Yeast high-expression promoter and application thereof

Abstract

The invention provides polynucleotides with promoters with high transcriptional activity in yeast, including constitutive and inducible high-expression promoters under different nitrogen source, temperature and carbon source culture conditions, and application of the polynucleotides in enhancement of target gene expression. The promoter element is provided for application of yeast synthetic biology, so that for example, the expression capability of yeast protein can be improved, and a foundation is laid for promoting application of yeast in the field of microbial protein biological manufacturing.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of synthetic biology and biotechnology, and specifically relates to a yeast expression promoter and application thereof. BACKGROUND

[0002] By 2050, the global population will approach 10 billion, and protein demand will increase by more than 50%. Microbial protein fermentation technology is considered the most promising new protein manufacturing technology due to its high protein and essential amino acid content, rapid reproduction, high production efficiency, low environmental footprint, and strong sustainability. In 2023, the total output value of feed microbial protein products reached approximately $9.4 billion. It is estimated that by 2035, microbial protein will account for 22% of the edible alternative protein market, with a size of $63.8 billion. Microbial protein, as a new source of feed and edible protein, will play an increasingly important role.

[0003] Yeast, as a key host for single-cell protein and foreign protein expression, is increasingly valued. Factors affecting the efficient expression of specific proteins in yeast include promoter activity, gene copy number, translation efficiency, and post-translational modification. Among these factors, the level of promoter transcription is a key factor in regulating gene expression. Promoters can be divided into two categories: constitutive and inducible. The former is generally not affected by external factors, and high expression of constitutive promoters can consistently and constantly express target genes at high levels. The latter is regulated by external chemical or physical signals, which can significantly increase the transcription level of target genes. These two types of promoters have their own advantages and disadvantages in application and are often used as expression elements to improve the synthesis capacity of foreign proteins in yeast.

[0004] Kluyveromyces marxianus is a food-grade yeast, which has been approved as a new food raw material and feed additive in China, and has been certified as safe by the US GRAS (Generally Recognized as Safe) and the European QPS (Qualified Presumption Safety). Meanwhile, the yeast has the characteristics of fast growth and tolerance to high temperature of 45℃ to 50℃, and has the safety and potential for producing microbial proteins. At present, a variety of exogenous and endogenous proteins have been expressed in the strain, such as ferulic acid esterase (CN 108410870 B), saccharifying enzyme (CN 116987604 A), soybean hemoglobin (CN 116949086 A), polygalacturonase (CN 115976094 B) and the like. In recent years, more progress has been made in the mining of promoter elements derived from Kluyveromyces marxianus. A batch of promoters that can be applied to express target genes in the strain have been cloned (Metab Eng Commun, 2020, 11: p.e00145; Front Bioeng Biotechnol, 2019, 7: p.97; Metab Eng Commun, 2021, 12: p.e00160), such as the constitutive strong promoters ENO1, TDH3 and PGK1. However, compared with conventional protein expression hosts such as Saccharomyces cerevisiae and Pichia pastoris, the existing promoters of Kluyveromyces marxianus still have many problems in terms of quantity, expression amount and regulation mode. For example, the inulin-induced inulinase promoter (INU1), the galactose-induced promoter GAL1 and the xylose-induced promoter IMTIP1 have high expression intensity, but the strength of the promoters depends on the carbon source, and will be down-regulated under high glucose concentration. Therefore, further research and improvement are needed to obtain more strong promoters with high expression intensity and convenient regulation. SUMMARY

[0005] In view of the above problems of the prior art, the purpose of the present application is to provide strong constitutive and inducible promoters derived from Kluyveromyces marxianus, and to provide elements for improving yeast protein expression capacity.

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0007] In a first aspect, the present application provides a high transcription activity level promoter, the nucleotide sequence of which is any one of (a) to (b):

[0008] (a) a nucleotide sequence as set forth in any one of SEQ ID NOs: 1-6, or a nucleotide sequence truncated from the 5' end by not more than 950 nucleotides, or not more than 750 nucleotides, or not more than 510 nucleotides, or not more than 320 nucleotides, or not more than 260 nucleotides, or not more than 250 nucleotides, not more than 230 nucleotides, based on the nucleotide sequence as set forth in any one of SEQ ID NOs: 1-6;

[0009] (b) a nucleotide sequence having 80% homology to the nucleotide sequence of (a) above and having promoter activity in K. marxianus and in at least one more yeast species other than K. marxianus.

[0010] Preferably, the nucleotide sequence thereof is a nucleotide sequence truncated from the 5' end by not more than 228 nucleotides, based on the nucleotide sequence as set forth in SEQ ID NO: 1;

[0011] or the nucleotide sequence thereof is a nucleotide sequence truncated from the 5' end by not more than 256 nucleotides, based on the nucleotide sequence as set forth in SEQ ID NO: 2;

[0012] the nucleotide sequence thereof is a nucleotide sequence truncated from the 5' end by not more than 770 nucleotides, based on the nucleotide sequence as set forth in SEQ ID NO: 3;

[0013] the nucleotide sequence thereof is a nucleotide sequence truncated from the 5' end by not more than 928 nucleotides, or not more than 743 nucleotides, or not more than 505 nucleotides, based on the nucleotide sequence as set forth in SEQ ID NO: 4;

[0014] the nucleotide sequence thereof is a nucleotide sequence truncated from the 5' end by not more than 249 nucleotides, based on the nucleotide sequence as set forth in SEQ ID NO: 5;

[0015] the nucleotide sequence thereof is a nucleotide sequence truncated from the 5' end by not more than 321 nucleotides, based on the nucleotide sequence as set forth in SEQ ID NO: 6.

[0016] More preferably, the nucleotide sequence thereof is a nucleotide sequence as set forth in SEQ ID NO: 1, or truncated from the 5' end by 228 nucleotides, based on the nucleotide sequence as set forth in SEQ ID NO: 1;

[0017] or the nucleotide sequence thereof is a nucleotide sequence as set forth in SEQ ID NO: 2, or truncated from the 5' end by 256 nucleotides, based on the nucleotide sequence as set forth in SEQ ID NO: 2;

[0018] or the nucleotide sequence thereof is a nucleotide sequence as set forth in SEQ ID NO: 3, or truncated from the 5' end by 770 nucleotides, based on the nucleotide sequence as set forth in SEQ ID NO: 2;

[0019] or a nucleotide sequence represented by SEQ ID NO: 4, or a nucleotide sequence obtained by truncating 928 nucleotides from the 5' end of a nucleotide sequence represented by SEQ ID NO: 4, or 743 nucleotides, or 505 nucleotides;

[0020] or a nucleotide sequence represented by SEQ ID NO: 5, or a nucleotide sequence obtained by truncating 249 nucleotides from the 5' end of a nucleotide sequence represented by SEQ ID NO: 5;

[0021] or a nucleotide sequence represented by SEQ ID NO: 6, or a nucleotide sequence obtained by truncating 321 nucleotides from the 5' end of a nucleotide sequence represented by SEQ ID NO: 6.

[0022] In the present application, the above-mentioned promoter is a constitutive strong promoter, and the nucleotide sequence thereof is any one of (a) to (d):

[0023] (a) a nucleotide sequence represented by SEQ ID NO: 3;

[0024] (b) a nucleotide sequence obtained by truncating not more than 770 nucleotides from the 5' end of a nucleotide sequence represented by SEQ ID NO: 3;

[0025] (c) a nucleotide sequence obtained by truncating the nucleotide sequence of the above-mentioned (a), wherein the truncated nucleotide sequence contains the nucleotide sequence of the above-mentioned (b);

[0026] (d) a nucleotide sequence having 80% homology with the nucleotide sequence of the above-mentioned (a) to (c) and having a promoter activity in K. marxianus and at least one or more yeasts other than K. marxianus.

[0027] In the present application, the above-mentioned promoter is a high-temperature inducible promoter, and the nucleotide sequence thereof is any one of (a) to (d):

[0028] (a) a nucleotide sequence represented by SEQ ID NO: 4;

[0029] (b) a nucleotide sequence obtained by truncating not more than 928 nucleotides, or not more than 743 nucleotides, or not more than 505 nucleotides from the 5' end of a nucleotide sequence represented by SEQ ID NO: 4;

[0030] (c) a nucleotide sequence obtained by truncating the nucleotide sequence of the above-mentioned (a), wherein the truncated nucleotide sequence contains the nucleotide sequence of the above-mentioned (b);

[0031] (d) a nucleotide sequence having 80% homology with the nucleotide sequence of (a) to (c) above and having a promoter activity in K. marxianus and at least one or more yeasts other than K. marxianus.

[0032] The above-mentioned promoter provided by the present application is an inorganic nitrogen source inducible promoter, and the nucleotide sequence thereof is any one of (a) to (d):

[0033] (a) a nucleotide sequence represented by SEQ ID NO: 1-2, 4-6;

[0034] (b) a nucleotide sequence obtained by truncating a 5' end of the nucleotide sequence represented by SEQ ID NO: 1 by not more than 228 nucleotides;

[0035] or a nucleotide sequence obtained by truncating a 5' end of the nucleotide sequence represented by SEQ ID NO: 2 by not more than 256 nucleotides;

[0036] a nucleotide sequence obtained by truncating a 5' end of the nucleotide sequence represented by SEQ ID NO: 4 by not more than 928 nucleotides, or not more than 743 nucleotides, or not more than 505 nucleotides;

[0037] a nucleotide sequence obtained by truncating a 5' end of the nucleotide sequence represented by SEQ ID NO: 5 by not more than 249 nucleotides;

[0038] a nucleotide sequence obtained by truncating a 5' end of the nucleotide sequence represented by SEQ ID NO: 6 by not more than 321 nucleotides;

[0039] (c) a nucleotide sequence obtained by truncating the nucleotide sequence of (a) above, wherein the nucleotide sequence obtained by truncating comprises the nucleotide sequence of (b) above;

[0040] (d) a nucleotide sequence having 80% homology with the nucleotide sequence of (a) to (c) above and having a promoter activity in K. marxianus and at least one or more yeasts other than K. marxianus.

[0041] The present application provides a transcription expression cassette, characterized in that the transcription expression cassette comprises the promoter.

[0042] Specifically, the transcription expression cassette further comprises a protein coding gene, which is operably linked to the polynucleotide having a promoter activity.

[0043] The present application provides an expression vector comprising the promoter or the transcription expression cassette. Preferably, the expression vector is a genomic integration expression vector.

[0044] The present application also provides a host cell containing the promoter or the transcription expression cassette or the expression vector. Specifically, the host cell is yeast.

[0045] Preferably, the host cell is Kluyveromyces marxianus, Kluyveromyces lactis, Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, Hansenula polymorpha, Issatchenkia orientalis, and Schizosaccharomyces pombe.

[0046] The present application also provides the use of the promoter or the transcription expression cassette, the expression vector, or the host cell in at least one of the following: (a) for enhancing the transcription level of a gene, or for preparing a reagent or a kit for enhancing the transcription level of a gene; (b) for preparing a protein, or for preparing a reagent or a kit for preparing a protein.

[0047] The present application provides a series of polynucleotide sequences with strong promoter activity isolated from Kluyveromyces marxianus, including constitutive promoters and high-temperature and inorganic nitrogen source inducible promoters. These polynucleotides with strong promoter activity can efficiently enhance the protein production capacity of yeast, provide new regulatory elements for the construction of yeast protein expression systems, and are suitable for high-temperature and inorganic nitrogen source fermentation conditions, which can save production costs and have great application potential. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 is the growth curve of Kluyveromyces marxianus under different culture conditions.

[0049] Figure 2 is a schematic diagram of the construction of expression plasmids with different promoters.

[0050] Figure 3 is the expression intensity of different promoters and their sequence variants under 8 culture conditions.

[0051] Figure 4 is the expression intensity of the truncated promoter under 8 culture conditions. DETAILED DESCRIPTION

[0052] The following is a specific embodiment, in order to better understand the present application, but does not constitute a limitation on the present application.

[0053] By comparing and analyzing the transcriptome of K. marxianus under different carbon sources (glucose, mixed sugar), different nitrogen sources (yeast powder organic nitrogen source, ammonium sulfate inorganic nitrogen source), and different temperature (30℃, 45℃) culture conditions, a batch of potential high, medium, and low transcriptional level constitutive and inducible promoters are screened. Then, through promoter sequence cloning, genome integration plasmid construction carrying fluorescent protein gene and transformation into K. marxianus, the transcriptional activity level of the candidate promoter is further evaluated by using fluorescent protein expression as an indicator, and the transcriptional activity level of the truncated promoter is evaluated. Thus, the constitutive and inducible strong promoters of K. marxianus are obtained.

[0054] Example 1: Screening of high expression intensity and inducible promoters under different temperature, nitrogen source and carbon source conditions by transcriptome analysis

[0055] (1) Yeast fermentation experiment and transcriptome sample preparation under different carbon sources, nitrogen sources and temperature conditions

[0056] Under different carbon sources, nitrogen sources and temperature conditions, a total of 8 yeast culture conditions were used, as shown in Table 1. Among them, 2 culture temperatures, i.e. 30 degrees and 45 degrees; 4 fermentation media composed of 2 nitrogen sources and 2 carbon sources are as follows.

[0057] 1) DD medium: Delft medium, with ammonium sulfate as inorganic nitrogen source and glucose as the only carbon source, its composition includes 20 g / L glucose, 7.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 2 mL / L trace element solution, 1 mL / L vitamin solution, pH adjusted to 5.0. 1 liter of trace element solution contains 3.0 g FeSO4·7H2O, 4.5 g ZnSO4·7H2O, 4.5 g CaCl2·2H2O, 1 g MnCl2·4H2O, 300 mg CoCl2·6H2O, 300 mg CuSO4·5H2O, 400 mg Na2MoO4·2H2O, 1 g H3BO3, 100 mg KI, 19 g Na2EDTA·2H2O. 1 liter of vitamin solution contains 50 mg d-biotin, 1.0 g d-calcium pantothenate, 1.0 g thiamin-HCl, 1.0 g thiamine hydrochloride, 1.0 g nicotinic acid, 0.2 g 4-aminobenzoic acid, 25 g m-inositol.

[0058] 2) DH medium: with mixed sugars as carbon source, mimicking the sugar composition of lignocellulose hydrolysis, the mixed sugars consisted of 12 g / L glucose, 6 g / L xylose, 3 g / L arabinose, 2 g / L galactose, 2 g / L mannose and 2 g / L cellobiose, the nitrogen source, other components and pH were the same as DD medium.

[0059] 3) YD medium: YPD medium, with yeast extract and peptone (YP) as organic nitrogen source, with glucose as sole carbon source, the composition included 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, pH adjusted to 5.0.

[0060] 4) YH medium: with mixed sugars as carbon source, the mixed sugars consisted of the same as DH medium, the nitrogen source composition and pH were the same as YD medium.

[0061] Table 1. Yeast growth performance under different culture conditions

[0062]

[0063] Kluyveromyces marxianus GQ7 (purchased from Beijing BNCC Biotech Research Institute, BNCC 191517) was selected from YPD plate and inoculated into 50-mL centrifuge tube containing 5 mL YPD medium, incubated at 30°C, 200 rpm, overnight to obtain seed broth. The seed broth was inoculated into 100-ml shake flask containing 50 mL fermentation medium with initial OD 600 of 0.2, and sampled every 2 hours to measure OD 600 . Three biological replicates were set for each culture condition. Origin 8.0 was used to fit the cell growth data with kinetics, and calculate the maximum cell growth and maximum specific growth rate. Specifically, Boltzmann function of Sigmoid model (“S” curve) was used for fitting.

[0064] The growth difference of yeast under different culture conditions was shown in Figure Figure 1 and Table 1. Compared with organic nitrogen source, K. marxianus GQ7 started to grow about 2 hours later on inorganic nitrogen source Figure 1 . When grown on YH medium (organic nitrogen source, mixed sugars) at 30°C, GQ7 showed the highest final OD 600 of 17.42, but the lowest maximum specific growth rate of 0.24 h -1 (Table 1). When GQ7 was grown on YPD medium at 30°C, it showed the highest maximum specific growth rate of 0.65 h -1 . In addition, when cells were grown on two inorganic nitrogen source media (DD and DH media) at 30°C, the final OD 600were all higher than 14.0, and the maximum specific growth rates were also observed higher than 0.50 h -1 Compared with 30℃, the cell accumulation of GQ7 was significantly reduced at 45℃. When comparing different carbon sources conditions, the final OD 600 and the maximum specific growth rate increased by 1.55-fold and decreased by 0.37-fold, respectively, in addition to this, mixed sugar seemed to have little effect on the cell growth of K. marxianus. These results indicated that inorganic nitrogen source mainly reduced the cell growth rate of K. marxianus GQ7, and high temperature largely hindered its cell accumulation. Therefore, temperature and nitrogen source had a greater impact on yeast growth, while carbon source had a smaller impact.

[0065] Transcriptome sample preparation: The transcriptome sampling time points (Table 1) were in the logarithmic growth phase, and the cells were grown to have similar growth rates under different culture conditions, with an average growth rate of 0.816 ± 0.004 OD 600 / h. The cells were collected by centrifugation and stored at -80℃ for later use.

[0066] (2) Transcriptome sequencing and analysis

[0067] Transcriptome data analysis: Total RNA extraction and transcriptome sequencing library construction were completed by Beijing Novogene Bioinformatics Technology Co., Ltd. (China, Beijing), the sequencing platform was NovaSeq 6000, and the sequencing data volume was 6G. The K. marxianus DMKU 3-1042 genome (NCBI RefSeq assembly accession number: GCF_001417885.1) was used as the reference genome. HISAT2 software was used to align the cleaned reads with the reference genome. Subsequently, the RNA-seq analysis platform of Novogene was used to quantify the gene transcription level and analyze the differentially expressed genes (DEGs). DEGs analysis was performed on 12 comparison groups under 8 different culture conditions.

[0068] According to the FPKM value (Fragments Per Kilobase of exon model per Million mapped fragments, which represents the transcription level of a gene) representing the transcription level of a gene, 26 candidate promoters were screened, and the gene information and transcription level under different culture conditions are shown in Table 2. There are 9 promoters with an average FPKM of the gene under different culture conditions ≥10000, which are likely to be potential high expression intensity promoters; 11 promoters with FPKM ≥4000 and <10000 are likely to be potential medium level expression promoters; 7 promoters with FPKM ≥1000 and <40000 are likely to be potential low expression level promoters.

[0069] Table 2 Transcription activity level of promoters under different temperature, nitrogen source and carbon source conditions

[0070]

[0071]

[0072] Table 3 shows high temperature, inorganic nitrogen source and mixed sugar inducible candidate promoters. According to the differential expression gene (DEG) analysis under different temperature, nitrogen source and carbon source conditions, according to the screening standard of expression difference fold ≥1.2 under at least one comparison condition, 4 high temperature inducible candidate promoters (P1, P15, P16, P25), 12 inorganic nitrogen source inducible candidate promoters (P3, P4, P5, P8, P10, P12, P13, P15, P16, P17, P18, P22) and 1 mixed carbon source inducible candidate promoter (P12) were screened.

[0073] Table 3 High temperature, inorganic nitrogen source and mixed sugar inducible candidate promoters and gene expression difference fold

[0074]

[0075] Example 2: Evaluation of transcription activity level of candidate promoters using fluorescence protein expression indication

[0076] (1) Construction of yeast genome integrated plasmid for detecting promoter expression intensity

[0077] The schematic process of plasmid construction is as follows: Figure 2The plasmid for constructing the green fluorescent protein eGFP gene expression cassette required for detecting the expression strength of the promoter is first constructed. On the one hand, the backbone plasmid pI2-MTU-DO-G418 is purchased from Addgene (item number: 160018), which carries the double-arm sequence of the K. marxianus I2 site integration and the G418 screening marker gene KanMX expression cassette. The fragment can be integrated into the yeast genome by traditional homologous recombination, and the transformants can be screened by YPD plates containing G418. On the other hand, the green fluorescent protein expression cassette without a promoter is obtained, in which the green fluorescent protein eGFP is codon-optimized and uses the PDC1 terminator (250 bp) of K. marxianus. The gene synthesis company Jinweizhi Biotechnology Co., Ltd. is used to synthesize the above-mentioned expression cassette, and the GFP dropout part sequence in pI2-MTU-DO-G418 is replaced, and the obtained plasmid is named as pI2-eGFPopt-T.

[0078] Using the pI2-eGFPopt-T plasmid as the backbone, different promoter sequences are inserted between the K. marxianus I2 L integration site and eGFPopt, to obtain genome integration plasmids carrying different promoters for detecting their expression strength. First, the genome of K. marxianus GQ7 (purchased from Beijing Beinaeliang Biotechnology Research Institute, BNCC 191517) is extracted (Yeast Genomic DNA Miniprep Kit, ZOMANBIO, item number: ZP302-2), and primers are designed according to the length of 1400 bp of the promoter to perform PCR amplification, to obtain different promoter sequences. The cloning method is performed according to the instructions of the homologous recombination rapid cloning kit pEASY-Basic Seamless Cloning and Assembly Kit (brand: Quanshijin, item number: CU201-03). The primers used are shown in Table 4. The obtained plasmid is verified by first-generation sequencing (sequencing primers in Table 4) whether it contains the target promoter fragment. Finally, 26 promoters with a total of 45 sequence polymorphic variants are obtained. The plasmids carrying these promoters are used for subsequent transformation of K. marxianus.

[0079] Table 4 Primers used for constructing genome integration plasmids for detecting the expression strength of the promoter

[0080]

[0081]

[0082] (2) Obtaining of yeast genome integration transformants and screening by flow cytometry

[0083] Obtaining of integration fragment: using primer pair I2-F and I2-R (Table 4), the DNA fragment that can be integrated at the I2 site of K. marxianus was amplified with the genomic integration plasmid carrying different promoter sequences as template. The composition of the integration fragment is shown in Figure B, including the left arm sequence of I2 integration site, the promoter to be detected, the codon-optimized eGFP coding sequence, the PDC1 terminator, the G418 selection gene (KanMX) expression cassette, and the right arm sequence of I2 integration site. Figure 2

[0084] Yeast transformation: K. marxianus GQ7 was transformed by chemical transformation method (kit: Frozen-EZ Yeast Transformation II Kit, item number: T2001, brand: Zymo), and the transformation product was plated on a YPD plate containing 200 μg / ml G418 (G418 selection plate) for screening. Incubate in a 30°C incubator for 2-4 days until single colonies grow.

[0085] Flow cytometry sorting of yeast genomic integration transformants: since the promoter to be detected in the yeast transformant can drive the expression of green fluorescent protein eGFP, the activity intensity of different promoters is proportional to the green fluorescence intensity of the transformant cells. In order to further separate the transformant cells with high promoter activity intensity, we used flow cytometry (BD FACSAria Fusion SORP) to screen and separate the transformants with high fluorescence intensity. After sorting, the plate was incubated at 30°C for 1-2 days until the cells grew.

[0086] (3) Detection of transcriptional activity level of different promoters under different temperature, nitrogen source and carbon source conditions

[0087] Detection method of eGFP green fluorescence intensity: take 50ul of bacterial liquid and add it to 150ul of sterile water, add it to a 96-well black enzyme plate, and place it in a BioTek multifunctional enzyme marker to detect fluorescence. Excitation light 479nm, absorption light 520nm, detect fluorescence value; wavelength 600nm to detect cell biomass. The fluorescence intensity value is the ratio of the measured fluorescence value to the cell biomass, and the final result is expressed as the fold increase of the fluorescence intensity value of different variants compared to the fluorescence intensity value of GQ7.

[0088] From the above sorting plate, randomly pick 8 well-grown transformants for culture in YPD liquid medium, grow to early logarithmic growth phase, sample and measure fluorescence value and cell biomass, calculate the fluorescence intensity per cell. Select the top 2 transformants with the highest fluorescence intensity value for subsequent detection of transcriptional activity level under different culture conditions.

[0089] ​Then, the cells were cultured under eight different culture conditions as described in Example 1, namely four different liquid culture media (DD, DH, YD, YH) and culture temperatures of 30°C and 45°C (Table 1). Once the cells reached the early logarithmic growth phase, samples were taken and fluorescence values ​​and cell biomass were measured. The fluorescence intensity per unit cell was calculated. Two biological replicates were set up for each culture condition.

[0090] The results are as follows Figure 3 As shown, compared with the control GQ7, the fluorescence intensity values ​​of six promoters, namely P3_v2 (v2 sequence variant of P3, SEQ ID NO: 1), P4_v2 (v2 sequence variant of P4, SEQ ID NO: 2), P9 (SEQ ID NO: 3), P16_v1 (v1 sequence variant of P16, SEQ ID NO: 4), P17 (SEQ ID NO: 5), and P19_v2 (v2 of P19, SEQ ID NO: 6), were significantly increased by more than other promoters and sequence variants, indicating that these nucleic acid sequences are strong promoters.

[0091] Table 5 shows the induction of these six promoters by high temperature, inorganic nitrogen sources, and mixed sugars. Promoter P16_v1 was induced by high temperature; compared to culture at 30℃, the fluorescence intensity expression value increased by 1.1 to 2.2 times under culture at 45℃. Inorganic nitrogen sources showed a more significant induction, increasing the fluorescence intensity expression value by 2.4 to 14.6 times compared to organic nitrogen sources. Promoters P3_v2, P4_v2, P17, and P19_v2 were significantly induced by inorganic nitrogen sources, with fluorescence intensity expression values ​​increasing by 1.3 to 4.4 times, 1.1 to 3.4 times, 2.0 to 7.6 times, and 2.6 to 9.5 times, respectively. Promoter P9 did not show significant induction under any of the eight culture conditions. Mixed sugars did not show significant induction of promoter expression.

[0092] Since there are few studies on the binding of the transcription factor of K. marxianus to the conserved sequence of the promoter, we analyzed the binding of the conserved sequence of the above-mentioned promoters to the high-expression, heat shock response and nitrogen source regulation-related transcription factors by referring to the analysis of the transcription factors of S. cerevisiae (Yeastract website, Find TF Binding Sites function). Among them, the above-mentioned promoters generally have multiple conserved sequences that are bound by the transcription factors Gcr1p and Gcr2p related to the regulation of the high expression of glycolytic genes, such as CTTCC, TTCCACTA, GAAGTAA, AAACAAAA, etc.; P16_v1 has multiple conserved sequences that are bound by the heat shock response transcription factor Hsf1p between 500 bp and 550 bp, such as NGAANNTTCN, GAANNTTCNNGAA, etc.; P16_v1 and P19_v2 have the conserved sequence GATTAG that is bound by the nitrogen source regulation transcription factor Gln3p, which is consistent with the result that the induction degree of these two promoters is obviously higher than that of the other four promoters.

[0093] Table 5 Induction of promoters and difference in expression value of fluorescence intensity

[0094]

[0095] Example 3: Evaluation of the transcriptional activity level of the truncated promoters

[0096] (1) Analysis of the sequence of the truncated promoter

[0097] The truncation strategy of the promoter is as follows: (1) Since there are differences in the fluorescence intensity values of different sequence variants of the same promoter, the conserved regions and variant regions among different sequence variants are analyzed; (2) Homologous promoters in different yeast species such as S. cerevisiae, K. lactis, G. candidum and S. pombe are searched, and sequence difference analysis among yeast species is performed. Combined with the two kinds of analysis, the position of truncation is determined. The promoter is shortened from the 5' end to the 3' end, and the truncation of the promoter is shown in Table 6.

[0098] (2) Construction of yeast genome integration plasmids carrying truncated promoters

[0099] In order to verify whether the truncated promoters still have high-level transcription or induced activity, the yeast genome integration plasmids carrying the sequence variants of the promoters P3_v2, P4_v2, P9, P16_v1, P17 and P19_v2 obtained in Example 2 were used as templates, the primers shown in Table 7 were used to obtain the truncated promoter fragments, and the yeast genome integration plasmids carrying the truncated promoters were constructed. The specific primer sequences are shown in Table 7. The obtained plasmids were verified by one-generation sequencing (sequencing primers in Table 7) to determine whether they contain the truncated target promoter fragments.

[0100] Table 6 Truncation of promoters

[0101] Truncated promoters Truncation P3_v2_t1 Truncated 228 bp from 5' of control promoter P3_v2 P4_v2_t1 Truncated 256 bp from 5' of control promoter P4_v2 P9-t1 Truncated 770 bp from 5' of control promoter P9 P16-v1-t1 Truncated 505 bp from 5' of control promoter P16-v1 P16-v1-t2 Truncated 743 bp from 5' of control promoter P16-v1 P16-v1-t3 Truncated 928 bp from 5' of control promoter P16-v1 P17-t1 Truncated 249 bp from 5' of control promoter P17 P19-v2-t1 Truncated 321 bp from 5' of control promoter P19-v2

[0102] Table 7 Primers for constructing genomic integration expression plasmid after promoter truncation

[0103]

[0104] (3) Obtaining of yeast genomic integration transformants and screening by flow cytometry

[0105] After obtaining the promoter expression vectors of different lengths, the I2L-I2R sequences containing the truncated promoters of different lengths were amplified using the primers in Table 4, and then integrated into the K. marxianus GQ7 genome to evaluate the promoter strength, in the same manner as in Example 2.

[0106] (4) Detection of transcriptional activity level of truncated promoters under different temperature, nitrogen source and carbon source conditions

[0107] Figure 4 The results of comparing the fluorescence intensity values of the truncated promoters cultured under 8 culture conditions to the control GQ7 showed that the expression strength of the truncated promoters was lower than that before truncation, and the activity of the truncated promoter P16-v1-t1 was reduced, but still maintained a high activity. After the other promoters were truncated to different lengths, the expression strength was reduced, and the strong promoter activity was lost, indicating that the truncated nucleic acid sequences were required to maintain the high transcriptional activity of the promoters.

[0108] The induction of different truncated promoters by high temperature and inorganic nitrogen source is shown in Table 8. The truncated promoter P16-v1-t1 was induced by high temperature to a similar degree as P16-v1 before truncation, and the degree of induction by inorganic nitrogen source was slightly lower than before truncation, indicating that the truncated sequence still had the function of responding to high temperature induction, but part of the nucleotide sequence required to respond to inorganic nitrogen source induction was truncated; the truncated promoters P16-v1-t2 and P16-v1-t3 lost the high temperature induction activity under some culture conditions, and the inorganic nitrogen source induction activity was significantly reduced, indicating that the truncated nucleic acid sequences were required to maintain the response of the promoters to high temperature and inorganic nitrogen source induction. For promoters P3-v2, P4-v2, P17 and P19-v2, at least half or more of the inorganic nitrogen source induction activity was lost after truncation, indicating that the region of the different promoters that was truncated may be an active functional region, and therefore appropriately extending the length of the promoter is beneficial to maintaining its induction activity strength.

[0109] Table 8 Induction of different length truncated promoters by high temperature and inorganic nitrogen source

[0110]

Claims

1. A promoter with a high level of transcriptional activity, characterized in that, The nucleotide sequence is any one of (a) to (b): (a) the nucleotide sequence shown in any one of SEQ ID NOs: 1-6, or a nucleotide sequence which is truncated from the 5' end of the nucleotide sequence shown in any one of SEQ ID NOs: 1-6 by not more than 950 nucleotides, or not more than 750 nucleotides, or not more than 510 nucleotides, or not more than 320 nucleotides, or not more than 260 nucleotides, or not more than 250 nucleotides, not more than 230 nucleotides; (b) a nucleotide sequence which has 80% homology with the nucleotide sequence of (a) above and has a promoter activity in K. marxianus and at least one more yeast other than K. marxianus.

2. The promoter of high level of transcription activity according to claim 1, characterized in that, The nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 1 truncated from the 5' end by not more than 228 nucleotides; or the nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 2 truncated from the 5' end by not more than 256 nucleotides; The nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 3 truncated from the 5' end by not more than 770 nucleotides; The nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 4 truncated from the 5' end by not more than 928 nucleotides, or not more than 743 nucleotides, or not more than 505 nucleotides; The nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 5 truncated from the 5' end by not more than 249 nucleotides; The nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 6 truncated from the 5' end by not more than 321 nucleotides.

3. The promoter of high level of transcriptional activity according to claim 2, characterized in that, The nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 1, or is obtained by truncating the nucleotide sequence shown in SEQ ID NO: 1 from the 5' end by 228 nucleotides; or the nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 2, or is obtained by truncating the nucleotide sequence shown in SEQ ID NO: 2 from the 5' end by 256 nucleotides; or the nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 3, or is obtained by truncating the nucleotide sequence shown in SEQ ID NO: 2 from the 5' end by 770 nucleotides; or the nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 4, or is obtained by truncating the nucleotide sequence shown in SEQ ID NO: 4 from the 5' end by 928 nucleotides, or 743 nucleotides, or 505 nucleotides; or the nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 5, or is obtained by truncating the nucleotide sequence shown in SEQ ID NO: 5 from the 5' end by 249 nucleotides; or the nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 6, or is obtained by truncating the nucleotide sequence shown in SEQ ID NO: 6 from the 5' end by 321 nucleotides.

4. A transcription expression cassette, comprising, The transcription expression cassette comprises the promoter of any one of claims 1 to 3.

5. The transcription expression cassette of claim 4, wherein, The transcription expression cassette further comprises a protein coding gene operably linked to the polynucleotide having promoter activity.

6. An expression vector, characterized by, The expression vector comprises the promoter of any one of claims 1 to 3 or the transcription expression cassette of claim 4 or 5; Preferably, the expression vector is a genomic integration expression vector.

7. A host cell, wherein, The host cell comprises the promoter of any one of claims 1 to 3 or the transcription expression cassette of claim 4 or 5, or the expression vector of claim 6.

8. The host cell of claim 5, wherein The host cell is a yeast.

9. The host cell of claim 8, wherein The host bacteria is *Max Kluyveromyces* ( Kluyveromyces marxianus Kluyveromyces lactis ( Kluyveromyces lactis ), brewer's yeast ( Saccharomyces cerevisiae ), Yarrowia lipolytica ( Yarrowia lipolytica Pichia pastoris ( Pichia pastoris ), Hansenula polymorpha (( Hansenula polymorpha ), Oriental Isaac yeast ( Issatchenkia orientalis ), Saccharomyces cerevisiae ( Schizosaccharomyces pombe ).

10. Use of the promoter of any one of claims 1 to 3 or the transcription expression cassette of claim 4 or 5, the expression vector of claim 6, or the host cell of any one of claims 7 to 9 in at least one of: (a) enhancing the transcription level of a gene, or in the preparation of a reagent or kit for enhancing the transcription level of a gene; (b) in the preparation of a protein, or in the preparation of a reagent or kit for the preparation of a protein.

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