Engineering biological containment system and method

Abstract

An engineered biological containment system comprising a first nucleic acid comprising at least a portion of a fragment of a first essential gene; a second nucleic acid comprising at least a portion of a fragment of a second essential gene; a third nucleic acid that activates transcription of the first nucleic acid; and a fourth nucleic acid encoding a non-natural amino acid encoding tool wherein the second nucleic acid comprises one or more nucleotide sequences encoding a first termination codon and optionally a nucleotide sequence encoding a second termination codon at the end position of the second essential gene coding region.

Description

Engineered biocontainment systems and methods Technical Field

[0001] The present application relates to the field of bioengineering technology, and in particular to an engineered biological containment system and method, engineered organisms obtained by the biological containment method, and applications thereof. Background Art

[0002] The rapid development of engineered organisms and synthetic biology has greatly promoted the development and application of biotechnology. Currently, artificially genetically modified organisms have shown important application prospects in industrial production, clinical medicine, and environmental remediation. For example, as a milestone in the development of artificially synthesized organisms, scientists have obtained brewer's yeast containing multiple chromosomes designed and synthesized from scratch, which can be used to develop high-yield strains that optimize the adaptation of exogenous metabolic pathways to chassis strains. However, while these engineered organisms have injected vitality into the development of scientific research and industry, they have also brought about problems such as the leakage of genetic information of artificially genetically modified organisms. These potential safety issues may have a negative impact on the natural environment ecology and human health.

[0003] In order to prevent engineered organisms from escaping into the external environment, containment strategies in related technologies include nutrient-deficiency biocontainment strategies, inducible biocontainment strategies, and gene flow barrier strategies. The nutrient-deficiency biocontainment strategy usually involves knocking out the genes required for the synthesis of essential substances in the engineered organism, making the growth of the engineered organism dependent on exogenously provided nutrients. Once the engineered organism escapes, it will not be able to grow due to the lack of corresponding nutrients in the environment. However, due to the widespread presence of various essential metabolic molecules in the natural environment, the nutrient-deficient strain can continue to survive, rendering the biocontainment strategy ineffective. The inducible biocontainment strategy not only requires a large amount of experimental verification, but also cannot prevent the engineered organism from escaping into the external environment by changing its own metabolic pathways and horizontal gene transfer. The gene flow barrier strategy achieves biocontainment through toxic protein-antitoxic protein, so when the toxic protein or antitoxic protein undergoes genetic mutation, the biocontainment strategy may fail.

[0004] Therefore, there is an urgent need to develop simple, effective and widely applicable biocontainment methods to improve human control over genetically modified organisms.

[0005] Summary of the Invention

[0006] To this end, the first embodiment of the present application provides an engineered biocontainment system, comprising: a first nucleic acid, wherein the first nucleic acid comprises at least a portion of a first essential gene; a second nucleic acid, wherein the second nucleic acid comprises at least a portion of a second essential gene; a third nucleic acid, wherein the third nucleic acid activates transcription of the first nucleic acid; and a fourth nucleic acid, wherein the fourth nucleic acid encodes a non-natural amino acid encoding tool, wherein the second nucleic acid comprises one or more nucleotide sequences encoding a first stop codon and, optionally, a nucleotide sequence encoding a second stop codon located at the end position of the coding region of the second essential gene.

[0007] In some embodiments, the first essential gene is the same as the second essential gene. In other embodiments, the first essential gene is different from the second essential gene.

[0008] In some embodiments, the third nucleic acid is an inducible promoter, preferably an inducible promoter pGAL and / or CaMV35S.

[0009] In some embodiments, the first stop codon encodes an unnatural amino acid.

[0010] In some embodiments, the nucleotide sequence encoding the first stop codon replaces a nucleotide sequence of one or more amino acids in the first 20 amino acids at the N-terminus of the protein encoded by the second essential gene. In some embodiments, the nucleotide sequence encoding the first stop codon replaces a nucleotide sequence of one or more amino acids in the 4th to 20th amino acids at the N-terminus of the protein encoded by the second essential gene.

[0011] In some embodiments, the second essential gene includes a gene whose expression level is at a medium to low level. Optionally, the second essential gene includes at least one of the following: CDC27 and CDC4.

[0012] In some embodiments, the nucleotide sequence encoding the first stop codon replaces the nucleotides encoding any one of the following sites or a combination thereof of CDC4: F4, P5, L6, A7, E8, F9, P10, L11, R12, D13, I14, P15, V16, P17, Y18, S19 and Y20; and / or the nucleotide sequence encoding the first stop codon replaces the nucleotides encoding any one of the following sites or a combination thereof of CDC27: N4, P5, E6, L7, A8, P9, F10, T11, L12, S13, R14, G15, I16, P17, S18, F19 and D20.

[0013] In some embodiments, the nucleotide sequence encoding the first stop codon replaces the nucleotides encoding any one of the following sites or a combination thereof of CDC4: F9, R12 and S19; the nucleotide sequence encoding the first stop codon replaces the nucleotides encoding any one of the following sites or a combination thereof of CDC27: P5, L7, A8, T11, R14, P17, F9 and D20.

[0014] In some embodiments, the second essential gene comprises at least one of: CDC27 and CDC4.

[0015] In some embodiments, the nucleotide sequence encoding the first stop codon replaces the nucleotide sequence encoding the 520th amino acid of CDC27, and / or the nucleotide sequence encoding the first stop codon replaces the nucleotide sequence encoding the 325th amino acid of CDC4.

[0016] In some embodiments, the unnatural amino acid encoding tool comprises a methoxytyrosyl-tRNA synthetase / leucine tRNA orthogonal pair (LeuOmeRS / tRNA CUA ) tool; the unnatural amino acid encoded by the first stop codon includes O-methyl-L-tyrosine (OmeY).

[0017] In some embodiments, the first essential gene is selected from the group consisting of RPC11, SKP1, and RPS3.

[0018] In some embodiments, the engineered biocontainment system further comprises a vector selected from the group consisting of a eukaryotic expression vector and a prokaryotic expression vector, optionally comprising: pXF412, pXF413, pXF414, pXF415, pXF294, pXF259, pDR196, pHISi, pESP-3, pESP-2, pESP-1, pHiSi-1, pGAG424, p53his, p RS426gal, pRS41H, pRS413, pRS416, pGBT9 and pAUR123; optionally, the prokaryotic cell expression vector includes: pET28a-TagRFP-N, pTrc-CKS, pET-DsbA, pET-Trx, pET-28a(+)-GFP, pET-28a(+)-sumo, pET-3c-sumo, pET-35b(+), pTXB1 and pCWori.

[0019] In some embodiments, the vector comprises a first vector, a second vector, and a third vector, wherein the first vector comprises the second nucleic acid and optionally the first nucleic acid; the second vector comprises the third nucleic acid; and the third vector comprises the fourth nucleic acid.

[0020] In some embodiments, the vector further comprises a replication origin, a screening marker and an enzyme cleavage site, optionally, the replication origin is at least one of the following: ARS and Ori; optionally, the screening marker is a nucleotide sequence encoding at least one of the following proteins: His, Ura, Leu, Amp and Kan; optionally, the enzyme cleavage site is at least one of the following: ApaI, BamHI, BglII, EcoRI, HindIII, KpnI, NcoI, NdeI, NheI, NotI, SacI, SalI, SphI, XbaI and XhoI.

[0021] The second aspect of the present application also proposes a method for engineered organism containment, comprising: introducing a third nucleic acid and a fourth nucleic acid into the engineered organism to be modified, wherein the third nucleic acid activates the transcription of a first essential gene of the engineered organism to be modified, and the fourth nucleic acid encodes a non-natural amino acid encoding tool; and making the second essential gene of the engineered organism to be modified contain one or more nucleotide sequences encoding a first termination codon and, optionally, a nucleotide sequence encoding a second termination codon located at the end position of the coding region of the second essential gene.

[0022] In some embodiments, the first essential gene is the same as the second essential gene. In other embodiments, the first essential gene is different from the second essential gene.

[0023] In some embodiments, the third nucleic acid is an inducible promoter, preferably an inducible promoter pGAL and / or CaMV35S. In some embodiments, the first stop codon encodes an unnatural amino acid.

[0024] The third embodiment of the present application further provides an engineered organism, comprising an engineered organism containment system as described in any embodiment of the first aspect of the present application.

[0025] In some embodiments, the engineered organisms include eukaryotic cells and prokaryotes, the eukaryotic cells include yeast, animal cells and plant cells, the animal cells include mammalian cells and insect cells, the plant cells include tobacco cells, Arabidopsis cells and goldfish vine cells; the prokaryotes include Escherichia coli and Bacillus subtilis.

[0026] The fourth embodiment of the present application further proposes a use of the engineered organism as described in any embodiment of the third aspect of the present application in producing sustainable biomass, wherein the sustainable biomass includes food, feed, medicine, biofuel and materials.

[0027] The fifth aspect of the present application also proposes a method for screening an engineered organism containment system, comprising: using a nucleotide sequence encoding a first stop codon to replace a nucleotide sequence of one or more amino acids in the first 20 amino acids at the N-terminus of a protein encoded by a second essential gene of the engineered organism to obtain a mutant library of the engineered organism, wherein the first stop codon encodes a non-natural amino acid; introducing a fourth nucleic acid into the mutant library of the engineered organism, wherein the fourth nucleic acid encodes a non-natural amino acid encoding tool; and screening the mutant library of the engineered organism based on the first culture condition to obtain the engineered organism containment system.

[0028] In some embodiments, the first culture condition is an unnatural amino acid.

[0029] In some embodiments, the screening method for the engineered organism containment system further comprises: introducing a third nucleic acid into a mutant library of the engineered organism, wherein the third nucleic acid activates transcription of a first essential gene in the engineered organism, and optionally, the third nucleic acid is an inducible promoter, preferably the inducible promoter pGAL and / or CaMV35S. Optionally, the first essential gene and the second essential gene are the same or different.

[0030] In some embodiments, the method for screening the engineered organism containment system further comprises: determining a second culture condition based on the type of the inducible promoter; and screening the mutant library of the engineered organism based on the first culture condition and the second culture condition to obtain the engineered organism containment system.

[0031] In some embodiments, the second culture condition is (provides) Gal and Glu.

[0032] In some embodiments, the screening method for the engineered organism containment system further comprises: using the nucleotide sequence encoding the first stop codon to replace the nucleotide sequence of one or more amino acids from the 4th to the 20th amino acids at the N-terminus of the protein encoded by the second essential gene of the engineered organism, to obtain a mutant library of the engineered organism.

[0033] The biocontainment method proposed in this application has the following beneficial technical effects:

[0034] (1) The possibility of engineered yeast escaping is greatly reduced, and the escape rate is lower than the limit of the detection method (10 -9 );

[0035] (2) The constructed target protein N-terminal library covers any residue at the N-terminus of the target essential protein. Through subsequent growth curve and sequencing analysis, the essential gene and the specific replacement position can be quickly determined, and the growth of the strain is ensured to be unaffected;

[0036] (3) It is universal and applicable to essential genes in most cells and other organisms including Saccharomyces cerevisiae. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0038] FIG1 is a schematic diagram of pGAL promoter replacement according to an embodiment of the present application;

[0039] FIG2 is a schematic diagram showing the principle of a biocontainment method for co-regulation of transcription and translation according to an embodiment of the present application;

[0040] FIG3 is a flow chart of the target protein N-terminal library screening site according to an embodiment of the present application;

[0041] FIG4 is a schematic diagram of genome reorganization of a transcriptional regulatory strain according to an embodiment of the present application;

[0042] FIG5 is a gradient dot plate diagram of yXF239, yXF240, yXF241 and WT according to an embodiment of the present application;

[0043] FIG6 is a graph showing the results of measuring the growth activity of a transcriptional regulatory strain according to an embodiment of the present application;

[0044] FIG7 is a graph showing the results of a growth activity assay of a transcription-translation co-regulated strain according to an embodiment of the present application;

[0045] FIG8 is a diagram illustrating the transcription-translation co-regulation escape rate verification according to an embodiment of the present application;

[0046] FIG9 is a PCR gel image of CDC4 and CDC27 gene fragments according to an embodiment of the present application;

[0047] FIG10 is a schematic diagram of the target protein N-terminal library construction and screening process according to an embodiment of the present application;

[0048] FIG11 is a diagram showing the distribution of CDC4 library conversion ratios according to an embodiment of the present application;

[0049] FIG12 is a diagram showing the distribution of CDC27 library conversion ratios according to an embodiment of the present application;

[0050] FIG13 is a graph showing the doubling time of strains with different CDC4 substitution positions according to an embodiment of the present application;

[0051] FIG14 is a graph showing the doubling time results of strains with different CDC27 substitution positions according to an embodiment of the present application. DETAILED DESCRIPTION

[0052] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0053] This application is made based on the following knowledge of the inventors:

[0054] The biocontainment strategies for engineered organisms in related technologies have prominent problems such as gene mutation, supplementation of metabolites by the natural environment, and horizontal gene transfer, which can render the biocontainment strategies ineffective. The efficiency of previous biocontainment methods is not ideal. Under non-permissive conditions, the escape rate of engineered organisms is higher than 10. -8 At the same time, under permitted conditions, the growth activity of the engineered organisms also decreased significantly with the engineering modification. In addition, the selection of the codon location for the introduction of non-natural amino acids also requires complex and careful design and extensive testing, which is time-consuming and labor-intensive.

[0055] In this regard, the present application proposes an engineered biocontainment system, which comprises a first nucleic acid, which comprises at least a portion of a first essential gene; a second nucleic acid, which comprises at least a portion of a second essential gene; a third nucleic acid, which activates the transcription of the first nucleic acid; and a fourth nucleic acid, which encodes a non-natural amino acid encoding tool, wherein the second nucleic acid comprises one or more nucleotide sequences encoding a first stop codon and, optionally, a nucleotide sequence encoding a second stop codon located at the end of the coding region of the second essential gene.

[0056] In some embodiments, the first nucleic acid comprises at least a portion of a first essential gene, and the third nucleic acid is an inducible promoter capable of activating transcription of the first nucleic acid under a first permissive condition. The second nucleic acid comprises at least a portion of a second essential gene and one or more nucleotide sequences encoding a first stop codon, and the fourth nucleic acid encodes a non-natural amino acid encoding tool. With the aid of the non-natural amino acid encoding tool, the fourth nucleic acid can introduce the non-natural amino acid into the translation process of the second essential gene under the second permissive condition, thereby achieving total synthesis of the protein encoded by the second essential gene in the organism.

[0057] In the embodiments of the present application, "essential genes" refer to genes that cause a lethal phenotype when deleted. Essential genes are genes that play a decisive role in the survival and development of a species under certain conditions. It is understandable that although different species have different genome sizes and gene compositions, these very different genomes all contain their own set of essential genes to maintain key cellular functions. Essential genes reveal the most basic biological mechanisms of a species and are necessary conditions for the survival of the species. The essential genes vary between different species. For example, in yeast, essential genes include CDC27, CDC4, MET30, RPC11, SKP1 and RPS3.

[0058] In some embodiments, the first essential gene and the second essential gene may be the same or different. It is understood that when the first essential gene and the second essential gene are the same, both transcriptional and translational suppression are applied to the same essential gene, i.e., the expression of the essential gene is controlled by an introduced exogenous promoter, and a protein-coding nucleotide in the coding region of the essential gene is replaced with an unnatural amino acid to achieve simultaneous suppression at both the transcriptional and translational levels. In some embodiments, the second essential gene contains a promoter upstream that directly regulates the transcription of the essential gene, in which case the first essential gene and the second essential gene may be the same. In some embodiments, the first essential gene contains a promoter upstream, while the second essential gene does not contain a promoter upstream, in which case the first essential gene and the second essential gene may be different genes.

[0059] In some embodiments, the first essential gene is selected from the group consisting of CDC27, CDC4, MET30, RPC11, SKP1, and RPS3. In some embodiments, the first essential gene is selected from the group consisting of RPC11, SKP1, and RPS3. In some embodiments, the first essential gene is RPS3.

[0060] In some embodiments, the second essential gene includes at least one of the following: CDC27, CDC4, MET30, RPC11, SKP1, and RPS3. In some embodiments, the second essential gene includes at least one of the following: CDC27 and CDC4. In some embodiments, the nucleotide sequence encoding the first stop codon replaces the nucleotide sequence encoding amino acid position 520 of CDC27, and / or the nucleotide sequence encoding the first stop codon replaces the nucleotide sequence encoding amino acid position 325 of CDC4.

[0061] In the embodiments of the present application, the "first nucleic acid" and the "second nucleic acid" comprise at least a portion of a first or second essential gene. It is understood that the "at least a portion" can be any portion of an essential gene. In some embodiments, the "at least a portion" can be a full-length fragment of an essential gene. In some embodiments, the "at least a portion" can be a fragment of the coding region of an essential gene.

[0062] In the embodiments of the present application, the "third nucleic acid" is an inducible promoter. "Inducible promoter" refers to a promoter that can activate or significantly increase the transcription level of a gene under the stimulation of certain specific physical or chemical signals, including natural inducible promoters such as light-induced expression gene promoters, heat-induced expression gene promoters, wound-induced expression gene promoters, fungal-induced expression gene promoters, and symbiotic bacteria-induced expression gene promoters; artificially constructed inducible promoters, such as pGAL promoter, CaMV35S, etc. The full name of the pGAL promoter is the galactokinase gene (GAL1) promoter. It is an inducible promoter in Saccharomyces cerevisiae, and its expression efficiency is regulated by Gal and Glu. Under culture conditions rich in Glu, the promoter is turned off, while under culture conditions that do not contain Glu and contain Gal, it is activated. Under this model, galactose and glucose correspond to the functional elements that turn on and off the promoter, respectively, thereby turning on and off the expression of genes downstream of the promoter. The method proposed in the examples of the present application utilizes this characteristic of the promoter and loads it onto essential genes, thereby specifically controlling the expression and shutdown of essential genes, and then controlling the survival and death of cells, thereby achieving the purpose of biological escape prevention.

[0063] It is understood that the first permissive condition depends on the type of inducible promoter. In some embodiments, the inducible promoter is a pGAL inducible promoter, and the first permissive condition is the presence of only galactose (Gal) in the culture medium, at which point the promoter is activated, thereby initiating expression of the first essential gene; the non-permissive condition is the presence of glucose (Glu) in the culture medium, at which point transcription of the first essential gene cannot be activated, thereby inhibiting the growth of the engineered organism.

[0064] In an embodiment of the present application, the nucleotide sequence encoding the first stop codon replaces the nucleotide sequence encoding any one or more of the first 20 amino acids at the N-terminus of a protein in a second essential gene. In some embodiments, the nucleotide sequence encoding the first stop codon replaces the nucleotide sequence encoding any one or more of the 4th to 20th amino acids at the N-terminus of a protein in a second essential gene. The inventors have discovered that by modifying the initial portion of the 4th to 20th amino acids at the front end of the N-terminus of most essential proteins, the structure and function of the essential proteins are not affected. Therefore, the biocontainment system proposed in the embodiments of the present application can effectively ensure the normal vitality of the engineered organisms and achieve normal survival and production.

[0065] In some embodiments, the nucleotide sequence encoding the first stop codon replaces the nucleotides encoding any one of the following sites or a combination thereof of CDC4: F4, P5, L6, A7, E8, F9, P10, L11, R12, D13, I14, P15, V16, P17, Y18, S19 and Y20; and / or the nucleotide sequence encoding the first stop codon replaces the nucleotides encoding any one of the following sites or a combination thereof of CDC27: N4, P5, E6, L7, A8, P9, F10, T11, L12, S13, R14, G15, I16, P17, S18, F19 and D20,

[0066] In this example, the first stop codon encodes an unnatural amino acid. Unnatural amino acids do not exist under natural conditions, so the second essential gene cannot be fully translated. In the presence of an unnatural amino acid, the unnatural amino acid encoding tool can introduce the unnatural amino acid into the translation process of the second essential gene, enabling normal protein synthesis. This achieves biocontainment at the translational level.

[0067] In the embodiments of the present application, the "second stop codon" encodes a naturally occurring stop codon located at the 3' end of the mRNA transcribed by the coding region sequence of the essential gene. The "first stop codon" refers to a stop codon located at other positions of the essential gene, except for the "second stop codon" in the natural state located at the 3' end of the mRNA. In some embodiments, the first stop codon and the second stop codon include: UAG, UAA, UGA. The nucleotide sequence encoding the first stop codon and the second stop codon includes: TAG, TAA, TGA. In some embodiments, the first stop codon is UGA, and the nucleotide sequence encoding the first stop codon is TAG.

[0068] "Unnatural amino acid" refers to an amino acid that is not encoded by the existing 64 genetic codons. In an embodiment of the present application, the unnatural amino acid includes O-methyl-L-tyrosine (OmeY).

[0069] In the embodiments of the present application, the "fourth nucleic acid" encodes the above-mentioned non-natural amino acid encoding tool. The "non-natural amino acid encoding tool" comprises a tRNA synthetase (RS) and tRNA of a certain type of non-natural amino acid. The tRNA synthetase in the tool can specifically recognize a certain type of non-natural amino acid and catalyze the loading of the non-natural amino acid onto the tRNA, thereby achieving the activation of the tRNA. The activated tRNA can specifically recognize the codon encoding the non-natural amino acid. In the embodiments of the present application, the non-natural amino acid encoding tool includes: methoxytyrosyl-tRNA synthetase / leucine tRNA orthogonal pairing (LeuOmeRS / tRNA CUA )tool.

[0070] It is understood that the second permissive condition depends on the non-natural amino acid encoded by the stop codon. In certain embodiments, the stop codon is an amber codon (TAG), and the second permissive condition is the introduction of an exogenous non-natural amino acid. In this case, the non-amino acid encoding tool is an orthogonal pairing of methoxytyrosyl-tRNA synthetase / leucine tRNA (LeuOmeRS / tRNACUA). As a non-natural amino acid encoding tool, the LeuOmeRS in the encoding tool can specifically recognize non-natural amino acids O-methyl-L-tyrosine (OmeY) and tRNA CUA , and catalyzes the loading of OmeY onto tRNA CUA 3'-CCA of tRNA CUA Activation, activated tRNA CUA By reading through the stop codon, the full-length essential protein is produced, allowing the organism to survive. Unnatural amino acids do not exist in the natural environment, so escape strains cannot produce full-length essential proteins and can only survive by relying on artificially provided unnatural amino acids, thereby reducing the possibility of engineered organisms escaping.

[0071] In some embodiments, the system further comprises a vector selected from the group consisting of a eukaryotic cell expression vector and a prokaryotic cell expression vector.

[0072] In the present application embodiment, "vector" refers to a nucleic acid molecule, preferably an artificial nucleic acid molecule. The vector in the context of the present application embodiment is suitable for incorporating or carrying a desired nucleic acid sequence, such as a nucleic acid sequence comprising an open reading frame. Such a vector can be a storage vector, an expression vector, a cloning vector, a transfer vector, etc. A storage vector is a vector that allows for convenient storage of nucleic acid molecules (such as mRNA molecules). Therefore, the vector can include a sequence corresponding to, for example, a desired mRNA sequence or a portion thereof, such as a sequence corresponding to an open reading frame and 3'-UTR of the mRNA. An expression vector can be used to produce an expression product such as RNA, such as mRNA, or a peptide, polypeptide, or protein. For example, an expression vector can include a sequence required for transcribing a vector sequence fragment, such as a promoter sequence, such as an RNA polymerase promoter sequence. A cloning vector is typically a vector containing a cloning site, which can be used to incorporate a nucleic acid sequence into a vector. A cloning vector can be, for example, a plasmid vector or a phage vector. A transfer vector can be a vector suitable for transferring a nucleic acid molecule into a cell or an organism, such as a viral vector. The vector in the context of the present application embodiment can be, for example, a eukaryotic expression vector or a prokaryotic expression vector. In some embodiments, eukaryotic expression vectors include: pDR196, pHISi, pESP-3, pESP-2, pESP-1, pHiSi-1, pGAG424, p53his, pRS426gal, pRS41H, pRS413, pRS416, pGBT9, and pAUR123; prokaryotic expression vectors include: pET28a-TagRFP-N, pTrc-CKS, pET-DsbA, pET-Trx, pET-28a(+)-GFP, pET-28a(+)-sumo, pET-3c-sumo, pET-35b(+), pTXB1, and pCWori. Preferably, the vector is a eukaryotic vector, such as pRS413 or pRS416. Preferably, the vector in the sense of the present application contains restriction sites, a selection marker, and sequences suitable for vector propagation, such as an origin of replication. Preferably, the vector in the context of the embodiments of the present application is a plasmid vector. Optionally, the origin of replication is at least one of the following: ARS and Ori; optionally, the selection marker is a nucleotide sequence encoding at least one of the following proteins: His, Ura, Leu, Amp, and Kan; optionally, the restriction enzyme cleavage site is at least one of the following: ApaI, BamHI, BglII, EcoRI, HindIII, KpnI, NcoI, NdeI, NheI, NotI, SacI, SalI, SphI, XbaI, and XhoI.

[0073] In some embodiments, the vector comprises a first vector, a second vector, and a third vector, wherein the first vector comprises the second nucleic acid; the second vector comprises the third nucleic acid; and the third vector comprises the fourth nucleic acid.

[0074] In some embodiments, the first vector comprises a second nucleic acid and a first nucleic acid.

[0075] In summary, the first permissive condition and the second permissive condition can only be met simultaneously when the inducer of the inducible promoter and the non-natural amino acid are present at the same time; the conditions commonly found in nature (absence of galactose / presence of glucose and natural amino acids) are non-permissive conditions. Under these conditions, the engineered organisms with the engineered biocontainment system of the present application cannot grow, ultimately achieving a safe, efficient, easy-to-operate, and widely applicable biocontainment effect.

[0076] In a second aspect, embodiments of the present application provide a method for engineered organism containment, comprising introducing a third nucleic acid and a fourth nucleic acid into an engineered organism to be modified, wherein the third nucleic acid activates transcription of a first essential gene of the engineered organism to be modified, and the fourth nucleic acid encodes a non-natural amino acid encoding tool; and causing a second essential gene of the engineered organism to be modified to include one or more nucleotide sequences encoding a first stop codon and, optionally, a nucleotide sequence encoding a second stop codon located at the end of the coding region of the second essential gene. The method for engineered organism containment of embodiments of the present application can achieve safe, efficient, easy-to-operate, and widely applicable biocontainment.

[0077] In a third aspect, an embodiment of the present application provides an engineered organism, characterized in that the engineered organism comprises the engineered organism containment system described in any embodiment of the first aspect.

[0078] In the examples of this application, "engineered organisms" refer to bacteria or cell lines engineered through targeted modification and large-scale cultivation to produce large quantities of useful metabolites or to exert unique physiological functions. It is understood that the unnatural amino acid encoding system and biocontainment strategy proposed in this application are universally applicable, and thus any chassis strain used in production can be modified using this system and strategy to prevent biological escape. This application is not intended to limit the specific type of engineered organism.

[0079] In some embodiments, the engineered organisms include eukaryotic cells and prokaryotes, the eukaryotic cells include yeast, animal cells and plant cells, the animal cells include mammalian cells and insect cells, the plant cells include tobacco cells, Arabidopsis cells and goldfish vine cells; the prokaryotes include Escherichia coli and Bacillus subtilis.

[0080] In a fourth aspect, embodiments of the present application provide uses of the engineered organisms described in any embodiment of the third aspect in producing sustainable biomass, wherein the sustainable biomass includes food, feed, medicine, biofuel, and materials.

[0081] In a fifth aspect, an embodiment of the present application provides a method for screening an engineered organism containment system, comprising replacing a nucleotide sequence encoding a first stop codon with a nucleotide sequence of one or more amino acids in the first 20 amino acids at the N-terminus of a protein encoded by a second essential gene of the engineered organism to obtain a mutant library of the engineered organism, wherein the first stop codon encodes a non-natural amino acid; introducing a fourth nucleic acid into the mutant library of the engineered organism, wherein the fourth nucleic acid encodes a non-natural amino acid encoding tool; and screening the mutant library of the engineered organism based on a first culture condition to obtain the engineered organism containment system.

[0082] In some embodiments, the method further comprises introducing a third nucleic acid into the mutant library of the engineered organism, wherein the third nucleic acid activates transcription of a first essential gene in the engineered organism.

[0083] In some embodiments, the method further comprises determining a second culture condition based on the type of the inducible promoter; and screening the mutant library of the engineered organism based on the first culture condition and the second culture condition to obtain the engineered organism containment system.

[0084] It should be noted that screening the mutant library of the engineered organisms is performed by verifying the growth activity and escape rate of the engineered organisms. Those skilled in the art will appreciate that the doubling time of a cell can be used to reflect the growth activity of a strain. Strains with high growth activity have shorter doubling times, whereas strains with low growth activity have longer doubling times. By continuously culturing and continuously monitoring the biomass within the culture system, the doubling time of the strain can be accurately calculated, thereby quantifying the growth activity of the strain.

[0085] Those skilled in the art will recognize that the escape rate can be used to measure biocontainment effectiveness. There is a correlation between the volume of bacterial solution and the amount of bacteria applied. When cells in a solution are plated under permissive conditions, all cells can grow on the plate, while cells grown under non-permissive conditions circumvent biocontainment. Therefore, the escape rate can be calculated by taking the number of clones grown when a large amount of bacterial solution is applied to a plate under non-permissive conditions as the denominator and the number of clones grown when a fixed amount of bacterial solution is applied to a plate under permissive conditions as the numerator. In some specific embodiments, the escape rate is calculated as follows: m mL of bacterial solution is applied to an SC solid culture medium plate for 2 days, and the number of single colonies grown is x; the bacterial solution is applied to an SC-Leu-Ura solid culture medium plate at a gradient concentration, and the minimum amount of bacteria applied to grow clones, n, is determined. The number of single colonies is y. The escape rate of the strain is then calculated as (y × n) / (x × m).

[0086] Since screening the mutant library of the engineered organism is performed by verifying the growth activity and escape rate of the engineered organism, and the number of monoclonal clones under non-permissive conditions and permissive conditions needs to be recorded when calculating the escape rate. Therefore, in some embodiments, the first culture condition is two culture media with and without the addition of non-natural amino acids. In some embodiments, the second culture condition is two culture media with and without the addition of Gal.

[0087] In some embodiments, the method further comprises: using the nucleotide sequence encoding the first stop codon to replace the nucleotide sequence of one or more amino acids from the 4th to the 20th amino acids at the N-terminus of the protein encoded by the second essential gene of the engineered organism to obtain a mutant library of the engineered organism.

[0088] The screening method for the engineered biocontainment system provided in the examples of the present application can quickly screen suitable sites for the introduction of unnatural amino acids, saving a lot of time, energy and costs.

[0089] According to the engineered organisms proposed in the examples of the present application, since they are modified to contain the non-natural amino acid encoding system described in the above examples, the engineered organisms are highly controllable. Therefore, by controlling the engineered organisms, that is, by biocontainment of the engineered organisms to prevent their escape, a high degree of control over the use of the engineered organisms in the production of sustainable biomass is achieved, thereby effectively ensuring the production of safe, green and sustainable biomass.

[0090] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0091] Unless otherwise specified, the quantitative tests in the following examples were performed three times, and the results were averaged.

[0092] Example 1

[0093] This example tests the effectiveness of a biocontainment method at the transcriptional level based on an inducible promoter (pGAL1, sequence number in the NCBI database: NM_001178368).

[0094] 1.1 Preparation of promoter fragment

[0095] Multiple PCR reactions were used to prepare inducible promoter fragments corresponding to different first essential genes and fused with the KanMX gene fragment (GenBank: LT726876.1, Range: 3800 to 4609, a total of 810 bp). This fragment can be used to screen successfully integrated yeast in subsequent genomic integration.

[0096] (1) Using pXF259 and pXF294 plasmids as templates, the KanMX gene fragment and the pGAL promoter gene fragment were synthesized by PCR, respectively. At the same time, in this PCR reaction, based on primers with homology arm sequences of the recombinant genes, the homology arms of the corresponding recombinant genes were introduced at the 5' end of the KanMX gene fragment and the 3' end of the pGAL promoter gene fragment, respectively. The primer sequences are shown in Table 1 (wherein lowercase letters represent homology arm sequences, and uppercase letters represent sequences that bind to the template);

[0097] Table 1

[0098] (2) The PCR product of step (1) was purified and recovered using the Qiagen PCR product purification kit.

[0099] (3) OE-PCR primers were designed based on different first essential genes RPC11 (sequence number in the NCBI database: NM_001018632), SKP1 (sequence number in the NCBI database: NM_001021365), and RPS3 (sequence number in the NCBI database: NM_001023783). The primer sequences are shown in Tables 2, 3, and 4.

[0100] Table 2

[0101] Table 3

[0102] Table 4

[0103] (4) Using the primers in step (3), the KanMX gene fragment purified in step (2) and the pGAL promoter gene fragment were fused by OE-PCR reaction to obtain fusion fragments KanMX-pGAL1, KanMX-pGAL2, and KanMX-pGAL3 corresponding to different first essential genes. The OE-PCR reaction system and reaction conditions are shown in Tables 5 and 6;

[0104] Table 5

[0105] Table 6

[0106] (5) The OE-PCR product in step (4) was purified and recovered using the Qiagen PCR product purification kit.

[0107] 1.2 Yeast transformation

[0108] Based on the principle of yeast gene homologous recombination, the purified fusion fragments KanMX-pGAL1-3 obtained in step 1.1 (5) were integrated into the yeast Wt genome respectively. The specific method is as follows.

[0109] (1) A wild-type yeast single clone (deposited in China Center for Type Culture Collection, accession number: CCTCC NO: M2014434) was inoculated into 4 mL of yeast-enriched medium YPD and cultured in a shaker at 30°C and 200 rpm for 24 h.

[0110] (2) Take 1 ml of the bacterial solution obtained in step (1) and use a spectrophotometer to measure the bacterial density and record the OD 600 value;

[0111] (3) According to the OD measured in step (2) 600 Take an appropriate amount of bacterial solution and add it to a triangular flask containing 20mL YPD to make the OD value per ml of bacterial solution 600 is 0.1;

[0112] (4) The bacterial solution in step (3) was cultured in a shaking incubator at 30°C and 200 rpm until the OD value per ml of bacterial solution was 600 0.6-1;

[0113] (5) Centrifuge the bacterial solution in step (4) at 2000 rpm / min for 5 min, then remove the supernatant and collect the bacterial cells;

[0114] (6) Add 10 mL of sterile ddH2O to the centrifuge tube containing the bacterial cells to resuspend the cells, then centrifuge at 2000 rpm / min for 5 min, remove the supernatant, and collect the bacterial cells;

[0115] (7) Add 0.1 M LipoAc to the centrifuge tube containing the bacterial cells to resuspend the cells, then centrifuge at 2000 rpm / min for 5 min, remove the supernatant, and collect the bacterial cells;

[0116] (8) Add 0.1 M LipoAc to the centrifuge tube containing the bacteria and resuspend the cells to obtain competent cells;

[0117] (9) Prepare the transformation solution using the purified fusion fragment KanMX-pGAL1-3 obtained in step 1.1(5) and add it to the transformation tube. The transformation solution system is shown in Table 7;

[0118] Table 7

[0119] (10) Add 100 μL of competent cells prepared in step (8) to the transformation tube;

[0120] (11) Mix by inversion and incubate at 30°C for 30 min;

[0121] (12) Add 50 μL of DMSO and quickly invert to mix;

[0122] (13) Incubate at 42°C for 15 min and then place on ice for 1 min;

[0123] (14) Centrifuge at 8000 rpm / min for 5 min, remove the supernatant, and collect the cells;

[0124] (15) Add 400 μL of 5 mM CaCl 2 to the centrifuge tube containing the bacteria to resuspend the cells and incubate at room temperature for 10 min.

[0125] (16) Spread 100 μL of bacterial solution evenly on the defective culture medium and culture for 2-3 days until a single colony grows;

[0126] (17) Extract the monoclonal strain obtained in step (16) and perform sequencing verification;

[0127] (18) After correct verification, strains yXF239, yXF240, and yXF241 for biocontainment were obtained.

[0128] 1.3 Growth activity assay

[0129] This example uses the strain obtained in 1.2 to measure growth activity. Those skilled in the art will appreciate that the doubling time of a cell can be used to reflect the growth activity of a strain. Strains with high growth activity have shorter doubling times, while strains with low growth activity have longer doubling times. By continuously culturing and continuously measuring the biomass within the culture system, the doubling time of the strain can be accurately calculated, thereby quantifying the growth activity of the strain.

[0130] (1) Add the strain for biocontainment obtained in 1.2 to the defective liquid culture medium (SC+Gal) and culture at 30°C and 200 rpm in a shaking incubator until the OD value per ml of bacterial liquid is 0. 600 is 2-3;

[0131] (2) Add the bacterial solution into the culture well and add the defective liquid culture medium to a final concentration of OD 600 =0.01, final volume 200 μL;

[0132] (3) Place the well plate in a bioscreen continuous incubator for incubation and data collection. The incubator program settings are shown in Table 8.

[0133] Table 8

[0134] (4) Calculate the doubling time of the strain based on the collected data. The calculation method is based on the relevant literature (Fernandez-Ricaud et al. BMC Bioinformatics (2016) 17:249).

[0135] In this example, the doubling time of yeast with the Wt genome was measured to be 3.1 hours, yXF239 to be 3.2 hours, yXF240 to be 3.1 hours, and yXF240 to be 3.2 hours. Growth activity assays conducted on the strains used for biocontainment showed that replacing the promoter of the essential gene with the inducible promoter pGAL had no effect on the growth of the strains ( Figure 6 ). That is, even after replacing the promoter with the inducible promoter, the strains used for biocontainment in this example retained their original growth capacity, demonstrating that subsequent biocontainment can be performed based on the introduction of the inducible promoter.

[0136] 1.4 Biocontainment Effectiveness Verification

[0137] This example uses the strain obtained in 1.2 to qualitatively and quantitatively verify the biocontainment effect. The inducer SC+Gal was added to the culture medium. Galactose culture medium is the permissive condition, while SC+Glu culture medium is the non-permissive condition.

[0138] 1.4.1 Qualitative Verification of Biocontainment Effectiveness

[0139] yXF239, yXF240, and yXF241 were spread onto solid culture media containing SC+Gal and SC+Glu, respectively, at varying concentrations and incubated at 30°C for 2 days. Qualitative verification of the biocontainment effect is shown in Figure 5. Under permissive conditions, strains yXF239, yXF240, and yXF241 all grew normally, whereas under non-permissive conditions, the growth of strains yXF239, yXF240, and yXF241 was inhibited, demonstrating that the inducible promoter in this example achieved a biocontainment effect.

[0140] 1.4.2 Quantitative verification of biocontainment effectiveness

[0141] This embodiment quantitatively verifies the biocontainment effect through an escape rate test. Those skilled in the art will know that there is a correlation between the volume of bacterial solution and the amount of bacteria applied. When cells in the solution are plated under permissive conditions, all cells can grow on the plate, while cells grown under non-permissive conditions evade biocontainment. Therefore, the number of clones grown by applying a large amount of bacterial solution on a plate under non-permissive conditions is used as the denominator, and the number of clones grown by applying a quantitative bacterial solution on a plate under permissive conditions is used as the numerator, then the corresponding escape rate can be calculated (escape rate calculation method, for example: 10 -4 After culturing for two days, the number of colonies growing on a plate containing 10-2 mL of bacterial solution on SC solid medium is x. On the eighth day, if no colonies grow on 10-2 mL and 10-1 mL of SC-Leu-Ura solid medium, but a colony grows on 1 mL and 10 mL of solid medium containing unnatural amino acids, the plate with the least colonies is counted as y. The escape rate of this strain is then (y × 10-4) / (x × 1).

[0142] (1) Add strains yXF239, yXF240, and yXF241 into SC+Gal liquid medium respectively, so that the initial OD 600 =0.1, and cultured in a shaking incubator at 30°C and 200 rpm until the late logarithmic phase, OD 600 =0.1 is about 2-3;

[0143] (2) Wash the cells three times and dilute to OD 600 =1;

[0144] (3) 10 -4 mL of yXF239, yXF240, and yXF241 bacterial suspension was spread on SC+Gal solid medium and cultured in a 30°C incubator for 2 days under permissive conditions;

[0145] (4) Count the number of colonies grown on the plate and record them as x;

[0146] (5) 10 -2 mL, 10 -1 mL, 1 mL, and 10 mL of bacterial solution were applied to SC+Glu solid culture medium, respectively, and cultured in a 30°C incubator for 8 days under non-permissive conditions;

[0147] (6) Determine the minimum amount of bacteria that can grow colonies, n, and count the colonies grown on the corresponding plate, which is recorded as y;

[0148] (7) Calculate the strain escape rate, escape rate = (y × n) / (x × 1), the results are shown in Table 9.

[0149] Table 9

[0150] As shown in Table 9, all transcriptional regulatory strains exhibited biorepression effects, among which yXF241 had the best repression effect, reaching the order of 1E-7, which shows that it is feasible to use it for biorepression.

[0151] In Example 1 of the present application, the essential gene promoter is replaced with an inducible pGAL promoter to construct a switch device for controlling the expression of the essential gene. The expression of the target essential gene is regulated by adding the promoter inducer Gal, thereby achieving the effect of biorepression. This example tested three different essential genes and found that the introduction of the pGAL promoter can successfully construct strains that are biorepressed at the transcriptional level without significantly affecting the growth of the strain. In order to better evaluate the effect of this method, this example tested the escape rate of the transcriptionally regulated strain and found that the best biorepression effect can be achieved by replacing the wild promoter of the RPS3 gene with the inducible pGAL promoter. Therefore, the inducible pGAL promoter can effectively inhibit the growth of engineered organisms at the transcriptional level, providing an effective biorepression method.

[0152] Example 2

[0153] This example tests the effectiveness of a biocontainment method based on the introduction of an inducible promoter (pGAL1) and a stop codon for transcriptional and translational co-regulation. Specifically, the unnatural amino acid encoding tool used is a methoxytyrosyl-tRNA synthetase / leucine tRNA orthogonal pair (LeuOmeRS pair, LeuOmeRS / tRNA CUA , ACS Synth.Biol.2018,7,9,2256–2269), namely providing a LeuOmeRS / tRNA comprising the above CUAThe test was conducted by using the pXF231 vector, and using F9, P5, H520, and F325 in the proteins encoded by the yeast essential genes CDC27 and CDC4 as the introduction sites for unnatural amino acids, respectively. The wild-type coding sequences of CDC27 and CDC4 are shown in SEQ ID No: 11 and SEQ ID No: 12, respectively.

[0154] Wild-type coding sequence of CDC27 SEQ ID No: 11:

[0155] Wild-type coding sequence of CDC4 SEQ ID No: 12:

[0156] 2.1 Obtaining transcriptional and translational co-regulatory strains

[0157] (1) Using yXF241 as a template, the purified fusion fragment KanMX-pGAL was prepared. The PCR primer sequences are shown in Table 10 (for specific steps and procedures, see Example 1.1);

[0158] Table 10

[0159] Note: The underlined region is the homologous region to the RPS3 gene.

[0160] (2) The purified fusion fragment KanMX-pGAL was integrated into the genomes of yeast yXF222, yXF098, yXF218, and yXF097 by yeast homologous recombination (see Example 1.2 for details). Compared with the yeast wild type, the 9th amino acid from the N-terminus encoded by the CDC4 gene of the yXF222 strain was replaced by F to the first stop codon (i.e., non-essential amino acid), and the replacement resulted in a CDC4 (F9amb) gene sequence with TAG; similarly, the 325th amino acid from the N-terminus encoded by the CDC4 gene of yXF098 was replaced by F to the first stop codon (i.e., non-essential amino acid) based on the wild type, and the replacement resulted in a CDC4 (F325amb) gene with TAG; similarly, the replacement resulted in a CDC27 (P5amb) gene sequence with TAG in yXF218; and the replacement resulted in a CDC27 (H520amb) gene sequence with TAG in yXF097.

[0161] (3) Sequencing verification: If the sequence is correct, the transcription-translation co-regulatory strains yXF251, yXF252, yXF253, and yXF254 are obtained (see Table 16 for correspondence).

[0162] 2.2 Growth activity assay

[0163] (1) In this example, the growth activity of the strain obtained in 2.2 was determined. The growth activity determination method is shown in Example 1.3.

[0164] The results of the growth activity assays for the co-regulated transcription and translation strains are shown in Figure 7. As shown in Figure 7, there is no significant difference in the doubling time of yeast with the Wt genome and that of yXF251, yXF252, yXF253, and yXF254, indicating that the modifications at the transcription and translation levels in this example did not affect the normal growth activity of the strains, demonstrating the feasibility of the biocontainment strategy for co-regulated transcription and translation.

[0165] 2.3 Biocontainment effectiveness verification

[0166] This example uses the strain obtained in 2.2 to qualitatively and quantitatively verify the biocontainment effect. Adding the inducer SC-leu+OmeY+Gal to the culture medium is the permissive condition, while SC+leu medium is the non-permissive condition.

[0167] 2.3.1 Qualitative verification of biocontainment effectiveness

[0168] Apply gradient concentrations of yXF251, yXF252, yXF253, and yXF254 to SC-leu+OmeY+Gal and SC+leu culture media, respectively. For details, refer to 1.4.1 for qualitative verification of biocontainment efficacy.

[0169] The qualitative verification of the biocontainment effect is shown in Figure 8 . Under non-permissive conditions, single clones of strains yXF251, yXF252, yXF253, and yXF254 could not grow, indicating a good biocontainment effect.

[0170] 2.3.2 Quantitative verification of biocontainment effectiveness

[0171] Refer to step 1.4.2 to quantitatively verify the biocontainment effect of yXF251, yXF252, yXF253, and yXF254, where n = 100 ml (the number of viable bacteria is about 10 9 ).

[0172] Table 11

[0173] The quantitative verification results of the biocontainment effect are shown in Table 11. When the biocontainment method of transcriptional and translational co-regulation was used, even if the number of viable bacteria at the time of plating reached 10 9 (measurement limit), under non-permissive conditions, no clones could be grown.

[0174] This example utilizes an inducible promoter (pGAL1) and a stop codon to simultaneously regulate two essential genes at the transcriptional and translational levels, constructing a biocontainment strategy that co-regulates transcription and translation. This strategy did not affect normal growth of the strain under permissive conditions (culture medium supplemented with both OmeY and Galactoose) and inhibited growth of the target yeast under non-permissive conditions (culture medium lacking both OmeY and Galactoose and supplemented with Glucose). Furthermore, no escape clones were detected under the extreme limits of existing testing methods, demonstrating excellent biocontainment efficacy.

[0175] Example 3

[0176] In related technologies, identifying essential genes and specific substitution locations is challenging, requiring complex and meticulous design or extensive testing. In this embodiment of the present invention, a target protein N-terminal library was constructed. This method can quickly and efficiently identify essential genes and specific substitution locations that have the least impact on strain growth. The specific steps are as follows.

[0177] 3.1 Preparation of transcriptional regulatory strains

[0178] Preparation of transcriptional regulatory strain yXF241 strain, its anti-escape effect has been verified to be the best in transcriptional regulatory strains (see Example 1 for details).

[0179] 3.2 Preparation of terminator fragments

[0180] In this example, 17 codon positions N4 to D20 and F4 to Y20 in the N-terminal region of the proteins encoded by the yeast essential genes CDC27 and CDC4 were used as sites for the introduction of unnatural amino acids (TAG), and terminator fragments corresponding to the coding positions were prepared.

[0181] (1) Using the pXF414 plasmid as a template, 17 CDC27 gene fragments and 17 CDC4 gene fragments were synthesized by PCR, wherein the nucleotide sequences corresponding to the 17 codons N4 to D20 and F4 to Y20 in the N-terminal region of the CDC27 and CDC4 proteins were replaced one by one by the terminator TAG. The PCR reaction system and conditions are shown in Tables 13 and 14.

[0182] Table 13

[0183] Table 14

[0184] (2) A strain with URA3 inserted before the CDC4 and CDC27 genes was used as a template to obtain the Ura3 gene fragment (GenBank: MG833229.1, Range: 171 to 1341, a total of 1171 bp) for screening genes during genomic integration. The primer sequence information in steps (1) and (2) is shown in Table 15;

[0185] Table 15

[0186] (3) Purifying and recovering the PCR products in steps (1) and (2) using a Macherey-Nagel PCR product purification kit;

[0187] (4) fusing the Ura3 gene fragment purified in step (3) with the CDC27 / CDC4 gene fragment by OE-PCR to obtain a CDC27-Ura3 / CDC4-Ura3 fragment;

[0188] (5) The PCR product in step (4) was purified and recovered using a Macherey-Nagel PCR product purification kit. The experimental PCR gel image is shown in Figure 9.

[0189] 3.3 Construction of target protein N-terminal library

[0190] In this example, yeast gene homologous recombination was used to integrate 17 CDC27-Ura3 / CDC4-Ura3 gene fragments into the yeast BY4741-pGAL-RPS3 (yXF241) genome, and clones successfully assembled into the genome were screened using Ura3. For specific methods, see Section 1.2 of Example 1. A schematic diagram of genome integration is shown in Figure 10.

[0191] 3.4 Growth activity assay

[0192] (1) When the strain obtained in 3.3 was applied to non-permissive conditions without OmeY, clones in which the terminator TAG successfully replaced the amino acid coding position could not grow. However, under permissive conditions with OmeY, they could grow normally, indicating that the biocontainment strategy in this example is effective.

[0193] (2) 124 CDC4-encoding strains and 146 CDC27-encoding strains were selected from the target protein N-terminal mutation library for sequencing.

[0194] (3) The OD obtained in 3.3 600 = 0.01 strain was added to SC-Ura-Leu + OmeY liquid medium, placed in a bioscreen growth curve analyzer at 30°C, and OD was measured every 20 minutes.600 , until the strain growth reached the plateau phase, the total measurement time was 48 hours, in order to determine the growth curve of all selected strains. The strain doubling time calculation method was referenced to (PRECOG: A tool for automated extraction and visualization of fitness components in microbial growth phenomics, BMC Bioinformatics. 2016.17:249).

[0195] Results

[0196] The first generation sequencing showed that CDC4 and CDC27 basically covered the diversity of the library, and the results were shown in Figures 11 and 12, indicating that the method for constructing the mutant library proposed in this embodiment has shown a significant improvement in the screening basis and screening efficiency of the biocontainment system. In addition, it was calculated that the doubling time of the starting strain was 2.9 hours, and the doubling time results of the remaining strains were shown in Figures 13 and 14. Thus, through the determination of the growth curve, it was shown that the method proposed in this embodiment, on the basis of ensuring the effectiveness of the biocontainment strategy of co-regulation of transcription and translation, can obtain anti-escape strains with low escape rate and good growth by further testing and screening sites that can introduce non-natural amino acids at the N-terminus of the protein. At the same time, through data analysis, the growth of these strains was almost indistinguishable from the original strain, indicating that the viability of the strains screened out by the mutant library constructed in this embodiment was not subject to the pressure stress of exogenous starting elements and non-natural amino acids, and can be well used for biological production.

[0197] In the N-terminal library screening of CDC4, the three sites F9, R12 and S19 can still maintain a doubling time similar to that of the original strain after the introduction of non-natural amino acids. In the N-terminal library screening of CDC27, among the 17 sites in the library, 8 sites (P5, L7, A8, T11, R14, P17, F9, D20) can maintain a similar growth rate to the original strain after the introduction of non-natural amino acids. These results show that by constructing the target protein N-terminal library, this embodiment does not need to rely on complex preliminary design. The essential genes and specific replacement positions can be quickly determined by screening the library at the N-terminus of the protein, which is of great significance for the biosafety of engineered organisms.

[0198] The strain information used in the examples of this application is shown in Table 16.

[0199] Table 16

[0200] The plasmid information used in the examples of this application is shown in Table 17.

[0201] Table 17

[0202] The instrument information used in the examples of this application is shown in Table 18.

[0203] Table 18

[0204] The preparation methods of the reagents used in the examples of this application are as follows.

[0205] 50× OmeY: Add 0.5 g of OmeY to 50 mL of ultrapure water and filter sterilize (OmeY cannot be autoclaved).

[0206] 1000×Carb (100 mg / mL): Add 1 g of Carb to 10 mL of ultrapure water and filter sterilize.

[0207] 2×Agar (4%): Add 10 g of Agar to 250 mL with water.

[0208] 40% glucose: add water to 200 g of glucose and make up to 500 mL.

[0209] 100×Ura: Add 2.24g of Ura to 1L with water

[0210] 100×His: Add 21 g of His to 1 L of water.

[0211] 50×Leu: Add 13 g of Leu to 1 L of water.

[0212] 2×SC-3: Yeast Nitrogen base w / o aa's and amm.sμLfate 3.4g, Amino acid drop out mix (cloneTech Aamix) 1.3g, Ammonium sulfate 10g, add water to make up to 1L.

[0213] SC-Ura liquid medium: 2×SC-3 250 mL, 40% glucose 25 g, 100×His 5 mL, 50×Leu 10 mL, add water to 500 mL.

[0214] SC-Ura solid medium: 2×SC-3 250 mL, 40% glucose 25 g, 100×His 5 mL, 50×Leu 10 mL, add 2×Agar (4%) to make up to 500 mL.

[0215] SC-His liquid culture medium: 2×SC-3 250 mL, 40% glucose 25 g, 100×Ura 5 mL, 50×Leu 10 mL, add water to 500 mL.

[0216] SC-His solid medium: 2×SC-3 250 mL, 40% glucose 25 g, 100×Ura 5 mL, 50×Leu 10 mL, add 2×Agar (4%) to make up to 500 mL.

[0217] SC-Leu-Ura liquid medium: 2×SC-3 250 mL, 40% glucose 25 g, 100×His 5 mL, add water to make up to 500 mL.

[0218] SC-Leu-Ura solid culture medium: 2×SC-3 250 mL, 40% glucose 25 g, 100×His 5 mL, add 2×Agar (4%) to make up to 500 mL.

[0219] SC-Leu-Ura+OmeY liquid culture medium: 2×SC-3 250 mL, 40% glucose 25 g, 100×His 5 mL, 50×OmeY 10 mL, add water to make up to 500 mL.

[0220] SC-Leu-Ura+OmeY solid culture medium: 2×SC-3 250 mL, 40% glucose 25 g, 100×His 5 mL, 50×OmeY 10 mL, add 2×Agar (4%) to make up to 500 mL.

[0221] SC-3 + OmeY liquid medium: 2× SC-3 250 mL, 40% glucose 25 g, 50× OmeY 10 mL, add water to 500 mL.

[0222] SC-3 solid culture medium: 2×SC-3 250 mL, 40% glucose 25 g, add 2×Agar (4%) to make up to 500 mL.

[0223] LB liquid medium: Add water to 10 g of LB Broth and make up to 400 mL.

[0224] LB+Carb solid medium: LB Broth 10 g, agar 6 g, 1000×Carb 0.4 mL, add water to make up to 400 mL.

[0225] LB+Carb liquid medium: LB Broth 10 g, 1000×Carb 0.4 mL, add water to 400 mL.

[0226] All reagents were sterilized at 121°C for 20 minutes.

[0227] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean 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 invention. 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 can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0228] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. An engineered biocontainment system, characterized in that: Include: a first nucleic acid comprising at least a portion of a first essential gene; a second nucleic acid comprising at least a portion of a second essential gene; a third nucleic acid that activates transcription of the first nucleic acid; and a fourth nucleic acid encoding a non-natural amino acid encoding tool, wherein the second nucleic acid comprises one or more nucleotide sequences encoding a first stop codon and optionally a nucleotide sequence encoding a second stop codon located at the end of the coding region of the second essential gene, Optionally, the first essential gene is the same as or different from the second essential gene.

2. The engineered biocontainment system according to claim 1, characterized in that: The third nucleic acid is an inducible promoter, preferably an inducible promoter pGAL and / or CaMV35S.

3. The engineered biocontainment system according to claim 1, characterized in that: The first stop codon encodes an unnatural amino acid.

4. The engineered biocontainment system of claim 1, wherein: The nucleotide sequence encoding the first stop codon replaces a nucleotide sequence of one or more amino acids in the first 20 amino acids at the N-terminus of the protein encoded by the second essential gene, Preferably, the nucleotide sequence encoding the first stop codon replaces the nucleotide sequence of one or more amino acids from the 4th to the 20th amino acids at the N-terminus of the protein encoded by the second essential gene.

5. The engineered biocontainment system of claim 4, wherein: The second essential gene includes genes whose expression levels are at medium to low levels. Optionally, the second essential gene includes at least one of the following: CDC27 and CDC4.

6. The engineered biocontainment system of claim 5, wherein: The nucleotide sequence encoding the first stop codon replaces the nucleotides encoding any of the following sites or a combination thereof of CDC4: F4, P5, L6, A7, E8, F9, P10, L11, R12, D13, I14, P15, V16, P17, Y18, S19 and Y20; and / or the nucleotide sequence encoding the first stop codon replaces the nucleotides encoding any of the following sites or a combination thereof of CDC27: N4, P5, E6, L7, A8, P9, F10, T11, L12, S13, R14, G15, I16, P17, S18, F19 and D20, Preferably, the nucleotide sequence encoding the first stop codon replaces any of the following sites in CDC4: or a combination thereof: F9, R12 and S19; the nucleotide sequence encoding the first stop codon replaces the nucleotides encoding any one of the following sites or a combination thereof of CDC27: P5, L7, A8, T11, R14, P17, F9 and D20.

7. The engineered biocontainment system of claim 1, wherein: The second essential gene includes at least one of the following: CDC27 and CDC4, The nucleotide sequence encoding the first stop codon replaces the nucleotide sequence encoding the 520th amino acid of CDC27, and / or the nucleotide sequence encoding the first stop codon replaces the nucleotide sequence encoding the 325th amino acid of CDC4.

8. An engineered biocontainment system according to any one of claims 1 to 7, characterized in that: The unnatural amino acid encoding tool includes a methoxytyrosyl-tRNA synthetase / leucine tRNA orthogonal pair (LeuOmeRS / tRNA CUA )tool; The unnatural amino acid encoded by the first stop codon includes O-methyl-L-tyrosine (OmeY).

9. The engineered biocontainment system of claim 1, wherein: The first essential gene is selected from the group consisting of RPC11, SKP1 and RPS3.

10. The engineered biocontainment system according to any one of claims 1 to 9, characterized in that: The invention also comprises a vector, wherein the vector is selected from the group consisting of a eukaryotic cell expression vector and a prokaryotic cell expression vector, and optionally, the eukaryotic cell expression vector comprises: pXF412, pXF413, pXF414, pXF415, pXF294, pXF259, pDR196, pHISi, pESP-3, pESP-2, pESP-1, pHiSi-1, pGAG424, p53his, pRS426gal, pRS41H, pRS413, pRS416, pGBT9 and pAUR123; optionally, the prokaryotic cell expression vector includes: pET28a-TagRFP-N, pTrc-CKS, pET-DsbA, pET-Trx, pET-28a(+)-GFP, pET-28a(+)-sumo, pET-3c-sumo, pET-35b(+), pTXB1 and pCWori.

11. The engineered biocontainment system of claim 10, wherein: The vector comprises a first vector, a second vector, and a third vector, wherein the first vector comprises the second nucleic acid and optionally the first nucleic acid; the second vector comprises the third nucleic acid; and the third vector comprises the fourth nucleic acid.

12. The engineered biocontainment system of claim 10, wherein: The vector also contains a replication origin, a screening marker and an enzyme cutting site. Optionally, the replication origin is at least one of: ARS and Ori; Optionally, the selection marker is a nucleotide sequence encoding at least one of the following proteins: His, Ura, Leu, Amp and Kan; Optionally, the restriction enzyme cleavage site is at least one of the following: ApaI, BamHI, BglII, EcoRI, HindIII, KpnI, NcoI, NdeI, NheI, NotI, SacI, SalI, SphI, XbaI and XhoI.

13. An engineered biocontainment method, characterized in that: include: Introducing a third nucleic acid and a fourth nucleic acid into the engineered organism to be modified, wherein the third nucleic acid activates transcription of a first essential gene of the engineered organism to be modified, and the fourth nucleic acid encodes a non-natural amino acid encoding tool; and The second essential gene of the engineered organism to be modified comprises one or more nucleotide sequences encoding a first stop codon and an optional nucleotide sequence encoding a second stop codon located at the end of the coding region of the second essential gene, Optionally, the first essential gene is the same as or different from the second essential gene.

14. The method according to claim 13, characterized in that The third nucleic acid is an inducible promoter, preferably an inducible promoter pGAL and / or CaMV35S; Optionally, the first stop codon encodes an unnatural amino acid.

15. An engineered organism, characterized in that: The engineered organism comprises an engineered biocontainment system as described in any one of claims 1-12. Optionally, the engineered organisms include eukaryotic cells and prokaryotes, the eukaryotic cells include yeast, animal cells and plant cells, the animal cells include mammalian cells and insect cells, the plant cells include tobacco cells, Arabidopsis cells and goldfish vine cells; the prokaryotes include Escherichia coli and Bacillus subtilis.

16. Use of the engineered organism in producing sustainable biomass according to claim 15, characterized in that: The sustainable biomass includes food, feed, medicine, biofuel and materials.

17. A method for screening an engineered biocontainment system, characterized in that: include: Substituting a nucleotide sequence encoding a first stop codon for a nucleotide sequence of one or more amino acids in the first 20 amino acids at the N-terminus of a protein encoded by a second essential gene of the engineered organism to obtain a mutant library of the engineered organism, wherein the first stop codon encodes a non-natural amino acid; introducing a fourth nucleic acid into the mutant library of the engineered organism, wherein the fourth nucleic acid encodes a non-natural amino acid encoding tool; and Based on the first culture condition, the mutant library of the engineered organism is screened to obtain the engineered biological containment system, Optionally, the first culture condition is an unnatural amino acid.

18. The method according to claim 17, characterized in that The method further comprises: introducing a third nucleic acid into the mutant pool of the engineered organism, wherein the third nucleic acid activates transcription of a first essential gene in the engineered organism, Optionally, the third nucleic acid is an inducible promoter, preferably an inducible promoter pGAL and / or CaMV35S, Optionally, the first essential gene is the same as or different from the second essential gene.

19. The method according to claim 18, characterized in that The method further comprises: determining the second culture condition according to the type of the inducible promoter; and Based on the first culture condition and the second culture condition, the mutant library of the engineered organism is screened to obtain the engineered organism containment system, Optionally, the second culture condition is Gal and Glu.

20. The method according to any one of claims 17 to 19, characterized in that The method further comprises: using the nucleotide sequence encoding the first stop codon to replace the nucleotide sequence of one or more amino acids from the 4th to the 20th amino acids at the N-terminus of the protein encoded by the second essential gene of the engineered organism, so as to obtain a mutant library of the engineered organism.

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