A set of high-strength artificial small promoters of Saccharomyces cerevisiae

Abstract

The present invention discloses a group of high-strength artificial small promoters of Saccharomyces cerevisiae. The present invention provides a library of artificial promoter mutants of Saccharomyces cerevisiae, which is obtained by random mutation of all or part of the 1-6th positions of the nucleotide sequence of the artificial promoter UASF-E-C-core1 of Saccharomyces cerevisiae from the 3' end. The artificial promoter mutants of Saccharomyces cerevisiae provided by the present invention are specifically any one of the following: 14 promoter mutants whose nucleotide sequences are shown in SEQ ID No.10-23. The present invention provides a group of high-strength artificial small promoters of Saccharomyces cerevisiae, wherein the strongest small promoter has a strength of about 3.3 times that of the natural strong promoter TDH3 and 7 times that of the starting promoter. The strength range of artificial small promoters is significantly broadened, which contributes to the better application of promoter engineering in gene expression regulation of metabolic pathways of Saccharomyces cerevisiae.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a group of high-strength artificial small promoters of Saccharomyces cerevisiae. Background Art

[0002] As a eukaryotic model organism, Saccharomyces cerevisiae is widely used in synthetic biology due to its advantages such as rapid growth, easy cultivation, high safety, and clear genetic background. Currently, Saccharomyces cerevisiae cell factories have been developed for the production of recombinant proteins, biofuels, and fine chemicals. Promoters are important transcriptional regulatory elements and play an important role in the regulation of gene expression in gene pathways and metabolic pathways of Saccharomyces cerevisiae. The construction of metabolic pathways often involves the expression of multiple genes, and the expression levels of these genes span several orders of magnitude. The fine regulation of these genes makes it challenging to select suitable promoters.

[0003] At present, a series of constitutive promoters endogenous to yeast are commonly used to express foreign genes in Saccharomyces cerevisiae cell factories, such as pPGK1, pTEF1, pFBA11, pGPD / pTDH3 and the inducible strong promoter pGAL1. However, it is worth noting that these endogenous promoters of yeast 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 yeast has developed rapidly, especially in the model microorganism Saccharomyces cerevisiae. However, the problem is that the promoter skeletons currently used for promoter modification are all from endogenous promoters of yeast. The new promoters obtained are generally large and have poor sequence diversity, resulting in poor stability after multiple recombination. The promoters obtained by hybridization have significantly improved this problem of poor sequence diversity, but there are also defects such as the obtained promoters are too large and rely on endogenous sequences such as UAS (upstream activation sequence) to function. In response to these problems, Alper et al. obtained a series of small promoters of 125bp in size that are independent of endogenous promoters of yeast by total synthesis. Its length is shortened by 80% compared with the integrated promoter. However, the strongest small promoter strength reported is about 70% of the strong endogenous yeast promoter TDH3, and this strength range is still difficult to meet the needs of researchers. Summary of the invention

[0004] The purpose of the present invention is to provide a group of high-strength artificial small promoters of Saccharomyces cerevisiae.

[0005] In a first aspect, the present invention claims a Saccharomyces cerevisiae artificial promoter mutant library.

[0006] The Saccharomyces cerevisiae artificial promoter mutant library claimed in the present invention is obtained by random mutation of all or part of the nucleotide sequence of the Saccharomyces cerevisiae artificial promoter UASF-EC-core1 at positions 1 to 6 from the 3' end.

[0007] The nucleotide sequence of the artificial promoter UASF-EC-core1 of Saccharomyces cerevisiae is shown in positions 29-153 of SEQ ID No.4.

[0008] In a specific embodiment of the present invention, the nucleotide sequence of the artificial promoter UASF-EC-core1 of Saccharomyces cerevisiae (positions 29-153 of SEQ ID No. 4) from the 1st to 6th positions from the 3' end are randomly mutated by PCR, and the nucleotide sequence of the artificial promoter UASF-EC-core1 of Saccharomyces cerevisiae (positions 29-153 of SEQ ID No. 4) from the 1st to 6th positions from the 3' end in the primers is NNNNNN, and N is A, T, C or G.

[0009] In a second aspect, the present invention claims a Saccharomyces cerevisiae artificial promoter mutant.

[0010] The artificial promoter mutant of Saccharomyces cerevisiae claimed in the present invention may be any of the following:

[0011] (A1) The nucleotide sequence is shown in SEQ ID No. 19 (corresponding to Figure 1 528 of );

[0012] (A2) The nucleotide sequence is shown in SEQ ID No. 13 (corresponding to Figure 1 510);

[0013] (A3) The nucleotide sequence is shown in SEQ ID No. 11 (corresponding to Figure 1 503 in );

[0014] (A4) The nucleotide sequence is shown in SEQ ID No. 10 (corresponding to Figure 1 501 in the

[0015] (A5) The nucleotide sequence is shown in SEQ ID No. 12 (corresponding to Figure 1 507);

[0016] (A6) The nucleotide sequence is shown in SEQ ID No. 14 (corresponding to Figure 1 512 in );

[0017] (A7) The nucleotide sequence is shown in SEQ ID No. 20 (corresponding to Figure 1 532 in );

[0018] (A8) The nucleotide sequence is shown in SEQ ID No. 21 (corresponding to Figure 1 536);

[0019] (A9) The nucleotide sequence is shown in SEQ ID No. 16 (corresponding to Figure 1 517);

[0020] The nucleotide sequence of (A10) is shown in SEQ ID No. 18 (corresponding to Figure 1 525 in );

[0021] The nucleotide sequence of (A11) is shown in SEQ ID No. 15 (corresponding to Figure 1 514);

[0022] The nucleotide sequence of (A12) is shown in SEQ ID No. 17 (corresponding to Figure 1 523 in );

[0023] The nucleotide sequence of (A13) is shown in SEQ ID No. 22 (corresponding to Figure 1 540 in );

[0024] (A14) The nucleotide sequence is shown in SEQ ID No. 23 (corresponding to Figure 1 545 in the above table).

[0025] In a third aspect, the present invention claims protection for the use of the artificial promoter mutant of Saccharomyces cerevisiae described in the second aspect above in promoting the expression of a target gene in Saccharomyces cerevisiae.

[0026] In a fourth aspect, the present invention claims protection for the use of the artificial promoter mutant of Saccharomyces cerevisiae described in the second aspect above in increasing the expression level of a target gene in Saccharomyces cerevisiae.

[0027] In a fifth aspect, the present invention claims protection for an expression cassette comprising the artificial promoter mutant of Saccharomyces cerevisiae described in the second aspect above.

[0028] The expression cassette consists of the artificial promoter mutant of Saccharomyces cerevisiae, the target gene and the terminator connected in sequence.

[0029] In a specific embodiment of the present invention, the terminator is specifically an SPG5 terminator.

[0030] Furthermore, the nucleotide sequence of the SPG5 terminator is shown in SEQ ID No. 23-213.

[0031] In a sixth aspect, the present invention claims protection for a recombinant vector containing the expression cassette described in the fifth aspect above.

[0032] In a specific embodiment of the present invention, the backbone vector of the recombinant vector is the universal expression vector pRS313 of Saccharomyces cerevisiae.

[0033] In a seventh aspect, the present invention claims protection for a recombinant Saccharomyces cerevisiae containing the recombinant vector described in the sixth aspect above.

[0034] In a specific embodiment of the present invention, the recipient Saccharomyces cerevisiae is specifically BY4742. Of course, it is also theoretically feasible to use other Saccharomyces cerevisiae.

[0035] In an eighth aspect, the present invention claims protection for the use of the artificial promoter mutant library of Saccharomyces cerevisiae described in the first aspect, the artificial promoter mutant of Saccharomyces cerevisiae described in the second aspect, the expression cassette described in the fifth aspect, the recombinant vector described in the sixth aspect, or the recombinant Saccharomyces cerevisiae described in the seventh aspect in the regulation of gene expression in metabolic pathways of Saccharomyces cerevisiae.

[0036] The present invention provides a group of high-strength artificial small promoters of Saccharomyces cerevisiae, among which the strongest small promoter is about 3.3 times stronger than the natural strong promoter TDH3 and 7 times stronger than the starting promoter. This significantly broadens the strength range of artificial small promoters and contributes to the better application of promoter engineering in gene expression regulation of metabolic pathways of Saccharomyces cerevisiae. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Comparison of the strength of a high-strength artificial small promoter and a strong endogenous yeast promoter. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0039] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0040] Example 1: Construction of YPL001 plasmid as blank control

[0041] 1. Amplification of GFP gene and SPG5 terminator

[0042] The GFP gene was synthesized by GenScript, and the synthetic plasmid (the GFP gene shown in positions 25-741 of SEQ ID No. 1 was inserted into pUC57) was used as a template, and PCR amplification was performed using primers GFPzeo-up2 / GFPzeo-down2 (see Table 1). The amplification system was TAKARA DNA polymerase 5×Buffer 10μl, Dntp mix 4μl, primers (see Table 1) 1μl each, cDNA, template 0.5μl, PrimerSTAR HS polymerase (2.5U / μL) 0.5μl, add distilled water to a total volume of 50μl. Amplification conditions are: 98℃ pre-denaturation for 2 minutes (1 cycle); 98℃ denaturation for 10 seconds, 56℃ annealing for 15 seconds, 72℃ extension for 1 minute (30 cycles); 72℃ extension for 8 minutes (1 cycle). The obtained amplification product was named GFP (SEQ ID No.1, the fragment contains 24bp plasmid pRS313 homology region, 20bp SPG5 homology region, and GFP gene sequence). The obtained PCR amplification product was purified using the PCR product purification kit of Shanghai Shenggong Biotechnology Co., Ltd., and the purified product was used for later use. The terminator SPG5 was amplified using the genome of Saccharomyces cerevisiae BY4742 as a template using primers SPG5-up2 / SPG5-down2 (see Table 1) (the method is the same as above), and the amplified product was named SPG5 (SEQ ID No. 2, the fragment contains a 22 bp GFP homology region, a 22 bp plasmid pRS313 homology region, and an SPG5 terminator sequence). The obtained PCR amplification product was purified using a PCR product purification kit from Shanghai Shenggong Biotechnology Co., Ltd., and the purified product was used for later use.

[0043] 2. PCR amplification of expression vector

[0044] The target expression vector was amplified using primers 313-up2 / 313-down2 (see Table 1) using the universal expression vector pRS313 of Saccharomyces cerevisiae (from Addgene, containing His3 marker) as a template. The amplification system and conditions were the same as those in step 1 of Example 1. The amplified product was named pRS313-His-vector (SEQ ID No. 3, the fragment was the pRS313 vector backbone, with 24 bp GFP homology region and 22 bp SPG5 homology region). The PCR amplified product was recovered and purified using the PCR gel recovery kit of Shanghai Shenggong Biotechnology Co., Ltd., and the purified product was set aside.

[0045] 3. Construction of YPL001 plasmid using CPEC method

[0046] CPEC system: Phusion High-Fidelity DNA polymerase 5×Buffer 5μl, Dntp mix 1, DMSO 0.75μl, two purified fragments (GFP gene and SPG5 terminator amplified purified fragments obtained in step 1) and pRS313-His-vector obtained in step 2 were added in equal moles, Phusion High-Fidelity DNA polymerase (2U / μL) 0.5μl, and distilled water was added to a total volume of 25μl. Amplification conditions were: 98℃ 30 seconds (1 cycle); 98℃ 10 seconds, 55℃ 30 seconds, 72℃ 15 seconds (15 cycles); 72℃ extension for 10 minutes (1 cycle). 5μl of PCR product was transferred into Trans1-T1 competent cells and placed on ice for 30 minutes, heat-shocked at 42℃ for 30 seconds, and immediately placed on ice for 2 minutes. Add 800 μl LB medium, incubate at 250 rpm and 37 °C for 1 hour, apply the bacterial solution on the LB plate containing ampicillin, and after overnight culture, use primers YpL001-YZ-up / YpL001-YZ-down (see Table 1) for PCR screening, and the positive clone band size is 1504 bp. The positive clones were cultured in liquid, and the positive clone plasmids were extracted for sequencing verification. The sequencing results showed that the target fragment was inserted into the vector pRS313-His to obtain plasmid YPL001. Use the plasmid extraction kit of Aisjin Company to extract the target plasmid YPL001 for use.

[0047] Table 1. Primers for constructing YPL001 and YPL002 plasmids

[0048]

[0049]

[0050] Example 2: Construction of YPL002 plasmid as a positive control

[0051] The strongest artificial small promoter UASF-EC-core1 (SEQ ID No. 4, position 29-153) reported by the Alper team was synthesized by GenScript. Primers Core11-up / Core11-down (see Table 1) were used for amplification, and the method was the same as step 1 of Example 1 to obtain a PCR product named UASF-EC-core1 (SEQ ID No. 4, the fragment contained a 20bp plasmid pRS313 homology region, a 24bp GFP homology region, and UASF-EC-core1). The YPL001 plasmid constructed in Example 1 was used as a template, and primers YpL001-up / YpL001-down (see Table 1) were used for PCR amplification to obtain the obtained amplified product named pRS313-His-GFP-SPG5 (SEQ ID No. 5, the fragment contained a pRS313 vector backbone, a GFP gene, and an SPG5 terminator). The obtained PCR amplification product was recovered and purified using a PCR gel recovery kit of Shanghai Shenggong Biotechnology Co., Ltd., and the purified product was used for standby. The above two fragments were connected by CPEC method (the method is the same as step 3 of Example 1). 5 μl of PCR product was transferred into Trans1-T1 competent cells and iced for 30 minutes, heat-shocked at 42°C for 30 seconds, and immediately placed on ice for 2 minutes. 800 μl of LB medium was added, incubated at 250rpm and 37°C for 1 hour, and the bacterial solution was applied to an LB plate containing ampicillin. After overnight culture, PCR screening was performed with primers Core11-up / GFPzeo-down2 (see Table 1), and the positive clone band size was 890 bp. The positive clones were liquid cultured and the positive clones were liquid cultured. The positive clone plasmids were extracted for sequencing verification. The sequencing results showed that the target fragment was inserted into the vector pRS313 to obtain plasmid YPL002. The plasmid extraction kit of Aisijin was used to extract the target plasmid YPL002 for standby use.

[0052] Example 3: Construction of strains Yp1001, Yp1002, Yp1007, and Yp1008

[0053] 1. Construction of YPL007 and YPL008 plasmids

[0054] 1. Amplification of pGPD / pTDH3 and pTEF1 promoters

[0055] The genome of Saccharomyces cerevisiae BY4742 was used as a template, and PCR amplification was performed using primers GPD-Dai-up / GPD-Dai-down and TEF1-Dai-up / TEF1-Dai-down (see Table 2). The amplification system was TAKARA DNA polymerase 5×Buffer 10μl, Dntp mix 4μl, primers 1μl each, cDNA, template 0.5μl, PrimerSTAR HS polymerase (2.5U / μL) 0.5μl, distilled water was added to the total volume of 50μl. Amplification conditions were: 98℃ pre-denaturation for 2 minutes (1 cycle); 98℃ denaturation for 10 seconds, 56℃ annealing for 15 seconds, 72℃ extension for 1 minute (30 cycles); 72℃ extension for 8 minutes (1 cycle). The amplified products were named pTDH3 (SEQ ID No.6, the fragment contains 20bp plasmid pRS313 homology region, 24bp GFP homology region and TDH3 promoter sequence), pTEF1 (SEQ ID No.7, the fragment contains 20bp plasmid pRS313 homology region, 24bp GFP homology region and TEF1 promoter). The obtained PCR amplification product was purified using a PCR product purification kit from Shanghai Shenggong Biotechnology Co., Ltd., and the purified product was set aside.

[0056] Table 2. Primers for constructing YPL007 and YPL008 plasmids

[0057] Primer name Sequence (5'-3') GPD-Dai-up GGAGAAAATACCGCATCAGGATACTAGCGTTGAATGTTAGCG GPD-Dai-down GAAAAGTTCTTCTCCTTTACTCATTTTGTTTGTTTATGTGTGTTTTATTC TEF1-Dai-up GGAGAAAATACCGCATCAGGAGTGATCCCCCACACACCATAG TEF1-Dai-down GAAAAGTTCTTCTCCTTTACTCATTTTGTAATTAAAACTTAGATTAG YpL001-YZ-up CCAAAGGTGTTCTTATGTAGTG YpL001-YZ-down CTTTAGGGTTCCGATTTAGTGC

[0058] 2. Amplify the expression vector

[0059] Using YPL001 constructed in Example 1 as a template, PCR amplification was performed using primers YpL001-YZ-up / YpL001-YZ-down (see Table 1), and the obtained amplified product was named pRS313-His-GFP-SPG5 (SEQ ID No. 5, the fragment contains the pRS313 vector backbone, GFP gene and SPG5 terminator). The obtained PCR amplified product was recovered and purified using a PCR gel recovery kit of Shanghai Shenggong Biotechnology Co., Ltd., and the purified product was set aside.

[0060] 3. Construction of YPL007 and YPL008 plasmids using CPEC method

[0061] CPEC system: Phusion High-Fidelity DNA polymerase 5×Buffer 5μl, Dntp mix 1, DMSO 0.75μl, purified fragment (pTDH3 or pTEF1 obtained in step 1) and pRS313-His-GFP-SPG5 were added in equal moles, Phusion High-Fidelity DNA polymerase (2U / μL) 0.5μl, and distilled water was added to a total volume of 25μl. Amplification conditions were: 98℃ 30 seconds (1 cycle); 98℃ 10 seconds, 55℃ 30 seconds, 72℃ 15 seconds (15 cycles); 72℃ extension for 10 minutes (1 cycle). 5μl of PCR product was transferred into Trans1-T1 competent cells and placed on ice for 30 minutes, heat-shocked at 42℃ for 30 seconds, and immediately placed on ice for 2 minutes. Add 800 μl LB medium, incubate at 250 rpm and 37 °C for 1 hour, apply the bacterial solution on the LB plate containing ampicillin, and after overnight culture, use primers GPD-Dai-up or TEF1-Dai-up / GFPzeo-down2 (see Table 1 and Table 2) for PCR screening, and the positive clone band size is 1557 bp or 1187 bp. The positive clones were cultured in liquid culture, and the positive clone plasmids were extracted for sequencing verification. The sequencing results showed that the target fragment was inserted into the vector pRS313-His-GFP-SPG5, and the plasmids YPL007 (corresponding to pTDH3) and YPL008 (corresponding to pTEF1) were obtained. Use the plasmid extraction kit of Aisijin Company to extract the target plasmid for use.

[0062] 2. Construction of Ypl001, Ypl002, Ypl007, and Ypl008 strains

[0063] Starting from the yeast BY4742 (saccharomyces cerevisiae BY4742, described in Carrie bakerbrachmann et al., 1998, Yeast, 14: 115-132, available to the public from Tianjin Industrial Biotechnology Institute), SD-Ura-His-Leu-Trp (Beijing Fanjinuo (Functional Genomics) Technology Co., Ltd.), 2% glucose, 0.005% His., 0.01% Leu., 0.01% Ura., 0.01% Trp. (each percentage sign represents g / 100mL). Take 1mL (OD about 0.6-1.0) and dispense it into a 1.5mL EP tube, centrifuge at 4°C, 10000g for 1min, discard the supernatant, wash the precipitate with sterile water (4°C), centrifuge under the same conditions, and discard the supernatant. Add 1 mL of treatment solution (10 mM LiAc; 10 mM DTT; 0.6 M sorbitol; 10 mM Tris-HCl (pH 7.5, add DTT when using the treatment solution) to the cells and place at 25°C for 20 min. Centrifuge, discard the supernatant, add 1 mL of 1 M sorbitol (sterilized by 0.22 μm water membrane) to the cells and resuspend, centrifuge, discard the supernatant (resuspend twice with 1 M sorbitol), and the final volume is about 80 μL. 1 μL of YPL001, YPL002, YPL007 and YPL008 plasmids were added respectively, mixed and transferred to an electroporation cup, electroshocked at 2.7 kv for 5.6 ms, and 1 mL of 1 M sorbitol was added. The culture was revived at 30°C for 1 h and spread on a screening medium plate (formula: 0.8% yeast selection medium SD-Ura-His-Leu-Trp, 2% glucose, 0.01% Leu., 0.01% Ura., 0.01% Trp. (each percentage sign represents g / 100 mL). The screening culture conditions were: 30°C, cultured for more than 36 h, and the strains were named Ypl001, Ypl002, Ypl007 and Ypl008 according to the different transferred plasmids.

[0064] Example 4: Construction of an artificial small promoter Kozak library and screening to obtain high-strength artificial small promoters

[0065] 1. Construction of Saccharomyces cerevisiae artificial promoter Kozak library

[0066] 1. Amplification of YPL-Kozak-mut fragment

[0067] The strongest artificial small promoter UASF-EC-core1 (SEQ ID No. 4, position 29-153) reported by the Alper team was synthesized by GenScript and PCR amplified using primers CoreKM-up / Core11 KM-down (see Table 3). The amplification system and conditions were the same as those in step 1 of Example 1. The obtained amplified product was named YPL-Kozak-mut (SEQ ID No. 8, the fragment includes 50bp plasmid pRS313 homology arms, UASF-EC-core1 and Kozakmut). The obtained PCR amplification product was recovered and purified using a PCR gel recovery kit of Shanghai Shenggong Biotechnology Co., Ltd., and the purified product was used for later use.

[0068] 2. Homologous recombination in Saccharomyces cerevisiae to construct a Saccharomyces cerevisiae artificial promoter Kozak library

[0069] Prepare the competent cells of Saccharomyces cerevisiae BY4742 (the method is the same as step 2 of Example 3). Add 2 μL of YPL-Kozak-mut fragment and pRS313-His-GFP-SPG5 vector fragment respectively, transfer to an electroporation cup after mixing, electroshock at 2.7 kv for 5.6 ms, add 1 mL of 1 M sorbitol, resuscitate at 30°C for 1 h, and spread on a screening medium plate (formula: 0.8% yeast selection medium SD-Ura-His-Leu-Trp, 2% glucose, 0.01% Leu., 0.01% Ura., 0.01% Trp. (each percentage sign represents g / 100 mL). The screening culture conditions are: 30°C, culture for more than 36 h.

[0070] Among the recombinant Saccharomyces cerevisiae obtained by screening, a recombinant vector formed by homologous recombination of the YPL-Kozak-mut fragment and the pRS313-His-GFP-SPG5 vector fragment is contained. The nucleotide sequence of the recombinant vector is shown in SEQ ID No.9, wherein N is A, C, T or G.

[0071] Table 3. Primers for constructing Kozak library

[0072]

[0073]

[0074] Note: N represents A or T or C or G.

[0075] 2. Screening of Kozak Library of Saccharomyces cerevisiae Artificial Promoter

[0076] 1. Flow cytometry screening of the Saccharomyces cerevisiae artificial promoter Kozak library

[0077] The strains obtained in the above steps were collected into 1.5 ml centrifuge tubes, washed twice with sterilized PBS buffer, sorted by flow cytometry, and 2% of the strains with higher fluorescence intensity were collected and cultured in 96 deep-well plates (with corresponding liquid culture medium). The screening and culture conditions were: temperature 30°C, humidity 80%, and rotation speed 800 rpm for more than 24 hours.

[0078] 2. 96-well plate rescreening

[0079] Use a 200μL dispenser to transfer the bacterial solution from the 96-well plate to a 96-well plate with a white bottom and black edges. Then perform fluorescence measurement with an excitation light of 395nm, an absorption light of 507nm, and OD measurement with a wavelength of 600nm. Calculate the fluorescence / OD600 ratio, and perform test tube rescreening on 38 clones that are significantly enhanced.

[0080] 3. Test tube rescreening

[0081] The 38 clones screened above were added to the corresponding liquid selection medium (formula: liquid yeast selection medium SD-Ura-His-Leu-Trp, 2% glucose; each percentage sign represents g / 100 mL) to prepare seed solution (30°C, 250 rpm, 12 h), and then the initial OD was calculated. 600nm =0.1 for transfer and continued cultivation (30°C, 250rpm, 12h), followed by fluorescence measurement, with excitation light at 395nm, absorption light at 507nm and OD measurement, with a wavelength of 600nm. Calculate the fluorescence / OD600 ratio, and finally retain fourteen small promoters, and the corresponding strains are named 501, 503, 507, 510, 512, 514, 517, 523, 525, 528, 532, 536, 540, 545 (SEQ ID No.10-SEQ ID No.23)

[0082] 3. Comparison of the strength of high-strength artificial small promoter and yeast endogenous strong promoter

[0083] Fluorescence measurement experiment: 4 strains Ypl001, Ypl002, Ypl007, Ypl008 and 14 strains obtained by screening in step 2 were activated in the corresponding solid selection medium (formula: solid yeast screening medium SD-Ura-His-Leu-Trp, 2% glucose, 1.5% agar; each percentage sign represents g / 100 mL), a total of 18 Saccharomyces cerevisiae strains were prepared in the corresponding liquid selection medium (formula: liquid yeast screening medium SD-Ura-His-Leu-Trp, 2% glucose; each percentage sign represents g / 100 mL) (30°C, 250rpm, 12h), and then the culture was transferred and continued according to the initial OD600nm=0.1 (30°C, 250rpm, 12h), and then the fluorescence measurement was performed under the conditions of excitation light 395nm, absorption light 507nm and OD measurement, and the measurement conditions were wavelength 600nm. The results are shown in Figure 1 .

[0084] The above test results show that by building a library of artificial small promoter Kozak regions and undergoing multiple rounds of screening, 14 high-strength artificial small promoters were finally obtained. Except for 545, whose activity was slightly lower than the endogenous strong promoters pTDH3 and pTEF1 of Saccharomyces cerevisiae, the activities of the remaining 13 artificial small promoters were higher than pTDH3 and pTEF1. Among them, the strongest artificial small promoter 528 was 3.3 times stronger than pTDH3 and pTEF1, and 7 times stronger than the strongest small promoter reported by Alper, which significantly broadened the dynamic range of artificial small promoters of Saccharomyces cerevisiae.

[0085] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims. <110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences <120> A set of high-strength artificial small promoters of Saccharomyces cerevisiae <130> GNCLN210181 <160> twenty three <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 ttgtaacag ctgctgggat tacacatggc 720 atggatgac tatacaata gcaagacgt tgtttcatcg c 761 <210> 2 <211> 258 <212> DNA <213> Artificial Sequence <400> 2 catggatgaa ctatacaaat agcaagacg ttgtttcatc gcgctattac caagaaggtt 60 actttacttg ttcttgcaca tggacgcacg tgtgtgttc atatatatat atatatatat 120 atatatatat ttgtgcttgt ttcattgtc tctatagtta atacattcta tttttatcgt 180 tatatttgca ttctctcgc aaaaactt catgaaaatt cggcagaaaa taagcaatt 240 gtaaacgtta atattttg 258 <210> 3 <211> 5013 <212> DNA <213> Artificial Sequence <400> 3 gaaaattcgg cagaaataa gcaattgta aacgttaata tttgttaaa attcgcgtta 60 aatttttgtt aaatcagctc atttttac caataggccg aaatcggcaa aatcccttat 120 aaatcaaag atagaccga gatagggttg agtgttgttc cagtttggaa caagtcca 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 gcgcagaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 1860 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga tttcaaaaa ggatcttcac 1920 ctagatcctt ttaaattaaa aatgaagtttt 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 tagttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg 2340 tatggcttca ttcagctccg gttcccacg atcaggcga gttacatgat ccccatgtt 2400 gtgaaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc 2460 agtgttatca ctcatggtta tggcagcact gcatattct cttactgtca tgccatccgt 2520 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg 2580 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac 2640 tttaaaagtg ctcatcattg gaaacgttc ttcggggcga aaactctca ggatcttacc 2700 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt 2760 tactttcacc agcgttctg ggtgagcaa aacaggaagg caaatgccg CAaaaaggg 2820 aaagggcg acacggaat gttgaatact catactctc cttttcaat attattgaag 2880 catttatcag ggttattgtc tcatgagcgg atacatattt gatgtattt agaaaaataa 2940 aaataggg gttccgcgca cattccccg aaagtgcca cctgggtcct ttcatcacg 3000 tgcttaaaaattattaattattaaattt fattaaaaaaaatagaa 3060 agtaaaaaaaaaaaataag aaaaaatagtt tttgttc cgagatgta aaagactcta 3120 gggggatcgc siaaatac taccttttat cttgctctc ctgctctcag gtattaatgc 3180 cgaattgttt catcttgtct gtgtagaaga ccacacacga aaatcctgtg attttacatt 3240 ttacttatcg ttaatcgaat gtatatctat ttaatctgct tttctgtct aaaatata 3300 tatgtaagt acgctttttg ttgaaatttt ttaaacctt gtttattt ttttcttcat 3360 tccgtaactc ttctaccttc tttatttact ttctaaaatc CAatacaa actaaaaat 3420 aaaaacac agagtaatt cccaattat tccatcatta aagatacga ggcgcgtgta 3480 agttacaggc aagcgatccg tcctaagaaa ccattattat catgacatta acctataaaa 3540 ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct 3600 gabacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac 3660 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg 3720 catcagagca gattgtactg agagtgcacc atattccgt 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 ttaacaaatga aaccaagatt cagattgcga tctctttaaa 4140 gggtggtccc ctagcgatag agcactcgat cttcccagaa aaagaggcag aagcagtagc 4200 agaacaggcc acacatcgc 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 tggttgcaa agggagaaag taggagatct ctcttgcgag atgatcccgc atttctttga 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 gctttttcttt ttttttctc 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 tatattcta atcaagctta taaaagagca ctgttgggcg tgagtggagg 120 cgccggaaaa agcatcgaa aaatctaga aaatgagta aaggaga acttttc 177 <210> 5 <211> 5937 <212> DNA <213> Artificial sequence <400> 5 catcgaaaaa atctgaaaa atgagtaag gagagaact ttcactgga gttgtcccaa 60 ttcttgttga attagatggt gatgttaatg ggcacaaatt ttcttcagt ggagaggggtg 120 aaggtgatgc aacatacgga aaacttaccc taatttat ttgcactact ggaaactac 180 ctgttccatg gcacactt gtcacactt ctcttatgg tgttcatgc tgcacactt 240 acccagatca tatgaaacgg catgacttt tcaagagtgc catgcccgaa ggttatgtac 300 aggaaagaac tatattttc aagatgacg ggaactacaa gacgtgct gaagtcaagt 360 ttgaaggtga tacccttgtt atagaatcg agttaaaagg tattgattt aaagagatg 420 gaaacattct tggacacaaa ttggaataca actataacc acacaatgta tacatcatgg 480 540 gaagcgttca actagcagac cattatcaac aaatactcc aattggcgat ggccctgtcc 600 ttttaccaga caccattac ctgtccacac aatctgccct ttcgaagat cccaacgaaa 660 agagagacca catggtcctt cttgagttg taacagctgc tgggattaca catggcatgg 720 atgaactata siaatagca agacgttgtt tcatcgcgct attack aggttacttt 780 acttgttctt gcacatggac gcacgttgtg tgttcatata tatatatata tatatatata 840 tatattgtg cttgttttca ttgtctctat agttaataca ttctttttt atcgttatat 900 ttgcattctc ttcgcataaa aacttcatga aaattcggca gaaataagc aaattgtaaa 960 cgttaatatt ttgttaaaat tcgcgttaaa ttttgttaa atcagctcat ttttacca 1020 ataggccgaa atcggcaaa tcccttataa atcaaagaa tagaccgaga taggttgag 1080 tgttgttcca gtttggaaca agagtccact attaaagac gtggactcca acgtcaagg 1140 gcgaaaaacc gtctatcagg gcgatggccc actacgtga ccatcaccct atcaagttt 1200 tttggggtcg aggtgccgta aagcactaaa tcggaccct 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 atattctct tactgtcatg ccatccgtaa gatgctttc tgtgactggt gagtactcaa 3480 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 3540 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 3600 cggggcgaaa actctcagg atcttaccgc tgttgagatc cagttcgatg taacccactc 3660 gtgcacccaa ctgatcttca gcatcttta ctttcaccag cgtttctggg tgagcaaaaa 3720 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaatgt tgaatactca 3780 tactctctcct ttttcaat tattgagca tttcaggg tattgtctc tattgcggat 3840 acatattga atgtatttag aaaaataac aaataggggt tccgcgcaca ttcccccgaa 3900 aagtgccacc tgggtccttt tcatcacgtg ctataaaaat aattataattt taaattttt 3960 Daddy daddy daddy aaaaaaaghaaaaaaaaaaaaaaaaaaaacttttt 4020 ttgttttccg aagatgtaaa agactctagg gggatcgcca acaaatacta ccttttatct 4080 tgctcttcct gctctcaggt attaatgccg aattgtttca tcttgtctgt gtagaagacc 4140 acacacgaaa atcctgtgat tttacatttt acttatcgtt aatcgaatgt atacttattt 4200 aatctgcttt tcttgtctaa taaatatata tgtaaagtac gctttttgtt gaaattttt 4260 aaacctttgt ttattttt ttcttcattc cgtaactctt ctaccttctt tatttacttt 4320 ctaaaatcca aatacaaaac ataaaaataa ataaacacag agtaaattcc caaattattc 4380 catcattaaa agatacgagg cgcgtgtaag ttacaggcaa gcgatccgtc ctaagaaacc 4440 attattca 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 ctttttttt cttagcgatt ggcattatca cataatgaat 4920 tatacattat ataaagtaat gtgatttctt cgaagaatat actaaaaaaat 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 ttttcttttt ttttctctt 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 gaatcattttt 120 gaataaaaaa cacgcttttt cagttcgagt ttatcattat caatactgcc atttcaaaga 180 atacgtaaat aattaatagt agtgatttc ctaactttat ttagtcaaaa aattagcctt 240 ttaattctgc tgtaacccgt acatgcccaa atagggggc gggttacaca gaatatataa 300 catcgtaggt gtctgggtga acagtttatt cctggcatcc actaaatata atggagcccg 360 cttttaagc tggcatccag aaaaaaaag aatcccagca ccaaaatatt gttttcttca 420 ccaaccatca gttcataggt ccattctctt agcgcaacta cagagaacag gggcacaaac 480 aggcaaaaa cgggcacaac ctcaatggag tgatgcaacc tgcctggagt aaatgatgac 540 acaaggcaat tgacccacgc atgtatctat ctcattttct tacaccttct attaccttct 600 gctctctctg atttggaaaa agctgaaaaa aaaggttgaa accagttccc tgaaatttatt 660 cccctacttg actaataagt atataaagac ggtaggtatt gattgtaatt ctgtaaatct 720 atttcttaaa cttcttaaat tctactttta tagttagtct ttttttagt 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 attaacttg tatattcta 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 attaacttg tatattcta atcaagctta 120 taaaagagca ctgttgggcg tgagtggagg cgccggaaaa aagcatcgaa aaaatctnnn 180 nnnatgagta aaggagaaga acttttcact ggagttgtcc caattcttgt tgaattagat 240 ggtgatgtta atgggcacaa atttctgtc 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 gatccccgg gctccaggaa 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 ccgcttccat 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 gggttatgt ctcatgagcg gatacatatt tgaatgtatt 4020 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgggtcc 4080 ttttcatcac gtgctataaa aataattata atttaaattt tttaatataa atatataaat 4140 taaaataga aagtaaaaa agaaattaaa gaaaaaatag ttttgtttt ccgaagatgt 4200 aaaagactct aggggatcg ccaacaaata ctacctttta tcttgctctt cctgctctca 4260 ggtattaatg ccgaattgtt tcatcttgtc tgtgtagaag accacacacg aaaatcctgt 4320 gattttacat tttacttatc gttaatcgaa tgtatatcta tttaatctgc ttttcttgtc 4380 taataaatat atatgtaaag taacgctttt gttgaaattt tttaaacctt tgtttatttt 4440 tttttcttca ttccgtaact cttctacctt ctttatttac tttctaaaat ccaaatacaa 4500 aacataaaaa taaataaaaca 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 ggtcaagctt ttaagaggc cctactggcg cgtggagtaa aaaggtttgg atcaggattt gcgccttttgg 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 gaagccacct cgcccaatgg taccaacgat gttccctcca ccaaaggtgt tcttatgtag tgacaccgat father tgcagcatac father father catgtgtata father father fathergtc father father gtatgatact gaagatgaca tcattctata cgtgtcattc tgaacgaggc gcgctttcct tttttcttt 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 aaaaatctt 120 page 125 <210> 11 <211> 125 <212> DNA <213> Artificial sequence <400> 11 cctccttgaa actgaaattt tagcatgtga ttaattaact tgtaatattc taatcaagct 60 tataaaagag cactgttggg cgtgagtgga ggcgccggaa aaaagcatcg aaaaatctc 120 125 horses <210> 12 <211> 125 <212> DNA <213> Artificial sequence <400> 12 cctccttgaa actgaaattt tagcatgtga ttaattaact tgtaatattc taatcaagct 60 tataaaagag cactgttggg cgtgagtgga ggcgccggaa aaaagcatcg aaaaatctg 120 page 125 <210> 13 <211> 125 <212> DNA <213> Artificial sequence <400> 13 cctccttgaa actgaaattt tagcatgtga ttaattaact tgtaatattc taatcaagct 60 tataaaagag cactgttggg cgtgagtgga ggcgccggaa aaaagcatcg aaaaatcta 120 tacc 125 <210> 14 <211> 125 <212> DNA <213> Artificial sequence <400> 14 cctccttgaa actgaaattt tagcatgtga ttaattaact tgtaatattc taatcaagct 60 tataaaagag cactgttggg cgtgagtgga ggcgccggaa aaaagcatcg aaaaatcta 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 aaaaatcta 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 aaaaatctg 120 dcaac 125 <210> 17 <211> 125 <212> DNA <213> Artificial sequence <400> 17 cctccttgaa actgaaattt tagcatgtga ttaattaact tgtaatattc taatcaagct 60 tataaaagag cactgttggg cgtgagtgga ggcgccggaa aaaagcatcg aaaaatcta 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 aaaaatctc 120 page 125 <210> 19 <211> 125 <212> DNA <213> Artificial sequence <400> 19 cctccttgaa actgaaattt tagcatgtga ttaattaact tgtaatattc taatcaagct 60 tataaaagag cactgttggg cgtgagtgga ggcgccggaa aaaagcatcg aaaaatctg 120 chart 125 <210> 20 <211> 125 <212> DNA <213> Artificial sequence <400> 20 cctccttgaa actgaaattt tagcatgtga ttaattaact tgtaatattc taatcaagct 60 tataaaagag cactgttggg cgtgagtgga ggcgccggaa aaaagcatcg aaaaatctt 120 page 125 <210> 21 <211> 125 <212> DNA <213> Artificial sequence <400> 21 cctccttgaa actgaaattt tagcatgtga ttaattaact tgtaatattc taatcaagct 60 tataaaagag cactgttggg cgtgagtgga ggcgccggaa aaaagcatcg aaaaatctc 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 aaaaatcta 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 aaaaatcta 120 ttatt 125

Claims

1. A Saccharomyces cerevisiae artificial promoter mutant, the nucleotide sequence of which is shown in SEQ ID No.

19.

2. Use of the artificial promoter mutant of Saccharomyces cerevisiae according to claim 1 in promoting the expression of a target gene in Saccharomyces cerevisiae.

3. Use of the artificial promoter mutant of Saccharomyces cerevisiae according to claim 1 in increasing the expression level of a target gene in Saccharomyces cerevisiae.

4. An expression cassette containing the artificial promoter mutant of Saccharomyces cerevisiae according to claim 1, which is composed of the artificial promoter mutant of Saccharomyces cerevisiae, a target gene and a terminator connected in sequence.

5. A recombinant vector containing the expression cassette according to claim 4.

6. A recombinant Saccharomyces cerevisiae containing the recombinant vector according to claim 5.

7. Use of the artificial promoter mutant of Saccharomyces cerevisiae according to claim 1, the expression cassette according to claim 4, the recombinant vector according to claim 5, or the recombinant Saccharomyces cerevisiae according to claim 6 in the regulation of gene expression in a metabolic pathway of Saccharomyces cerevisiae.

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