Saccharomyces cerevisiae artificial promoter mutants
By random mutation and PCR amplification of the artificial promoter of Saccharomyces cerevisiae, a high-intensity artificial small promoter of Saccharomyces cerevisiae was obtained, which solved the problem of the deficiency of the endogenous promoter of Saccharomyces cerevisiae in metabolic engineering and realized genes Significant improvement in expression and expansion of promoter engineering applications.
Patent Information
- Application Number
- CN202411736835.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2021-02-23
- Publication Date
- 2025-01-14
AI Technical Summary
The existing endogenous promoters of Saccharomyces cerevisiae have problems such as insufficient number of characteristics, poor dynamic range and susceptible to interference from endogenous substances in metabolic engineering, resulting in insufficient stability and intensity of gene expression regulation, which is difficult to meet research needs.
By randomly mutation of the nucleotide sequence of UASF-E-C-core1, the artificial promoter of Saccharomyces cerevisiae, the specific site was removed and PCR amplified, a set of high-intensity small artificial promoters of Saccharomyces cerevisiae was obtained, and the strength reached the natural strong promoter. 3.3 times TDH3 significantly broadens the application scope of promoter engineering.
The significant increase in the expression of the target gene in Saccharomyces cerevisiae was achieved, and a set of high-intensity artificial small promoters of Saccharomyces cerevisiae was provided, which met the needs of gene expression regulation in metabolic pathways and broadened the application scope of promoter engineering.
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Abstract
Description
[0001] This application is a divisional application with application number 202110200235.5, application date 2021.02.23, and invention name “A group of high-strength artificial small promoters of brewer's yeast”. Technical Field
[0002] The invention relates to the field of biotechnology, in particular to a group of artificial promoter mutants of saccharomyces cerevisiae. Background Art
[0003] 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.
[0004] 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 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 reported strongest small promoter strength is about 70% of the yeast endogenous strong promoter TDH3, and this strength range is still difficult to meet the needs of researchers. Summary of the invention
[0005] The purpose of the present invention is to provide a group of high-strength artificial small promoters of Saccharomyces cerevisiae.
[0006] In a first aspect, the present invention claims a Saccharomyces cerevisiae artificial promoter mutant.
[0007] The artificial promoter mutant of Saccharomyces cerevisiae claimed in the present invention is obtained by randomly mutating some nucleotides in positions 1 to 6 from the 3' end of the nucleotide sequence of the artificial promoter UASF-EC-core1 of Saccharomyces cerevisiae.
[0008] The above-mentioned artificial promoter mutant of Saccharomyces cerevisiae is obtained by randomly mutating at least one of the nucleotides at positions 148, 149, 152 and 153 of the nucleotide sequence shown in SEQ ID No. 4, and removing the nucleotides at positions 1-28 and 154-177. Further, the mutant is obtained by mutating the nucleotide at position 152 of the nucleotide sequence shown in SEQ ID No. 4 from A to C, randomly mutating at least one of the nucleotides at positions 148, 149 and 153, and removing the nucleotides at positions 1-28 and 154-177. Further, the mutant is obtained by mutating the nucleotide at position 152 of the nucleotide sequence shown in SEQ ID No. 4 from A to C, the nucleotide at position 153 from A to C or G, the nucleotide at position 149 from G to C or T, the nucleotide at position 148 from A to T or C or G, and removing the nucleotides at positions 1-28 and 154-177.
[0009] 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 the 6th position from the 3' end is 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 the 6th position from the 3' end in the primers is NNNNNN, and N is A, T, C or G.
[0010] In a second aspect, the present invention claims a Saccharomyces cerevisiae artificial promoter mutant.
[0011] The artificial promoter mutant of Saccharomyces cerevisiae claimed in the present invention may be any of the following: (A1) The nucleotide sequence is shown in SEQ ID No. 19 (corresponding to Figure 1 528 in ); (A2) The nucleotide sequence is shown in SEQ ID No. 13 (corresponding to Figure 1 510 in ); (A3) The nucleotide sequence is shown in SEQ ID No.11 (corresponding to Figure 1 503 in ); (A4) The nucleotide sequence is shown in SEQ ID No. 10 (corresponding to Figure 1 501 in ); (A5) The nucleotide sequence is shown in SEQ ID No. 12 (corresponding to Figure 1 507 in ); (A6) The nucleotide sequence is shown in SEQ ID No. 14 (corresponding to Figure 1 512 in ); (A7) The nucleotide sequence is shown in SEQ ID No. 20 (corresponding to Figure 1 (532 in ); (A8) The nucleotide sequence is shown in SEQ ID No. 21 (corresponding to Figure 1 (536 in ); (A9) The nucleotide sequence is shown in SEQ ID No. 16 (corresponding to Figure 1 517 of ); (A10) The nucleotide sequence is shown in SEQ ID No. 18 (corresponding to Figure 1 (525 in ); (A11) The nucleotide sequence is shown in SEQ ID No.15 (corresponding to Figure 1 514 in ); (A12) The nucleotide sequence is shown in SEQ ID No. 17 (corresponding to Figure 1 523 in ); (A13) The nucleotide sequence is shown in SEQ ID No. 22 (corresponding to Figure 1 (540 in ); (A14) The nucleotide sequence is shown in SEQ ID No. 23 (corresponding to Figure 1 545 in the table).
[0012] 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.
[0013] 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.
[0014] 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.
[0015] The expression cassette consists of the artificial promoter mutant of Saccharomyces cerevisiae, the target gene and the terminator connected in sequence.
[0016] In a specific embodiment of the present invention, the terminator is specifically an SPG5 terminator.
[0017] Furthermore, the nucleotide sequence of the SPG5 terminator is shown in SEQ ID No. 23-213.
[0018] In a sixth aspect, the present invention claims protection for a recombinant vector containing the expression cassette described in the fifth aspect above.
[0019] In a specific embodiment of the present invention, the backbone vector of the recombinant vector is the universal expression vector pRS313 of Saccharomyces cerevisiae.
[0020] In a seventh aspect, the present invention claims protection for a recombinant Saccharomyces cerevisiae containing the recombinant vector described in the sixth aspect above.
[0021] 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.
[0022] 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.
[0023] 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
[0024] Figure 1 Comparison of the strength of a high-strength artificial small promoter and a strong endogenous yeast promoter. DETAILED DESCRIPTION
[0025] 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.
[0026] 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.
[0027] Example 1: Construction of YPL001 plasmid as blank control 1. GFP Amplification of gene and SPG5 terminator GFP The gene was synthesized by GenScript Corporation to synthesize plasmid (the 25th to 741st positions of SEQ ID No. GFP The gene was inserted into pUC57) as a template and PCR amplification was performed with primers GFPzeo-up2 / GFPzeo-down2 (see Table 1). The amplification system was TAKARA PrimeSTAR®HS DNA polymerase 5×Buffer 10μl, Dntp mix 4μl, primers (see Table 1) 1μl each, cDNA, template 0.5μl, PrimerSTAR HS polymerase (2.5 U / μL) 0.5μl, and distilled water was added to a total volume of 50μl. The 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 obtained amplified product was named GFP (SEQID No.1, the fragment contains 24bp plasmid pRS313 homologous region, 20bp SPG5 homologous region, and GFP gene sequence). The obtained PCR amplification product was purified using a PCR product purification kit of Shanghai Shenggong Biotechnology Co., Ltd., and the purified product was used for future use. The terminator SPG5 was amplified using the primers SPG5-up2 / SPG5-down2 (see Table 1) using the genome of Saccharomyces cerevisiae BY4742 as a template (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 of Shanghai Shenggong Biotechnology Co., Ltd., and the purified product was used for future use.
[0028] 2. PCR amplification of expression vector The target expression vector was amplified using the universal expression vector pRS313 of Saccharomyces cerevisiae (from Addgene, containing His3 marker) as a template and primers 313-up2 / 313-down2 (see Table 1). 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.
[0029] 3. Construction of YPL001 plasmid using CPEC method CPEC system: Phusion High-Fidelity DNA polymerase 5× Buffer 5μl, Dntp mix 1, DMSO 0.75μl, two purified fragments (obtained in step 1 GFP Gene and SPG5 terminator amplification and purification fragment) and pRS313-His-vector obtained in step 2 were added in equal moles, Phusion High-Fidelity DNA polymerase (2 U / μL) 0.5μl, and distilled water was added to a total volume of 25μl. Amplification conditions are: 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 800μl LB medium, incubate at 250 rpm and 37℃ for 1 hour, apply the bacterial solution on 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 1504bp. The positive clone was cultured in liquid, and the positive clone plasmid was 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 Aisin to extract the target plasmid YPL001 for use.
[0030] Table 1. Primers for constructing YPL001 and YPL002 plasmids
[0031] Example 2: Construction of YPL002 plasmid as a positive control 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. The PCR product was named UASF-EC-core1 (SEQ ID No. 4, the fragment contained 20bp plasmid pRS313 homology region, 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 pRS313 vector backbone, GFP gene and SPG5 terminator). The obtained PCR amplification product was recovered and purified using the 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 placed in an ice bath 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 250 rpm and 37°C for 1 hour, and the bacterial solution was spread on an LB plate containing ampicillin. After overnight culture, primers Core11-up / GFPzeo-down2 (see Table 1) were used for PCR screening, and the positive clone band size was 890 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 to obtain plasmid YPL002. The plasmid extraction kit of Aisijin was used to extract the target plasmid YPL002 for standby use.
[0032] Example 3: Construction of strains Yp1001, Yp1002, Yp1007, and Yp1008 1. Construction of YPL007 and YPL008 plasmids 1. Amplification of pGPD / pTDH3 and pTEF1 promoters 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 PrimeSTAR®HS DNA polymerase 5×Buffer 10μl, Dntp mix 4μl, 1μl of each primer, cDNA, template 0.5μl, PrimerSTAR HS polymerase (2.5 U / μL) 0.5μl, and distilled water was added to a total volume of 50μl. The 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 20 bp plasmid pRS313 homology region, 24 bp GFP homology region and TDH3 promoter sequence) and pTEF1 (SEQ ID No. 7, the fragment contains 20 bp plasmid pRS313 homology region, 24 bp GFP homology region and TEF1 promoter). The obtained PCR amplified products were purified using the PCR product purification kit of Shanghai Shenggong Biotechnology Co., Ltd., and the purified products were used for later use.
[0033] Table 2. Primers for constructing YPL007 and YPL008 plasmids
[0034] 2. Amplify the expression vector 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 from Shanghai Shenggong Biotechnology Co., Ltd., and the purified product was set aside.
[0035] 3. Construction of YPL007 and YPL008 plasmids using CPEC method 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 (2 U / 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 800μl LB medium, incubate at 250 rpm and 37℃ for 1 hour, apply the bacterial solution on 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. The band size of positive clones is 1557bp or 1187bp. 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 plasmids YPL007 (corresponding to pTDH3) and YPL008 (corresponding to pTEF1) were obtained. Use the plasmid extraction kit of Aisin to extract the target plasmid for use.
[0036] 2. Construction of Ypl001, Ypl002, Ypl007, and Ypl008 strains Saccharomyces cerevisiae BY4742 ( saccharomyces cerevisiaeBY4742, 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 / 100 mL). Take 1 mL (OD about 0.6-1.0) and dispense it into a 1.5 mL EP tube, centrifuge at 4℃, 10000g for 1 min, discard the supernatant, wash the precipitate with sterile water (4℃), 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 passing through a 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, electroporated at 2.7 kv for 5.6 ms, and 1 mL of 1 M sorbitol was added. The cells were revived at 30 °C for 1 h, and then spread on screening medium plates (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, culture for more than 36 h, and the strains were named Ypl001, Ypl002, Ypl007, and Ypl008 according to the different transferred plasmids.
[0037] Example 4: Construction of an artificial small promoter Kozak library and screening to obtain high-strength artificial small promoters 1. Construction of Saccharomyces cerevisiae artificial promoter Kozak library 1. Amplification of YPL-Kozak-mut fragment The strongest artificial small promoter UASF-EC-core1 (SEQ ID No. 4, 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 the PCR gel recovery kit of Shanghai Shenggong Biotechnology Co., Ltd., and the purified product was used for later use.
[0038] 2. Homologous recombination in Saccharomyces cerevisiae to construct a Saccharomyces cerevisiae artificial promoter Kozak library Prepare the competent cells of Saccharomyces cerevisiae BY4742 (the method is the same as step 2 of Example 3). Add 2 μL of the YPL-Kozak-mut fragment and the 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 conditions for screening culture are: 30 ° C, culture for more than 36 h.
[0039] 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.
[0040] Table 3. Primers for constructing Kozak library
[0041] Note: N represents A or T or C or G.
[0042] 2. Screening of Kozak Library of Saccharomyces cerevisiae Artificial Promoter 1. Flow cytometry screening of the Saccharomyces cerevisiae artificial promoter Kozak library 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 culture conditions were: temperature 30 °C, humidity 80%, and rotation speed 800 rpm for more than 24 h.
[0043] 2. 96-well plate rescreening 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.
[0044] 3. Test tube rescreening 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 ℃, 250 rpm, 12 h), and then the initial OD 600nm =0.1 for transfer and continued cultivation (30 ℃, 250 rpm, 12 h), followed by fluorescence measurement, with excitation light at 395 nm, absorption light at 507 nm and OD measurement at a wavelength of 600 nm. The fluorescence / OD600 ratio was calculated, and fourteen small promoters were finally retained. The corresponding strains were named 501, 503, 507, 510, 512, 514, 517, 523, 525, 528, 532, 536, 540, 545 (SEQ ID No.10- SEQ ID No.23) 3. Comparison of the strength of high-strength artificial small promoter and yeast endogenous strong promoter Fluorescence assay experiment: Ypl001, Ypl002, Ypl007, Ypl008 and 14 strains obtained 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 ℃, 250 rpm, 12 h), and then the culture was transferred and continued according to the initial OD600nm=0.1 (30 ℃, 250 rpm, 12 h), and then the fluorescence assay was performed under the conditions of excitation light 395 nm, absorption light 507 nm and OD assay at a wavelength of 600 nm. The results are shown in Figure 2. Figure 1 .
[0045] The above test results show that by building a library of artificial small promoter Kozak regions and undergoing multiple rounds of screening, 14 high-intensity artificial small promoters were finally obtained, except that the activity of 545 was slightly lower than that of the endogenous strong promoter of Saccharomyces cerevisiae. pTDH3 and pTEF1 Except for the above, the activities of the other 13 artificial small promoters were higher than pTDH3 and pTEF1 Among them, the strongest artificial small promoter 528 has a strength of pTDH3 and pTEF1 The protein expression of the proposed small promoter is 3.3 times that of the strongest small promoter reported by Alper and 7 times that of the strongest small promoter reported by Alper, which significantly broadens the dynamic range of artificial small promoters in Saccharomyces cerevisiae.
[0046] 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.
Claims
1. A Saccharomyces cerevisiae artificial promoter mutant, characterized in that: The mutant is obtained by randomly mutating at least one of the nucleotides at positions 148, 149, 152 and 153 of the nucleotide sequence shown in SEQ ID No. 4, and removing the nucleotides at positions 1-28 and 154-177.
2. The artificial promoter mutant of Saccharomyces cerevisiae according to claim 1, characterized in that: The mutant is obtained by mutating the nucleotide at position 152 of the nucleotide sequence shown in SEQ ID No. 4 from A to C, randomly mutating at least one of the nucleotides at positions 148, 149 and 153, and removing the nucleotides at positions 1-28 and 154-177.
3. The artificial promoter mutant of Saccharomyces cerevisiae according to claim 1 or 2, characterized in that: The mutant is obtained by mutating the nucleotide at position 152 of the nucleotide sequence shown in SEQ ID No. 4 from A to C, the nucleotide at position 153 from A to C or G, the nucleotide at position 149 from G to C or T, the nucleotide at position 148 from A to T or C or G, and removing the nucleotides at positions 1-28 and 154-177.
4. The artificial promoter mutant of Saccharomyces cerevisiae according to claim 1 or 2, characterized in that: The mutant is any of the following: (A1) the nucleotide sequence is shown in SEQ ID No. 13; (A2) the nucleotide sequence is shown in SEQ ID No. 11; (A3) the nucleotide sequence is shown in SEQ ID No. 10; (A4) the nucleotide sequence is shown in SEQ ID No. 12; (A5) The nucleotide sequence is shown in SEQ ID No.
18.
5. Use of the artificial promoter mutant of Saccharomyces cerevisiae according to any one of claims 1 to 4 in promoting the expression of a target gene in Saccharomyces cerevisiae.
6. Use of the artificial promoter mutant of Saccharomyces cerevisiae according to any one of claims 1 to 4 in increasing the expression level of a target gene in Saccharomyces cerevisiae.
7. An expression cassette containing the artificial promoter mutant of Saccharomyces cerevisiae according to any one of claims 1 to 4, which is composed of the artificial promoter mutant of Saccharomyces cerevisiae, a target gene and a terminator connected in sequence.
8. A recombinant vector containing the expression cassette according to claim 7.
9. A recombinant Saccharomyces cerevisiae containing the recombinant vector according to claim 8.
10. Use of the artificial promoter mutant of Saccharomyces cerevisiae according to any one of claims 1 to 4, the expression cassette according to claim 7, the recombinant vector according to claim 8, or the recombinant Saccharomyces cerevisiae according to claim 9 in the regulation of gene expression in a metabolic pathway of Saccharomyces cerevisiae.