WO2024250157A1 - Fully synthetic yeast inducible promoter and construction method therefor - Google Patents

Abstract

Provided are a fully synthetic yeast inducible promoter and a construction method therefor. By means of modularizing the promoter architecture, the present method splits the upstream activation region and core promoter region thereof, and uses rational design, computer model assistance and high-throughput library construction to screen spacer sequences. Promoter architecture using Saccharomyces cerevisiae as the chassis microorganism is explored, and a fully synthetic non-natural yeast inducible promoter sequence is obtained. The problems of there being an insufficient number of well-characterized promoters due to high background leakage of promoters, insufficient induction strength and limited types, or of homologous recombination in Saccharomyces cerevisiae easily occurring, are solved.

Description

A fully synthetic yeast inducible promoter and its construction method Technical Field

The present invention relates to the technical field of synthetic biology, and in particular to a fully synthetic yeast inducible promoter and a construction method thereof.

Background Art

With the rapid development of synthetic biology, the application of Saccharomyces cerevisiae as a chassis microorganism to express enzymes related to metabolic pathways and to synthesize natural products or popular compounds has emerged one after another. For example, by modifying the promoter of Saccharomyces cerevisiae to slow down or accelerate certain metabolic pathways, the product can have a better yield or purity.

Currently, Saccharomyces cerevisiae has been widely used in metabolic engineering and gene circuit design, and promoters, as key elements of gene regulation, play an important role in these applications. In metabolic engineering, the expression level of target genes can be precisely controlled by using inducible promoters, the balance and optimization of metabolic pathways can be achieved, or gene circuits can be established to control cell behavior. In gene circuit design, promoters can be used to construct biosensors that respond to external signals and output the required data.

According to the mode of action of Saccharomyces cerevisiae promoters, they can be divided into constitutive promoters and inducible promoters. Among them, the activity of constitutive promoters is relatively stable and is not changed by stimulation, such as pTDH3 and pCYC1. In most cases, the activity of this type of promoter is coupled to the growth rate of the strain and is related to the carbon source content in the environment. The activity of the inducible promoter depends on chemical or physical stimulation. By adding a certain stimulus at a regular and quantitative time, the activity of the promoter can be enhanced or weakened and the transcription level of the gene can be changed. Common inducers used for stimulation include galactose, inorganic phosphates, copper, etc. Commonly used inducible promoters include pGAL1, pPHO5, pCUP1, etc. Because inducible promoters can avoid the influence of exogenous gene expression on the growth of early Saccharomyces cerevisiae, they are widely used in metabolic engineering. In addition, inducible promoters can also affect This affects the sensitivity and overall performance of the biosensor.

However, existing endogenous inducible promoters are usually difficult to meet the requirements of optimizing metabolic flux. The main reasons are as follows: First, the dynamic range of existing inducible promoters is poor and is often not orthogonal to the native regulation of yeast, so there are not enough inducible promoters with good characteristics. Second, the repeated use of the same promoter or core promoter region is prone to homologous recombination, which in turn leads to instability of the synthetic pathway of Saccharomyces cerevisiae. For example, pGAL1 is often repeatedly used to express different enzymes to construct metabolic pathways. In order to compensate for the lack of promoter strength, the gene copy number must be increased, and the use of too many galactose-inducible promoters may deplete the transcription activator Gal4p, thereby interfering with galactose metabolism; third, as a component of excellent biosensors, most natural inducible promoters have defects, such as excessive background leakage expression and insufficient induction strength. Fourth, the design of exogenous transferases is still unclear. Therefore, it is necessary to develop more inducible synthetic promoters to increase the yield of target products and reduce the interference of host fitness loss.

With the rapid development of genomics and the application of machine learning in synthetic biology, some studies have constructed promoter libraries and used convolutional neural network (CNN) technology to establish mathematical models to predict promoter activity by inputting sequences. However, this screening method for establishing promoter libraries is extremely labor-intensive and the number of useful promoters is small. In contrast, many useful sequences have been discovered by mining new natural promoters using gene mining technology, but the number of inducible promoters contained in the genome is still limited.

In addition, the technologies used to develop new Saccharomyces cerevisiae promoters also include site-selective mutagenesis and hybrid promoter technology. Among them, site-selective mutagenesis regulates the expression of target genes by establishing a promoter library with fine-tuned strength, such as error-prone PCR (ep-PCR) and saturation mutagenesis, but the promoters obtained have similar sequences, which may lead to sequence instability and homologous recombination. Hybrid promoter technology adopts an assembly replacement strategy to connect various result components of the required functions together, or replace the core promoter region to obtain new hybrid promoters with multiple functions. For example, multiple upstream activation sequences can be replaced. Polymerization creates a promoter with a repeating operator to recruit more transcription factors for interaction. Some researchers tandemly linked the upstream activation sequence ZF and found that when eight tandem upstream activation sequence ZFs were included, the transcriptional output of the system increased by nearly 60 times, increasing the strength of the promoter.

Although some of the above methods can be used to construct non-natural inducible promoters, some sequences of the constructed promoters are still derived from the natural sequences of Saccharomyces cerevisiae, which has certain limitations. First, there are not many Saccharomyces cerevisiae promoters with good characteristics, such as insufficient promoter strength, few types, strong homology, etc.; second, the promoters are not suitable for gene circuit design, such as long sequences, small response range, and excessive background expression. Therefore, it is particularly important to develop a design scheme for an efficient and fully synthetic inducible promoter architecture.

Summary of the invention

The purpose of the present invention is to overcome the shortcomings of the prior art and solve at least one defect in the above-mentioned background technology.

To achieve the above object, the present invention provides the following technical solutions:

In a first aspect of the present invention, a method for constructing a fully synthetic yeast inducible promoter is provided, comprising: determining the basic structure of the promoter, wherein the promoter comprises, in order from 5′→3′, the following operably connected sequences: an upstream activation sequence, a first spacer sequence, a nucleosome unfavorable sequence, a second spacer sequence, a TATA box, a third spacer sequence, and a transcription start site; screening the sequence bases of the first spacer sequence, the second spacer sequence, and the third spacer sequence using a rational design method, a model library construction method, and/or a high-throughput library construction method; and combining the first spacer sequence, the second spacer sequence, and the third spacer sequence to obtain the promoter.

In some embodiments, the screening of the sequence bases of the first spacer sequence, the second spacer sequence, and the third spacer sequence comprises: screening the sequence bases of the first spacer sequence by a rational design method; screening the sequence bases of the second spacer sequence by a model library construction method; and screening by a high-throughput library construction method. The sequence bases of the third spacer sequence.

Furthermore, the method of screening the sequence bases of the second spacer sequence by the model library building method includes: optimizing the transcription model and randomly generating a promoter fragment containing the second spacer sequence; using the optimized transcription model to predict the strength of the promoter fragment containing the second spacer sequence; and screening the target promoter containing the second spacer sequence.

Furthermore, the optimized transcription model includes: inputting a sequence combination into the transcription model, wherein the sequence combination is obtained by placing sequences of different lengths at different positions; and screening the sequence combination based on the model prediction accuracy to obtain an optimized sequence position and length.

Furthermore, the screening of sequence bases of the third spacer sequence by a high-throughput library construction method includes: analyzing restriction sites and designing degenerate primers, obtaining a promoter library containing the third spacer sequence by amplification; constructing a recombinant vector using the promoter library containing the third spacer sequence; and transforming the recombinant vector into a host and screening a target promoter containing the third spacer sequence.

The second aspect of the present invention provides a fully synthetic yeast-inducible promoter, wherein the fully synthetic yeast-inducible promoter is obtained by the construction method of the fully synthetic yeast-inducible promoter.

In some embodiments, the number of upstream activating sequences in the promoter is 1, 2, 3 or 4.

The third aspect of the present invention provides a recombinant vector, which includes the fully synthetic yeast inducible promoter.

A fourth aspect of the present invention provides a recombinant bacterium, wherein the genome of the recombinant bacterium comprises the recombinant vector.

The fifth aspect of the present invention provides the use of the fully synthetic yeast-inducible promoter, the recombinant vector or the recombinant bacteria in regulating the metabolism of Saccharomyces cerevisiae.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The fully synthetic yeast-inducible promoter and its construction method provided by the present invention can solve the problem of insufficient number of promoters with good characteristics due to insufficient promoter strength and small number of types in metabolic engineering and cell circuit design.

(2) The fully synthetic yeast-inducible promoter and its construction method provided by the present invention can avoid the high similarity between sequences, thereby solving the problem of homologous recombination that is prone to occur in Saccharomyces cerevisiae.

(3) The fully synthetic yeast inducible promoter and its construction method provided by the present invention are applied to the inducible system, and a series of synthetic promoters with low background expression and high induction multiple can be screened out, thereby being applied to gene circuit design.

(4) The method for constructing a fully synthetic yeast inducible promoter provided by the present invention can be used in different induction systems, thereby improving overall adaptability.

(5) The method for constructing a fully synthetic yeast-inducible promoter provided by the present invention obtains a series of spacer sequences of different strengths through model design. Compared with traditional high-throughput library construction and screening methods, this method has the advantages of saving time and cost.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a basic schematic diagram of the design of a fully synthetic inducible promoter for Saccharomyces cerevisiae in an embodiment;

FIG2 is a basic structural diagram of a xylose-inducible promoter of Saccharomyces cerevisiae in an embodiment;

FIG3 is a schematic diagram of a standard test carrier in one embodiment;

FIG4 is a schematic diagram of a high-throughput test carrier in an embodiment;

FIG5 is a schematic diagram of a standard test system in one embodiment;

FIG6 is a diagram showing the effect of different spacer sequences sp.A on promoter strength in an embodiment, wherein FIG6a and FIG6b respectively show the promoter relative strength RPU and the induction fold Fold change;

FIG. 7 is a technical roadmap for promoter model design in one embodiment;

FIG8 is a diagram showing the effect of different spacer sequences sp.B on promoter strength in an embodiment, wherein FIG8a and FIG8b respectively show the promoter relative strength RPU and the induction fold Fold change;

FIG9 is a comparison diagram of the predicted value and the measured value of the promoter strength of the spacer sequence sp.B in an embodiment;

FIG10 is a schematic diagram of a high-throughput testing system in an embodiment;

FIG. 11 is a diagram showing the effect of different spacer sequences sp.C on promoter strength in one embodiment, wherein FIGS. 11a-11c respectively represent the cases where the spacer sequences are sp.CI, sp.CII and sp.CIII;

FIG12 is a diagram showing the effect of different spacer sequences sp.C on promoter strength in an embodiment, wherein FIG12a and FIG12b respectively show the promoter relative strength RPU and the induction fold Fold change;

Figure 13 is a diagram showing the effect of different sequences on promoter strength in an embodiment, wherein Figures 13a and 13b respectively represent the promoter relative strength RPU and the induction fold Fold change.

DETAILED DESCRIPTION

The technical solution of this patent will be further described in detail below in conjunction with specific implementation methods. It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by ordinary technicians in the technical field to which this application belongs.

The 5'→3' involved in this application refers to the directionality of DNA or RNA molecules. The 5' end refers to the position where the fifth carbon atom from the end of the DNA or RNA is connected to a phosphate group, and the 3' end refers to the oxygen atom on the third carbon atom from the end of the DNA or RNA, and this oxygen atom is usually connected to the phosphate group of the next nucleotide. Therefore, the different composition of the 5' end and the 3' end determines the directionality of the DNA and RNA molecules, that is, from the 5' end to the 3' end is the end-to-end direction of the molecule. In the processes of DNA replication, transcription and translation, the 5'→3' direction is more often used to describe the extension direction of the DNA chain and the synthesis direction of the RNA chain. For example, in the process of DNA replication, DNA polymerase will use the single-stranded template as the basis and start from the template. The DNA strand is read from the 3' end to the 5' end of the plate and a new DNA strand is synthesized in the 3'→5' direction, that is, it is extended from the 5' end to the 3' end. Similarly, RNA polymerase reads the single-stranded DNA template from the 3' end to the 5' end when transcribing RNA molecules, and the direction of synthesizing the RNA strand is 5'→3'.

Promoters refer to a series of DNA sequences located near the start point of eukaryotic gene transcription, which can bind to RNA polymerase II, transcription factors and other regulatory factors to initiate gene transcription. Generally speaking, yeast promoters include nucleosomes and open chromatin regions, where nucleosomes are complex protein molecules with a diameter of about 10 nanometers that form high-level structures on chromosomes and are composed of two pairs of acidic proteins and two pairs of basic proteins. The proportion of nucleosome occupancy affects the compactness and accessibility of chromatin. Nucleosomes with low occupancy rates help transcription factors bind to regulatory DNA sequences to regulate promoter activity and promote transcription. Relatively speaking, nucleosomes with high occupancy rates limit the accessibility of promoter regions, thereby affecting gene expression. Polyadenylation/polythymine (polyA/polyT) is a nucleosome-unfavorable sequence that is ubiquitous in eukaryotes and is generally between 10-20 bp in length. These sequences can help maintain the stability of chromatin and are common in natural Saccharomyces cerevisiae promoters, such as pTDH3, pHIS3, pADH2, etc.

The open chromatin region in the promoter chromatin structure usually includes the upstream element sequence (The Upstream Element) and the core promoter region (The Core Promoter). The distance between these two regions may be hundreds of base pairs or more. Among them, the upstream element regulates genes by recruiting transcription factors (TF). When the transcription factor binds to the transcription factor binding site (TFBS) in the upstream element sequence, the promoter can regulate gene expression. According to the effect of the upstream element on the intensity of gene expression, it can be divided into upstream activation sequence (UAS) and upstream repression sequence (URS). The former enhances gene expression, while the latter weakens gene expression. The core promoter is independent of any regulation and directly interacts with RNA polymerase II (pol-II) and universal transcription factors to form a pre-initiation complex (PIC) to initiate transcription. Core promoters are regions that carry the minimum information required to initiate transcription. They generally include a TATA box and a transcription start site (TSS). Analyzing the sequence structure of these promoters helps us understand their regulatory mechanisms, which is very important for metabolic engineering and gene circuit design of Saccharomyces cerevisiae.

The present invention provides a method for constructing a fully synthetic yeast-inducible promoter, which solves the problem of insufficient number of promoters with good characteristics due to excessive background leakage of promoters, insufficient induction strength, and few types in metabolic engineering and cell circuit design. In addition, the method can avoid high similarity between sequences, thereby solving the problem of homologous recombination that is prone to occur in Saccharomyces cerevisiae.

The construction method of the fully synthetic yeast inducible promoter includes: determining the basic structure of the promoter, wherein the promoter includes, in order from 5′→3′, the following operably connected sequences: an upstream activation sequence, a first spacer sequence, a nucleosome unfavorable sequence, a second spacer sequence, a TATA box, a third spacer sequence, and a transcription start site; screening the sequence bases of the first spacer sequence, the second spacer sequence, and the third spacer sequence by a rational design method, a model library construction method, and/or a high-throughput library construction method; and combining the first spacer sequence, the second spacer sequence, and the third spacer sequence to obtain the promoter.

The present invention also provides a fully synthetic yeast-inducible promoter, which is obtained by the construction method of the fully synthetic yeast-inducible promoter.

The present invention also provides a recombinant vector, which comprises the fully synthetic yeast inducible promoter.

The present invention also provides a recombinant bacterium and the recombinant vector in the genome of the recombinant bacterium.

The present invention also provides the use of the fully synthetic yeast-inducible promoter, the recombinant vector or the recombinant bacteria in regulating the metabolism of Saccharomyces cerevisiae.

The present invention will be further described below through specific experimental examples.

Example 1: Determination of the basic structure of the xylose-inducible promoter pxlnR of Saccharomyces cerevisiae

(1) Determination of basic elements. The upstream activation sequence UAS, polyadenylation polyA, TATA box and transcription start site TSS on the promoter were determined through literature research.

(2) Determination of the basic structure of the Saccharomyces cerevisiae promoter. As shown in Figure 1, the optimal distance between the upstream activation sequence UAS and polyadenylation polyA is 24 bp; the optimal distance between polyadenylation polyA and the TATA box is 62 bp; the sequence between the TATA box and the transcription start site TSS is derived from the promoter pADH2, at which the background strength is the weakest; the sequence after the transcription start site TSS is replaced by the kozak sequence, which is conducive to high expression after promoter induction, where the nucleotide sequence of the kozak sequence is 5′-TAAATAAAAAAA-3′ (SEQ ID NO: 1).

(3) Determination of the basic structure of the xylose-inducible promoter pxlnR of Saccharomyces cerevisiae. As shown in Figure 2, the induction system of the eukaryotic transcription factor xlnR is introduced on the basic structure of step (2), that is, the xylose operon xlnO is placed at the upstream activation sequence UAS, so that the promoter is activated by xylose induction, wherein the nucleotide sequence of the xlnO sequence is 5′-gaatttaggctaaagaaagatc-3′ (SEQ ID NO: 2). The sequence of the obtained xylose-inducible promoter proxlnR of Saccharomyces cerevisiae is shown in SEQ ID NO: 3, and the sequence of the transcription factor xlnR acting on the upstream activation sequence xlnO is shown in SEQ ID NO: 4.

It should be understood that this embodiment uses the xylose operon xlnO as an example to introduce the specific implementation of the present application, but in the circuit components of the existing sensor and receiver set, sequences such as lexO and RpaO that bind to transcription factors can also be selected as upstream activation sequences UAS, that is, in addition to being applicable to the xylose-induced xlnR system, this embodiment can also be applied to other induction systems such as the β-estradiol-induced Er system. In addition, the number of upstream activation sequences UAS can also be changed, such as setting 2, 3 or 4.

Example 2: Construction of standard test vector pXJH1

As shown in Figure 3, the yellow fluorescent protein YFP was used as a reporter gene, tENO2 was used as a terminator, and a lethal gene CCDB for screening promoters was placed in front of the yellow fluorescent protein YFP to construct a standard test vector pXJH1, thereby establishing a promoter strength detection system, wherein the sequence of the standard test vector pXJH1 is shown in SEQ ID NO: 5. Among them, MAR4_CaURA3 represents uracil, which is used to screen yeast monoclones that can survive in a yeast defective medium lacking uracil (SD-Δura); ori represents the replication initiation site of the prokaryotic gene plasmid, which is used for DNA replication; Chl ^R represents chloramphenicol, which is used to screen Escherichia coli monoclones that can survive on a chloramphenicol resistance plate.

Specifically, as shown in Table 1, using plasmid CY386 as a template and pXJH1-1-f/pXJH1-1-r as a primer pair for amplification, PCR fragment No. 1 was obtained; using plasmid PY114 as a template and pXJH1-2-f/pXJH1-2-r as a primer pair for amplification, PCR fragment No. 2, i.e., the expression frame of the gene, was obtained; using plasmid CY637576int as a template and pXJH1-3-f/pXJH1-3-r as a primer pair for amplification, PCR fragment No. 3, i.e., the yellow fluorescent protein YFP and terminator T25, was obtained; using plasmid CY386 as a template and pXJH1-4-f/pXJH1-4-r as a primer pair for amplification, PCR fragment No. 4, i.e., the lethal gene CCDB, was obtained. Then, the four PCR fragments were homologously recombined using the ClonExpress Ultra One Step Cloning Kit to obtain the recombinant vector pXJH1. The kit was purchased from Novezan.

Table 1 Primer sequence list

It should be understood that this example uses the endogenous terminator tENO2 of Saccharomyces cerevisiae as an example to introduce the specific implementation mode of the present application, but terminators from other sources can also be selected, and the specific types of the selected terminators include but are not limited to TTPI1P, TFBA1, THXT7, TPGI1, TCYC1, TTEF1, TADH1, and TGPM1.

Example 3: Construction of high-throughput testing vectors pXJH200, pXJH201 and pXJH203

(1) Division of spacer sequences. As shown in Figure 2, the spacer sequences between all characteristic sequences were divided and named separately. Among them, the spacer sequence between xlnR and polyA is the spacer sequence sp.A, the spacer sequence between polyA and TATA box is the spacer sequence sp.B, and the spacer sequence between TATA box and TSS is the spacer sequence sp.C. In addition, the 90 bp spacer sequence sp.C was equally divided into three parts, named sp.CI, sp.CII and sp.CIII respectively.

(2) Construction of high-throughput test vector pXJH200. As shown in Figure 4, the yellow fluorescent protein YFP was used as a reporter gene, tENO2 was used as a terminator, and the red fluorescent protein RFP was used as a reference gene. The lethal gene CCDB was placed at the spacer sequence sp.CI of the original promoter pxlnR to construct a high-throughput test vector pXJH200 with the spacer sequence sp.CI, and its nucleotide sequence is shown in SEQ ID NO:14. Among them, the lethal gene CCDB is used to screen different spacer sequences sp.CI; yfp represents yellow fluorescent protein; MAR2_K1LEU2 represents leucine, which is used to screen yeast clones that can survive in a yeast defective medium (SD-Δleu) lacking leucine; mRuby2 represents red fluorescent protein RFP, which is used to detect whether the entire gene expression frame is integrated into the yeast genome; ori represents the replication start site of the prokaryotic gene plasmid, Used for DNA replication; Chl ^R stands for chloramphenicol, used to screen for E. coli single clones that can survive on chloramphenicol resistance plates.

Specifically, the plasmid pXJH1 successfully constructed in Example 2 was used as a template, and pXJH200-1-f/pXJH200-1-r was used as a primer pair for amplification to obtain PCR fragment No. 1; plasmid pXJH1 was used as a template, and pXJH200-2-f/pXJH200-2-r was used as a primer pair for amplification to obtain PCR fragment No. 2; plasmid pXJH123 was used as a template, and pXJH200-3-f/pXJH200-3-r was used as a primer pair for amplification to obtain PCR fragment No. 7. Then, the three PCR fragments were transformed by homologous recombination using the ClonExpress Ultra One Step Cloning Kit to obtain the recombinant vector pXJH200, wherein the kit was purchased from Novezan.

(3) Construction of high-throughput test vector pXJH201. Similar to step (2), a high-throughput vector pXJH201 for screening the spacer sequence sp.CII was constructed, and its nucleotide sequence is shown in SEQ ID NO:15.

Specifically, the plasmid pXJH1 successfully constructed in Example 2 was used as a template, and pXJH200-1-f/pXJH201-1-r was used as a primer pair for amplification to obtain PCR fragment 3; plasmid pXJH1 was used as a template, and pXJH201-2-f/pXJH202-2-r was used as a primer pair for amplification to obtain PCR fragment 4; plasmid pXJH123 was used as a template, and pXJH201-3-f/pXJH200-3-r was used as a primer pair for amplification to obtain PCR fragment 8. Then, the three PCR fragments were transformed by homologous recombination using the ClonExpress Ultra One Step Cloning Kit to obtain the recombinant vector pXJH201, wherein the kit was purchased from Novezan.

(4) Construction of high-throughput test vector pXJH202. Similar to step (2), a high-throughput vector pXJH202 for screening spacer sequence sp.CIII was constructed, and its nucleotide sequence is shown in SEQ ID NO:16.

Specifically, the plasmid pXJH1 successfully constructed in Example 2 was used as a template and the primer pair pXJH200-1-f/pXJH202-1-r was used for amplification to obtain PCR fragment 5; the plasmid pXJH1 was used as a template and the primer pair pXJH202-2-f/pXJH202-2-r was used for amplification to obtain PCR fragment 6; the plasmid pXJH123 was used as a template and the primer pair pXJH202-3-f/pXJH200-3-r was used for amplification to obtain PCR fragment 9. Then The three PCR fragments were transformed by homologous recombination using the ClonExpress Ultra One Step Cloning Kit to obtain the recombinant vector pXJH202. The kit was purchased from Novezan.

In addition, the primer sequences involved in steps (2)-(4) are shown in Table 2.

Table 2 Primer sequence list

Example 4: Rational design of spacer sequence sp.A and promoter strength test

(1) Rational design of the spacer sequence sp.A. Randomly generate and screen a 24 bp sequence of the same length as the original spacer sequence sp.A to obtain a series of spacer sequence sp.A muts designed by the rational design method, thereby verifying the effect of different spacer sequence sp.A sequences on promoter strength.

Specifically, considering that the spacer sequence sp.A between the transcription factor binding site xlnO and polyA does not belong to the core promoter region, it is believed that the specificity of its sequence has little effect on the promoter, so the following screening conditions are set: first, the GC% content should not be too high and should be maintained between 25-50%; second, the length of the continuous base A in the sequence should not exceed 5. The sp.A sequence generated and screened on the random sequence webpage is shown in Table 3, where the original sequence is sp.A0.

Table 3 Rational design table of spacer sequence sp.A

(2) Construction of promoters containing different sp.A sequences. Long primers containing sp.A sequences were designed for template-free PCR amplification to obtain a series of different PCR fragments containing sp.A sequences, which were used as the first PCR fragment. Using plasmid pXJH120 as a template, a primer pair was designed for amplification to obtain the second PCR fragment.

The first PCR fragment and the second PCR fragment were fused by overlap PCR to obtain a complete promoter sequence containing the sp.A sequence, and the lethal gene CCDB on the vector pXJH1 successfully constructed in Example 2 was replaced with the above complete promoter sequence using the golden gate technology, wherein the golden gate system and conditions are shown in Table 4. Finally, the plasmids pXJH318-pXJH337 with correct sequencing and containing promoters of different sp.A sequences were obtained by E. coli transformation and sequencing.

Table 4 Golden Gate system and conditions

(3) Construction of a standard test system. As shown in Figure 5, the test plasmids pXJH318 to pXJH337 constructed in step (2) were digested with restriction endonuclease BsaI to obtain transformed enzyme fragments, which contained ura3 homology arms and a complete test promoter, yellow fluorescent protein YFP, and a YFP expression frame consisting of terminator tENO1 obtained by overlap PCR fusion in step (2). Among them, xyl represents xylose, which is used as an inducer; xlnR represents a transcription factor that acts on the upstream activation sequence xlnO; P _test represents the promoter of the test; aTc represents anhydrotetracycline hydrochloride, which is used as an inducer; P _tet represents a promoter. After adding the inducer aTc, the P _tet promoter begins to function and the downstream gene begins to express; TetR represents a promoter that is often expressed in the background strain. The expressed tetracycline inhibitor acts on the P _tet promoter to prevent the expression of downstream genes; ChXV (his3::) indicates the histidine site integrated into the ChXV chromosome; IIA.ChV (ura3::) indicates the histidine site integrated into the IIA.ChV chromosome; yfp indicates yellow fluorescent protein.

The competent strain sXJH110 was then prepared using the zymo Frozen-EZ Yeast Transformation II Kit ^TM yeast transformation kit. Specifically, the fragment obtained after the above enzyme digestion and purification was integrated into the ura3 site of the Saccharomyces cerevisiae chromosome IIA.ChV, and the fragment containing the xlnR expression frame was integrated into the His3 site of the Saccharomyces cerevisiae chromosome ChXV to obtain yeast transformants.

The yeast transformants were inoculated into 96 deep-well plates containing 500 μL of the corresponding defective medium and cultured in an incubator at 30°C and 800 rpm. After culturing for 24-48 hours, they were transferred to a new 96 deep-well plate containing defective medium at a ratio of 1:200, and dehydrated tetracycline atc at a working concentration of 100 ng/mL and xylose xyl at a working concentration of 10 mM were added as inducers. As a control, yeast strains CYE72, CYE72/CY671, and CYE72/CY637 should also be added to each test, and the transferred yeast strain CYE72/CY637 needs to be added with dehydrated tetracycline atc at a working concentration of 100 ng/mL. After culturing the strain for 16 hours, the bacterial solution was diluted with an appropriate amount of 1xPBS buffer to obtain the test bacterial solution to be tested.

(4) Test of the effect of different spacer sequences sp.A on promoter strength. The HTS mode of the flow cytometry analyzer BD FACSCelesta ^TM was used to detect the expression of yellow fluorescent protein YFP in the bacterial solution, where the fluorescence of 10,000 cells was detected for each sample, and the yellow fluorescent protein YFP was measured using the FITC-A channel. The obtained data were processed by FlowJo to obtain the median fluorescence Flu (in au). Among them, the relative promoter strength RPU = (YFP _test -YFP _CYE72 ) ÷ (YFP _CYE72/cy671 -YFP _CYE72 ), and the induction fold Fold change = (YFP _{after induction} -YFP _CYE72 ) ÷ (YFP _background -YFP _CYE72 ).

Results: Figure 6 shows the relative strength and corresponding induction multiples of promoters containing different spacer sequences sp.A after induction. The ordinate of Figure 6a represents the expression data of yellow fluorescent protein YFP. The relative promoter strength RPU obtained after treatment, the horizontal axis represents the specific spacer sequence sp.A contained in the promoter, the gray column corresponding to the negative sign shown in the horizontal axis represents the background expression of the promoter, and the black column corresponding to the positive sign shown in the horizontal axis represents the expression after the promoter is induced; the vertical axis of Figure 6b represents the induction fold Fold change, and the horizontal axis represents the specific spacer sequence sp.A contained in the promoter.

It can be seen that the rationally designed different spacer sequences sp.A have some influence on the strength of the inducible promoter. In general, the strength of the promoter background is positively correlated to a certain extent with the strength of the promoter after induction. For example, the strength of the promoter after induction containing spacer sequences such as sp.A7, sp.A8, and sp.A15 is stronger, and the corresponding promoter background expression is higher; the promoter background strength decreases, and the promoter strength after induction also decreases. In addition, the promoter background containing spacer sequences such as sp.A9, sp.A10, and sp.A12 is lower than the control group sp.A0, and the induction multiple changes are greater, which is more suitable for cell circuit design.

Example 5: Computational model design and promoter strength test of spacer sequence sp.B

This embodiment develops a method for generating sequences based on target output by improving the existing yeast promoter model. As shown in Figure 7, based on the existing yeast promoter model, the wet experiment data is combined to optimize the calculation of the strength of the inducible yeast promoter, and the model accuracy is verified. Randomly generate sequences, and use the above calculation model to predict the promoter strength, so as to screen the target sequence, that is, a series of spacer sequences sp.B with different strengths are obtained through model design. Compared with traditional high-throughput library construction and screening methods, this method has the advantages of saving time and cost.

(1) Establishment of computational model.

First, we analyze the algorithm. Assume that the sum of all possible binding sites of a transcription factor x in the promoter is BS _xsi . Among them, i represents the position, s represents the chain, C _x represents the fixed transcription factor concentration, and K _dxsi represents the minimum binding equilibrium constant, which is calculated by calculating the position weight matrix PW of the transcription factor TF motif. M got it.

The sum of all transcription factor binding sites on the yeast promoter, DB _x , was calculated, that is, the sum of binding sites on all transcription factors.

Predict promoter accessibility. DB×P represents the weighted estimate of DNA binding transcription factor TF in the absence of chromatin, _Cp and e are constants, and Ω values are different in different chromatin backgrounds.

Taking into account that nucleosomes prevent TF binding, the estimated TF binding to promoters in different chromatin backgrounds was scaled by Ω.
CB＝DB×Ω

The predicted expression level EL is the weighted value of the estimated value on the learned value plus a constant. The predicted expression level is multiplication in linear space and addition in logarithmic space.
EL＝CB×A+ _ce

Secondly, the model is trained. The model is trained using a dataset with glucose as the medium. The dataset mainly covers the following contents: First, the transcriptome data of yeast cells in glucose medium, including gene numbers and gene expression data; second, the growth curve data of yeast in different concentrations of glucose medium, including time and absorbance (Optical Density, OD value) data in different concentrations of glucose medium. The transcription model is implemented through the tensorflow framework, and AdamOptimizer is used to minimize the mean square error between the prediction and measurement of expression levels. In addition, 1024 promoter sequences are learned in batches, and the first one is learned. The activity and synergy of transcription factor TF, then the concentration of transcription factor TF, and then the activity parameters of specific positions. The sequence length of model training is 110bp, and the actual input sequence is 80bp. The excess part of the sequence exceeding 80bp is deleted, and the sequence less than 80bp is filled with a random sequence. Further screening of the data set during the model training stage and constructing a training set that is more in line with the characteristics of sp.B can enhance the model's learning ability for the characteristics of sp.B sequences. In addition, adjusting and modifying some parameters of the model, such as learning rate and training batches, can improve the model's prediction accuracy.

Finally, the model is optimized. Sequence ordering and composition will significantly affect the prediction accuracy of the model. Generally speaking, the binding of transcription factor TF has obvious positional bias on Azf1, Mga1, Mot3, Skn7, Ume6 and poly-A motifs. When using the pTpA promoter scaffold model as the basic model for model training, 17bp and 13bp flanking sequences are usually added upstream and downstream of the input sequence, respectively, so that the actual sequence length sent to the model is 110bp, of which the 17bp left flank sequence is TGCATTTTTTTCACATC (SEQ ID NO: 52), and the 13bp right flank sequence is GGTTACGGCTGTT (SEQ ID NO: 53). Referring to the characteristics of the model training data set, it was found that the data set was a TATA-box upstream sequence. Then, combined with the promoter architecture actually used by the research group, the TATA-box upstream sequence was analyzed in detail, the motif components were refined, and the predicted sequence components were tiled and arranged. The binding of transcription factors TF was estimated respectively, and the best combination of sp.B+neutral sequence and poly-A+spacer1 was screened. In addition, the model was verified using other published yeast promoter data.

In the further model optimization stage, in order to study the sequence length and order and analyze the position bias of the motif, this solution uses the combination of sequences of different lengths placed in different positions as input, screens the position order and corresponding sequence length with the best prediction effect and the most suitable model algorithm, and improves the prediction accuracy of the model. Then, using the trained model to predict and sort the randomly generated sequence output, it can reduce the manpower, material and financial resources of high-throughput experiments and save time costs.

(2) Design of computational model for spacer sequence sp.B. The randomly generated target sequence of specified length is placed in the computational model obtained in step (1) for prediction, and a series of ideal sequences with low background and high induction are obtained. The top n sequences with the best results are screened for subsequent experimental verification. The sp.B sequences obtained by screening in this example and the output results of the model calculation are shown in Table 5.

Table 5 Design table of calculation model for spacer sequence sp.B

(3) Construction of promoters containing different sp.B sequences. Using PCR technology, the original sequence sp.B0 on pxlnR was replaced with a series of sp.B spacer sequences shown in Table 6 to obtain a complete promoter sequence containing sp.B sequence.

Specifically, the promoter was first divided into sp.B upstream, sp.B and sp.B downstream, and pxlnR was used as a template to design different primer pairs for amplification to obtain sp.B upstream fragments and sp.B downstream fragments; long primers containing sp.B sequences were designed for template-free PCR amplification to obtain sp.B fragments; and then sp.B upstream, sp.B and sp.B downstream were used as templates and amplified with primer pairs to obtain a complete promoter containing sp.B sequences.

Using the golden gate technology, the lethal gene CCDB on the vector pXJH1 successfully constructed in Example 2 was replaced with the above complete promoter sequence, wherein the golden gate system and conditions are similar to those shown in Table 5 in step (2) of Example 4. Finally, through E. coli transformation and sequencing, a plasmid with correct sequencing and containing different sp.B sequence promoters was obtained.

(4) Construction of a standard test system. Similar to step (3) of Example 4, the 24 test plasmids constructed in step (3) of this example were used to obtain enzyme fragments with transformation, and then yeast transformants were transformed and cultured to obtain the test bacterial solution to be detected.

(5) Test on the influence of different spacer sequences sp. B on promoter strength. Similar to step (4) of Example 4, the expression level of fluorescent protein in the bacterial solution obtained in step (4) of this example was detected by flow cytometry, and data processing was performed.

Results: Figure 8 shows the relative strength and corresponding induction fold of promoters containing different spacer sequences sp.B after induction. The ordinate of Figure 8a represents the relative promoter strength RPU obtained after processing the yellow fluorescent protein YFP expression data, the abscissa represents the specific spacer sequence sp.B contained in the promoter, the gray column corresponding to the negative sign shown in the abscissa represents the background expression of the promoter, and the black column corresponding to the positive sign shown in the abscissa represents the expression of the promoter after induction; the ordinate of Figure 8b represents the induction fold Fold change, and the abscissa represents the specific spacer sequence sp.B contained in the promoter.

It can be seen that different spacer sequences sp.B designed by the computational model have different effects on the strength of the inducible promoter. The background expression of sequences such as sp.B1, sp.B5, sp.B8, sp.B23 is lower than that of the original sequence sp.B0, and the induction multiple is also much higher than that of the original promoter. Therefore, the performance of the sequence screened by this embodiment is better than the original promoter pxlnR. In addition, the relative intensity of the background of sequences such as sp.B1, sp.B5, sp.B8, sp.B23 is lower than 0.01, which is more suitable for cell circuit design.

(6) Matching test of model design. Determine the matching degree between actual test data and calculation through linear regression analysis. The correlation of the prediction results of the machine model.

Results: Figure 9 shows the matching of the actual test data and the computer model prediction results, where the horizontal axis represents the relative promoter strength RPU of the actual test, and the vertical axis represents the predicted value of the relative promoter strength RPU. It can be seen that the R value between the measured value and the predicted value reached 0.821, which shows that the linear fit is good and the model prediction is relatively accurate. Therefore, it is feasible to predict the sp.B sequence using the computational model established in this embodiment.

Example 6: High-throughput library design and promoter strength test of spacer sequence sp.C

(1) High-throughput library design of the spacer sequence sp.C. The spacer sequence sp.C was divided into sp.CI, sp.CII and sp.CIII, and a series of high-throughput vector libraries of the three spacer sequences were obtained by random primer design and golden gate technology, which will be placed in a high-throughput test system for testing.

Specifically, according to the sequence characteristics of the core promoter region, three pairs of degenerate primers were designed for library construction of sp.CI, sp.CII and sp.CIII spacer sequences: Random fragment 1 of spcI was amplified with primer-pXJH200-spacer2a-f/XJH-HT-1-r primer pair. Random fragment 2 of spcI was amplified with primer-pXJH200-spacer2b-f/XJH-HT-1-r primer pair. Random fragment 3 of spcI was amplified with primer-pXJH200-spacer2c-f/XJH-HT-1-r primer pair. Among them, random bases W/Y were selected as much as possible for primer design, and the primer design of the spacer sequence sp.CIII minimized the content of A base to avoid premature transcription.

The primer sequences are as follows:

primer-pXJH200-spacer2a-f: 5′-GACCCTGAAGACAAAATANSNSYSYWSSNSYYNWYYWYNWYWNNYYNWAAATATGTCTTCCATGACAGAGTGCCTGGTGATGTTAATGGTCACAA-3′(SEQ ID NO:79);

primer-pXJH200-spacer2b-f: 5′-GACCCTGAAGACAACTCGWWWYYWYY NNWNWYYNNNWNNYYNWNWYWWTTTAATGTCTTCCATGACAGAGTGCCTGGTGATGTTAATGGTCACAA-3′ (SEQ ID NO: 80);

primer-pXJH200-spacer2c-f: 5′-GACCCTGAAGACAATGTTWWYYWWYYNNWWWYYNNNNYYWWNNWWYWWAGAAATGTCTTCCATGACAGAGTGCCTGGTGATGTTAATGGTCACAA-3′(SEQ ID NO:81);

XJH-HT-1-r: 5′-TTGTGACCATTAACATCACCAGGCACT-3′ (SEQ ID NO:82).

Using the optimized golden gate technology, the lethal gene CCDB on the vector pXJH200 successfully constructed in Example 3 was replaced with random fragment 1 to obtain a reaction product, wherein the golden gate system and conditions are shown in Table 6. The above reaction product was transformed into the competent state trans10 by conventional large intestine transformation to obtain large intestine transformants. Subsequently, the culture medium with corresponding resistance was dripped onto the transformation plate, that is, LB culture medium containing chloramphenicol was dripped onto the plate, and all the large intestine transformants were hung into the culture medium with a coating rod. Finally, the conventional plasmid extraction step was performed to obtain a new promoter plasmid library library.sp.CI muts with different spacer sequences sp.CI.

Table 6 Golden Gate system and conditions

Similarly, the lethal gene CCDB on the vectors pXJH201 and pXJH202 successfully constructed in Example 3 was replaced with random fragment 2 and random fragment 3, respectively, and after transformation and plasmid extraction, plasmids with different A new promoter plasmid library library.sp.CII muts with the spacer sequence sp.CII and a new promoter plasmid library library.sp.CIII muts with a different spacer sequence sp.CII I.

(2) Construction of high-throughput test system As shown in FIG10 , the plasmid library library.sp.CI muts constructed in step (1) was digested with restriction endonuclease BsaI to obtain transformed restriction fragments, which contained the entire expression cassette at both ends of the leu2 homology arm. Among them, xyl represents xylose, which is used as an inducer; xlnR represents a transcription factor acting on the upstream activation sequence xlnO; P _test represents a tested promoter; aTc represents anhydrotetracycline hydrochloride, which is used as an inducer; P _tet represents a promoter. After adding the inducer aTc, the P _tet promoter begins to function and the downstream gene begins to express; TetR represents a tetracycline inhibitor commonly expressed in the background strain, which acts on the P _tet promoter to prevent the expression of downstream genes; ChXV (his3::) represents a histidine site integrated into the ChXV chromosome; IIB.ChIII (leu2::) represents a leucine site integrated into the IIB.ChIII chromosome; YFP represents a yellow fluorescent protein; MAR2_K1LEU2 represents leucine, which is used to screen yeast monoclonal clones that can survive in a yeast defective medium (SD-Δleu) lacking leucine; RFP represents a red fluorescent protein; RPU standard Cassette represents the standard expression cassette for expressing red fluorescent protein RFP, which is used to detect whether the entire expression cassette is integrated into the leucine site.

Then, the competent strain sXJH110 was prepared using the zymo Frozen-EZ Yeast Transformation II Kit ^TM. Specifically, the strain sXJH110 contained an xlnR expression cassette integrated at the His3 site. The fragment obtained after the above enzyme digestion and purification was integrated into the leu2 site of Saccharomyces cerevisiae chromosome IIB.Ch III to obtain yeast transformants.

96 yeast transformants were randomly selected from the transformation plate and inoculated into a 96-deep-well plate containing 500 μL of yeast defective medium (SD-ΔHis-ΔLeu medium) lacking histidine and leucine, and cultured in an incubator at 30°C and 800 rpm. After culturing for 24-48 hours, the transformants were transferred to a 96-deep-well plate containing a new defective medium at a ratio of 1:200 and anhydrotetracycline at a working concentration of 100 ng/mL was added. and xylose at a working concentration of 10 mM were added as inducers. As controls, yeast strains CYE72, CYE72/CY671 and CYE72/CY637 should also be added to each test, among which the transfer yeast strain CYE72/CY637 needs to add anhydrotetracycline atc at a working concentration of 100 ng/mL. After culturing the strain for 16 hours, the bacterial solution was diluted with an appropriate amount of 1xPBS buffer to obtain a high-throughput test strain for the spacer sequence sp.CI.

Similarly, the plasmid libraries library.sp.CII muts and library.sp.CIII muts constructed in step (1) were respectively digested, and transformed and conventionally induced by flow cytometry were performed to obtain high-throughput test strains of the spacer sequences sp.CII and sp.CIII.

(3) High-throughput testing of promoter strength in different plasmid libraries. The HTS mode of the flow cytometry analyzer BD FAC SCelesta ^TM was used to detect the expression of yellow fluorescent protein YFP and red fluorescent protein RFP in the bacterial solution. The fluorescence of 10,000 cells was detected for each sample, and the yellow fluorescent protein YFP and red fluorescent protein RFP were measured using FITC-A and PE Texas-A channels, respectively. The data of strains expressing both red fluorescent protein RFP and yellow fluorescent protein YFP were selected for processing to understand the expression of yellow fluorescent protein YFP when red fluorescent protein RFP was constantly expressed.

Results: Figure 11 shows the effect of different spacer sequences on promoter strength, where Figures 11a-11c represent the situations of spacer sequences sp.CI, sp.CII and sp.CIII respectively. The ordinates are the induction intensity, i.e. the expression level of yellow fluorescent protein YFP after induction, and the abscissas are the different yeast strains selected on the transformation plate. The dotted line represents the situation of the control strain, i.e. the induction intensity when the promoter is pxlnR.

It can be seen that after adding inducers to the same plasmid library, the expression levels of the yellow fluorescent protein YFP in different yeast strains are different. Among them, the expression intensity of the yellow fluorescent protein YFP in the strains in the library.sp.CI muts and library.sp.CII muts are concentrated between 4k-6.7k au, of which 7 strains in the library.sp.CI muts have higher intensities than the control strains after induction, and 14 strains in the library.sp.CII muts have higher intensities than the control strains after induction. Compared with the library.sp.CI muts and library. sp.CII muts, the induction strength of strains in library.sp.CIII muts was polarized: the expression intensity of yellow fluorescent protein YFP in some strains was concentrated between 3.6k-6.6k au, while that in others was less than 200 a.u. This large variation in promoter strength may be related to the change in the transcription start site. In addition, there were 16 strains in library.sp.CIII muts with higher induction strength than the control strain.

Fifteen strains with high expression of yellow fluorescent protein YFP after induction in the three libraries were selected for background expression test. It was found that the background expression levels of yellow fluorescent protein YFP in the yeast strains in these three libraries were concentrated between 80 and 150 a.u., which was slightly higher than the background expression level of yellow fluorescent protein YFP in the control strain.

At the same time, the genome of the above strain was used as a template, seq1-f/sequence-r was used as a primer pair for amplification, and the obtained fragments were sequenced. The results are shown in Table 7. The primer sequences are as follows:

seq1-f: 5′-CATTTTCCTCTACGAGCTGTCACCG-3′ (SEQ ID NO:83);

sequence-r: 5′-GGCCATGGAACTGGCAATTTACCA-3′ (SEQ ID NO:84).

Table 7 Base sequence table of different spacer sequences sp.CI muts, sp.CII muts and sp.CIII muts

(4) Construction of promoters containing different sp.C sequences. The strains of library.sp.CI muts, library.sp.CII muts, and library.sp.CIII muts were selected and their sequences were combined to construct sp.C muts with new spacer sequences. The construction method can be to select strains with low background but high expression after induction from the three libraries for combination, or to randomly combine existing spacer sequences sp.C. The obtained sp.C sequences are shown in Table 8, where the original sequence is sp.C0.

Table 8 High-throughput library design for spacer sequence sp.C

Using PCR technology, the original sequence sp.C0 on pxlnR was replaced with a series of sp.C spacer sequences shown in Table 10 to obtain a complete promoter sequence containing the sp.C sequence. Specifically, the promoter was first divided into sp.C upstream and sp.C, and pGSH7 was used as a template and amplified with a primer pair to obtain the sp.C upstream fragment; then a long primer containing the sp.C sequence was designed for template-free PCR amplification to obtain the sp.C fragment.

Using the golden gate technology, the lethal gene CCDB on the vector pXJH1 successfully constructed in Example 2 was replaced with the above complete promoter sequence, wherein the golden gate system and conditions are similar to those shown in Table 5 in step (2) of Example 4. Finally, through E. coli transformation and sequencing, a plasmid with correct sequencing and containing different sp.C sequence promoters was obtained.

(5) Construction of standard test system. Similar to step (3) of Example 4, the 28 test plasmids constructed in step (4) of this example were used to obtain enzyme fragments with transformation, and then yeast transformants were transformed and cultured to obtain the test bacterial solution to be detected.

(6) Test on the influence of different spacer sequences sp. C on promoter strength. Similar to step (4) of Example 4, the expression level of fluorescent protein in the bacterial solution obtained in step (5) of this example was detected by flow cytometry, and data processing was performed.

Results: Figure 12 shows the relative strength of promoters containing different spacer sequences sp.C after induction and the corresponding Induction fold. The ordinate of Figure 12a represents the relative promoter strength RPU obtained after processing the yellow fluorescent protein YFP expression data, the abscissa represents the specific spacer sequence sp.C contained in the promoter, the gray column corresponding to the negative sign shown in the abscissa represents the background expression of the promoter, and the black column corresponding to the positive sign shown in the abscissa represents the expression after the promoter is induced; the ordinate of Figure 12b represents the induction fold Fold change, and the abscissa represents the specific spacer sequence sp.C contained in the promoter.

It can be seen that the different spacer sequences sp.C obtained by high-throughput library design have some influence on the strength of the inducible promoter. When the relative promoter strength RPU of the promoter background is distributed between 0.01-1, the relative promoter strength RPU after induction is distributed between 7-16, among which the background relative strength of the promoter containing sp.C6 and sp.C13-sp.C24 sequences is lower than that of the control group sp.C0, and the background expression of the promoter containing sp.C20 sequence is lower than 0.001. In addition, the induction fold change of most promoters is greater than that of the original promoter pxlnR, among which the induction fold of the promoter containing sp.C18 sequence exceeds 500.

Example 7: Construction of non-natural synthetic promoter and promoter strength test

(1) Construction of synthetic promoters. The spacer sequences sp.A, sp.B and sp.C obtained in Examples 4-6 were screened according to the expression level of the background yellow fluorescent protein YFP, and then the construction of the promoter pxlnR was referred to. With xlnR as the upstream activation sequence UAS, the three spacer sequences were randomly combined to obtain a series of new xylose-inducible promoter sequences, the composition structure of which is shown in Table 9. The specific sequences are shown in SEQ ID NO: 160-SEQ ID NO: 182.

Table 9 New promoter sequences and names

The promoter p205-227 was obtained by PCR. Specifically, the promoter was first divided into three fragments, namely, upper, middle and lower fragments, namely fragment 1, fragment 2 and fragment 3. Then, fragment 1 and fragment 3 were amplified by PCR with templates, and fragment 2 was amplified by PCR without template. Finally, fragment 1, fragment 2 and fragment 3 were used as templates and amplified with primer pairs to obtain the complete promoter.

Using the golden gate technology, the lethal gene CCDB on the vector pXJH1 successfully constructed in Example 2 was replaced with the above complete promoter sequence, wherein the golden gate system and conditions are similar to those shown in Table 5 in step (2) of Example 4. Finally, the plasmid with correct sequencing was obtained by E. coli transformation and sequencing.

(2) Construction of standard test system. Similar to step (3) of Example 4, the 23 test plasmids constructed in step (1) of this example were used to obtain enzyme fragments with transformation, and then yeast transformants were transformed and cultured to obtain the test bacterial solution to be detected.

(3) Promoter strength test. Similar to step (4) of Example 4, the promoter strength was tested by flow cytometry. The fluorescent protein expression level of the bacterial solution obtained in step (3) of this embodiment is measured and data processing is performed.

Results: Figure 13 shows the relative strength and corresponding induction fold of different promoters after induction. The ordinate of Figure 13a represents the relative promoter strength RPU obtained after processing the yellow fluorescent protein YFP expression data, the abscissa represents the specific promoter sequence contained in the promoter, the gray column represents the background expression of the promoter, and the black column represents the expression after the promoter is induced; the ordinate of Figure 13b represents the induction fold Fold change, and the abscissa represents the specific promoter sequence contained in the promoter.

It can be seen that most of the new promoters constructed in this scheme showed good characteristics. The background expression of the promoters was low, and their relative promoter strength RPU was basically lower than 0.1. The background relative strength RPU of promoters p205 and p206 was less than 0.001, and the relative promoter strength RPU after induction was between 4 and 12. At the same time, except for promoter p215, the induction folds of the new promoters were basically greater than the induction folds of the original promoter pxlnR, among which the induction folds of promoters p208 and p223 were higher than 600.

In summary, the present invention provides a fully synthetic yeast-inducible promoter and a construction method thereof. The method modularizes the promoter architecture, splits its upstream activation region and core promoter region, and uses rational design, computer model assistance, and high-throughput library construction methods to screen the spacer sequence, explores the architecture mode of the promoter using Saccharomyces cerevisiae as the chassis microorganism, obtains a new non-natural yeast-inducible promoter sequence, and solves the problem of insufficient number of well-characterized promoters due to insufficient promoter strength and few types, or the problem of homologous recombination that is easy to occur in Saccharomyces cerevisiae.

The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.

Claims (10)

A method for constructing a fully synthetic yeast-inducible promoter, characterized by comprising: Determine the basic structure of the promoter, wherein the promoter includes, from 5′→3′, operably connected: an upstream activation sequence, a first spacer sequence, a nucleosome unfavorable sequence, a second spacer sequence, a TATA box, a third spacer sequence, and a transcription start site; Using a rational design method, a model library construction method and/or a high-throughput library construction method to screen the sequence bases of the first spacer sequence, the second spacer sequence and the third spacer sequence; and The first spacer sequence, the second spacer sequence and the third spacer sequence are combined to obtain the promoter. The method for constructing a fully synthetic yeast-inducible promoter according to claim 1, characterized in that the sequence bases for screening the first spacer sequence, the second spacer sequence and the third spacer sequence include: Screening the sequence bases of the first spacer sequence by a rational design method; Screening the sequence bases of the second spacer sequence by a model library building method; and The sequence bases of the third spacer sequence are screened by a high-throughput library construction method. The method for constructing a fully synthetic yeast-inducible promoter according to claim 2, characterized in that the screening of sequence bases of the second spacer sequence by the model library construction method comprises: Optimizing the transcription model and randomly generating a promoter fragment containing the second spacer sequence; The optimized transcription model is used to predict the strength of the promoter fragment containing the second spacer sequence; and the target promoter containing the second spacer sequence is screened. The method for constructing a fully synthetic yeast inducible promoter according to claim 3, wherein the optimized transcription model comprises: Inputting a sequence combination into the transcription model, wherein the sequence combination is obtained by placing sequences of different lengths at different positions; and The sequence combinations are screened based on the model prediction accuracy to obtain optimized sequence positions and lengths. The method for constructing a fully synthetic yeast-inducible promoter according to claim 3 or 4, characterized in that the sequence bases of the third spacer sequence screened by a high-throughput library construction method include: Analyze the restriction site and design degenerate primers to obtain the promoter containing the third spacer sequence by amplification. Library; constructing a recombinant vector using the promoter library containing the third spacer sequence; and The recombinant vector is transformed into a host and the target promoter containing the third spacer sequence is selected. A fully synthetic yeast-inducible promoter, characterized in that the fully synthetic yeast-inducible promoter is obtained by the construction method of the fully synthetic yeast-inducible promoter according to any one of claims 1 to 5. The fully synthetic yeast inducible promoter according to claim 6, characterized in that the number of upstream activation sequences in the promoter is 1, 2, 3 or 4. A recombinant vector, characterized in that the recombinant vector comprises the fully synthetic yeast inducible promoter according to claim 6 or 7. A recombinant bacterium, characterized in that the genome of the recombinant bacterium contains the recombinant vector according to claim 8. Use of the fully synthetic yeast-inducible promoter according to claim 6 or 7, the recombinant vector according to claim 8 or the recombinant bacteria according to claim 9 in regulating the metabolism of Saccharomyces cerevisiae.