WO2024250157A1 - Promoteur inductible de levure entièrement synthétique et son procédé de construction - Google Patents

Promoteur inductible de levure entièrement synthétique et son procédé de construction Download PDF

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WO2024250157A1
WO2024250157A1 PCT/CN2023/098388 CN2023098388W WO2024250157A1 WO 2024250157 A1 WO2024250157 A1 WO 2024250157A1 CN 2023098388 W CN2023098388 W CN 2023098388W WO 2024250157 A1 WO2024250157 A1 WO 2024250157A1
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sequence
promoter
spacer sequence
spacer
fully synthetic
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陈业
熊婧卉
栗曾理
郭淑慧
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Shenzhen Institute of Advanced Technology of CAS
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/30—Detection of binding sites or motifs
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B5/00—ICT specially adapted for modelling or simulations in systems biology, e.g. gene-regulatory networks, protein interaction networks or metabolic networks
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    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/85—Saccharomyces
    • C12R2001/865—Saccharomyces cerevisiae

Definitions

  • 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.
  • 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.
  • Saccharomyces cerevisiae by modifying the promoter of Saccharomyces cerevisiae to slow down or accelerate certain metabolic pathways, the product can have a better yield or purity.
  • 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.
  • 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.
  • promoters can be used to construct biosensors that respond to external signals and output the required data.
  • Saccharomyces cerevisiae promoters 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.
  • 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.
  • the gene copy number 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.
  • promoter libraries and used convolutional neural network (CNN) technology to establish mathematical models to predict promoter activity by inputting sequences.
  • CNN convolutional neural network
  • this screening method for establishing promoter libraries is extremely labor-intensive and the number of useful promoters is small.
  • 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.
  • the technologies used to develop new Saccharomyces cerevisiae promoters also include site-selective mutagenesis and hybrid promoter technology.
  • 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.
  • 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.
  • the present invention provides the following technical solutions:
  • a method for constructing a fully synthetic yeast inducible promoter 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.
  • 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 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 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.
  • 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.
  • 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.
  • 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.
  • the present invention has the following beneficial effects:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • polyA/polyT Polyadenylation/polythymine
  • 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.
  • the upstream element regulates genes by recruiting transcription factors (TF).
  • TF transcription factor binding site
  • the promoter can regulate gene expression.
  • UAS upstream activation sequence
  • URS upstream repression sequence
  • 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.
  • 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.
  • Example 1 Determination of the basic structure of the xylose-inducible promoter pxlnR of Saccharomyces cerevisiae
  • the upstream activation sequence UAS, polyadenylation polyA, TATA box and transcription start site TSS on the promoter were determined through literature research.
  • 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).
  • step (3) Determination of the basic structure of the xylose-inducible promoter pxlnR of Saccharomyces cerevisiae.
  • 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.
  • 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 ⁇ -estradiol-induced Er system. In addition, the number of upstream activation sequences UAS can also be changed, such as setting 2, 3 or 4.
  • the yellow fluorescent protein YFP was used as a reporter gene
  • tENO2 was used as a terminator
  • 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.
  • MAR4_CaURA3 represents uracil, which is used to screen yeast monoclones that can survive in a yeast defective medium lacking uracil (SD- ⁇ ura); 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.
  • 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.
  • 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
  • spacer sequences (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.
  • 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- ⁇ leu) 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.
  • 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.
  • step (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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the plasmids pXJH318-pXJH337 with correct sequencing and containing promoters of different sp.A sequences were obtained by E. coli transformation and sequencing.
  • 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).
  • 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.
  • ChXV his3:: indicates the histidine site integrated into the ChXV chromosome
  • IIA.ChV 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 ⁇ L 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.
  • 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.
  • 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.
  • the rationally designed different spacer sequences sp.A have some influence on the strength of the inducible promoter.
  • the strength of the promoter background is positively correlated to a certain extent with the strength of the promoter after induction.
  • 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.
  • 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.
  • 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.
  • DB ⁇ P represents the weighted estimate of DNA binding transcription factor TF in the absence of chromatin, Cp and e are constants, and ⁇ values are different in different chromatin backgrounds.
  • the predicted expression level EL is the weighted value of the estimated value on the learned value plus a constant.
  • 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.
  • 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.
  • adjusting and modifying some parameters of the model such as learning rate and training batches, can improve the model's prediction accuracy.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • Example 2 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.
  • step (3) of Example 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.
  • step (4) of Example 4 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.
  • 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
  • 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
  • the abscissa represents the specific spacer sequence sp.B contained in the promoter.
  • 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
  • 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.
  • 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).
  • 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.
  • 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.
  • the conventional plasmid extraction step was performed to obtain a new promoter plasmid library library.sp.CI muts with different spacer sequences sp.CI.
  • 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.
  • 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.
  • 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.
  • 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::
  • IIB.ChIII leu2::
  • YFP represents a yellow fluorescent protein
  • MAR2_K1LEU2 leucine, which is used to screen yeast monoclonal clones that can survive in a yeast defective medium (SD- ⁇ leu) 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.
  • 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 ⁇ L of yeast defective medium (SD- ⁇ His- ⁇ Leu 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.
  • yeast defective medium SD- ⁇ His- ⁇ Leu medium
  • anhydrotetracycline at a working concentration of 100 ng/mL was added.
  • xylose at a working concentration of 10 mM were added as inducers.
  • 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.
  • 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.
  • 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.
  • the expression levels of the yellow fluorescent protein YFP in different yeast strains are different.
  • 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.
  • 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
  • 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.
  • 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.
  • Example 2 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.
  • step (4) of this example 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.
  • step (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.
  • 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
  • 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
  • the abscissa represents the specific spacer sequence sp.C contained in the promoter.
  • the different spacer sequences sp.C obtained by high-throughput library design have some influence on the strength of the inducible promoter.
  • 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.
  • 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.
  • 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.
  • Example 2 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.
  • step (1) of this example was used to obtain enzyme fragments with transformation, and then yeast transformants were transformed and cultured to obtain the test bacterial solution to be detected.
  • step (3) of Example 4 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.
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

L'invention concerne un promoteur inductible à la levure entièrement synthétique et son procédé de construction. En modularisant l'architecture du promoteur, le procédé actuel divise la région d'activation en amont et la région centrale du promoteur, et utilise la conception rationnelle, l'assistance de modèles informatiques et la construction de banques à haut débit pour cribler les séquences d'espacement. L'architecture de promoteur utilisant Saccharomyces cerevisiae en tant que micro-organisme de châssis est explorée, et une séquence de promoteur inductible de levure non naturelle entièrement synthétique est obtenue. Les problèmes liés à l'insuffisance du nombre de promoteurs bien caractérisés en raison d'une fuite importante des promoteurs, d'une force d'induction insuffisante et de types limités, ou à la recombinaison homologue qui se produit facilement chez Saccharomyces cerevisiae, sont résolus.
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Citations (4)

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US5063154A (en) * 1987-06-24 1991-11-05 Whitehead Institute For Biomedical Research Pheromone - inducible yeast promoter
US20170275635A1 (en) * 2016-03-24 2017-09-28 The Board Of Trustees Of The Leland Stanford Junior University Inducible production-phase promoters for coordinated heterologous expression in yeast
CN107236732A (zh) * 2017-06-13 2017-10-10 青岛百慧智业生物科技有限公司 一种新型酵母诱导型启动子及其应用
US20190062730A1 (en) * 2017-04-04 2019-02-28 Wisconsin Alumni Research Foundation Methods of designing programmable inducible promoters

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Publication number Priority date Publication date Assignee Title
US5063154A (en) * 1987-06-24 1991-11-05 Whitehead Institute For Biomedical Research Pheromone - inducible yeast promoter
US20170275635A1 (en) * 2016-03-24 2017-09-28 The Board Of Trustees Of The Leland Stanford Junior University Inducible production-phase promoters for coordinated heterologous expression in yeast
US20190062730A1 (en) * 2017-04-04 2019-02-28 Wisconsin Alumni Research Foundation Methods of designing programmable inducible promoters
CN107236732A (zh) * 2017-06-13 2017-10-10 青岛百慧智业生物科技有限公司 一种新型酵母诱导型启动子及其应用

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KOTOPKA BENJAMIN J., SMOLKE CHRISTINA D.: "Model-driven generation of artificial yeast promoters", NATURE COMMUNICATIONS, NATURE PUBLISHING GROUP, UK, vol. 11, no. 1, UK, XP093246095, ISSN: 2041-1723, DOI: 10.1038/s41467-020-15977-4 *

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