Cells with environmentally regulated viability

Abstract

To provide a cell comprising viability controlled by an environment, comprising viability controlled by an environment for biological encapsulation or the like, and having high genetic stability of a circuit related to the same control.SOLUTION: There is provided a cell transformant, the cell transformant including: a system which may exhibit in a guiding state, a gene essential for survival of the cell. In a non-guiding condition of the cell expression, by leakage expression of the survival essential gene, viability is maintained. In a guiding condition of the cell expression, by overexpression of the survival essential gene, viability is suppressed, and the survival essential gene is expressed from only the expression guiding system.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to cells with environmentally controlled viability, for example, to transformants of biologically containable microorganisms that maintain viability under specific culture conditions but whose viability is suppressed outside those conditions (such as the natural environment). [Background technology]

[0002] Genetically modified microorganisms have a wide range of applications, and their importance is increasing year by year. For example, such microorganisms are used as a site for synthesizing various physiologically active substances (insulin, interferon, etc.) and antigens (vaccines), making significant contributions to the field of biopharmaceuticals.

[0003] On the other hand, if genetically modified microorganisms were to spread into the natural environment, there are concerns that they could have a negative impact on the environment. Therefore, there are several conditions that must be met before such microorganisms can be used, one of which is that they must be able to survive under specific culture conditions but be unable to survive outside of those conditions (such as the natural environment) (biological containment).

[0004] A classic method of biological containment involves disrupting the genes responsible for synthesizing essential nutrients that are not abundant in the natural environment, rendering the microorganism auxotrophic; under human care, the microorganism can survive because the nutrients are artificially provided, but in a natural environment, the microorganism dies due to lack of nutrients (Non-Patent Document 1). However, in most cases attempted to date, environmental niches exist where such nutrients are available, resulting in survival, and this method is considered to be incomplete.

[0005] Another method involves introducing an artificial genetic circuit and genetically programming it so that it dies in a natural environment (Non-Patent Document 1). For example, a genetic circuit that constitutively expresses a toxin is introduced, and an inducer is added to artificially express an antitoxin that neutralizes the toxin, allowing the organism to survive under human care. However, in a natural environment, the expression of the antitoxin ceases, the organism becomes toxic, and the organism dies. However, artificial genetic circuits are genetically unstable. That is, the circuit can be damaged by genetic mutations and easily disabled by subculture. Recently, a method has been reported to reduce the genetic instability of these biological containment circuits by introducing a genetic circuit that expresses a toxin under the control of a low-temperature-inducible promoter and a genetic circuit that constitutively expresses the antitoxin (Non-Patent Document 2). However, this method does not prevent the circuit from being damaged or removed, and is still insufficient. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Ramos, HJO et al., Soil Biol. Biochem., 2011, Vol. 43, No. 8, pp. 1626-1638 [Non-patent document 2] Stirling, F. et al., Mol. Cell, 2017, Vol. 68, No. 4, pp. 686-697 [Non-patent document 3] Kitagawa, M. et al., DNA Res., 2005, Vol. 12, pp. 291-299 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention has been made in consideration of the problems associated with the prior art, and aims to provide cells that can be used for biological containment, etc., that have environmentally controlled viability, and that have high genetic stability in the circuits involved in said control. [Means for solving the problem]

[0008] As a result of extensive research to achieve the above-mentioned objective, the inventors conceived of utilizing genes essential for the survival of cells (Escherichia coli), which had previously been comprehensively identified (Non-Patent Document 3), in a biological containment circuit.

[0009] Specifically, although these genes are normally essential for survival (Fig. 1A), if their overexpression conversely results in toxicity, we hypothesized that they could be used for biological containment by expressing these opposing properties related to survival in an environment-dependent manner (Fig. 1C).

[0010] On the other hand, although cell viability is lost by deleting the endogenous gene, we also considered that viability could be complemented by leakage and expression of an exogenous gene essential for survival from the biological containment circuit, as shown in Figure 1B. By adopting such a configuration, even if the circuit is damaged by genetic mutation or other factors, the cell will no longer be able to survive, and it is therefore anticipated that it will be possible to construct a containment circuit that is genetically maintained in a stable manner.

[0011] Therefore, we first overexpressed each of the essential survival genes that we had comprehensively identified in E. coli and evaluated their colony-forming ability. As a result, we were able to select 105 genes, including tyrS, that were essential for survival at normal expression levels but were toxic when overexpressed.

[0012] Furthermore, when a plasmid containing an exogenous tyrS gene that can be expressed under the control of an inducible promoter was introduced into E. coli and the cells were subjected to repeated dilution culture, the plasmid was completely lost after six dilution cultures. On the other hand, when the plasmid was introduced into E. coli cells lacking endogenous tyrS and the cells were subjected to repeated dilution cultures, the plasmid was completely retained even after 12 dilution cultures. In other words, the researchers demonstrated that a biological containment circuit capable of inducibly expressing an essential survival gene that exerts toxicity when overexpressed is genetically robust and stable in cells lacking the endogenous gene, leading to the completion of the present invention.

[0013] Thus, the present invention provides the following: <1> A transformant of a cell, a system capable of inducibly expressing a gene essential for the survival of the cell, Under non-inducible conditions of gene expression, viability is maintained by leaky expression of the survival-essential gene; Under conditions for inducing gene expression, overexpression of the survival-essential gene suppresses viability; and A transformant in which the survival-essential gene is expressed only from the expression induction system. <2> The survival-essential gene is at least one gene selected from the following gene group: <1> The transformant according to Gene cluster: tyrS, dnaX, ftsK, mrdA, mrdB, mukB, rpsD, lexA, pheT, prfA, lepB, polA, leuS, ftsI, yaeL, secA, parC, ftsQ, rplP, dnaC, mviN, lolE, yejM, dnaA, rplD, rpoD , ftsH, secY, hemH, rpoH, lgt, ftsL, rplS, ribF, rpsB, rpsC, ftsX, yhbN, ygjE, ileS, rplB, rpsA, lpxB, pgsA, tilS, msbA, ftsW, rpsS, degS, infB, mreC, trmU, yfiO, dnaB, lpxC, zipA, yjgP, yrbK, lspA, murG, yrfF, proS, lolA, mreD, plsB, dxs, ssb, frr, mraY, rho, accB, argS, ftsE, secD, yidC, grpE, psd, ftsY, groL , lnt, rplQ, secE, hemA, rpsM, accC, plsC, fmt, ubiA, ftsB, nusG, secF, rlpB, rpoC, rpsG, glyS, rpsH, rplN, dnaE, holB, rpsR, rne, hemG, kdtA, rplJ, and mreB. <3> Further comprising a DNA construct for expressing a desired gene, <1> or <2> The transformant described in <4> the cell is a microorganism; <1> ~ <3> The transformant according to any one of the above. [Effects of the Invention]

[0014] According to the present invention, it is possible to provide cells having a genetic circuit with strong genetic stability, for example, cells having a biological containment circuit with strong genetic stability, and a biological containment method using the cells. [Brief explanation of the drawings]

[0015] [Figure 1A] 1 is a diagram for explaining an outline of one embodiment of the present invention, which shows that in a cell (wild-type), viability is maintained by expression of an endogenous gene (e.g., tyrS) that is essential for survival. [Figure 1B] 1 is a diagram showing an overview of one embodiment of the present invention. As shown in the diagram, in the cells (transformants) of the present invention, the function of an (endogenous) survival-essential gene on the genome is suppressed, but under non-inducible conditions of an inducible promoter (e.g., a T5 promoter / lac operator (lacI) system), the viability is maintained by leaky expression of an (exogenous) survival-essential gene in an introduced DNA construct (e.g., a plasmid). [Figure 1C] 1 is a diagram showing an overview of one embodiment of the present invention, and as shown in the diagram, under conditions for inducing the promoter (e.g., in the presence of IPTG), the exogenous survival-essential gene is overexpressed, resulting in toxicity and suppressing the viability of the cells of the present invention. [Figure 2]This photograph shows the effectiveness of a biological containment circuit (ASKA-tyrS) using an essential survival gene (tyrS) that can be overexpressed under the control of an inducible promoter. Under non-inducing conditions (in the absence of IPTG), E. coli (ΔtyrS) that retains the circuit but lacks endogenous tyrS maintains viability, grows normally, and forms colonies (left side of the figure). On the other hand, under inducing conditions (in the presence of IPTG), the essential survival gene is overexpressed, causing toxicity and no detectable colony formation (right side of the figure). [Figure 3] This graph shows the results of repeatedly diluting and culturing E. coli that contains the biological containment circuit (ASKA-tyrS) but lacks endogenous tyrS (denoted "ΔtyrS" in the figure) and E. coli that contains the circuit but does not lack endogenous tyrS (denoted "wt" in the figure), and analyzing the maintenance rate of the circuit (plasmid). This shows that a biological containment circuit that can inducibly express a survival-essential gene that exerts toxicity when overexpressed is genetically robustly stable in microorganisms lacking the endogenous gene. DETAILED DESCRIPTION OF THE INVENTION

[0016] As shown in the Examples below, the inventors selected genes that are essential for cell survival when expressed at normal levels, but whose overexpression suppresses cell viability. They then found that biological containment is possible by controlling the expression of such genes according to the environment. Furthermore, the inventors demonstrated that suppressing the function of the endogenous genes results in a loss of cell viability, but that viability can be complemented by the leakage and expression of exogenous genes essential for cell survival from the biological containment circuit. In other words, they demonstrated that the biological containment circuit is genetically robust and stable.

[0017] Therefore, the present invention provides A transformant of a cell, a system capable of inducibly expressing a gene essential for the survival of the cell, Under non-inducible conditions of gene expression, viability is maintained by leaky expression of the survival-essential gene; Under conditions for inducing gene expression, overexpression of the survival-essential gene suppresses viability; and The present invention provides a transformant in which the essential survival gene is expressed only from the expression induction system.

[0018] <cell> The "cells" of the present invention may be prokaryotic or eukaryotic cells, including, for example, microorganisms, plant cells, insect cells, and animal cells. The "microorganisms" of the present invention may be, for example, prokaryotes (eubacteria, archaea) or eukaryotes (algae, protists, fungi, and slime molds). More specifically, prokaryotes include bacteria of the genus Escherichia, such as Escherichia coli; Bacillus, such as Bacillus subtilis; Pseudomonas, such as Pseudomonas putida; and Rhizobium, such as Rhizobium meliloti. Furthermore, eukaryotes include yeasts, such as Saccharomyces cerevisiae and Schizosaccharomyces pombe.

[0019] <Essential genes for survival> In the present invention, "a gene essential for survival (a gene essential for survival)" means a gene whose cell viability is suppressed not only when the function of the gene is suppressed, but also when the gene is overexpressed.

[0020] Here, "inhibition of viability" means killing the cells and / or inhibiting cell proliferation. For example, the cell proliferation rate can be reduced to at least one-tenth, preferably at least one-hundredth, and more preferably at least one-thousandth of that of a normal cell (e.g., a microorganism before the suppression of a gene essential for survival, a cell before the overexpression of the gene, or a cell (parent strain) before transformation). Also, the cell death rate can be reduced to at least 10 times, preferably at least 100 times, and more preferably at least 1000 times that of a normal cell.

[0021] On the other hand, "maintaining viability" means, for example, that the cell proliferation rate or survival rate is 0.2 times or more, preferably 0.5 times or more, and more preferably 0.8 times or more compared to normal. It also means that the cell death rate is equivalent to normal (for example, 0.8 to 1.2 times the normal death rate).

[0022] More specific examples of the "essential survival genes" according to the present invention include the genes shown in Tables 1 to 5 below, which were selected in the Examples described below.

[0023] [Table 1]

[0024] [Table 2]

[0025] [Table 3]

[0026] [Table 4]

[0027] [Table 5]

[0028] In Tables 1 to 5, these genes derived from Escherichia coli (wild-type) and the proteins they encode are identified by specific sequences (sequences defined by UniProt IDs), but the survival-essential genes of the present invention are not limited to those identified by these typical sequences.

[0029] Furthermore, mutations in nucleotide sequences can occur in nature, and the amino acids they encode can also change accordingly. Therefore, essential survival genes according to the present invention also include genes that encode proteins (variants) consisting of amino acid sequences in which one or more amino acids have been substituted, deleted, added, and / or inserted in the typical wild-type amino acid sequence derived from E. coli.

[0030] With regard to the survival-essential genes of the present invention, the term "multiple" is not particularly limited, but may refer to, for example, within 300 amino acids (within 250 amino acids, within 200 amino acids, etc.), within 150 amino acids (within 140 amino acids, within 130 amino acids, within 120 amino acids, within 110 amino acids, etc.), within 100 amino acids (within 90 amino acids, within 80 amino acids, within 70 amino acids, within 60 amino acids, etc.), within 50 amino acids (within 40 amino acids, within 30 amino acids, within 20 amino acids, etc.), within 10 amino acids (within 9 amino acids, within 8 amino acids, within 7 amino acids, within 6 amino acids, etc.), or within several amino acids (within 5 amino acids, within 4 amino acids, within 3 amino acids, within 2 amino acids).

[0031] Furthermore, with the current state of the art, those skilled in the art can use the genetic information (e.g., nucleotide sequence) to identify homologous genes from the same or other cells. Methods for identifying homologous genes include, for example, hybridization techniques (Southern, EM, J. Mol. Biol., 98:503, 1975) and polymerase chain reaction (PCR) techniques (Saiki, RK, et al. Science, 230:1350-1354, 1985; Saiki, RK, et al. Science, 239:487-491, 1988). To identify homologous genes, hybridization reactions are typically performed under stringent conditions. Examples of stringent hybridization conditions include 6 M urea, 0.4% SDS, and 0.5x SSC, or hybridization conditions of equivalent stringency. If more stringent conditions are used, for example, 6 M urea, 0.4% SDS, and 0.1×SSC, it is expected that genes with higher homology will be isolated.

[0032] Therefore, the survival-essential gene according to the present invention also includes a protein encoded by a DNA that hybridizes under stringent conditions with the DNA consisting of the typical wild-type nucleotide sequence derived from E. coli.

[0033] Furthermore, the protein encoded by the identified homologous gene usually has high homology (high similarity), preferably high identity, with that of the bacterium. Here, "high" means at least 30% or more, preferably 40% or more, more preferably 50% or more, even more preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, and even more preferably 85% or more (e.g., 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more).

[0034] Therefore, the survival-essential genes of the present invention also include genes encoding proteins consisting of amino acid sequences that have at least 30% homology (similarity) or identity to the typical wild-type amino acid sequence derived from E. coli.

[0035] Sequence homology can be determined using the BLAST program (Altschul et al. J. Mol. Biol., 215:403-410, 1990). This program is based on the BLAST algorithm by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87:2264-2268, 1990; Proc. Natl. Acad. Sci. USA, 90:5873-5877, 1993). For example, when analyzing an amino acid sequence using BLAST, the parameters are, for example, score = 50 and word length = 3. When analyzing an amino acid sequence using the Gapped BLAST program, the procedure can be as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific techniques for these analysis methods are known.

[0036] Furthermore, the present invention may target one type of essential survival gene, but may target multiple types from the viewpoint of performing biological containment, etc. with greater precision and further increasing the genetic stability of the genetic circuit used for said containment, etc. (such as the expression induction system and DNA construct according to the present invention described below). Here, the multiple types are not particularly limited, and examples include two or more types (for example, three, four, five, six, seven, eight, or nine types), and ten or more types.

[0037] <Expression induction system> In the present invention, a "system capable of inducibly expressing a gene essential for survival (expression induction system)" refers to a system capable of inducing the expression of a gene essential for survival under specific conditions. The "gene expression" controlled here may be at the transcription level, the post-transcription level (such as the translation level), or both.

[0038] As will be shown in the Examples below, suppressing the expression of an endogenous essential survival gene results in a loss of cell viability, but viability is complemented by leakage expression of the essential survival gene from the expression induction system of the present invention. Therefore, the expression induction system is genetically highly stable. In other words, to achieve such high genetic stability, the essential survival gene must be expressed only from the expression induction system in the transformant of the present invention.

[0039] Furthermore, the expression induction system of the present invention is not particularly limited as long as it can induce the expression of a survival-essential gene, and examples include: [1] a system in which a survival-essential gene can be inducibly expressed under the control of an inducible promoter; [2] a system in which a survival-essential gene can be inducibly expressed under the control of a riboswitch; and [3] a combination of a system in which a molecule that suppresses the function of a survival-essential gene can be inducibly expressed under the control of an inducible promoter and a system in which a survival-essential gene can be constitutively expressed.

[0040] [1] A system in which essential genes for survival can be inducibly expressed under the control of an inducible promoter. More specifically, embodiments of the present invention that use a system in which a survival-essential gene can be inducibly expressed under the control of an inducible promoter include: A transformant of a cell, a DNA construct in which a gene essential for the survival of the cell is operably linked to an inducible promoter; The function of the endogenous essential survival gene is suppressed, Under non-inducing conditions of gene expression, viability is maintained by leaky expression of the essential survival gene; and A transformant in which viability is suppressed due to overexpression of the essential survival gene under conditions for inducing the expression of the gene. Examples include:

[0041] In another embodiment, a transformant of a cell, an endogenous promoter of a gene essential for the survival of the cell is replaced with an inducible promoter, and the inducible promoter and the essential survival gene are operably linked; Under non-inducing conditions of gene expression, viability is maintained by leaky expression of the essential survival gene; and A transformant in which viability is suppressed due to overexpression of the essential survival gene under conditions for inducing the expression of the gene. Other examples include:

[0042] <Inducible promoter> An "inducible promoter" according to the present invention refers to a control region that induces the expression (transcription) of a gene operably linked downstream thereof under predetermined conditions. Here, "predetermined conditions" include the presence or absence of compounds (sugars, amino acids, peptides, proteins, antibiotics, nutrients, metabolites, etc.), the presence or absence of metals, the presence or absence of light irradiation, high or low temperatures, and the presence or absence of gas (oxygen, etc.).

[0043] The "inducible promoter" capable of inducing expression according to such conditions is not particularly limited, and examples thereof include the lac promoter induced by IPTG, lactose, allolactose, etc., the promoter of the Tet-on system / Tet-off system induced in the presence or absence of tetracycline or its derivatives (doxycycline, etc.), the Trp promoter induced in the absence of tryptophan, the Tac promoter consisting of a combination of the lac promoter and the Trp promoter, the GAL1 promoter and the GAL10 promoter induced by galactose, etc., the araBAD promoter induced by arabinose, the CUP1 promoter induced by copper ions, the promoters of genes encoding heat shock proteins (HSPs) such as HSP10, HSP60, and HSP90 induced at high temperatures, the cspA promoter induced at low temperatures, and the temperature-sensitive promoter P RPromoter and P L Examples of promoters include the promoter of the ribulose bisphosphate carboxylase small subunit (rbcS) gene, which is a light-inducible promoter.

[0044] Furthermore, some of these inducible promoters have an operator sequence, such as the lac promoter and promoters of the Tet-on system / Tet-off system. Under non-inducing conditions, expression is suppressed by binding of a repressor to this operator sequence, but under inducing conditions, the repressor dissociates from the operator sequence, allowing expression. Therefore, the "inducible promoter" of the present invention also includes a constitutive promoter equipped with an operator sequence. Such operator sequences are not particularly limited, but examples include the lac operator and the Tet operator. For more information on "constitutive promoters," see below.

[0045] By using such an "inducible promoter," the system overexpresses the essential survival gene under inducing conditions, suppressing cell viability, while leaking expression (leaking transcription) of the essential survival gene to the extent that cell viability is maintained under non-inducing conditions.

[0046] [2] A system in which essential genes for survival can be inducibly expressed under the control of a riboswitch Next, an embodiment of the present invention will be described, in which a system in which essential survival genes can be inducibly expressed under the control of a riboswitch is used as an expression induction system. More specifically, such an embodiment includes: A transformant of a cell, The gene essential for the survival of the cell is operably linked to a constitutive promoter and includes a DNA construct capable of being inducibly expressed under the control of a riboswitch; The function of the endogenous essential survival gene is suppressed, Under non-inducing conditions of gene expression, viability is maintained by leaky expression of the essential survival gene; and A transformant in which viability is suppressed due to overexpression of the essential survival gene under conditions for inducing the expression of the gene. Examples include:

[0047] In another aspect, A transformant of a cell, The endogenous promoter of a gene essential for the survival of the cell is replaced with a constitutive promoter, and the essential gene for survival can be inducibly expressed under the control of a riboswitch; Under non-inducing conditions of gene expression, viability is maintained by leaky expression of the essential survival gene; and A transformant in which viability is suppressed due to overexpression of the essential survival gene under conditions for inducing the expression of the gene. Other examples include:

[0048] <Riboswitch> A "riboswitch" according to the present invention refers to an RNA that selectively binds to a specific compound. In the absence of the compound, a secondary structure is formed by RNA base pairing. If a ribosome binding site is present downstream of the riboswitch, the riboswitch inhibits ribosome binding to the site, thereby preventing translation of the mRNA of a gene located further downstream (in the present invention, an essential gene for survival). On the other hand, in the presence of the compound, the secondary structure associated with the binding of the compound is dissolved, allowing the ribosome to bind to the ribosome binding site. Therefore, in the presence of the compound, the mRNA of the gene is translated, and expression of the gene can be induced.

[0049] Furthermore, some riboswitches promote translation upon binding of such specific compounds (translation-promoting, ON switches), while others conversely inhibit the binding of a specific compound to a riboswitch by inhibiting the binding of the downstream ribosome-binding site to the ribosome, thereby preventing the translation of mRNA of a gene located further downstream (translation-repressing, OFF switches). In the present invention, both translation-promoting and translation-repressing riboswitches are suitable for use.

[0050] Furthermore, there are no particular limitations on the "compound" that induces translation of a downstream gene by binding or not binding to the riboswitch of the present invention, and examples include sugars, amino acids, peptides, proteins, antibiotics, nutrients, etc. Furthermore, the "riboswitch" of the present invention may be any RNA that undergoes a secondary structural change depending on the presence or absence of the binding of the aforementioned compound, and is not particularly limited. Examples include, but are not limited to, a theophylline-responsive riboswitch, an adenosylcobalamin-responsive riboswitch, a cyclic di-GMP-responsive riboswitch, a flavin mononucleotide-responsive riboswitch, a glucosamine-6-phosphate-responsive riboswitch, a glutamine-responsive riboswitch, a glycine-responsive riboswitch, a lysine-responsive riboswitch, a preQ1-responsive riboswitch, a purine-responsive riboswitch, an S-adenosylhomocysteine-responsive riboswitch, an S-adenosylmethionine-responsive riboswitch, an S-adenosylhomocysteine ​​& S-adenosylmethionine-responsive riboswitch, a tetrahydrofolate-responsive riboswitch, a thiamine pyrophosphate-responsive riboswitch, a molybdenum-responsive riboswitch, and an adenine-responsive riboswitch.

[0051] In this system, under conditions where the "riboswitch" represses the translation of mRNA of an essential survival gene that is overtranscribed by a constitutive promoter (described below) (the non-induction conditions of the gene expression), cell viability is maintained by the protein encoded by the leaky expressed (leaky translated) essential survival gene. On the other hand, under conditions where the riboswitch promotes translation (the induction conditions of the gene expression), the mRNA of the essential survival gene that is overtranscribed by a constitutive promoter (described below) is translated without being repressed, thereby repressing cell viability.

[0052] [3] A combination of a system in which a molecule that suppresses the function of a survival-essential gene can be inducibly expressed under the control of an inducible promoter and a system in which a survival-essential gene can be constitutively expressed. Next, we will explain an embodiment of the present invention that uses a combination of an expression induction system that can inducibly express a molecule that suppresses the function of a survival-essential gene under the control of an inducible promoter and a system that can constitutively express the survival-essential gene. More specifically, such an embodiment can be mentioned as follows.

[0053] A transformant of a cell, a system capable of inducibly expressing a molecule that suppresses the function of a gene essential for the survival of the cell, and a system capable of constitutively expressing the gene essential for the survival of the cell, Under conditions of induction of expression of said molecule, viability is maintained by leaky expression of said survival-essential gene; and A transformant in which viability is suppressed due to overexpression of the essential gene for survival under non-inducible conditions for the expression of the molecule. Other examples include:

[0054] In the present invention, the "molecule that suppresses the function of a gene essential for survival" may be any molecule that targets the gene and has the activity of suppressing its function (transcription, translation), etc., as described below, and examples thereof include small RNA (sRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), antisense RNA, and RNA with ribozyme activity, as described below. Further examples include genome editing systems containing site-specific nucleases and / or guide RNAs, as described below.

[0055] Furthermore, the "system capable of constitutively expressing a survival-essential gene" is not particularly limited and may be, for example, a DNA construct containing the survival-essential gene operably linked to a constitutive promoter described below. Alternatively, the system may be an endogenous survival-essential gene in which the endogenous promoter has been replaced with a constitutive promoter described below.

[0056] In addition, when the DNA construct is present, unlike the systems [1] and [2] above, it is not necessary to suppress the function of the endogenous essential survival gene in order to express the essential survival gene only from the expression induction system of the present invention. However, from the viewpoint of further enhancing genetic stability, it is desirable to suppress the function.

[0057] In this system, the expression of the "molecule that suppresses the function of the essential survival gene" is induced by the inducible promoter described above, and under conditions where the expression of the essential survival gene is suppressed (conditions where the gene expression is not induced), the cell viability is maintained by the leaked expression of the essential survival gene. On the other hand, under conditions where the expression of the molecule is not induced and the essential survival gene is overexpressed (conditions where the gene expression is induced), the cell viability is suppressed.

[0058] Overexpression and leaky expression In the present invention, "overexpression" means that the expression level (mRNA level and / or protein level) of a survival-essential gene of the present invention induced by the expression induction system of the present invention is greater than the expression level induced by its endogenous promoter, and is so high that it can suppress the viability of a cell transformant of the present invention. The degree of expression depends on the type of survival-essential gene being induced, but is usually 5-fold or more, more preferably 10-fold or more, even more preferably 20-fold or more, more preferably 50-fold or more, even more preferably 100-fold or more, more preferably 200-fold or more, even more preferably 500-fold or more, and more preferably 1000-fold or more, compared to the expression level of a survival-essential gene of the present invention induced by its endogenous promoter.

[0059] In addition, "leakage expression" means that the expression level of the essential gene for survival according to the present invention is equivalent to that induced by its endogenous promoter, and is an expression level that can maintain the viability of the transformant of the cell of the present invention. The degree of such expression depends on the type of essential gene for survival to be induced, etc. Compared with the expression level of the essential gene for survival according to the present invention induced by its endogenous promoter, it is usually 0.2 to 3 times, preferably 0.5 to 2 times, more preferably 0.8 to 1.5 times. The expression intensity by such a promoter or the like can be evaluated by those skilled in the art using known methods (for example, the methods described in Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1995, Vol. 1, pp. 105-128).

[0060] <constitutive promoter> The "constitutive promoter" of the present invention that enables such overexpression may be any control region that constitutively and strongly induces the expression (transcription) of an essential gene for survival operably linked thereto, and there is no particular limitation. Examples of promoters available in prokaryotic cells such as Escherichia coli include the T5 promoter, T3 promoter, and T7 promoter. Examples of constitutive promoters available in eukaryotic cells such as yeast include the ADH1 promoter, PGK1 promoter, ENO promoter, and PYK1 promoter.

[0061] <DNA construct> In the present invention, the "DNA construct" introduced into a cell as a biological containment circuit or the like is not particularly limited as long as it can induce the expression of an essential gene for survival or the like in the cell. For example, a self-replicating vector, that is, a plasmid that exists as an extrachromosomal independent entity and whose replication does not depend on chromosomal replication can be constructed. Further, the vector may be one that is integrated into the genome of the cell when introduced into the cell and is replicated together with the chromosome into which it is integrated.

[0062] Examples of such vectors include plasmids and phage DNA. Examples of plasmids include Escherichia coli-derived plasmids (pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, pCA24N, etc.), yeast-derived plasmids (YEp13, YEp24, YCp50, etc.), and Bacillus subtilis-derived plasmids (pUB110, pTP5, etc.). Examples of phage DNA include λ phage (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.). Procedures and methods commonly used in the field of genetic engineering can be used for constructing such vectors. For example, to insert the essential survival gene of the present invention into a vector, a method may be used in which purified DNA (essential survival gene) is first cleaved with an appropriate restriction enzyme, inserted into a restriction enzyme site or multicloning site of an appropriate vector, and then ligated to the vector.

[0063] The "DNA construct" of the present invention may, if necessary, contain an expression-controlling sequence other than an inducible promoter, such as a terminator sequence, enhancer sequence, splicing signal sequence, poly A addition signal sequence, or ribosome binding sequence (SD sequence), which can be utilized in the cells into which it is to be introduced.

[0064] Furthermore, the "DNA construct" according to the present invention may contain a selection marker gene, if necessary. The selection marker gene may be appropriately selected depending on the method for selecting transformed cells, and examples thereof include drug resistance genes such as a kanamycin resistance gene, a chloramphenicol resistance gene, an ampicillin resistance gene, a tetracycline resistance gene, and a neomycin resistance gene, genes involved in the intracellular biosynthesis of nutrients such as amino acids and nucleic acids (genes that complement auxotrophy), genes related to chemiluminescence such as luciferase, and genes encoding fluorescent proteins such as GFP.

[0065] The "essential survival gene" contained in the DNA construct may have DNA encoding another protein added thereto so that the protein encoded by the essential survival gene is expressed as a fused protein. The site of addition is not particularly limited; it may be either the 5'-end (the N-terminus of the protein encoded by the essential survival gene) or the 3'-end (the C-terminus of the protein encoded by the essential survival gene), or both. However, the DNA encoding the essential survival gene and the other protein must be added in the same reading frame. The "other protein" fused in this manner is also not particularly limited, and examples include purification tag proteins such as polyhistidine (His) tag proteins, FLAG-tag proteins (registered trademark, Sigma-Aldrich), and glutathione S-transferase (GST); detection tag proteins such as fluorescent proteins such as GFP and chemiluminescent proteins such as luciferase.

[0066] When the present invention targets multiple essential survival genes, each essential survival gene may be inserted into a DNA construct, or multiple essential survival genes may be inserted into a single vector. When multiple essential survival genes are inserted into a single vector, these essential survival genes preferably form an operon. Here, an "operon" is a nucleic acid sequence unit consisting of one or more genes transcribed under the control of the same promoter.

[0067] The DNA construct of the present invention is introduced into cells using a known method, which can be appropriately selected by those skilled in the art depending on the type of cell, etc. Examples of such an introduction method include the calcium chloride method, heat shock method, calcium phosphate method, electroporation method, spheroplast method, lithium acetate method, conjugative transfer method, and method using calcium ions.

[0068] <Suppression of essential genes for survival> As will be shown in the Examples below, from the viewpoint of enabling biological containment with high genetic stability, it is desirable that the cell transformant of the present invention contains the above-mentioned DNA construct while suppressing the function of an endogenous gene essential for survival.

[0069] In the present invention, "suppression of the function of an essential gene for survival" includes both complete suppression (inhibition) and partial suppression of the function. It also includes suppression of the expression of an essential gene for survival, as well as suppression of the activity of the protein encoded by the gene. Such suppression can be achieved, for example, by introducing a mutation into the coding region, non-coding region, expression control region (endogenous promoter region), etc. of the essential gene for survival.

[0070] In the present invention, the mutations introduced into essential survival genes are not particularly limited as long as they suppress the function of the gene, and examples include nucleotide substitutions, deletions, additions, and / or insertions, with nonsense mutations, frameshift mutations, and null mutations being preferred. Furthermore, the number of mutations introduced into essential survival genes is also not particularly limited as long as they suppress the function of the gene, and may be one or multiple (e.g., 2, 3 or less, 5 or less, 10 or less, 20 or less, 30 or less, 40 or less, 50 or less).

[0071] Such a mutation does not necessarily result in the loss of the entire amino acid sequence encoded by the survival-essential gene; it is sufficient to introduce a mutation into the gene so that only a portion of the amino acid sequence is lost or altered. For example, a nucleotide mutation may be introduced that results in the alteration or deletion of at least 10% (preferably 20% or more, more preferably 30% or more, even more preferably 50% or more, more preferably 70% or more, even more preferably 80% or more, more preferably 90% or more, and particularly preferably 100%) of the amino acid sequence encoded by the survival-essential gene.

[0072] There are no particular limitations on the region in which the amino acid sequence is changed or deleted in this way, as long as the function is suppressed. For example, a person skilled in the art can identify functional domains in the amino acid sequence encoded by a gene essential for survival based on information from Uniprot, etc., and can then introduce mutations targeting the nucleotide sequence encoding that domain.

[0073] Those skilled in the art can introduce mutations into essential genes by known mutagenesis methods, including, but not limited to, genome editing, physical mutagenesis, chemical mutagens, transposon-based genomic DNA introduction, and transcriptional targeting using sRNA, siRNA, shRNA, antisense RNA, and RNA with ribozyme activity.

[0074] Genome editing is a method for modifying target genes using site-specific nucleases (e.g., zinc finger nucleases (ZFNs), transcription activation-like effector nucleases (TALENs), DNA double-strand break enzymes such as CRISPR-Cas9). For example, fusion proteins such as ZFNs (U.S. Patent Nos. 6,265,196, 8,524,500, 7,888,121, European Patent No. 1,720,995), TALENs (U.S. Patent Nos. 8,470,973, 8,586,363), and nuclease domain-fused PPR (pentatricopeptide repeat) (Nakamura et al., Plant Cell Physiol 53:1171-1179 (2012)), CRISPR-Cas9 (U.S. Patent No. 8,697,359, International Publication No. 2013 / 176772), CRISPR-Cpf1 (Zetsche B. et al., Cell, 163(3):759-71, (2015)), and Target-AID (K. Nishida et al., Targeted nucleotide editing using hybrid prokaryotic and Examples include methods that use guide RNA and protein complexes such as those described in "Vertebrate Adaptive Immune Systems, Science, DOI:10.1126 / science.aaf8729,(2016)."

[0075] Physical mutagenesis methods include, for example, heavy ion beam (HIB) irradiation, fast neutron irradiation, gamma ray irradiation, and ultraviolet irradiation (see Hayashi et al., Cyclotrons and Their Applications, 2007, 18th International Conference, pp. 237-239, and Kazama et al., Plant Biotechnology, 2008, Vol. 25, pp. 113-117).

[0076] Examples of methods using chemical mutagens include treatment with chemical mutagens (see, for example, Zwar and Chandler, Planta, 1995, vol. 197, pp. 39-48). Chemical mutagens are not particularly limited, but include ethyl methanesulfonate (EMS), N-ethyl-N-nitrosourea (ENU), N-methyl-N-nitrosourea (MNU), sodium azide, sodium bisulfite, hydroxylamine, N-methyl-N'-nitro-N-nitroguanidine (MNNG), N-methyl-N'-nitrosoguanidine (NTG), O-methylhydroxylamine, nitrous acid, formic acid, and nucleotide analogs.

[0077] Methods for introducing transposons or the like into genomic DNA include, for example, the method described in H. Mori et al., Research in Microbiology, 2000, Vol. 151, No. 2, pp. 121-128, and the method described in S.Y. Gerdes et al., J. Bacteriol., 2003, Vol. 185, No. 19, pp. 5673-5684.

[0078] For cells into which a mutation has been introduced by the above-mentioned methods, the introduction of a mutation into the essential survival gene can be confirmed by known methods. Examples of such known methods include DNA sequencing (next-generation sequencing, etc.), PCR, microarray analysis, Southern blotting, and Northern blotting. Using these methods, it is possible to determine whether a mutation has been introduced into the essential survival gene by comparing the sequence or length of the gene before and after the mutation. Furthermore, by using Northern blotting, RT-PCR, Western blotting, ELISA, microarray analysis, etc., if a decrease in the expression level of the transcription product or translation product of the essential survival gene is observed in a cell into which a mutation has been introduced, the cell can be confirmed as a cell into which a mutation has been introduced into the essential survival gene.

[0079] Another method for confirming that a mutation has been introduced into an essential gene for survival is TILLING (Targeting Induced Local Lesions IN Genomes) (see Slade et al., Transgenic Res., 2005, Vol. 14, pp. 109-115, and Comai et al., Plant J., 2004, Vol. 37, pp. 778-786). In particular, when a non-selective mutation is introduced into the genome using the aforementioned heavy ion beam irradiation or chemical mutagen, an essential gene for survival or a portion thereof can be amplified by PCR, and individuals having a mutation in the amplification product can be selected by the aforementioned TILLING or the like.

[0080] Furthermore, as shown in the Examples below, the function of a gene essential for survival can also be suppressed by homologous recombination, which can be performed using, for example, the lambda Red recombination system (T. Baba et al., Mol. Syst. Biol., 2006, 2, 20060008), Cre / Lox, attB / attP, or other integrase systems.

[0081] In the present invention, since the gene whose function is to be suppressed is essential for survival, it is preferable that the function be suppressed in response to a predetermined condition. For example, the above-mentioned inducible promoter can be used for such condition-dependent suppression.

[0082] Furthermore, the transformant of the cell of the present invention has high genetic stability and can be subjected to biological containment, etc., and therefore can be used as a site for synthesizing various useful substances. That is, the transformant of the present invention may further contain a DNA construct for expressing a desired gene.

[0083] In the present invention, the "desired gene" is not particularly limited as long as it is a gene that encodes a protein or RNA that is desired to be expressed and produced in a transformant of the cell of the present invention, and examples thereof include genes that encode physiologically active substances such as insulin and interferon, and antigens (proteins, RNAs, etc.) that can be used in vaccines. Furthermore, the transformant of the present invention may contain only one type of desired gene, or may contain multiple types of desired genes.

[0084] A "DNA construct" for expressing a desired gene can be selected and prepared by a person skilled in the art from known sources, as appropriate, in the same way as for expressing the above-mentioned essential survival genes. The promoter in the DNA construct may be an inducible promoter or a constitutive promoter.

[0085] A "DNA construct" for expressing desired genes may contain each desired gene inserted individually, or multiple genes may be inserted into one DNA construct. When multiple desired genes are inserted into a vector, it is preferable that these genes form an operon when multiple genes are inserted into one DNA construct. Furthermore, the desired gene and DNA encoding the above-mentioned essential survival gene or a molecule that suppresses the function of the gene may be contained in the same DNA construct, or they may form an operon. [Example]

[0086] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples.

[0087] Example 1: Identification of essential genes whose overexpression exerts toxicity We targeted the open reading frames (ORFs) of all essential genes (300 genes) derived from the E. coli W3110 strain (Non-patent Document 3) and attempted to identify genes that exert toxicity when overexpressed.

[0088] Specifically, first, polynucleotides (6xHis fusion ORFs) encoding fusion proteins in which an epitope tag consisting of six consecutive histidine residues (6xHis) was added to the N-terminus of the protein encoded by each ORF were prepared.

[0089] Next, these 6xHis fusion ORFs were each cloned into the plasmid pCA24N for expression under the control of a strong T5 promoter. In this plasmid, a lacO sequence was inserted between the T5 promoter and the 6xHis fusion ORF. Therefore, expression of the 6xHis fusion ORF was suppressed by LacI, the product of the lacIq gene, which is constitutively expressed on the plasmid. However, in the presence of isopropyl β-D-1-thiogalactopyranoside (IPTG), overexpression of the 6xHis fusion ORF was expected, overcoming repression by LacI / lacO.

[0090] Next, the prepared plasmids were introduced into E. coli AG1 to establish transformants harboring the plasmids corresponding to the individual ORFs, and each transformant was cultured overnight in LB liquid medium containing chloramphenicol (50 μg / ml), a selection marker for the plasmid.

[0091] The culture medium was then 3 The culture was diluted 1:1, and 250 μL of the diluted solution was inoculated onto an LB agar medium containing IPTG (3 mM) and chloramphenicol (50 μg / ml), and the formed colonies were detected. 6 The mixture was diluted 2-fold and inoculated onto an agar medium containing only chloramphenicol, and the colonies formed were detected.

[0092] The number of bacterial individuals that formed colonies even when the ORF was overexpressed was calculated from the former colony count, and the total number of bacterial individuals inoculated was calculated from the latter colony count. From these two results, the incidence of bacterial individuals (escapers) that formed colonies even when the ORF was overexpressed was calculated.

[0093] As a result, although not shown in the figure, the following 105 genes were selected as ORFs with an escaper incidence rate of <2%, which are essential for the survival of E. coli but which exert toxicity when overexpressed. tyrS, dnaX, ftsK, mrdA, mrdB, mukB, rpsD, lexA, pheT, prfA, lepB, polA, leuS, ftsI, yaeL, secA, parC, ftsQ, rplP, dnaC, mviN, lolE, yejM, dnaA, rplD, rpoD , ftsH, secY, hemH, rpoH, lgt, ftsL, rplS, ribF, rpsB, rpsC, ftsX, yhbN, ygjE, ileS, rplB, rpsA, lpxB, pgsA, tilS, msbA, ftsW, rpsS, degS, infB, mreC, trmU , yfiO, dnaB, lpxC, zipA, yjgP, yrbK, lspA, murG, yrfF, proS, lolA, mreD, plsB, dxs, ssb, frr, mraY, rho, accB, argS, ftsE, secD, yidC, grpE, psd, ftsY, gro L, lnt, rplQ, secE, hemA, rpsM, accC, plsC, fmt, ubiA, ftsB, nusG, secF, rlpB, rpoC, rpsG, glyS, rpsH, rplN, dnaE, holB, rpsR, rne, hemG, kdtA, rplJ, mreB.

[0094] Example 2: Biocontainment with robust genetic stability Among the genes identified in Example 1 that exert toxicity when overexpressed, tyrS was selected to demonstrate a genetically stable biological containment method.

[0095] Specifically, the aforementioned plasmid (pCA24N-6xHis-fused tyrS) expressing the 6xHis-fused tyrS ORF was first introduced into E. coli BL21-AI to prepare transformants. The endogenous tyrS gene present in the genome of the transformants was then replaced with a kanamycin resistance gene using λred recombinase. This is expected to ensure that the TyrS gene essential for viability is supplied solely from the exogenous tyrS gene secreted from the plasmid.

[0096] In fact, when the transformants containing the pCA24N-6xHis-fused tyrS and kanamycin resistance gene prepared in this manner were cultured in the presence of IPTG, the expression of exogenous tyrS was induced and the E. coli cells died (right side of the figure), as shown in Figure 2. On the other hand, in the absence of IPTG, despite the lack of endogenous tyrS and the lack of induction of exogenous tyrS expression, the cells maintained viability and formed colonies due to the leaky expression of tyrS (left side of the figure).

[0097] Next, the transformant containing the pCA24N-6xHis fused tyrS and kanamycin resistance gene was cultured overnight in LB liquid medium containing kanamycin (25 μg / mL) and chloramphenicol (50 μg / mL). After washing twice with fresh LB liquid medium, it was cultured overnight in a medium containing only kanamycin. 3 The cells were diluted 1:1 and cultured for another 24 hours. 6 The resulting solution was diluted 1:1 and 250 μL was inoculated onto LB solid medium containing kanamycin (25 μg / mL). After overnight incubation, the resulting colonies were inoculated onto LB solid medium containing chloramphenicol (50 μg / mL). Chloramphenicol resistance was used as an indicator to check whether the plasmid was maintained. A portion of the solution was also taken and re-inoculated into 10 mL of LB solid medium. 3 The diluted culture and plasmid maintenance assay were repeated 12 times. One dilution culture corresponds to approximately 10 generations.

[0098] We also performed a similar experiment on the parent strain that retains tyrS in its genome, except that repeated dilution cultures were performed in LB liquid medium containing tetracycline (1.25 μg / mL as tetracycline hydrochloride), the selection marker for BL21-AI.

[0099] As a result, as shown in Figure 3, the pCA24N-6xHis fused tyrS was almost completely lost in the parent strain after six dilution cultures, but the test strain in which tyrS was deleted from the genome retained the plasmid even after 12 dilution cultures. [Industrial Applicability]

[0100] As described above, according to the present invention, it is possible to provide cells that can be used for biological containment, etc., that have environmentally controlled viability, and that have high genetic stability in the circuits used for such control (biological containment circuits, etc.).

[0101] Therefore, according to the present invention, even in an environment without physical containment as specified by biosafety level (BSL) or the like, it is possible to completely contain cell transformants, and therefore the present invention is useful in the production of biopharmaceuticals, etc.

Claims

1. A transformant of a cell, a system capable of inducibly expressing a gene essential for the survival of the cell, Under non-inducible conditions of gene expression, viability is maintained by leaky expression of the survival-essential gene; Under conditions for inducing gene expression, viability is suppressed due to overexpression of the survival-essential gene; the survival-essential gene is expressed only from the expression induction system; the cell is Escherichia coli that is deficient in the endogenous gene essential for survival, The survival essential gene is the tyrS gene, and The expression induction system is a system that can inducibly express the survival-essential gene under the control of a T5 promoter and a Lac operator. Transformants.

2. The transformant according to claim 1, further comprising a DNA construct for expressing a desired gene.

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