Growth-coupled yeast-based directed evolution screening method and device and use

Abstract

A growth-coupled yeast-based directed evolution screening method and a device and the use. The provided growth-coupled yeast carries a cytoplasmic linear plasmid, TP-DNApol error-prone DNA polymerase and a plurality of growth-related genes capable of being integrated into a genome and regulatory molecules thereof. A regulatory molecule and an evolved biomacromolecule are functionally coupled to induce the expression and repression of a downstream gene to achieve the aims of screening and enrichment. By means of the method, more types of proteins can be evolved, positive and negative screening of evolved proteins can be realized in the same system, and automatic screening of evolved strains can be realized.

Description

A growth-coupled yeast directed evolution screening method, device and application Technical Field

[0001] The present disclosure relates to the technical field of directed evolution, and in particular to a growth-coupled yeast directed evolution screening method, device and application. Background Art

[0002] In 2018, half of the Nobel Prize in Chemistry was awarded to Frances H. Arnold, a scientist who used directed evolution to evolve enzymes. Arnold's representative achievements include evolving cytochrome P450 to catalyze chemical reactions not found in living organisms, such as the production of chiral cis-cyclopropane, a precursor to antidepressants (Wang, ZJ, et al. (2014). Improved cyclopropanation activity of histidine-ligated cytochrome P450 enables the enantioselective formal synthesis of levomilnacipran. Angew Chem Int Ed Engl 53(26):6810-3.). Her outstanding achievement lies in using directed evolution to modify the gene sequence of proteases, improving the catalytic efficiency of chemical reactions or creating new chemical reactions, which can be applied in the pharmaceutical, new energy, and environmental fields.

[0003] Before the Nobel Prize was awarded for directed evolution, the technology had already undergone three or four decades of development. Major technological advances were concentrated in the 1980s and 1990s, such as Error-prone PCR (Leung, DW, E. Chen, and D.V. Goeddel (1989). A Method for Random Mutagenesis of a Defined DNA Segment Using a Modified Polymerase Chain Reaction, Technique 1.), which remains the most mainstream evolution method. This method uses a low-fidelity Taq DNA polymerase to perform PCR amplification on a DNA fragment, generating a library of DNA fragments containing point mutations. These DNA fragments are then constructed into vectors, expressed in Escherichia coli, and screened to obtain the desired function. Another example is DNA shuffling (Stemmer, WP (1994). Rapid evolution of a protein in vitro by DNA shuffling. Nature 370(6488):389-91.), another directed evolution technique that complements Error-prone PCR. DNaseI is used in the PCR reaction to break up the DNA fragments. Primer mismatch may occur in the next round of PCR reaction, so recombination may occur between DNA fragments. This method can change the order of DNA bases and the length of DNA fragments, making it more likely to create new genes.

[0004] Directed evolution has become a powerful technique, widely used in numerous biological and chemical fields, including protein engineering, for both scientific research and practical applications. The current full process of directed evolution involves mutation introduction, gene library enrichment, and screening, with these three steps potentially repeated multiple times depending on demand. Mutation introduction and enrichment are typically performed in vitro, but over the past decade, methods for mutation introduction and enrichment in vivo have been gradually developed and promoted. Directed evolution in vivo offers numerous advantages: Directed evolution directly in vivo is more closely aligned with practical application environments. However, in vitro evolution requires consideration of the capacity of the mutation library and considerable effort in screening the library. The resulting mutants may not be suitable for practical applications. Directly generating mutation libraries in cells, on the other hand, is simpler, requires less effort, and allows the resulting mutants to be directly adapted to in vivo application environments.

[0005] The principle of directed evolution in organisms still relies on the use of low-fidelity Taq DNA polymerases. However, these low-fidelity enzymes are typically expressed within the organism, with the DNA fragments also present within the organism as plasmids or inserted into the genome. The low-fidelity enzymes introduce point mutations at the target site in vivo, and the mutated proteins are then produced using the organism's own transcription and translation systems. These proteins are then screened within the organism, and only those that produce the target function survive. Organisms that fail to produce the desired mutant protein are killed. Common organisms used as platforms include Escherichia coli, yeast, and mammalian cells. For example, the PACE (phage-assisted continuous evolution) technique, developed by David R. Liu's laboratory, uses phage-assisted directed evolution (Esvelt, Kevin M., Jacob C. Carlson, and David R. Liu (2011). A system for the continuous directed evolution of biomolecules. Nature 472(7344):499-503). This technology uses bacteriophage as a medium to engineer Escherichia coli, into which AP and MP plasmids are transferred. The AP plasmid expresses gene III, which is essential for phage growth, and the MP plasmid expresses low-fidelity DNA polymerase. Phage is used to infect the engineered Escherichia coli, and the phage injects its own genes into the E. coli, expressed as SP. The low-fidelity DNA polymerase on the MP plasmid performs error-prone replication of the genes on the SP plasmid. Only when the protein expressed by SP meets the target function can it initiate the expression of gene III on the AP plasmid, thereby helping to assemble into new phage with infectious activity. The entire process occurs in a continuously flowing lagoon system. Phage that has not accumulated favorable mutations and cannot be assembled into infectious phage will be washed away with the liquid flow.

[0006] The PACE system has become a commonly used system for directed evolution in vivo. To address the problem of dispersed mutation regions in the PACE system, David V. Schaffer and John E. Dueber developed the EvolvR system (Halperin, SO, et al. (2018). CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window. Nature 560(7717): 248-252.), which applies the advantages of CRISPR-cas9 positioning to the field of directed evolution, concentrating mutations within the cas9 targeting region.

[0007] Evolution within eukaryotic cells has a wider range of application scenarios and is more advanced. Currently, the chassis cells used in domestic in vivo directed evolution systems are mostly Escherichia coli and viruses, which are a series of technologies based on the transformation of the PACE system. Pharmaceuticals, vaccines, and other biological products targeting eukaryotic organisms are less compatible in such directed evolution systems, and the evolution of disease-related biomacromolecules is difficult to achieve through prokaryotic directed evolution. Compared to E. coli, proteins screened in yeast or mammalian cells can be directly applied in disease prevention and treatment.

[0008] One eukaryotic evolutionary system is VEGAS (English, JG, et al. (2019). VEGAS as a Platform for Facile Directed Evolution in Mammalian Cells. Cell 178(3):748-761.e17.). Similar to the PACE system, it uses viruses to infect mammals and exploits the high mutation rate of the sindbis virus for directed evolution.

[0009] Another eukaryotic directed evolution system, orthorep (Zhong, Z., Ravikumar, A., & Liu, CC (2018). Tunable Expression Systems for Orthogonal DNA Replication. ACS synthetic biology, 7 (12), 2930–2934.) has unique advantages. The Orthorep system is a directed evolution system that uses Saccharomyces cerevisiae as a base cell. The cytoplasmic linear plasmid from Kluyveromyces lactis is transferred into Saccharomyces cerevisiae. Since the cytoplasmic linear plasmid carries a full set of elements for DNA replication, transcription, and translation, it can independently express proteins in the cytoplasm and can exist stably. Due to the different source species, the elements of the linear plasmid and the elements in the genome are orthogonal to each other. Therefore, the DNA polymerase carried by the linear plasmid can be modified to achieve low-fidelity DNA replication, and point mutations can be performed on the linear plasmid DNA to form a mutation library. The modified DNA polymerase will not replicate genes on the genome, so it can achieve site-directed mutations in the organism. This method does not require the viral transfection steps of the PACE system and VEGAS system. The method is simple and integrates the function of the locked region of the EvolvR system. In addition, the Orthorep system is a eukaryotic system and can evolve proteins that can only be expressed in eukaryotic systems. It has huge application space.

[0010] The current system has the following flaws:

[0011] Lack of an integrated screening system. The system currently focuses on using yeast itself to generate mutant libraries, which require a complex screening process. Different screening methods may be required for different proteins, requiring significant modifications to the entire system. Currently, the system can evolve proteins whose functional changes have a significant phenotype, such as antibiotic resistance proteins and catalytic enzymes. However, it is difficult to evolve protein functions with less obvious phenotypes, such as protein-DNA binding, protein-protein binding, and protein-small molecule binding.

[0012] It is not possible to perform positive and negative screening on proteins with complex functions. Currently, the system's evolutionary path for proteins is usually single-threaded. For example, to evolve an enzyme to catalyze a certain substrate more efficiently, it is only necessary to continuously screen for enzymes that can produce more products in the same amount of time. However, it is difficult to evolve enzymes that recognize different substrates. Therefore, the evolved enzyme obtained may respond to other substrates at the same time. In addition, proteins usually have multiple domains. When evolving a domain, it is likely to have an impact on other domains of the protein, thereby weakening the overall function of the protein.

[0013] The process cannot be automated. Evolution within an automated fluidics system, like the PACE system, would save significant labor costs. Currently, there is no precedent for automated evolution of this system.

[0014] Therefore, there is an urgent need to develop an automated directed evolution protein screening system that can realize integrated screening of different functional proteins and positive and negative screening of functionally complex proteins.

[0015] Summary of the Invention

[0016] The present disclosure aims to solve one of the technical problems in the related art at least to a certain extent.

[0017] A first aspect of the present disclosure provides a growth-coupled yeast directed evolution screening method, the method comprising:

[0018] (A) Construction of an exogenous plasmid containing the target sequence;

[0019] (B) Construction of error-prone DNA polymerase expression plasmid;

[0020] (C) Construction of growth-related gene expression plasmids;

[0021] (D) transferring the exogenous plasmid described in step (A), the error-prone DNA polymerase expression plasmid described in step (B), and the growth-related gene expression plasmid described in step (C) into yeast,

[0022] The biomacromolecule expressed by the evolution target sequence can drive / inhibit the expression of the growth-related gene in step (C) through the regulatory molecule of the growth-related gene.

[0023] (E) placing the yeast under a growth stress environment, wherein the target sequence to be evolved contained in the yeast that survives under the growth stress environment is a target sequence that conforms to the direction of directed evolution.

[0024] The growth-coupled yeast directed evolution screening method provided by the present disclosure can achieve integrated screening. The growth-coupled evolution screening method can enrich for favorable mutations and simplify the intermediate process. Currently, the screening and enrichment process for existing international and domestic patents is relatively complex, and multiple other screening methods must be used in conjunction to obtain truly useful mutants. The present disclosure can couple function with growth in a personalized manner based on protein characteristics. Yeast growth-related genes are regulated by evolved proteins, and logical judgments can be made about the protein's function. For example, after the activity of a certain enzyme is improved through evolution, it can produce more small molecules or interact more strongly with other downstream molecules, inducing the expression of other genes. This gene can interact with the regulatory sequence preceding the growth-related gene to regulate the expression of the growth-related gene. Only strains that evolve proteins with the expected function can express more growth-related genes and thus reproduce and survive. Strains that fail to evolve will stagnate in growth and gradually dilute and disappear with elution or passage. The small molecules here can be regulatory molecules that directly participate in the expression and expression strength of growth-related genes, or they can be intermediate products in the biological reaction process.

[0025] According to a specific embodiment of the present disclosure, the target sequence includes at least a portion of the sequence of one or more genes.

[0026] According to a specific embodiment of the present disclosure, the biomacromolecule is a protein, which includes a protein expressed by a single gene or a fusion protein co-expressed by multiple genes.

[0027] According to a specific embodiment of the present disclosure, the molecular weight of the protein is 4-400KD.

[0028] According to a specific embodiment of the present disclosure, the molecular weight of the protein is 8-200KD.

[0029] According to a specific embodiment of the present disclosure, the protein includes any one selected from enzymes, receptors, and transcriptional regulators, or the protein includes a fusion protein composed of any one of enzymes, receptors, and transcriptional regulators.

[0030] According to a specific embodiment of the present invention, the fusion protein is a fusion protein composed of a combination of multiple different enzymes.

[0031] According to a specific embodiment of the present invention, the fusion protein is a fusion protein composed of a combination of multiple different receptors.

[0032] According to a specific embodiment of the present invention, the fusion protein is a fusion protein composed of a combination of multiple different transcription regulatory factors.

[0033] According to a specific embodiment of the present invention, the fusion protein is a fusion protein of at least two proteins selected from enzymes, receptors or transcriptional regulators.

[0034] According to a specific embodiment of the present disclosure, the enzyme is an enzyme with catalytic activity or an enzyme associated with gene expression.

[0035] According to a specific embodiment of the present disclosure, the enzyme associated with gene expression includes enzymes selected from DNA polymerases, transcriptases, gene editing enzymes, capping enzymes and enzymes associated with epigenetic modification.

[0036] According to a specific embodiment of the present disclosure, the gene editing enzyme includes at least one selected from Cas family proteins such as Cas9, Cas12, ZFP, and TALEN.

[0037] According to a specific embodiment of the present disclosure, the enzyme associated with epigenetic modification includes at least one selected from histone acetyltransferase or deacetylase, histone methyltransferase or demethylase, ubiquitinase or deubiquitinase, DNA methylase or demethylase.

[0038] According to a specific embodiment of the present disclosure, the transcriptional regulatory factor includes at least one selected from LysR, AraC / XylS, TetR, LuxR, LacI, ArsR, IcIR, MerR, AsnC, MarR, NtrC (EBP), OmpR, DeoR, Cold shock, GntR, Crp, C2H2, bZIP, dCas9-based, ZFP-based, and TALEN-based transcriptional regulatory factors.

[0039] According to a specific embodiment of the present disclosure, the receptor includes any one selected from membrane receptors, cytoplasmic receptors, and nuclear receptors.

[0040] According to a specific embodiment of the present disclosure, the receptor is a membrane receptor, and the membrane receptor includes any one selected from ion channel-shaped receptors, G protein-coupled receptors, and tyrosine kinase-coupled receptors.

[0041] According to a specific embodiment of the present disclosure, the exogenous plasmid is derived from a cytoplasmic linear plasmid of a non-host eukaryotic organism.

[0042] According to specific embodiments of the present disclosure, the non-host eukaryotic organism comprises Kluyveromyces lactis.

[0043] According to a specific embodiment of the present disclosure, the growth-related gene includes at least one selected from antibiotic resistance genes and nutritional marker genes.

[0044] According to a specific embodiment of the present disclosure, the antibiotic resistance gene includes at least one selected from the group consisting of bleomycin resistance gene, kanamycin resistance gene, hygromycin resistance gene, nourseothricin, and amphotericin.

[0045] According to a specific embodiment of the present disclosure, the nutritional marker gene includes at least one selected from URA, LEU, HIS, ADE2, TRP1, and MET17.

[0046] According to a specific embodiment of the present disclosure, the step (E) further comprises:

[0047] (1) the expression of the growth-related gene causes the yeast to die as a negative screening,

[0048] The expression of the growth-related gene allows the yeast to survive as a positive selection,

[0049] Obtaining yeast containing a target sequence that conforms to the direction of directed evolution through the negative screening and / or positive screening process;

[0050] (2) obtaining the target sequence that conforms to the directed evolution direction based on the yeast in step (1),

[0051] Among them, the growth-related gene whose expression causes the yeast to die is a negative screening gene, and the growth-related gene whose expression causes the yeast to survive is a positive screening gene.

[0052] According to a specific embodiment of the present disclosure, in step (E), negative screening and positive screening are simultaneously performed on the yeast under the growth stress environment, wherein the screening pressures of the negative screening and the positive screening are adjustable.

[0053] According to a specific embodiment of the present disclosure, the error-prone DNA polymerase and the exogenous plasmid are derived from the same species.

[0054] According to a specific embodiment of the present disclosure, the error-prone DNA polymerase only acts on the exogenous plasmid to perform error-prone replication.

[0055] According to a specific embodiment of the present disclosure, the yeast includes at least one selected from Saccharomyces cerevisiae, Saccharomyces paradoxica, Saccharomyces bayanus, Saccharomyces rioja and Saccharomyces pastorianus.

[0056] According to a specific embodiment of the present disclosure, the yeast is Saccharomyces cerevisiae.

[0057] According to a specific embodiment of the present disclosure, the regulatory molecule of the growth-related gene includes any one selected from a protein, a nucleic acid, and a first small molecule substance.

[0058] According to specific embodiments of the present disclosure, the nucleic acid comprises a DNA sequence.

[0059] According to a specific embodiment of the present disclosure, the DNA sequence includes at least one selected from a promoter, an insulator, and an enhancer.

[0060] According to a specific embodiment of the present disclosure, the first small molecule substance includes at least one selected from antibiotics, enzyme substrates, short peptides, oligopeptides, sugars, lipids, vitamins, inorganic substances, alkaloids, and metabolites.

[0061] The growth-coupled yeast directed evolution screening method provided by the present disclosure can realize positive and negative screening, and can effectively fix the function of the protein within a certain range, which meets the needs of practical applications. Usually, proteins are multifunctional proteins. Due to the structure of the protein, changes in a certain function may affect other functions. In order to maintain the other functions unchanged and specifically evolve one of the functions, the help of positive and negative screening systems is needed. The current positive and negative screening systems are mostly two separate screening systems, which are difficult to use at the same time. The efficiency of the separated screening is low and it increases manpower and material resources. The growth-coupled yeast directed evolution screening method provided by the present disclosure can realize positive and negative screening at the same time, without the need to perform genetic operations on the engineered bacteria in the middle, and maintain the continuity of evolution.

[0062] A second aspect of the present disclosure provides a method for directed evolution of the interaction ability between a target protein and a first protein, the method comprising:

[0063] Yeast is cultured and screened using the growth-coupled yeast directed evolution screening method, and a target protein having an ability to interact with the first protein or an increased or decreased ability to interact is obtained based on the yeast that survives the growth stress environment.

[0064] in,

[0065] The target protein is fused with the BD of the transcription factor of the growth-related gene in step (D) to form a first fusion protein, and the first protein is fused with the AD of the transcription factor of the growth-related gene to form a second fusion protein; or

[0066] The target protein is fused with the AD of the transcription factor of the growth-related gene in step (D) to form a first fusion protein, and the first protein is fused with the BD of the transcription factor of the growth-related gene to form a second fusion protein.

[0067] Wherein, the BD is the binding domain of the upstream activation sequence of the promoter of the growth-related gene, and the AD is the activation domain of the upstream activation sequence of the promoter of the growth-related gene.

[0068] A third aspect of the present disclosure provides a method for directed evolution of the ability of a target protein to interact with a cis-acting element, the method comprising:

[0069] Yeast is cultured and screened using the method described in the first aspect, and a target protein having an ability to interact with the cis-acting element or having an increased or decreased ability to interact is obtained based on yeast that survive the growth stress environment.

[0070] in,

[0071] The cis-acting element is on the growth-related gene expression plasmid in step (C), and the cis-acting element is the cis-acting element of the growth-related gene;

[0072] The sequence for expressing the target protein is the target sequence described in step (A).

[0073] According to a specific embodiment of the present disclosure, the target protein includes at least one selected from transcription factors, histones, DNA methylases, gene editing enzymes, and DNA binding proteins.

[0074] A fourth aspect of the present disclosure provides a method for directed evolution of the interaction ability of a target protein with a second small molecule, the method comprising:

[0075] Yeast is cultured and screened using the method described in the first aspect, and a target protein having an ability to interact with the second small molecule or having an increased or decreased ability to interact is obtained based on the yeast that survives the growth stress environment.

[0076] The sequence for expressing the target protein is the target sequence described in step (A), and the second small molecule substance is a switch for controlling the expression and strength of the growth-related gene.

[0077] According to a specific embodiment of the present disclosure, the second small molecule substance promotes / inhibits the binding of the target protein to the growth-related gene.

[0078] According to a specific embodiment of the present disclosure, the target protein comprises at least two binding sites.

[0079] According to a specific embodiment of the present disclosure, after the first binding site of the target protein binds to the second small molecule substance, the binding of the second binding site of the target protein to the regulatory molecule of the growth-related gene is turned on / off.

[0080] According to a specific embodiment of the present disclosure, after the first binding site of the target protein binds to the second small molecule substance, the binding of the second binding site of the target protein to the regulatory molecule of the growth-related gene is enhanced / weakened.

[0081] According to specific embodiments of the present disclosure, the first binding site is the same as or different from the second binding site.

[0082] According to a specific embodiment of the present disclosure, the second small molecule substance includes at least one selected from antibiotics, enzyme substrates, oligopeptides, sugars, lipids, vitamins, inorganic substances, alkaloids, hormones, oligonucleotides, metal ions, short peptides, drug molecules, and metabolites.

[0083] According to a specific embodiment of the present disclosure, the regulatory molecule includes a DNA sequence or a transcription factor.

[0084] According to a specific embodiment of the present disclosure, the DNA sequence includes any one selected from the group consisting of the lactose operon, the xylose operon, and the tetracycline operon.

[0085] According to a specific embodiment of the present disclosure, the transcription factor includes any one of lacI, xylR, and tetR.

[0086] A fifth aspect of the present disclosure provides a method for directed evolution of enzyme catalytic ability, the method comprising:

[0087] Yeasts are cultured and screened using the method described in the first aspect, and catalytic enzymes that conform to the direction of directed evolution are obtained based on yeasts that survive under the growth stress environment.

[0088] in,

[0089] The nucleic acid sequence expressing the catalytic enzyme is the target sequence described in step (A),

[0090] The enzyme substrate of the catalytic enzyme or the product after the catalytic enzyme catalyzes the enzyme substrate can activate / inhibit the function of the biosensor. The biosensor is the regulatory molecule of the growth-related gene in step (C). After the function of the biosensor is activated, it can promote the expression of the growth-related gene. It should be noted that the product after the catalytic enzyme catalyzes the enzyme substrate here can, under certain circumstances, also serve as a substrate for another enzyme. The product and the substrate are only different references in the reflection process and do not represent the final state of the substance.

[0091] According to a specific embodiment of the present disclosure, the enzyme substrate can activate the function of the biosensor. When the growth-related gene is a negative screening gene, the enzyme with a stronger ability to catalyze the enzyme substrate after evolution is better able to survive under the growth stress environment.

[0092] According to a specific embodiment of the present disclosure, the product of the catalytic enzyme after catalyzing the enzyme substrate can activate the function of the biosensor. When the growth-related gene is a positive screening gene, the enzyme with a stronger ability to catalyze the enzyme substrate after evolution can better survive under the growth stress environment.

[0093] According to a specific embodiment of the present disclosure, the evolved enzyme includes any one selected from synthetases, transferases, oxidoreductases, and hydrolases.

[0094] According to a specific embodiment of the present disclosure, the evolved enzyme is a synthetase, and the synthetase includes any one selected from tRNA synthetase, glutamine synthetase, and DNA ligase.

[0095] According to a specific embodiment of the present disclosure, the evolved enzyme is a transferase, and the transferase includes any one selected from methyltransferase and glutamyltransferase.

[0096] According to a specific embodiment of the present disclosure, the evolved enzyme is an oxidoreductase, and the oxidoreductase includes any one selected from dehydrogenase, peroxidase, oxygenase, and oxidase.

[0097] According to a specific embodiment of the present disclosure, the hydrolase comprises any one selected from trypsin and nuclease.

[0098] According to a specific embodiment of the present disclosure, the enzyme substrate is capable of entering the yeast.

[0099] According to a specific embodiment of the present disclosure, the regulatory molecule comprises a transcription factor.

[0100] A sixth aspect of the present disclosure provides an automatic evolution liquid flow device, the device comprising a sample inlet area, an evolution area, a sample outlet area, and an injection area.

[0101] in,

[0102] The sample introduction zone controls the culture medium to enter the evolution zone;

[0103] The evolution zone is connected to the sample introduction zone and receives the culture medium input from the sample introduction zone. The evolution zone contains yeast cultured using the above-mentioned growth-coupled yeast directed evolution screening method, the method for directed evolution of the interaction ability of a target protein with a first protein, the method for directed evolution of the interaction ability of a target protein with a cis-acting element, the method for directed evolution of the interaction ability of a target protein with a second small molecule, and the method for directed evolution of enzyme catalytic ability, as well as components for maintaining the growth state of the yeast;

[0104] The sample outlet area is connected to the evolution area and receives the culture medium discharged from the evolution area;

[0105] The injection zone is connected to the evolution zone, and the purpose of screening the evolved yeast is achieved by injecting small molecule substances for screening into the evolution zone.

[0106] The automated evolutionary flow device provided herein enables long-term yeast growth in a constant-temperature incubator. The open culture system allows for both inflow and outflow of liquid, maintaining yeast growth at a fixed growth stage. Antibiotics or other small molecules for growth regulation and screening can be gradually added to the culture medium. As evolution progresses, yeast growth accelerates, accelerating the inflow and outflow rates to increase yeast growth pressure and thereby regulating the evolutionary pressure of the evolutionary system from multiple perspectives. According to some embodiments of the present disclosure, the volume and rate of culture medium entering the evolution zone serve as part of the growth stress.

[0107] According to specific embodiments of the present disclosure, the volume and rate of culture medium entering the evolution zone is used as part of the growth stress.

[0108] According to a specific embodiment of the present disclosure, the volume and rate of small molecule substances used for screening into the evolution zone are used as part of the growth stress.

[0109] According to a specific embodiment of the present disclosure, the small molecule substance for screening includes at least one selected from antibiotics, isopropylthiogalactoside, and yeast counter-screening drugs.

[0110] In a seventh aspect, the present disclosure provides a gene product, which is produced by yeast expression obtained by screening a growth-coupled yeast directed evolution screening method, a method for directing the evolution of the ability of a target protein to interact with a first protein, a method for directing the evolution of the ability of a target protein to interact with a cis-acting element, a method for directing the evolution of the ability of a target protein to interact with a second small molecule, a method for directed evolution of enzyme catalytic ability, or the above-mentioned devices.

[0111] According to specific embodiments of the present disclosure, the gene product is a protein or RNA.

[0112] An eighth aspect of the present disclosure provides a nucleic acid molecule encoding the above-mentioned gene product.

[0113] A ninth aspect of the present disclosure provides a cell comprising the aforementioned gene product and / or the aforementioned nucleic acid molecule.

[0114] Additional aspects and advantages of the present disclosure will be set forth in part in the following description and, in part, will be obvious from the following description, or may be learned through practice of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0115] The above and / or additional aspects and advantages of the present disclosure will become apparent and readily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0116] FIG1 shows a schematic diagram of the growth-coupled yeast directed evolution screening process in one embodiment of the present disclosure;

[0117] FIG2 shows a schematic diagram of a growth-coupled yeast directed evolution screening method according to an embodiment of the present disclosure;

[0118] FIG3 shows a schematic diagram of the growth-coupled directed evolution of yeast enzyme catalytic capacity in one embodiment of the present disclosure;

[0119] FIG4 shows a schematic diagram of an automatic evolutionary liquid flow device according to an embodiment of the present disclosure;

[0120] FIG5 is a schematic diagram showing the effect of auxin on yeast growth in Example 1 of the present disclosure, using changes in IPTG concentration to simulate protein evolution levels, with the horizontal axis representing IPTG treatment time in hours (h) and the vertical axis representing OD600 values. Differently labeled lines represent different IPTG treatment concentrations in millimolar (mM);

[0121] FIG6 is a schematic diagram showing the effect of 5-FOA on yeast growth in Example 1 of the present disclosure, wherein the horizontal axis represents the 5-FOA treatment concentration in milligrams per milliliter (mg / ml), and the vertical axis represents the OD600 value;

[0122] Figures 7A-7C are schematic diagrams showing the effects of antibiotic and antibiotic resistance protein expression levels on yeast growth in Example 1 of the present disclosure. Figure 7A represents G418, Figure 7B represents hygbR, and Figure 7C represents bleoR. The horizontal axis represents IPTG treatment time in hours (h), and the vertical axis represents OD600. Different labeled lines represent different IPTG treatment concentrations in millimolar (mM). For the same antibiotic, six different antibiotic concentrations were used, namely 0, 0.05, 0.1, 0.2, 0.5, and 1 mg / ml.

[0123] Figures 8A-8C show schematic diagrams of three plasmids constructed in Example 2 of the present disclosure, wherein Figure 8A shows the integration plasmid of the P1 plasmid expressing the NP868R-T7 fusion protein, Figure 8B shows the error-prone TP-DNApol expression plasmid, and Figure 8C shows the bleoR gene expression plasmid driven by the T7 promoter;

[0124] FIG9 shows the artificial evolution process of the NP868R-T7 fusion protein in Example 2 of the present disclosure, wherein the horizontal axis represents the evolution time in days, and the vertical axis represents the concentration of the antibiotic in milligrams per milliliter (mg / ml);

[0125] FIG10 shows the final evolutionary sequencing results of the NP868R-T7 fusion protein in Example 2 of the present disclosure;

[0126] FIG11 is a schematic diagram showing the verification results of the mutant transcription using the RT-PCR method in Example 2 of the present disclosure;

[0127] FIG12 is a schematic diagram showing the results of protein expression verification of mutants using flow cytometry in Example 2 of the present disclosure, wherein T7WT represents the quantification of protein expression before mutation, and T7648722 represents the protein expression after mutation;

[0128] FIG13 shows the automatic evolution process of NP868R-T7 fusion protein in Example 3 of the present disclosure;

[0129] FIG14 shows the final evolution sequencing results of the automatically evolved NP868R-T7 fusion protein in Example 3 of the present disclosure;

[0130] FIG15 shows a diagram for verifying the expression of mutants using flow cytometry in Example 3 of the present disclosure. In the three comparison groups of the histogram, WT represents the yeast before mutation and is located on the left side of the histogram comparison group; NP868R-T7Q648LH772R represents the mutant obtained in Example 2 and is located in the middle of the histogram comparison group; NP868R-T7I244VQ648LH772R represents the mutant obtained on the basis of Example 2 and is located on the right side of the histogram comparison group;

[0131] FIG16 shows a schematic diagram of the construction method of the positive and negative screening evolution vector in Example 4 of the present disclosure;

[0132] FIG17 shows a schematic diagram of the positive and negative screening evolution method in Example 4 of the present disclosure;

[0133] FIG18 shows a schematic diagram of the positive and negative screening process in Example 4 of the present disclosure;

[0134] FIG19 shows a diagram of the interaction mechanism between proteins and small molecules in Example 5 of the present disclosure;

[0135] FIG20 shows a schematic diagram of the positive and negative screening process in Example 5 of the present disclosure;

[0136] FIG21 shows a schematic diagram of the statistical analysis of the ability of mutants to bind to nucleic acid sequences using flow cytometry in Example 5 of the present disclosure. Among the six comparison groups in the histogram, WT represents yeast before mutation and is located on the left side of the histogram comparison group; K54E represents a mutation at one site and is located in the middle of the histogram comparison group; E103G represents a mutation at another site and is located on the right side of the histogram comparison group;

[0137] Figure 22 shows a schematic diagram of the statistical analysis of the mutant's ability to bind lactate using flow cytometry in Example 5 of the present disclosure. Among the three comparison groups in the histogram, the left side represents the case when the IPTG concentration is 0 mM, and the right side represents the case when the IPTG concentration is 20 mM.

[0138] Detailed Description of the Invention

[0139] The embodiments of the present disclosure are described in detail below. The embodiments described below are exemplary and are only used to explain the present disclosure, and should not be understood as limiting the present disclosure.

[0140] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. Throughout the present disclosure, "plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0141] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0142] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the present disclosure belongs.

[0143] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present disclosure, but not excluding other contents.

[0144] Herein, the term "fusion protein" refers to the expression product of two genes recombined by DNA recombination technology.

[0145] In this article, the term "microfluidics" refers to the science and technology involved in systems that use microchannels (with dimensions of tens to hundreds of micrometers) to process or manipulate tiny fluids (with volumes of nanoliters to attoliters).

[0146] In this article, the term "5-fluoroorotic acid" (5-fluorouracil-6-carboxylic acid monohydrate; 5-FOA) is used in yeast molecular genetics studies to detect expression of the URA3 gene, which encodes orotidine-5'-monophosphate (OMP) dicarboxylase. Yeast with active URA3 (Ura+) can convert 5-FOA to cytotoxic fluorodeoxyuridine. Only yeast strains with mutations in the URA3 gene can grow in media containing 5-FOA when uracil is supplemented. 5-FOA is commonly used in experiments with Saccharomyces cerevisiae (URA3), Schizosaccharomyces pombe (URA4 and URA5), Candida albicans (URA3), and Escherichia coli (pyrF).

[0147] In this article, the term "Isopropyl-beta-D-thiogalactopyranoside" (IPTG) is an organic compound with the chemical formula C9H 18 O5S, an allolactose mimetic, can trigger the transcription of the lactose operon, thereby inducing the expression of proteins corresponding to genes downstream of the lactose operon.

[0148] As used herein, "G418" (Geneticin) is an aminosugar antibiotic structurally similar to neomycin, gentamicin, and kanamycin. It inhibits protein synthesis by inhibiting the genes of transposons Tn601 and Tn5, interfering with 80S ribosome function. It is toxic to both prokaryotic and eukaryotic cells, including bacteria, yeast, plant, and mammalian cells, as well as protozoa and worms. It is the most commonly used selective agent for stable transfection.

[0149] In this article, the term "hygbR" Hygromycin B is an aminoglycoside antibiotic produced by Streptomyces hygroscopicus. It inhibits protein synthesis by interfering with 70S ribosomal translocation and inducing misreading of mRNA templates, thereby killing prokaryotes (such as bacteria), eukaryotes (such as yeast, fungi) and higher mammalian eukaryotic cells.

[0150] In this article, the term "bleomycin" refers to a large family of antitumor glycopeptide antibiotics produced by the metabolism of Streptomyces verticillus. The most commonly used members of this family are the bleomycin and phleomycin families. The former are primarily used as antitumor compounds in combination with other anticancer agents to treat lymphomas, squamous cell carcinomas, and testicular cancer. The latter are primarily used as selective antibiotics in molecular genetic research to screen for stably transfected cells carrying resistance genes such as Sh ble.

[0151] In this article, the term "NP868R" is derived from African swine fever virus ASFV, which is associated with post-transcriptional modification of mRNA and can catalyze the process of mRNA capping.

[0152] In this article, small molecules are compounds with a chemical molecular weight of less than 900 Daltons, or small units of short peptides or nucleic acids. They can enter and exit yeast by free diffusion. The small molecules mentioned in the article are mainly divided into four categories: first small molecules, second small molecules, third small molecules, and small molecules for screening. Among them, the first small molecules are regulatory molecules of growth-related genes, that is, small molecules that can directly affect whether growth-related genes are expressed or not / the strength thereof, such as IPTG and lacI binding, DAPG and PhlF binding, aTc and tetR binding, xylose and xylR binding, etc. Here, IPTG, DAPG, aTc, and xylose are the aforementioned first small molecules. After these first small molecules bind to the latter, they will directly affect the latter's binding to DNA; the second small molecule is a small molecule that can directly interact with the target protein to be evolved, and it changes the conformation of the target protein by binding to the target protein. This will affect the interaction between the target protein and the regulatory molecules of growth-related genes, thereby achieving the purpose of affecting the expression of growth-related genes. For example, the ligand-receptor interaction of neurotransmitters, drugs or hormones, such as β-adrenaline and β-adrenaline receptor (β2AR), serotonin and serotonin receptor (5-HTR), etc. The evolved receptors induce the conduction of downstream signal pathways, such as the cAMP / PKA signaling pathway, JAK / STAT pathway, etc. STAT will induce gene expression, antagonists and protein interactions, such as ADH-1 (C22H34N8O6S2) and N-cadherin (N-cadherin) dherin), inhibition of PD-1 / PD-L1 by a variety of non-monoclonal antibody small molecule drugs, evolution of PD-1 / PD-L1, affecting the downstream PI3K-AKT signaling pathway, modification of proteins such as FOXO or CREB directly affects gene expression. When the regulatory molecule of a growth-related gene is itself the target of evolution, the first small molecule is the second small molecule. As mentioned above, when the binding ability of the target protein to the second small molecule is required, and the second small molecule is IPTG, that is, when the second small molecule itself is the regulatory molecule of a growth-related gene, the second small molecule here The third small molecule refers to the intermediate product in the biological reaction process, such as enzyme catalysis products, etc. Under certain conditions, this type of intermediate product may affect the regulation of the growth-related genes by the first type of small molecule. For example, in the glycolysis pathway, lactic acid is the final product and glucose is the starting substrate. The output of intermediate products including glucose hexaphosphate and pyruvate may affect the production of lactic acid. The fourth type is small molecules for screening, which are mainly added to the yeast growth environment, such as various antibiotics, so that the target molecules of the surviving yeast will produce mutations to adapt to the environment under stress.It should be noted that, under certain circumstances, the first small molecule substance partially overlaps with the second small molecule substance and the screening small molecule substance, such as antibiotics, toxic substances, etc. For example, when evolving bleomycin resistance protein, the protein is both a growth-related gene and a target protein, so bleomycin is both the second small molecule and the screening small molecule.

[0153] In this article, the term "biosensor" refers to an analytical device consisting of a biological sensing element and a signal transducer. The biological sensing element is used to identify target substances, mainly including biological substances such as antibodies, enzymes, nucleic acids, cells, etc., as well as some synthetic substances similar to biological substances, such as aptamers, peptides, and molecularly imprinted polymers. The signal transducer is used to convert the interaction between the biological sensing element and the target molecule into a different signal. For example, enzymes catalyze chemical reactions of specific substances and convert them into electrical signals; biological antibodies capture specific antigens and then convert them into optical signals through labeled fluorescence. The biosensor in this article is a protein-based biosensor that mainly changes its function through allosteric effects or forms polymers. After sensing the target substance, it affects the expression of downstream reporter genes through allosteric effects or forming polymers.

[0154] As used herein, the term "fusion protein" is a protein composed of at least two domains encoded by separate genes that have been linked together so that they are transcribed and translated as a single unit to produce a single polypeptide.

[0155] A growth-coupled yeast directed evolution screening method

[0156] According to some embodiments of the present disclosure, the present disclosure provides a growth-coupled yeast directed evolution screening method, as shown in FIG1 , comprising:

[0157] S110, constructing an exogenous plasmid containing the target sequence;

[0158] S120, construction of error-prone DNA polymerase expression plasmid;

[0159] S130, construction of growth-related gene expression plasmids;

[0160] S140, transferring the exogenous plasmid described in step S110, the error-prone DNA polymerase expression plasmid described in step S120, and the growth-related gene expression plasmid described in step S130 into yeast,

[0161] The biological macromolecules expressed by the evolution target sequence can drive / inhibit the expression of the growth-related gene in step S130 through the regulatory molecules of the growth-related gene.

[0162] S150, placing the yeast under a growth stress environment, wherein the target sequence to be evolved contained in the yeast surviving under the growth stress environment is a target sequence that conforms to the direction of directed evolution.

[0163] According to some embodiments of the present disclosure, the present disclosure provides a growth-coupled yeast directed evolution screening method. As shown in Figure 2, the yeast obtained by this method contains a cytoplasmic linear plasmid, TP-DNApol error-prone DNA polymerase, a variety of growth-related genes that can be integrated into the genome and their regulatory sequences. The regulatory sequences are coupled with the functions of the evolved biological macromolecules to induce the expression and repression of downstream genes to achieve the purpose of screening and enrichment. This method can be used for evolutionary screening of more active enzymes, interactions between proteins and nucleic acids, interactions between proteins, synergistic effects between multiple domains of proteins, etc. The changes in the functions of proteins or nucleic acids evolved by this method can be reflected in the expression of growth-related genes, and favorable mutations can be automatically and continuously enriched. Therefore, this method can be used in automated liquid flow systems, including microfluidic systems.

[0164] According to some embodiments of the present disclosure, all controllable factors that can affect the yeast growth rate, such as the concentration of antibiotics, the frequency of culture medium replacement, etc., can be used as part of this screening method. When such controllable factors are used as part of this screening method, they are also within the scope of protection of the present disclosure.

[0165] According to some embodiments of the present disclosure, the screening pressure can be adjusted, including increasing / decreasing antibiotic concentration, accelerating / slowing down the frequency of culture medium replacement, yeast flow rate, adding / reducing the types of antibiotics, and other variable factors known in the art in all yeast screening systems.

[0166] According to some specific embodiments of the present disclosure, screening for yeast growth-related genes can be performed by first knocking out endogenous yeast growth-related genes and then introducing new growth-related genes to accurately obtain the desired growth-related genes.

[0167] According to some embodiments of the present disclosure, any growth-related gene known in the art, including URA, LEU, HIS, bleoR, KanR, and hygbR genes, can be used as a single gene or as multiple genes in series. Regardless of the method of use, it can be used as part of the present screening method. When the growth-related gene is used as part of the present screening method, it is also within the scope of protection of the present disclosure.

[0168] According to some embodiments of the present disclosure, any method known in the art for connecting different genes, including gene fusion and gene concatenation, such as self-cleaving 2A peptide (2A), when used as part of the present screening method, is protected by the present disclosure.

[0169] According to some specific embodiments of the present disclosure, any growth-related gene known in the art that, upon expression, can interact with the specific growth environment of yeast and kill yeast can be used as a negative screening gene of the present disclosure. For example, yeast with URA3 gene activity (Ura+) can convert 5-FOA into cytotoxic fluorodeoxyuridine, thereby killing the yeast.

[0170] According to some specific embodiments of the present disclosure, any growth-related gene known in the art that, upon expression, enables yeast to survive in a specific growth environment can be used as a positive selection gene of the present disclosure. For example, yeast with KanR gene activity (Kan+) can survive in the presence of Kan antibiotics in the growth environment.

[0171] According to some specific embodiments of the present disclosure, when the negative screening gene and the positive screening gene are used in series, and the expression of both genes is regulated by the macromolecules expressed by the evolved target sequence, by changing the corresponding growth stress pressure, positive and negative screening can be carried out simultaneously in the same yeast.

[0172] According to some specific embodiments of the present disclosure, a growth-coupled yeast directed evolution screening method is provided by the present disclosure. When two or more growth-related genes and genes of the target sequence are transferred into the yeast, the simultaneous evolution of genes of multiple target sequences can be achieved by simply changing the corresponding growth stress pressure.

[0173] According to some embodiments of the present disclosure, the present disclosure provides a growth-coupled yeast directed evolution screening method, in which the biological macromolecule expressed by the evolved target sequence can bind to a nucleic acid sequence, and the nucleic acid sequence can be a known operator existing in nature or an artificial operator that has never existed in nature.

[0174] According to some embodiments of the present disclosure, when the culture time is long enough, the biomacromolecule can break away from the originally bound operator and bind to a new operator. For example, the favorable mutation is extracted through plasmid and then transfected, and accumulated from generation to generation until it meets the direction of the directed evolution.

[0175] According to some embodiments of the present disclosure, the present disclosure provides a growth-coupled yeast directed evolution screening method, in which the biological macromolecules expressed by the evolved target sequence can bind to proteins. For example, using the yeast two-hybrid principle, AD and BD are respectively fused with the target protein for expression, one of which is integrated with the exogenous plasmid, and the other can be integrated with the exogenous plasmid, or integrated into the yeast genome, preferably transferred to the yeast genome. If the evolved target proteins have stronger interactions, AD and BD are connected, thereby initiating the expression of downstream growth-related genes.

[0176] According to some embodiments of the present disclosure, the BD is the binding domain of the upstream activating sequence of the promoter of a growth-related gene, the AD is the activation domain of the upstream activating sequence of the promoter of a growth-related gene, and the upstream activating sequence is the upstream activating sequence (UAS) of the promoter of the galactose metabolic enzyme system GAL gene.

[0177] According to some embodiments of the present disclosure, the exogenous plasmid containing the target sequence can be subjected to directed evolution and screening as long as it can be transformed into yeast, and there is no obvious limitation on the length of the target sequence.

[0178] According to some embodiments of the present disclosure, a growth-coupled yeast directed evolution screening method is provided, wherein the biological macromolecule expressed by the evolved target sequence is an enzyme that can catalyze chemical reactions, for example, a catalytic enzyme in a metabolic process, and the catalytic product of the catalytic enzyme can induce the expression of downstream growth-related genes.

[0179] According to a specific embodiment of the present disclosure, the present disclosure provides a growth-coupled yeast directed evolution screening method, which can direct the evolution of the catalytic ability of an enzyme, wherein the nucleic acid sequence corresponding to the enzyme is the target sequence, the nucleic acid sequence corresponding to the protein catalyzed by the enzyme is integrated into the yeast genome, and molecules that interact with the product of the protein catalyzed by the enzyme serve as regulatory molecules of the growth-related genes, wherein enzymes that conform to the evolutionary direction can grow more yeast per unit time under the growth stress environment.

[0180] According to a more specific embodiment of the present disclosure, the present disclosure provides a growth-coupled yeast directed evolution screening method, which can direct the evolution of the catalytic ability of an enzyme, for example, improving the ability of EctABC to catalyze l-aspartate-β-semialdehyde (ASA): Halomonas elongata strain has a gene cluster EctABC, which can catalyze l-aspartate-β-semialdehyde (ASA) to ectoine, and the EctB enzyme is the key rate-limiting enzyme. The evolved EctB enzyme can produce more intermediate product l-2,4-diaminobutyric acid (DABA), thereby increasing the production of ectoine. AraC-ect can be used as a biosensor to sense ectoine. At the same time, AraC-ect is also a transcriptional activator. When the production of ectoine is increased, it can promote the expression of downstream genes (https: / / doi.org / 10.1016 / j.ymben.2015.05.004). As shown in FIG3 , in the present disclosure, EctB or EctABC can be recombined as the target sequence on the cytoplasmic P1 plasmid, and the AraC-ect biosensor can be integrated into the genome together with the growth-related genes. EctB or EctABC can be mutated to produce more Ectoine, which can be used together with AraC-ect to induce the expression of the growth gene, allowing yeast to survive under the stress environment corresponding to the growth gene. If a biosensor for l-aspartate-β-semialdehyde (ASA) is obtained, the expression of growth-related genes can be affected by sensing changes in the concentration of ASA. For example, when the concentration of ASA decreases, the biosensor signal becomes weaker, and the expression of downstream growth-related genes decreases. Growth-related genes are designed to be toxic proteins, and the toxicity of the proteins is reduced, resulting in better yeast growth.

[0181] According to a specific embodiment of the present disclosure, the present disclosure provides a growth-coupled yeast directed evolution screening method, which can direct the evolution of the target protein's ability to interact with a second small molecule. When the target protein binds to the second small molecule, the protein conformation changes, thereby making the protein's ability to bind to the regulatory molecule of the growth-related gene stronger or weaker. For example, when the growth-related gene is an antibiotic resistance gene and the regulatory molecule of the growth-related gene can express the antibiotic resistance gene, the target protein that conforms to the directed evolution direction is a target protein that can further activate the function of the regulatory molecule after binding to the second small molecule, and vice versa.

[0182] According to a more specific embodiment of the present disclosure, the present disclosure provides a growth-coupled directed evolution screening method for yeast, which can direct the evolution of the ability of the target protein to interact with a second small molecule. On the one hand, the directed evolution target protein can bind to the regulatory sequence of the growth-related gene to inhibit the expression of the growth-related gene. On the other hand, it can bind to the second small molecule. When it binds to the second small molecule, the protein conformation changes, so that the growth-related gene is expressed. By regulating the pressure of the growth stress environment, the binding ability of the protein to the second small molecule is enhanced, and the protein conforms to the evolution direction, so that the yeast survives the growth stress environment.

[0183] According to a more specific embodiment of the present disclosure, the present disclosure provides a growth-coupled yeast directed evolution screening method, which can direct the ability of the evolution protein to interact with a second small molecule, for example, using a reverse tetracycline-controlled transcription activation system (rtTA), wherein the second small molecule is Dox (a tetracycline derivative), the target protein to be evolved is rtTA, which is a fusion protein of a mutant tetR and VP16, and the regulatory molecule is TRE (composed of a plurality of tetR tetracycline response elements in series). When there is no Dox, rtTA does not bind to TRE. When Dox is present, it promotes rtTA to bind to TRE, thereby initiating the expression of downstream genes. The ability of yeast to survive in the growth stress environment can be improved by evolving the binding ability of the target protein rtTA to Dox.

[0184] According to another specific embodiment of the present disclosure, the present disclosure provides a growth-coupled yeast directed evolution screening method, which can direct the evolution of the ability of a protein to interact with a second small molecule. The directed evolution protein can simultaneously act on the regulatory molecules of two or more growth-related genes. When the transferred growth-related genes simultaneously contain positive screening genes and negative screening genes, the evolved protein can evolve different affinities with the regulatory molecules while evolving the ability to interact with the second small molecule.

[0185] Automatic evolution liquid flow device

[0186] According to some embodiments of the present disclosure, the present disclosure provides an automatic evolutionary liquid flow device, as shown in FIG4 , the device includes a sample inlet area, an evolution area, a sample outlet area, and an injection area;

[0187] in,

[0188] The sample introduction zone controls the culture medium to enter the evolution zone;

[0189] The evolution zone is connected to the sample introduction zone and receives the culture medium input from the sample introduction zone. The evolution zone contains the growth-coupled yeast and components for maintaining the growth state of the yeast.

[0190] The sample outlet area is connected to the evolution area and receives the culture medium discharged from the evolution area;

[0191] The small molecule injection zone is connected to the evolution zone, and the purpose of screening the evolved yeast is achieved by injecting small molecule substances for screening into the evolution zone.

[0192] According to some embodiments of the present disclosure, the evolution zone includes components for maintaining yeast growth, such as a temperature control device maintaining 30° C., a magnetic stirrer, a dedicated evolution bottle, a rubber stopper, and a filter membrane.

[0193] According to some embodiments of the present disclosure, the culture medium injection area and the sample outlet area include: a peristaltic pump, flexible tubes of multiple specifications, and injection needles of multiple specifications.

[0194] According to some embodiments of the present disclosure, the injection area includes: an injection pump, a syringe, flexible tubes of multiple specifications, and injection needles of multiple specifications.

[0195] According to some specific embodiments of the present disclosure, a peristaltic pump is used to squeeze air to draw culture medium from a bottle into the evolution zone. The rate of the peristaltic pump can be adjusted to control the inflow volume of the culture medium. The screening small molecules required for positive and negative screening can be injected through the injection zone. The volume of the inflowing small molecules can be controlled by the injection pump. The inflow volume can be proportionally matched to the volume of the evolution bottle in the evolution zone, and the amount of culture medium and screening small molecules flowing in can be increased or decreased to control the screening pressure. Both the concentration of the screening small molecule and the rate of inflow into the culture medium can be used as part of regulating the screening pressure. Compared to manual evolution, the rate of inflow into the culture medium is a unique advantage of automation.

[0196] According to some embodiments of the present disclosure, the small molecule substance for screening includes at least one selected from antibiotics, isopropylthiogalactoside, and yeast counter-screening drugs.

[0197] According to some embodiments of the present disclosure, the amount and rate of inflow of the culture medium and small molecules for screening can be controlled by microfluidics.

[0198] The scheme of the present disclosure will be explained below with reference to the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be regarded as limiting the scope of the present disclosure. If the specific techniques or conditions are not specified in the examples, they shall be carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. The reagents or instruments used without indicating the manufacturer are all conventional products that can be obtained commercially. It should be noted that the GAY319 yeast strain used in the experiments of this application is from UCI University (García-García, JD, et al., Using continuous directed evolution to improve enzymes for plant applications. Plant Physiol, 2022.188(2): p.971-983.).

[0199] Example 1 Screening of Yeast Growth-Related Genes

[0200] 1. Screening of Nutritional Marker Genes

[0201] IPTG was used to induce the expression of growth-related nutritional marker genes to simulate evolutionary states. The results obtained, shown in Figure 5, demonstrate that the growth rates of URA and LEU significantly change under IPTG induction, making them suitable for use as part of the growth-coupled yeast screening method disclosed herein. It should be noted that this experiment was conducted under IPTG induction. Different inducing agents may result in different growth-related genes being screened. Therefore, this does not mean that HIS genes are unsuitable for use in the present disclosure.

[0202] 2. Negative screening

[0203] Yeast (GAY319 strain) was treated with different concentrations of 5-fluoroorotic acid (5-FOA) while maintaining constant URA3 expression. The results are shown in Figure 6 , demonstrating that when URA3 is fixedly expressed in yeast, increasing 5-FOA concentration increases the toxicity to cells, making it suitable for negative screening.

[0204] 3. Screening of resistance genes

[0205] IPTG was used to induce the expression of growth-related nutritional marker genes to simulate the evolutionary state. The results are shown in Figures 7A to 7C. All antibiotic-related genes (G418, hygbR, and bleoR) were able to show a gradually increasing effect of repressing antibiotics as the target proteins evolved. In addition, the expression of antibiotic-resistance genes had different effects on yeast growth as the antibiotic concentration changed. The two variables acted together to produce a synergistic effect on yeast growth.

[0206] The present invention uses different IPTG concentrations to induce different states of target protein evolution. When the IPTG concentration increases, it means that the target protein has a higher degree of evolution and is closer to the target function, which can more effectively induce the expression of downstream growth-related genes.

[0207] Example 2 Evolution of NP868R-T7 fusion protein

[0208] Using bleoR as a growth-coupled selection pressure, we evolved an NP868R-T7 fusion protein with enhanced transcriptional and translational activity. The plasmids required for construction are shown in Figures 8A to 8C , including the P1 plasmid expressing the NP868R-T7 fusion protein (Figure 8A), the error-prone TP-DNApol expression plasmid (purchased from Addgene) (Figure 8B), and the T7 promoter-driven bleoR gene expression plasmid (Figure 8C).

[0209] After the three plasmids were successfully constructed, they were transformed into the GAY319 strain. The P1 plasmid, which expresses the NP868R-T7 fusion protein, underwent homologous recombination with the GAY319 genome and the P1 plasmid, then integrated into the GAY319 strain. The transformed strain evolved through the following pathways.

[0210] Evolution was performed using 96-well plates, with six replicates per well, and 500 μl of bacterial solution per well. The initial selection pressure for evolution was 0.05 mg / ml bleomycin. After five days of evolution, as shown in Figure 9, one well showed a significant increase in growth rate, indicating successful evolution. The selection pressure was gradually increased until the antibiotic concentration reached 1 mg / ml, at which point growth reached its maximum rate, indicating the maximum evolutionary potential had been reached.

[0211] The P1 plasmid was extracted from the final bacterial solution, and the extracted plasmid was sequenced after PCR, and two point mutations were found, as shown in Figure 10.

[0212] Functional validation of the mutant NP868R-T7Q648LH772R was performed. RT-PCR and flow cytometry were used to analyze the changes in transcriptional activity and protein expression of the mutant, respectively. The RT-PCR results are shown in Figure 11 (WT represents the transcriptional expression level before mutation, and mut represents the transcriptional expression level after mutation). The transcriptional expression level after mutation was significantly higher than that before mutation. The results of flow cytometry verification of protein expression before and after the mutation are shown in Figure 12 (T7WT represents the protein expression quantification before mutation, and T7648722 represents the protein expression level after mutation). As can be seen from Figures 11 and 12, the evolved NP868R-T7 has significantly improved transcriptional activity and protein expression levels compared to the unevolved NP868R-T7.

[0213] The present disclosure can use the expression of growth-related genes to screen the evolutionary state of the target protein, and can realize the mutation-screening integration in the organism. It saves the steps of selecting mutants, reconstructing the screening system, and large-scale screening. The final product of evolution can obtain favorable mutations within 6 holes, which is much more convenient than the screening of hundreds of mutants that may be needed by traditional mutation screening methods. For proteins with fewer mutation sites, the time required for finally screening favorable mutations is about one month (constructing the vector for 1 week, evolving for 2-3 weeks, and verifying the screening results for 1 week), which greatly saves the time to obtain mutants.

[0214] Example 3 Automatic evolution of NP868R-T7 fusion protein

[0215] The mutant obtained in Example 2 was used as the evolution substrate for this example, and automatic evolution was performed using an automated liquid flow system. The process is shown in Figure 13. After half a month of automatic evolution, the bacterial liquid in the final culture system was extracted according to the plasmid extraction method in Example 2, and PCR sequencing was performed. As shown in Figure 14, a new mutation site was obtained. The mutant NP868R-T7I244VQ648LH772R was then functionally verified and analyzed using a flow cytometer to observe changes in protein expression in the mutant. The results are shown in Figure 15 (in the three comparison groups of the histogram, WT represents yeast before mutation, located on the left side of the histogram comparison group; NP868R-T7Q648LH772R represents the mutant obtained in Example 2, located in the middle of the histogram comparison group; NP868R-T7I244VQ648LH772R represents a newly obtained mutant based on Example 2, located on the right side of the histogram comparison group). It can be seen from the fluorescence results that the new mutation obtained after automatic evolution enhances the expression function of the protein. Experiments have shown that after artificial evolution reaches the maximum screening space, the automatic evolution system can continue to increase the screening pressure to further enhance the function of the protein. The flow rate also has a certain impact on the growth of yeast and can also be used as a type of screening pressure.

[0216] The automated evolution fluidics system provides enhanced control over the flow rates of culture medium and small molecules, achieving higher screening pressures than manual evolution and greater precision than manual passaging. Automated evolution can be used when manual evolution is ineffective. This method requires no engineering of the yeast; the automated system can be directly set up, taking only 1-2 hours from setup to start of evolution. The total time from the start of evolution to obtaining favorable mutations is less than one month.

[0217] Example 4 Positive and negative screening of evolved LmrA proteins

[0218] LmrA is a transcription factor that can bind to two DNA sequences (operators) simultaneously. To develop LmrA as a more useful synthetic biology component, it is necessary to increase its orthogonality. To enable LmrA to bind to only one operator, the vector designed is shown in Figure 16.

[0219] The positive and negative screening method is shown in Figure 17. The bleomycin resistance gene (bleoR) is used for positive screening, and orotidine 5-phosphate decarboxylase (URA) is used for negative screening. The evolved LmrA has stronger binding to LmrO and weaker binding to QacO. Two operators (LmrO and QacO) are inserted into the non-conserved region of the standard promoter sequence. When LmrA binds to LmrO, the promoter is unable to drive expression of the downstream URA. URA can break down the substrate pentafluoroorotic acid (5-FOA) into the toxic substance pentafluorouracil (5-FU), potentially killing yeast. When LmrA binds to QacO, the promoter is unable to drive expression of bleoR, failing to inhibit bleomycin-induced yeast killing.

[0220] The positive and negative selection evolution process is shown in Figure 18. First, the yeast was treated with 0.5 mg / ml 5-FOA for 7 days. When yeast growth reached an OD600 of 0.7-0.8, the 5-FOA concentration was increased. This accelerated yeast growth, reaching the same OD value in just 4 days. Next, a negative selection was performed, followed by a positive selection. Growth reached an OD600 of 0.7-0.8 in just 1 day. Next, the 5-FOA concentration was increased while bleomycin was added. While maintaining the 5-FOA concentration, the bleomycin concentration was continuously increased while observing yeast growth. When yeast still reached the desired OD value within 1 day even at the highest bleomycin and 5-FOA concentrations, LmrA evolution was considered to have reached its maximum potential.

[0221] The evolution results were verified by flow cytometry. The other procedures for extracting yeast linear plasmids and PCR were the same as in Examples 2 and 3. LmrA protein expression was induced using 0 mM IPTG and 20 mM IPTG before and after evolution, respectively. The fluorescence values ​​under the two culture conditions were divided to obtain the fold change value. The fold change results of the evolution are shown in Table 1.

[0222] Table 1

[0223] The evolved LmrA was named LmrAD9. Table 1 shows that the binding strength of LmrA to QacO decreased 22-fold before and after evolution, and the binding strength of LmrAD9 to QacO almost disappeared. The binding strength of LmrA to LmrO decreased slightly, by 1.6-fold, but the effect was not significant. After evolution, the binding strength of LmrAD9 to the two operators differed by 35-fold, significantly widening the binding capacity of LmrA to the two operators and improving the orthogonality of LmrA.

[0224] Example 5 Interaction between proteins and small molecules

[0225] lldR is a lactate metabolism regulatory protein in prokaryotic cells. It binds to the two operator regions on the promoter and inhibits the expression of downstream genes. When lactate binds to it, it can change its conformation and enable the expression of downstream genes. In order to construct a lactate biosensor, lldR is made more sensitive to lactate.

[0226] The positive and negative screening methods are shown in Figure 19, using the bleomycin resistance gene (bleoR) for positive screening and orotidine 5-phosphate decarboxylase (URA) for negative screening.

[0227] The positive and negative screening evolution process is shown in Figure 20: First, the concentration of bleomycin was continuously increased from 0.05 mg / ml to 0.8 mg / ml in an environment containing 20 mM lactic acid for positive screening. Next, 0.5 mg / ml 5-FOA was added for negative screening in an environment without lactic acid, and the positive and negative screening were repeated twice. Finally, 1 mg / ml bleomycin was used for the final positive screening in an environment with 20 mM lactic acid until the OD value reached about 0.7.

[0228] Sequencing of the mutated yeast revealed the two sites with the largest mutation rates, K54E and E103G. The evolutionary results were verified by flow cytometry. The yeast linear plasmid extraction and PCR procedures were identical to those in Examples 2 and 3. The binding ability of lldR to the operator was observed in isolation without the addition of lactic acid. Expression of the lldR protein before and after evolution was induced using six concentration gradients of IPTG, ranging from 0 mM to 20 mM. The results, shown in Figure 21, indicate that after evolution, most mutants have lost some of their operator binding ability.

[0229] The results of the evolution were verified by flow cytometry. The other procedures for extracting yeast linear plasmids and PCR were the same as in Examples 2 and 3. The response of the two lldR mutants to lactic acid was observed when lactic acid was added: 0 mM IPTG and 20 mM IPTG were used to induce expression of the lldR protein before and after evolution, respectively. The results are shown in Figure 22, indicating that after evolution, the K54E mutant has a certain improvement in its sensitivity to lactic acid.

[0230] This evolution yielded a biosensor with enhanced lactate binding capacity, lldR. However, the overall results leave room for improvement. The loss of operator binding capacity may be due to issues during the evolution process, perhaps due to the low negative selection concentration used. However, data retrieval and modeling analysis indicate that if a biosensor with enhanced lactate sensitivity is to be evolved, conformational changes will inevitably result in a loss of some operator binding capacity.

[0231] This disclosure is the first to combine positive and negative screening, adding two small molecules to the system simultaneously to achieve simultaneous positive and negative screening of target proteins. The conversion of the positive and negative screening systems is very convenient. All evolution-related genes need to be transferred into the same yeast strain, eliminating the need to prepare multiple engineered bacteria for transformation. Simply treating a single strain with different small molecules can complete both positive and negative screening. Positive and negative screening can be performed simultaneously, with multiple small molecules treated at different concentrations. This simple operation greatly shortens the evolution process, saving manpower and time.

[0232] Compared to traditional evolutionary methods, which are primarily used to evolve enzymes, the present disclosure can be used to evolve a wider variety of proteins. It can be used to evolve proteins whose phenotypes are not readily apparent after mutation, such as protein-protein interactions, protein-small molecule interactions, protein-DNA interactions, extramembrane protein interactions, and protein biosensors. Because the present disclosure can transform protein function into inducing the expression of other genes, it can indirectly reveal the protein's phenotype, using yeast growth as a form of expression. The function of evolved proteins can change exponentially, up to several dozen times.

[0233] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present disclosure. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification and features of different embodiments or examples, unless they are mutually inconsistent.

[0234] Although the embodiments of the present disclosure have been shown and described above, it is understood that the above embodiments are illustrative and are not to be construed as limitations on the present disclosure. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present disclosure.

Claims

1. A growth-coupled yeast directed evolution screening method, characterized in that: The method comprises: (A) constructing an exogenous plasmid containing the target sequence; (B) Construction of error-prone DNA polymerase expression plasmid; (C) Construction of growth-related gene expression plasmid; (D), transferring the exogenous plasmid described in step (A), the error-prone DNA polymerase expression plasmid described in step (B), and the growth-related gene expression plasmid described in step (C) into yeast, Wherein, the biological macromolecule expressed by the evolution target sequence can drive / inhibit the expression of the growth-related gene in step (C) through the regulatory molecule of the growth-related gene, (E) placing the yeast under a growth stress environment, wherein the target sequence to be evolved contained in the yeast surviving under the growth stress environment is a target sequence that conforms to the direction of directed evolution.

2. The method according to claim 1, characterized in that The target sequence includes at least a portion of the sequence of one or more genes.

3. The method according to claim 1, characterized in that The biomacromolecule is a protein, which includes a protein expressed by a single gene or a fusion protein expressed by multiple genes.

4. The method according to claim 3, characterized in that: The molecular weight of the protein is 4-400KD.

5. The method according to claim 3, characterized in that: The molecular weight of the protein is 8-200KD.

6. The method according to claim 3, characterized in that The protein includes any one selected from enzymes, receptors, and transcriptional regulatory factors, or the protein includes a fusion protein composed of any one of enzymes, receptors, and transcriptional regulatory factors.

7. The method according to claim 6, characterized in that The enzyme includes an enzyme having catalytic activity or an enzyme associated with gene expression.

8. The method according to claim 7, characterized in that The enzymes associated with gene expression include those selected from DNA polymerases, transcriptases, gene editing enzymes, capping enzymes, and enzymes associated with epigenetic modification.

9. The method according to claim 8, characterized in that The gene editing enzyme includes at least one selected from Cas family proteins such as Cas9, Cas12, ZFP, and TALEN.

10. The method according to claim 8, characterized in that The enzyme associated with epigenetic modification includes at least one selected from histone acetyltransferase or deacetylase, histone methyltransferase or demethylase, ubiquitinase or deubiquitinase, DNA methylase or demethylase.

11. The method according to claim 6, characterized in that The receptor includes at least one selected from membrane receptors, cytoplasmic receptors, and nuclear receptors.

12. The method according to claim 6, characterized in that The receptor is a membrane receptor, and the membrane receptor includes any one selected from ion channel receptors, G protein coupled receptors, and tyrosine kinase coupled receptors.

13. The method according to claim 6, characterized in that The transcriptional regulatory factor includes at least one selected from LysR, AraC / XylS, TetR, LuxR, LacI, ArsR, IcIR, MerR, AsnC, MarR, NtrC (EBP), OmpR, DeoR, Cold shock, GntR, Crp, C2H2, bZIP, dCas9-based, ZFP-based, and TALEN-based transcriptional regulatory factors.

14. The method according to claim 1, characterized in that The exogenous plasmid is derived from a cytoplasmic linear plasmid of a non-host eukaryotic organism.

15. The method according to claim 14, characterized in that The non-host eukaryotic organism includes Kluyveromyces lactis.

16. The method according to claim 1, characterized in that The growth-related gene comprises at least one selected from antibiotic resistance genes and nutritional marker genes.

17. The method according to claim 16, characterized in that The antibiotic resistance gene includes at least one selected from the group consisting of a bleomycin resistance gene, a kanamycin resistance gene, a hygromycin resistance gene, a nourseoinhibitor gene, and an amphotericin gene.

18. The method according to claim 16, characterized in that The nutritional marker gene includes at least one selected from URA, LEU, HIS, ADE2, TRP1, and MET17.

19. The method according to claim 1, characterized in that Step (E) further comprises: (1) the expression of the growth-related gene causes the yeast to die as a negative screening, The expression of the growth-related gene allows the yeast to survive as a positive selection, Through the negative screening and / or positive screening process, yeast containing the target sequence that conforms to the direction of directed evolution is obtained; (2) obtaining the target sequence that conforms to the directed evolution direction based on the yeast in step (1), Among them, the growth-related gene whose expression causes the yeast to die is a negative screening gene, and the growth-related gene whose expression causes the yeast to survive is a positive screening gene.

20. The method according to claim 19, characterized in that In step (E), negative screening and positive screening are performed on the yeast simultaneously under the growth stress environment, wherein the screening pressures of the negative screening and the positive screening are adjustable.

21. The method according to claim 1, characterized in that The error-prone DNA polymerase and the exogenous plasmid are derived from the same species.

22. The method according to claim 1, characterized in that The error-prone DNA polymerase only acts on the exogenous plasmid to perform error-prone replication.

23. The method according to claim 1, characterized in that The yeast comprises at least one selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces paradoxica, Saccharomyces bayanus, Saccharomyces rio and Saccharomyces pastorianus.

24. The method according to claim 1, characterized in that The yeast is Saccharomyces cerevisiae.

25. The method according to claim 1, characterized in that The regulatory molecules of the growth-related genes include any one selected from proteins, nucleic acids, and first small molecules.

26. The method according to claim 25, characterized in that The nucleic acid includes a DNA sequence.

27. The method according to claim 26, characterized in that The DNA sequence includes at least one selected from a promoter, an insulator, and an enhancer.

28. The method according to claim 25, characterized in that The first small molecule substance includes at least one selected from antibiotics, enzyme substrates, short peptides, oligopeptides, sugars, lipids, vitamins, inorganic substances, alkaloids, and metabolites.

29. A method for directed evolution of the interaction ability between a target protein and a first protein, characterized in that: The method comprises: Cultivating and screening yeast using the method of any one of claims 1 to 28, obtaining a target protein having an ability to interact with the first protein or an increased or decreased ability to interact with the first protein based on the yeast surviving under the growth stress environment, in, The target protein is fused with the BD of the transcription factor of the growth-related gene in step (D) to form a first fusion protein, and the first protein is fused with the AD of the transcription factor of the growth-related gene to form a second fusion protein; or The target protein is fused with the AD of the transcription factor of the growth-related gene in step (D) to form a first fusion protein, and the first protein is fused with the BD of the transcription factor of the growth-related gene to form a second fusion protein. Wherein, the BD is the binding domain of the upstream activation sequence of the promoter of the growth-related gene, and the AD is the activation domain of the upstream activation sequence of the promoter of the growth-related gene.

30. A method for directed evolution of the ability of a target protein to interact with a cis-acting element, characterized in that: The method comprises: Cultivating and screening yeast using the method of any one of claims 1 to 28, obtaining a target protein having the ability to interact with the cis-acting element or having an increased or decreased ability to interact based on the yeast surviving under the growth stress environment, in, The cis-acting element is on the growth-related gene expression plasmid in step (C), and the cis-acting element is the cis-acting element of the growth-related gene; The sequence for expressing the target protein is the target sequence described in step (A).

31. The method according to claim 30, characterized in that The target protein includes at least one selected from transcription factors, histones, DNA methylases, gene editing enzymes, and DNA binding proteins.

32. A method for directed evolution of the interaction ability between a target protein and a second small molecule, characterized in that: The method comprises: Cultivating and screening yeast using the method of any one of claims 1 to 28, obtaining a target protein having an ability to interact with the second small molecule or an increased or decreased ability to interact with the second small molecule based on the yeast surviving under the growth stress environment, The sequence expressing the target protein is the target sequence in step (A), and the second small molecule substance is a switch for determining whether the growth-related gene is expressed and the strength of expression.

33. The method according to claim 32, characterized in that The second small molecule substance promotes / inhibits the binding of the target protein with the regulatory molecule of the growth-related gene.

34. The method according to claim 33, characterized in that The target protein comprises at least two binding sites.

35. The method according to claim 34, characterized in that After the first binding site of the target protein binds to the second small molecule, the binding between the second binding site of the target protein and the regulatory molecule of the growth-related gene is turned on / off.

36. The method according to claim 35, characterized in that After the first binding site of the target protein binds to the second small molecule substance, the binding between the second binding site of the target protein and the regulatory molecule of the growth-related gene is enhanced / weakened.

37. The method according to claim 35, characterized in that The first binding site is the same as or different from the second binding site.

38. The method according to claim 32, characterized in that The second small molecule substance includes at least one selected from antibiotics, enzyme substrates, oligopeptides, sugars, lipids, vitamins, inorganic substances, alkaloids, hormones, oligonucleotides, metal ions, short peptides, drug molecules, and metabolites.

39. The method according to claim 33, characterized in that The regulatory molecules include DNA sequences or transcription factors.

40. The method according to claim 39, characterized in that The DNA sequence comprises any one selected from the group consisting of a lactose operon, a xylose operon, and a tetracycline operon.

41. The method according to claim 39, characterized in that The transcription factor includes any one of lacI, xylR, and tetR.

42. A method for directed evolution of enzyme catalytic ability, characterized in that: The method comprises: Cultivating and screening yeast using the method of any one of claims 1 to 28, and obtaining a catalytic enzyme that meets the direction of directed evolution based on the yeast that survives under the growth stress environment, Wherein, the nucleic acid sequence expressing the catalytic enzyme is the target sequence described in step (A), The enzyme substrate of the catalytic enzyme or the product after the catalytic enzyme catalyzes the enzyme substrate can activate / inhibit the function of the biosensor. The biosensor is the regulatory molecule of the growth-related gene in step (C). After the function of the biosensor is activated, it can promote the expression of the growth-related gene.

43. The method according to claim 42, characterized in that The evolved enzyme comprises any one selected from synthetase, transferase, oxidoreductase and hydrolase.

44. The method according to claim 43, characterized in that The evolved enzyme is a synthetase, and the synthetase includes any one selected from tRNA synthetase, glutamine synthetase, and DNA ligase.

45. The method according to claim 43, characterized in that The evolved enzyme is a transferase, and the transferase includes any one selected from methyltransferase and glutamyltransferase.

46. ​​The method according to claim 43, characterized in that The evolved enzyme is an oxidoreductase, and the oxidoreductase includes any one selected from dehydrogenase, peroxidase, oxygenase, and oxidase.

47. The method according to claim 43, characterized in that The hydrolase comprises any one selected from trypsin and nuclease.

48. The method according to claim 42, characterized in that The enzyme substrate is able to enter the yeast.

49. The method according to claim 42, characterized in that The regulatory molecules are selected from transcription factors.

50. An automatic evolving liquid flow device, characterized in that: The device comprises a sample inlet area, an evolution area, a sample outlet area, and an injection area. in, The sample introduction zone controls the culture medium to enter the evolution zone; The evolution zone is connected to the injection zone, and receives the culture medium input from the injection zone. The evolution zone contains yeast cultured using the growth-coupled yeast directed evolution screening method according to any one of claims 1 to 28, the method for determining the ability of a directed evolution target protein to interact with a first protein according to claim 29, the method for determining the ability of a directed evolution target protein to interact with a cis-acting element according to claim 30 or 31, the method for determining the ability of a directed evolution target protein to interact with a second small molecule according to any one of claims 32 to 41, and the method for determining the catalytic ability of a directed evolution enzyme according to any one of claims 42 to 49, and components for maintaining the growth state of the yeast; The sample outlet area is connected to the evolution area and receives the culture medium discharged from the evolution area; The injection zone is connected to the evolution zone, and the purpose of screening the evolved yeast is achieved by injecting small molecule substances for screening into the evolution zone.

51. The device according to claim 50, characterized in that The volume and rate at which the medium enters the evolution zone is used as part of the growth stress.

52. The device according to claim 50, characterized in that Screening for entry into the evolution zone uses the volume and rate of small molecules as part of the growth stress.

53. The device according to claim 50, characterized in that The small molecule substance for screening includes at least one selected from antibiotics, isopropylthiogalactoside, and yeast counter-screening drugs.

54. A gene product, characterized in that The yeast cultured by the growth-coupled yeast directed evolution screening method described in any one of claims 1-28, the method for determining the ability of a directed evolution target protein to interact with a first protein described in claim 29, the method for determining the ability of a directed evolution target protein to interact with a cis-acting element described in claim 30 or 31, the method for determining the ability of a directed evolution target protein to interact with a second small molecule substance described in any one of claims 32-41, the method for determining the catalytic ability of an enzyme described in any one of claims 42-49, or the yeast expression obtained by screening using the device described in any one of claims 50-53.

55. The gene product according to claim 54, characterized in that The gene product is a protein or RNA.

56. A nucleic acid molecule encoding the gene product of claim 54 or 55.

57. A cell, characterized in that Comprising the gene product of claim 54 or 55 and / or the nucleic acid molecule of claim 56.

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