sorbitol) to a final volume of about 80 μ L. YPL001, YPL002, YPL007 and YPL008 plasmid 1 μ L were added, mixed well and transferred to an electric cuvette, shocked at 2.7kv for 5.6ms, added with 1mL of 1M sorbitol, revived at 30 ℃ for 1h, spread on a screening medium plate (formulation: 0.8% yeast selection medium SD-Ura-His-Leu-Trp, 2% glucose, 0.01% Leu., 0.01% Ura., 0.01% Trp. (each percentage number indicates g/100mL), screened and cultured under conditions of 30 ℃ for 36h or more, and each strain was named Ypl, 36002, Ypl007 and Ypl according to the difference of the transferred plasmids.
Example 4 construction of Artificial Small promoter Kozak library and screening to obtain high-Strength Artificial Small promoter
Construction of Saccharomyces cerevisiae artificial promoter Kozak library
1. Amplification of YPL-Kozak-mut fragment
The strongest artificial small promoter reported by Alper group, UASF-E-C-Core1 (positions 29-153 of SEQ ID No. 4), was synthesized by Kinsley and PCR amplified using the primers CoreKM-up/Core11 KM-down (see Table 3). The amplification system and conditions were the same as in step 1 of example 1. The resulting amplification product was designated YPL-Kozak-mut (SEQ ID No.8, which fragment includes the 50bp plasmid pRS313 homology arm, UASF-E-C-core1 and Kozakmut). And (3) recovering and purifying the obtained PCR amplification product by using a PCR glue recovery kit of Shanghai biological engineering Co.
2. Homologous recombination in saccharomyces cerevisiae body, construction of saccharomyces cerevisiae artificial promoter Kozak library
Saccharomyces cerevisiae BY4742 was made competent (same procedure as step two of example 3). YPL-Kozak-mut fragment and pRS313-His-GFP-SPG5 vector fragment were added in an amount of 2. mu.L each, mixed well, transferred to an electric cuvette, shocked at 2.7kv for 5.6ms, added with 1mL of 1M sorbitol, thawed at 30 ℃ for 1 hour, and spread on a screening medium plate (formulation: 0.8% yeast selection medium SD-Ura-His-Leu-Trp, 2% glucose, 0.01% Leu., 0.01% Ura, 0.01% Trp. (each percentage indicates g/100mL), screening culture was carried out at 30 ℃ for 36 hours or more.
The recombinant saccharomyces cerevisiae obtained by screening contains a recombinant vector formed by homologous recombination of a YPL-Kozak-mut fragment and a pRS313-His-GFP-SPG5 vector fragment, wherein the nucleotide sequence of the recombinant vector is shown as SEQ ID No.9, and N is A, C, T or G.
TABLE 3 construction of Kozak library primers
Note: n represents A or T or C or G.
Second, screening of Saccharomyces cerevisiae artificial promoter Kozak library
1. Flow cytometry initial screening of saccharomyces cerevisiae artificial promoter Kozak library
Collecting the strains obtained in the steps into a 1.5ml centrifuge tube, washing twice by using a sterilized PBS buffer solution, sorting by using a flow cytometer, collecting 2% strains with higher fluorescence intensity, culturing in a 96 deep-well plate (added with a corresponding liquid culture medium), and carrying out screening culture under the conditions as follows: culturing at 30 deg.C, humidity 80% and rotation speed of 800rpm for more than 24 h.
2. 96-hole plate double screen
Transfer the 96-deep-well plate bacterial liquid to the 96-well plate with white bottom and black edge by using 200 mu L of a row gun. Subsequently, fluorescence measurement was carried out under the conditions of 395nm for excitation light, 507nm for absorption light and 600nm for OD measurement. The fluorescence/OD 600 ratio was calculated and 38 clones with significant enhancement were rescreened in vitro.
3. Test tube rescreening
The seed solutions (30 ℃ C., 250rpm, 12 hours) were prepared by cloning 38 clones selected as described above in the corresponding liquid selection medium (formulation: liquid yeast selection medium SD-Ura-His-Leu-Trp, 2% glucose; each percentage number indicates g/100mL), followed by selection according to the initial OD 600nm The culture was continued by transfer (30 ℃, 250rpm, 12 hours) at 0.1, followed by fluorescence measurement under the conditions of 395nm for excitation light, 507nm for absorption light and 600nm for OD measurement. fluorescence/OD 600 ratios were calculated, and finally fourteen small promoters were retained, and the corresponding strains were named 501, 503, 507, 510, 512, 514, 517, 523, 525, 528, 532, 536, 540, 545(SEQ ID NO: S)ID No.10-SEQ ID No.23)
Thirdly, comparing the strength of the high-strength artificial small promoter with that of the yeast endogenous strong promoter
Fluorescence measurement experiment: the 4 strains Ypl001, Ypl002, Ypl007 and Ypl008 and a total of 14 strains of the saccharomyces cerevisiae strains obtained by screening in step two were activated in the corresponding solid selection medium (formulation: solid yeast screening medium SD-Ura-His-Leu-Trp, 2% glucose, 1.5% agar; each percentage number indicates g/100mL), a seed solution (30 ℃, 250rpm, 12h) was prepared in the corresponding liquid selection medium (formulation: liquid yeast screening medium SD-Ura-His-Leu-Trp, 2% glucose; each percentage number indicates g/100mL), followed by transfer culture (30 ℃, 250rpm, 12h) according to initial OD600nm 0.1 followed by fluorescence measurement under excitation light 395nm, absorption light 507nm and OD measurement under wavelength 600nm, the results of which are shown in fig. 1.
The above measurement results show that 14 high-strength artificial small promoters are finally obtained by constructing a library of the artificial small promoter Kozak region and performing multiple rounds of screening, and except 545 activity which is slightly lower than that of the saccharomyces cerevisiae endogenous strong promoters pTDH3 and pTEF1, the activity of the other 13 artificial small promoters is higher than that of pTDH3 and pTEF 1. The strength of the artificial small promoter 528 with the strongest strength is 3.3 times that of pTDH3 and pTEF1 and 7 times that of the strongest small promoter reported by Alper, so that the dynamic range of the saccharomyces cerevisiae artificial small promoter is remarkably widened.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific examples, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences
<120> a group of high-strength saccharomyces cerevisiae artificial small promoters
<130> GNCLN210181
<160> 23
<170> PatentIn version 3.5
<210> 1
<211> 761
<212> DNA
<213> Artificial sequence
<400> 1
gtaaggagaa aataccgcat caggatgagt aaaggagaag aacttttcac tggagttgtc 60
ccaattcttg ttgaattaga tggtgatgtt aatgggcaca aattttctgt cagtggagag 120
ggtgaaggtg atgcaacata cggaaaactt acccttaaat ttatttgcac tactggaaaa 180
ctacctgttc catggccaac acttgtcact actttctctt atggtgttca atgcttttca 240
agatacccag atcatatgaa acggcatgac tttttcaaga gtgccatgcc cgaaggttat 300
gtacaggaaa gaactatatt tttcaaagat gacgggaact acaagacacg tgctgaagtc 360
aagtttgaag gtgataccct tgttaataga atcgagttaa aaggtattga ttttaaagaa 420
gatggaaaca ttcttggaca caaattggaa tacaactata actcacacaa tgtatacatc 480
atggcagaca aacaaaagaa tggaatcaaa gttaacttca aaattagaca caacattgaa 540
gatggaagcg ttcaactagc agaccattat caacaaaata ctccaattgg cgatggccct 600
gtccttttac cagacaacca ttacctgtcc acacaatctg ccctttcgaa agatcccaac 660
gaaaagagag accacatggt ccttcttgag tttgtaacag ctgctgggat tacacatggc 720
atggatgaac tatacaaata gcaaagacgt tgtttcatcg c 761
<210> 2
<211> 258
<212> DNA
<213> Artificial sequence
<400> 2
catggatgaa ctatacaaat agcaaagacg ttgtttcatc gcgctattac caagaaggtt 60
actttacttg ttcttgcaca tggacgcacg ttgtgtgttc atatatatat atatatatat 120
atatatatat ttgtgcttgt tttcattgtc tctatagtta atacattcta tttttatcgt 180
tatatttgca ttctcttcgc ataaaaactt catgaaaatt cggcagaaaa taagcaaatt 240
gtaaacgtta atattttg 258
<210> 3
<211> 5013
<212> DNA
<213> Artificial sequence
<400> 3
gaaaattcgg cagaaaataa gcaaattgta aacgttaata ttttgttaaa attcgcgtta 60
aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat 120
aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca 180
ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc 240
ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta 300
aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg 360
gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg 420
gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcg 480
cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg 540
ctattacgcc agctggcgaa ggggggatgt gctgcaaggc gattaagttg ggtaacgcca 600
gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattgtaata cgactcacta 660
tagggcgaat tggagctcca ccgcggtggc ggccgctcta gaactagtgg atcccccggg 720
ctgcaggaat tcgatatcaa gcttatcgat accgtcgacc tcgagggggg gcccggtacc 780
cagcttttgt tccctttagt gagggttaat tccgagcttg gcgtaatcat ggtcatagct 840
gtttcctgtg tgaaattgtt atccgctcac aattccacac aacataggag ccggaagcat 900
aaagtgtaaa gcctggggtg cctaatgagt gaggtaactc acattaattg cgttgcgctc 960
actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg 1020
cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct 1080
gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt 1140
atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc 1200
caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctcggcc cccctgacga 1260
gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata 1320
ccaggcgttc ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 1380
cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg 1440
taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 1500
cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag 1560
acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt 1620
aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt 1680
atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg 1740
atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac 1800
gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 1860
gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac 1920
ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac 1980
ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt 2040
tcgttcatcc atagttgcct gactgcccgt cgtgtagata actacgatac gggagggctt 2100
accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt 2160
atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc 2220
cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa 2280
tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg 2340
tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt 2400
gtgaaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc 2460
agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt 2520
aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg 2580
gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac 2640
tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc 2700
gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt 2760
tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg 2820
aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag 2880
catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa 2940
acaaataggg gttccgcgca catttccccg aaaagtgcca cctgggtcct tttcatcacg 3000
tgctataaaa ataattataa tttaaatttt ttaatataaa tatataaatt aaaaatagaa 3060
agtaaaaaaa gaaattaaag aaaaaatagt ttttgttttc cgaagatgta aaagactcta 3120
gggggatcgc caacaaatac taccttttat cttgctcttc ctgctctcag gtattaatgc 3180
cgaattgttt catcttgtct gtgtagaaga ccacacacga aaatcctgtg attttacatt 3240
ttacttatcg ttaatcgaat gtatatctat ttaatctgct tttcttgtct aataaatata 3300
tatgtaaagt acgctttttg ttgaaatttt ttaaaccttt gtttattttt ttttcttcat 3360
tccgtaactc ttctaccttc tttatttact ttctaaaatc caaatacaaa acataaaaat 3420
aaataaacac agagtaaatt cccaaattat tccatcatta aaagatacga ggcgcgtgta 3480
agttacaggc aagcgatccg tcctaagaaa ccattattat catgacatta acctataaaa 3540
ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct 3600
gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac 3660
aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg 3720
catcagagca gattgtactg agagtgcacc ataattccgt tttaagagct tggtgagcgc 3780
taggagtcac tgccaggtat cgtttgaaca cggcattagt cagggaagtc ataacacagt 3840
cctttcccgc aattttcttt ttctattact cttggcctcc tctagtacac tctatatttt 3900
tttatgcctc ggtaatgatt ttcatttttt tttttccacc tagcggatga ctcttttttt 3960
ttcttagcga ttggcattat cacataatga attatacatt atataaagta atgtgatttc 4020
ttcgaagaat atactaaaaa atgagcaggc aagataaacg aaggcaaaga tgacagagca 4080
gaaagcccta gtaaagcgta ttacaaatga aaccaagatt cagattgcga tctctttaaa 4140
gggtggtccc ctagcgatag agcactcgat cttcccagaa aaagaggcag aagcagtagc 4200
agaacaggcc acacaatcgc aagtgattaa cgtccacaca ggtatagggt ttctggacca 4260
tatgatacat gctctggcca agcattccgg ctggtcgcta atcgttgagt gcattggtga 4320
cttacacata gacgaccatc acaccactga agactgcggg attgctctcg gtcaagcttt 4380
taaagaggcc ctactggcgc gtggagtaaa aaggtttgga tcaggatttg cgcctttgga 4440
tgaggcactt tccagagcgg tggtagatct ttcgaacagg ccgtacgcag ttgtcgaact 4500
tggtttgcaa agggagaaag taggagatct ctcttgcgag atgatcccgc attttcttga 4560
aagctttgca gaggctagca gaattaccct ccacgttgat tgtctgcgag gcaagaatga 4620
tcatcaccgt agtgagagtg cgttcaaggc tcttgcggtt gccataagag aagccacctc 4680
gcccaatggt accaacgatg ttccctccac caaaggtgtt cttatgtagt gacaccgatt 4740
atttaaagct gcagcatacg atatatatac atgtgtatat atgtatacct atgaatgtca 4800
gtaagtatgt atacgaacag tatgatactg aagatgacaa ggtaatgcat cattctatac 4860
gtgtcattct gaacgaggcg cgctttcctt ttttcttttt gctttttctt tttttttctc 4920
ttgaactcga cggatcatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac 4980
cgcatcagga tgagtaaagg agaagaactt ttc 5013
<210> 4
<211> 177
<212> DNA
<213> Artificial sequence
<400> 4
ggagaaaata ccgcatcagg ggcgcgcccc tccttgaaac tgaaatttta gcatgtgatt 60
aattaacttg taatattcta atcaagctta taaaagagca ctgttgggcg tgagtggagg 120
cgccggaaaa aagcatcgaa aaaatctaga aaaatgagta aaggagaaga acttttc 177
<210> 5
<211> 5937
<212> DNA
<213> Artificial sequence
<400> 5
catcgaaaaa atctagaaaa atgagtaaag gagaagaact tttcactgga gttgtcccaa 60
ttcttgttga attagatggt gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg 120
aaggtgatgc aacatacgga aaacttaccc ttaaatttat ttgcactact ggaaaactac 180
ctgttccatg gccaacactt gtcactactt tctcttatgg tgttcaatgc ttttcaagat 240
acccagatca tatgaaacgg catgactttt tcaagagtgc catgcccgaa ggttatgtac 300
aggaaagaac tatatttttc aaagatgacg ggaactacaa gacacgtgct gaagtcaagt 360
ttgaaggtga tacccttgtt aatagaatcg agttaaaagg tattgatttt aaagaagatg 420
gaaacattct tggacacaaa ttggaataca actataactc acacaatgta tacatcatgg 480
cagacaaaca aaagaatgga atcaaagtta acttcaaaat tagacacaac attgaagatg 540
gaagcgttca actagcagac cattatcaac aaaatactcc aattggcgat ggccctgtcc 600
ttttaccaga caaccattac ctgtccacac aatctgccct ttcgaaagat cccaacgaaa 660
agagagacca catggtcctt cttgagtttg taacagctgc tgggattaca catggcatgg 720
atgaactata caaatagcaa agacgttgtt tcatcgcgct attaccaaga aggttacttt 780
acttgttctt gcacatggac gcacgttgtg tgttcatata tatatatata tatatatata 840
tatatttgtg cttgttttca ttgtctctat agttaataca ttctattttt atcgttatat 900
ttgcattctc ttcgcataaa aacttcatga aaattcggca gaaaataagc aaattgtaaa 960
cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca 1020
ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag 1080
tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg 1140
gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt 1200
tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag 1260
agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc 1320
gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc 1380
gcttaatgcg ccgctacagg gcgcgtcgcg ccattcgcca ttcaggctgc gcaactgttg 1440
ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaagg ggggatgtgc 1500
tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac 1560
ggccagtgaa ttgtaatacg actcactata gggcgaattg gagctccacc gcggtggcgg 1620
ccgctctaga actagtggat cccccgggct gcaggaattc gatatcaagc ttatcgatac 1680
cgtcgacctc gagggggggc ccggtaccca gcttttgttc cctttagtga gggttaattc 1740
cgagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa 1800
ttccacacaa cataggagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga 1860
ggtaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt 1920
gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct 1980
cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 2040
cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 2100
acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 2160
ttttccatag gctcggcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 2220
ggcgaaaccc gacaggacta taaagatacc aggcgttccc ccctggaagc tccctcgtgc 2280
gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 2340
gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 2400
ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 2460
actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 2520
gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 2580
ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta 2640
ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 2700
gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 2760
tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 2820
tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 2880
aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 2940
aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctgcccgtcg 3000
tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 3060
gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 3120
agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 3180
aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 3240
gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 3300
caaggcgagt tacatgatcc cccatgttgt gaaaaaaagc ggttagctcc ttcggtcctc 3360
cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 3420
ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 3480
ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 3540
gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 3600
cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 3660
gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 3720
caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 3780
tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 3840
acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 3900
aagtgccacc tgggtccttt tcatcacgtg ctataaaaat aattataatt taaatttttt 3960
aatataaata tataaattaa aaatagaaag taaaaaaaga aattaaagaa aaaatagttt 4020
ttgttttccg aagatgtaaa agactctagg gggatcgcca acaaatacta ccttttatct 4080
tgctcttcct gctctcaggt attaatgccg aattgtttca tcttgtctgt gtagaagacc 4140
acacacgaaa atcctgtgat tttacatttt acttatcgtt aatcgaatgt atatctattt 4200
aatctgcttt tcttgtctaa taaatatata tgtaaagtac gctttttgtt gaaatttttt 4260
aaacctttgt ttattttttt ttcttcattc cgtaactctt ctaccttctt tatttacttt 4320
ctaaaatcca aatacaaaac ataaaaataa ataaacacag agtaaattcc caaattattc 4380
catcattaaa agatacgagg cgcgtgtaag ttacaggcaa gcgatccgtc ctaagaaacc 4440
attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg 4500
cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct 4560
tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc 4620
gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat 4680
aattccgttt taagagcttg gtgagcgcta ggagtcactg ccaggtatcg tttgaacacg 4740
gcattagtca gggaagtcat aacacagtcc tttcccgcaa ttttcttttt ctattactct 4800
tggcctcctc tagtacactc tatatttttt tatgcctcgg taatgatttt catttttttt 4860
tttccaccta gcggatgact cttttttttt cttagcgatt ggcattatca cataatgaat 4920
tatacattat ataaagtaat gtgatttctt cgaagaatat actaaaaaat gagcaggcaa 4980
gataaacgaa ggcaaagatg acagagcaga aagccctagt aaagcgtatt acaaatgaaa 5040
ccaagattca gattgcgatc tctttaaagg gtggtcccct agcgatagag cactcgatct 5100
tcccagaaaa agaggcagaa gcagtagcag aacaggccac acaatcgcaa gtgattaacg 5160
tccacacagg tatagggttt ctggaccata tgatacatgc tctggccaag cattccggct 5220
ggtcgctaat cgttgagtgc attggtgact tacacataga cgaccatcac accactgaag 5280
actgcgggat tgctctcggt caagctttta aagaggccct actggcgcgt ggagtaaaaa 5340
ggtttggatc aggatttgcg cctttggatg aggcactttc cagagcggtg gtagatcttt 5400
cgaacaggcc gtacgcagtt gtcgaacttg gtttgcaaag ggagaaagta ggagatctct 5460
cttgcgagat gatcccgcat tttcttgaaa gctttgcaga ggctagcaga attaccctcc 5520
acgttgattg tctgcgaggc aagaatgatc atcaccgtag tgagagtgcg ttcaaggctc 5580
ttgcggttgc cataagagaa gccacctcgc ccaatggtac caacgatgtt ccctccacca 5640
aaggtgttct tatgtagtga caccgattat ttaaagctgc agcatacgat atatatacat 5700
gtgtatatat gtatacctat gaatgtcagt aagtatgtat acgaacagta tgatactgaa 5760
gatgacaagg taatgcatca ttctatacgt gtcattctga acgaggcgcg ctttcctttt 5820
ttctttttgc tttttctttt tttttctctt gaactcgacg gatcatatgc ggtgtgaaat 5880
accgcacaga tgcgtaagga gaaaataccg catcaggggc gcgcccctcc ttgaaac 5937
<210> 6
<211> 844
<212> DNA
<213> Artificial sequence
<400> 6
ggagaaaata ccgcatcagg atactagcgt tgaatgttag cgtcaacaac aagaagttta 60
atgacgcgga ggccaaggca aaaagattcc ttgattacgt aagggagtta gaatcatttt 120
gaataaaaaa cacgcttttt cagttcgagt ttatcattat caatactgcc atttcaaaga 180
atacgtaaat aattaatagt agtgattttc ctaactttat ttagtcaaaa aattagcctt 240
ttaattctgc tgtaacccgt acatgcccaa aatagggggc gggttacaca gaatatataa 300
catcgtaggt gtctgggtga acagtttatt cctggcatcc actaaatata atggagcccg 360
ctttttaagc tggcatccag aaaaaaaaag aatcccagca ccaaaatatt gttttcttca 420
ccaaccatca gttcataggt ccattctctt agcgcaacta cagagaacag gggcacaaac 480
aggcaaaaaa cgggcacaac ctcaatggag tgatgcaacc tgcctggagt aaatgatgac 540
acaaggcaat tgacccacgc atgtatctat ctcattttct tacaccttct attaccttct 600
gctctctctg atttggaaaa agctgaaaaa aaaggttgaa accagttccc tgaaattatt 660
cccctacttg actaataagt atataaagac ggtaggtatt gattgtaatt ctgtaaatct 720
atttcttaaa cttcttaaat tctactttta tagttagtct tttttttagt tttaaaacac 780
caagaactta gtttcgaata aacacacata aacaaacaaa atgagtaaag gagaagaact 840
tttc 844
<210> 7
<211> 474
<212> DNA
<213> Artificial sequence
<400> 7
ggagaaaata ccgcatcagg agtgatcccc cacacaccat agcttcaaaa tgtttctact 60
ccttttttac tcttccagat tttctcggac tccgcgcatc gccgtaccac ttcaaaacac 120
ccaagcacag catactaaat ttcccctctt tcttcctcta gggtgtcgtt aattacccgt 180
actaaaggtt tggaaaagaa aaaagagacc gcctcgtttc tttttcttcg tcgaaaaagg 240
caataaaaat ttttatcacg tttctttttc ttgaaaattt ttttttttga tttttttctc 300
tttcgatgac ctcccattga tatttaagtt aataaacggt cttcaatttc tcaagtttca 360
gtttcatttt tcttgttcta ttacaacttt ttttacttct tgctcattag aaagaaagca 420
tagcaatcta atctaagttt taattacaaa atgagtaaag gagaagaact tttc 474
<210> 8
<211> 232
<212> DNA
<213> Artificial sequence
<220>
<221> misc_feature
<222> (178)..(183)
<223> n is a, c, g, or t
<400> 8
tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg ggcgcgcccc 60
tccttgaaac tgaaatttta gcatgtgatt aattaacttg taatattcta atcaagctta 120
taaaagagca ctgttgggcg tgagtggagg cgccggaaaa aagcatcgaa aaaatctnnn 180
nnnatgagta aaggagaaga acttttcact ggagttgtcc caattcttgt tg 232
<210> 9
<211> 6030
<212> DNA
<213> Artificial sequence
<220>
<221> misc_feature
<222> (178)..(183)
<223> n is a, c, g, or t
<400> 9
tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg ggcgcgcccc 60
tccttgaaac tgaaatttta gcatgtgatt aattaacttg taatattcta atcaagctta 120
taaaagagca ctgttgggcg tgagtggagg cgccggaaaa aagcatcgaa aaaatctnnn 180
nnnatgagta aaggagaaga acttttcact ggagttgtcc caattcttgt tgaattagat 240
ggtgatgtta atgggcacaa attttctgtc agtggagagg gtgaaggtga tgcaacatac 300
ggaaaactta cccttaaatt tatttgcact actggaaaac tacctgttcc atggccaaca 360
cttgtcacta ctttctctta tggtgttcaa tgcttttcaa gatacccaga tcatatgaaa 420
cggcatgact ttttcaagag tgccatgccc gaaggttatg tacaggaaag aactatattt 480
ttcaaagatg acgggaacta caagacacgt gctgaagtca agtttgaagg tgataccctt 540
gttaatagaa tcgagttaaa aggtattgat tttaaagaag atggaaacat tcttggacac 600
aaattggaat acaactataa ctcacacaat gtatacatca tggcagacaa acaaaagaat 660
ggaatcaaag ttaacttcaa aattagacac aacattgaag atggaagcgt tcaactagca 720
gaccattatc aacaaaatac tccaattggc gatggccctg tccttttacc agacaaccat 780
tacctgtcca cacaatctgc cctttcgaaa gatcccaacg aaaagagaga ccacatggtc 840
cttcttgagt ttgtaacagc tgctgggatt acacatggca tggatgaact atacaaatag 900
caaagacgtt gtttcatcgc gctattacca agaaggttac tttacttgtt cttgcacatg 960
gacgcacgtt gtgtgttcat atatatatat atatatatat atatatattt gtgcttgttt 1020
tcattgtctc tatagttaat acattctatt tttatcgtta tatttgcatt ctcttcgcat 1080
aaaaacttca tgaaaattcg gcagaaaata agcaaattgt aaacgttaat attttgttaa 1140
aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca 1200
aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga 1260
acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc 1320
agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc 1380
gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc 1440
cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg 1500
caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac 1560
agggcgcgtc gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc 1620
gggcctcttc gctattacgc cagctggcga aggggggatg tgctgcaagg cgattaagtt 1680
gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt gaattgtaat 1740
acgactcact atagggcgaa ttggagctcc accgcggtgg cggccgctct agaactagtg 1800
gatcccccgg gctgcaggaa ttcgatatca agcttatcga taccgtcgac ctcgaggggg 1860
ggcccggtac ccagcttttg ttccctttag tgagggttaa ttccgagctt ggcgtaatca 1920
tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatagga 1980
gccggaagca taaagtgtaa agcctggggt gcctaatgag tgaggtaact cacattaatt 2040
gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga 2100
atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc 2160
actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 2220
gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 2280
cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctcggc 2340
ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 2400
ctataaagat accaggcgtt cccccctgga agctccctcg tgcgctctcc tgttccgacc 2460
ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa 2520
tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 2580
cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 2640
aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 2700
gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 2760
agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 2820
ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 2880
cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 2940
tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 3000
aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 3060
tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 3120
atctgtctat ttcgttcatc catagttgcc tgactgcccg tcgtgtagat aactacgata 3180
cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 3240
gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 3300
gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 3360
tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc 3420
tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 3480
tcccccatgt tgtgaaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 3540
aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 3600
atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 3660
tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca 3720
catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 3780
aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 3840
tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 3900
gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 3960
tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 4020
tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgggtcc 4080
ttttcatcac gtgctataaa aataattata atttaaattt tttaatataa atatataaat 4140
taaaaataga aagtaaaaaa agaaattaaa gaaaaaatag tttttgtttt ccgaagatgt 4200
aaaagactct agggggatcg ccaacaaata ctacctttta tcttgctctt cctgctctca 4260
ggtattaatg ccgaattgtt tcatcttgtc tgtgtagaag accacacacg aaaatcctgt 4320
gattttacat tttacttatc gttaatcgaa tgtatatcta tttaatctgc ttttcttgtc 4380
taataaatat atatgtaaag tacgcttttt gttgaaattt tttaaacctt tgtttatttt 4440
tttttcttca ttccgtaact cttctacctt ctttatttac tttctaaaat ccaaatacaa 4500
aacataaaaa taaataaaca cagagtaaat tcccaaatta ttccatcatt aaaagatacg 4560
aggcgcgtgt aagttacagg caagcgatcc gtcctaagaa accattatta tcatgacatt 4620
aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg 4680
tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc 4740
cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct 4800
taactatgcg gcatcagagc agattgtact gagagtgcac cataattccg ttttaagagc 4860
ttggtgagcg ctaggagtca ctgccaggta tcgtttgaac acggcattag tcagggaagt 4920
cataacacag tcctttcccg caattttctt tttctattac tcttggcctc ctctagtaca 4980
ctctatattt ttttatgcct cggtaatgat tttcattttt ttttttccac ctagcggatg 5040
actctttttt tttcttagcg attggcatta tcacataatg aattatacat tatataaagt 5100
aatgtgattt cttcgaagaa tatactaaaa aatgagcagg caagataaac gaaggcaaag 5160
atgacagagc agaaagccct agtaaagcgt attacaaatg aaaccaagat tcagattgcg 5220
atctctttaa agggtggtcc cctagcgata gagcactcga tcttcccaga aaaagaggca 5280
gaagcagtag cagaacaggc cacacaatcg caagtgatta acgtccacac aggtataggg 5340
tttctggacc atatgataca tgctctggcc aagcattccg gctggtcgct aatcgttgag 5400
tgcattggtg acttacacat agacgaccat cacaccactg aagactgcgg gattgctctc 5460
ggtcaagctt ttaaagaggc cctactggcg cgtggagtaa aaaggtttgg atcaggattt 5520
gcgcctttgg atgaggcact ttccagagcg gtggtagatc tttcgaacag gccgtacgca 5580
gttgtcgaac ttggtttgca aagggagaaa gtaggagatc tctcttgcga gatgatcccg 5640
cattttcttg aaagctttgc agaggctagc agaattaccc tccacgttga ttgtctgcga 5700
ggcaagaatg atcatcaccg tagtgagagt gcgttcaagg ctcttgcggt tgccataaga 5760
gaagccacct cgcccaatgg taccaacgat gttccctcca ccaaaggtgt tcttatgtag 5820
tgacaccgat tatttaaagc tgcagcatac gatatatata catgtgtata tatgtatacc 5880
tatgaatgtc agtaagtatg tatacgaaca gtatgatact gaagatgaca aggtaatgca 5940
tcattctata cgtgtcattc tgaacgaggc gcgctttcct tttttctttt tgctttttct 6000
ttttttttct cttgaactcg acggatcata 6030
<210> 10
<211> 125
<212> DNA
<213> Artificial sequence
<400> 10
cctccttgaa actgaaattt tagcatgtga ttaattaact tgtaatattc taatcaagct 60
tataaaagag cactgttggg cgtgagtgga ggcgccggaa aaaagcatcg aaaaaatctt 120
caaca 125
<210> 11
<211> 125
<212> DNA
<213> Artificial sequence
<400> 11
cctccttgaa actgaaattt tagcatgtga ttaattaact tgtaatattc taatcaagct 60
tataaaagag cactgttggg cgtgagtgga ggcgccggaa aaaagcatcg aaaaaatctc 120
caacc 125
<210> 12
<211> 125
<212> DNA
<213> Artificial sequence
<400> 12
cctccttgaa actgaaattt tagcatgtga ttaattaact tgtaatattc taatcaagct 60
tataaaagag cactgttggg cgtgagtgga ggcgccggaa aaaagcatcg aaaaaatctg 120
caaag 125
<210> 13
<211> 125
<212> DNA
<213> Artificial sequence
<400> 13
cctccttgaa actgaaattt tagcatgtga ttaattaact tgtaatattc taatcaagct 60
tataaaagag cactgttggg cgtgagtgga ggcgccggaa aaaagcatcg aaaaaatcta 120
taacc 125
<210> 14
<211> 125
<212> DNA
<213> Artificial sequence
<400> 14
cctccttgaa actgaaattt tagcatgtga ttaattaact tgtaatattc taatcaagct 60
tataaaagag cactgttggg cgtgagtgga ggcgccggaa aaaagcatcg aaaaaatcta 120
cgaag 125
<210> 15
<211> 125
<212> DNA
<213> Artificial sequence
<400> 15
cctccttgaa actgaaattt tagcatgtga ttaattaact tgtaatattc taatcaagct 60
tataaaagag cactgttggg cgtgagtgga ggcgccggaa aaaagcatcg aaaaaatcta 120
tctag 125
<210> 16
<211> 125
<212> DNA
<213> Artificial sequence
<400> 16
cctccttgaa actgaaattt tagcatgtga ttaattaact tgtaatattc taatcaagct 60
tataaaagag cactgttggg cgtgagtgga ggcgccggaa aaaagcatcg aaaaaatctg 120
tcaac 125
<210> 17
<211> 125
<212> DNA
<213> Artificial sequence
<400> 17
cctccttgaa actgaaattt tagcatgtga ttaattaact tgtaatattc taatcaagct 60
tataaaagag cactgttggg cgtgagtgga ggcgccggaa aaaagcatcg aaaaaatcta 120
ctaca 125
<210> 18
<211> 125
<212> DNA
<213> Artificial sequence
<400> 18
cctccttgaa actgaaattt tagcatgtga ttaattaact tgtaatattc taatcaagct 60
tataaaagag cactgttggg cgtgagtgga ggcgccggaa aaaagcatcg aaaaaatctc 120
caagc 125
<210> 19
<211> 125
<212> DNA
<213> Artificial sequence
<400> 19
cctccttgaa actgaaattt tagcatgtga ttaattaact tgtaatattc taatcaagct 60
tataaaagag cactgttggg cgtgagtgga ggcgccggaa aaaagcatcg aaaaaatctg 120
caata 125
<210> 20
<211> 125
<212> DNA
<213> Artificial sequence
<400> 20
cctccttgaa actgaaattt tagcatgtga ttaattaact tgtaatattc taatcaagct 60
tataaaagag cactgttggg cgtgagtgga ggcgccggaa aaaagcatcg aaaaaatctt 120
cagca 125
<210> 21
<211> 125
<212> DNA
<213> Artificial sequence
<400> 21
cctccttgaa actgaaattt tagcatgtga ttaattaact tgtaatattc taatcaagct 60
tataaaagag cactgttggg cgtgagtgga ggcgccggaa aaaagcatcg aaaaaatctc 120
accaa 125
<210> 22
<211> 125
<212> DNA
<213> Artificial sequence
<400> 22
cctccttgaa actgaaattt tagcatgtga ttaattaact tgtaatattc taatcaagct 60
tataaaagag cactgttggg cgtgagtgga ggcgccggaa aaaagcatcg aaaaaatcta 120
tcgtc 125
<210> 23
<211> 125
<212> DNA
<213> Artificial sequence
<400> 23
cctccttgaa actgaaattt tagcatgtga ttaattaact tgtaatattc taatcaagct 60
tataaaagag cactgttggg cgtgagtgga ggcgccggaa aaaagcatcg aaaaaatcta 120
ttatt 125
Claims (9)
1. The saccharomyces cerevisiae artificial promoter mutant library is obtained by randomly mutating all or part of 1 st to 6 th positions from the 3' end of a nucleotide sequence of a saccharomyces cerevisiae artificial promoter UASF-E-C-core 1.
2. The saccharomyces cerevisiae artificial promoter mutant library of claim 1, wherein: the nucleotide sequence of the Saccharomyces cerevisiae artificial promoter UASF-E-C-core1 is shown as 29-153 th site of SEQ ID No. 4.
3. The saccharomyces cerevisiae artificial promoter mutant is any one of the following mutants:
(A1) the nucleotide sequence is shown as SEQ ID No. 19;
(A2) the nucleotide sequence is shown as SEQ ID No. 13;
(A3) the nucleotide sequence is shown as SEQ ID No. 11;
(A4) the nucleotide sequence is shown as SEQ ID No. 10;
(A5) the nucleotide sequence is shown as SEQ ID No. 12;
(A6) the nucleotide sequence is shown as SEQ ID No. 14;
(A7) the nucleotide sequence is shown as SEQ ID No. 20;
(A8) the nucleotide sequence is shown as SEQ ID No. 21;
(A9) the nucleotide sequence is shown as SEQ ID No. 16;
(A10) the nucleotide sequence is shown as SEQ ID No. 18;
(A11) the nucleotide sequence is shown as SEQ ID No. 15;
(A12) the nucleotide sequence is shown as SEQ ID No. 17;
(A13) the nucleotide sequence is shown as SEQ ID No. 22;
(A14) the nucleotide sequence is shown as SEQ ID No. 23.
4. The use of the Saccharomyces cerevisiae artificial promoter mutant of claim 3 to promote the expression of a gene of interest in Saccharomyces cerevisiae.
5. The use of the Saccharomyces cerevisiae artificial promoter mutant of claim 3 for increasing the expression level of a target gene in Saccharomyces cerevisiae.
6. The expression cassette containing the saccharomyces cerevisiae artificial promoter mutant as claimed in claim 3, which consists of the saccharomyces cerevisiae artificial promoter mutant, a target gene and a terminator which are connected in sequence.
7. A recombinant vector comprising the expression cassette of claim 6.
8. A recombinant Saccharomyces cerevisiae containing the recombinant vector of claim 7.
9. Use of the saccharomyces cerevisiae artificial promoter mutant library according to claim 1 or 2 or the saccharomyces cerevisiae artificial promoter mutant according to claim 3 or the expression cassette according to claim 6 or the recombinant vector according to claim 7 or the recombinant saccharomyces cerevisiae according to claim 8 for gene expression regulation of a metabolic pathway of saccharomyces cerevisiae.
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