Specific inversion system as well as construction method and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a specific inversion system and a construction method and application thereof. According to the invention, a new tool capable of carrying out efficient multi-site specific inversion in eukaryotes is obtained through recombinase directed evolution and inversion site screening. Test results show that the inversion system can be used in eukaryotes, realizes high-efficiency multi-site inversion, solves the disadvantage of death caused by deletion, makes up the vacancy of the eukaryote multi-site inversion system, has wide application prospects, and provides a new tool for cell barcodes, genetic circuits and metabolic engineering.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a specific inversion system, a construction method and an application thereof. Background Art

[0002] The site-specific inversion system is a recombination system that can mediate the directional inversion of the DNA sequence between two specific inversion sites. This inversion process is mediated by a specific inversion enzyme. Generally, site-specific inversion systems can form different degrees of complexity. The simplest system contains a pair of inversion sites and inversion enzymes, which can produce two genetic phenotypes, such as the Hin (H inversion) system, the Gin (G inversion) system, and the Cin (C inversion) system. More complex systems involve multiple pairs of inversion sites, which can produce many different genotypes, such as the Min system and the Shufflons system. However, so far, almost all known site-specific DNA inversion systems exist in bacteria, and no such inversion system has been identified in eukaryotes.

[0003] A similar research work is the construction of a single-pair site-specific inversion system in Saccharomyces cerevisiae. Han et al. directed evolution of the bacterial Rci recombinase in Saccharomyces cerevisiae, screened out the enzyme Rci8 with a very high ratio of inversion efficiency to deletion efficiency, and constructed a single-pair site inversion system Rci8 / sfxa101 in yeast. The system contains a pair of sfxa101 sites and the recombinase Rci8. In practical applications, by setting the sfxa101 site sequence in the opposite direction, only inversion can be mediated. However, genome editing often involves complex rearrangements. A single pair of inversion sites cannot undergo a large number of rearrangement events in a short period of time, which has limitations on the diversity of structural variations. In addition, in the yeast genome synthesis project (Sc2.0 project), the SCRaMbLE system will inevitably produce a large number of deletions of DNA between sites when mediating genome-scale rearrangements, and even cause strain lethality. Therefore, it is crucial to develop a stable, multi-site inversion and efficient transformation system. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a specific inversion system, a construction method and an application thereof.

[0005] The present invention provides a recombinase variant, which includes a recombinase with an amino acid sequence as shown in SEQ ID NO: 1, in which the alanine F at position 246 is mutated to valine V, the alanine at position 295 is mutated to serine S, and the alanine at position 330 is mutated to threonine T.

[0006] Furthermore, the recombinase variant includes amino acids such as those shown in SEQ ID NO: 1, in which the glutamine Q at position 30 is mutated to lysine K, the leucine L at position 109 is mutated to proline P, the alanine F at position 246 is mutated to valine V, the alanine A at position 295 is mutated to serine S and the alanine A at position 330 is mutated to threonine T.

[0007] The present invention screened and obtained the recombinase variants Rci51 and Rci51-5 in sequence through two rounds of directed evolution, wherein the multi-site reversal ability of the Rci51-5 mutant was greatly improved, and the problem of multi-site reversal causing lethality was avoided.

[0008] Furthermore,

[0009] The Rci51 is a recombinant enzyme with an amino acid sequence as shown in SEQ ID NO: 1, wherein the alanine F at position 246 is mutated to valine V, the alanine at position 295 is mutated to serine S, and the alanine at position 330 is mutated to threonine T;

[0010] The Rci51-5 is a recombinant enzyme with an amino acid sequence as shown in SEQ ID NO: 1, in which the glutamine Q at position 30 is mutated to lysine K, the leucine L at position 109 is mutated to proline P, the alanine F at position 246 is mutated to valine V, the alanine at position 295 is mutated to serine S, and the alanine at position 330 is mutated to threonine T.

[0011] The present invention provides an inversion system, which comprises the recombinase variant of the present invention and an inversion site;

[0012] The inversion site includes at least one of sfxa101, sfxa102, sfxa107, sfxa108, sfxa109 and / or sfxa112;

[0013] Furthermore, the inversion system of the present invention further includes at least one target gene.

[0014] The target gene includes, but is not limited to, a promoter, a terminator, a structural gene, an enhancer silencer and / or an inversion site.

[0015] Furthermore, the experimental results of the present invention show that the inversion efficiency of the sfxa101 site is the highest.

[0016] The inversion includes single-site inversion and / or multi-site inversion.

[0017] The principle of the inversion system provided by the present invention is: inserting the target gene between the inversion sites in opposite directions, and under the action of the recombinase, completing the conversion of the position and transcription direction of the target gene between the two inversion sites in opposite directions;

[0018] Specifically, the target gene may be single or multiple; the inversion site may be single or multiple, which is not limited in the present invention.

[0019] The inversion system described in the present invention can achieve single-site and multi-site inversion, and can be designed according to experimental requirements when using the recombinase variant described in the present invention and / or the inversion site is sfxa101.

[0020] The site-specific recombination system, i.e., the inversion system of the present invention, is a multifunctional genome editing tool. By setting the direction of the recombination site, the inversion or deletion of DNA between sites can be controlled, which is of great significance for the generation of genetic diversity. However, deletions in the recombination system may play a negative role. For example, in the yeast genome synthesis project (Sc2.0 project), the SCRaMbLE system will inevitably produce a large number of deletions of DNA between sites when mediating genome-scale rearrangements, and even cause strain lethality. Constructing a DNA recombination system with DNA inversion bias (tending to occur inversion between reverse sites rather than deletion between sites in the same direction) may effectively solve this problem.

[0021] The present invention obtains highly efficient inversion enzymes by designing a new screening system and two rounds of progressively stringent directed evolution, and creates for the first time in eukaryotes a multiple inversion system Rci51-5 / multi-sfxa101 that tends to undergo inversion rather than deletion by screening inversion sites. At the same time, compared with the commonly used site-specific recombination system Cre / multi-loxP, this system exhibits a significantly reduced deletion rate, and this unique advantage also enables the multiple inversion system to be cleverly applied to metabolic pathway promoter replacement.

[0022] The inversion system described in the present invention only requires a simple initial one-step assembly process to generate a diverse library, and also provides a unique tool for the fields of genome engineering, cell barcoding, etc. in eukaryotes.

[0023] Furthermore, the inversion system of the present invention expands the scale of the multi-gene promoter regulation system by increasing the number of promoters and exogenous genes to produce more diverse gene expression combinations, and can also replace exogenous gene modules to achieve multiple product optimizations.

[0024] The inversion system of the present invention enriches the types of site-specific recombination systems of eukaryotic organisms. The multiple inversion system produces complex structural rearrangements through random inversion of DNA between multiple sites, plays a key role in generating genetic diversity and phenotypic adaptation, and also provides new tools for cell barcoding, genetic circuits and metabolic engineering. At the same time, the invention also lays a solid foundation for further optimization and development of new recombinase systems and in-depth exploration of inversion mechanisms.

[0025] The present invention provides a biomaterial comprising at least one of the following A) to C):

[0026] A), a nucleic acid encoding the recombinase variant of the present invention and / or the inversion system of the present invention;

[0027] B), a recombinant vector containing the nucleic acid shown in A);

[0028] C), transforming or transfecting host cells with the recombinant vector shown in C);

[0029] D) The mixture obtained by culturing the host cells shown in C).

[0030] The nucleic acid encoding the recombinant antigen of the present invention may be DNA, RNA, cDNA or PNA. In an embodiment of the present invention, the nucleic acid is in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. Nucleic acid may include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). Nucleic acid may be linear or circular in topology. Nucleic acid may be a part of a vector (e.g., an expression or cloning vector), or a fragment. The nucleic acid may be obtained directly from a natural source, or may be prepared with the assistance of recombination, enzymatic methods or chemical techniques. The RNA form is mRNA obtained by gene transcription, etc.

[0031] In the present invention, the nucleic acid may be codon-optimized or non-codon-optimized, and these optimizations include but are not limited to: codon usage preference, elimination of secondary structures that are not conducive to expression (such as hairpin structures), changes in GC content, CpG dinucleotide content, secondary structure of mRNA, cryptic splicing sites, early polyadenylation sites, internal ribosome entry sites and binding sites, negative CpG islands, RNA unstable regions, repeat sequences (direct repeats, inverted repeats, etc.) and restriction sites that may affect cloning.

[0032] The present invention provides a recombinant vector, which comprises a vector backbone and the nucleic acid of the present invention.

[0033] Recombinant vector of the present invention refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule, comprising a desired coding sequence and an appropriate nucleic acid sequence or element that is essential for the expression of an operably connected coding gene in a specific host organism. Once transformed into a suitable host, the vector can be replicated and play a role independently of the host genome, or, in some cases, self-integrated into the genome. In this specification, "plasmid" and "vector" can sometimes be interchanged and general, because plasmid is the most commonly used vector form at present. However, the present invention is intended to include such other forms of expression vectors, which play an equivalent role, which is known or will become known in the art, including but not limited to: plasmid, phage particle, viral vector and / or only potential genome insert.

[0034] Furthermore, the sources of the vector skeleton described in the present invention include plants, animals, bacteria, fungi, bacteriophages, or viruses, which are not limited in the present invention.

[0035] The host cells provided by the present invention may be derived from plants, animals, bacteria, fungi, bacteriophages or viruses, which are not limited in the present invention. The present invention uses vectors constructed by recombinant DNA technology to transform or transfect host cells, so that the transformed host cells have the ability to replicate protein-encoding vectors, or express desired proteins, or exercise gene reversal capabilities.

[0036] Furthermore, the transformation methods include chemical transformation and electrotransformation; the transfection methods include calcium phosphate coprecipitation, artificial liposome method, and viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, and the like.

[0037] The present invention provides the use of at least one of the following a) to c) in target gene conversion:

[0038] a) the recombinase variant according to the present invention;

[0039] b) The reversal system of the present invention;

[0040] c) The biomaterial of the present invention.

[0041] The present invention provides a product of target gene conversion, which includes auxiliary materials and at least one of the following I) to III):

[0042] 1), the recombinase variant according to claim 1 or 2;

[0043] II), the inversion system according to any one of claims 3 to 6;

[0044] III), the biomaterial according to claim 8.

[0045] In the product of the present invention, the excipients include buffers, culture media, antibiotics, stabilizers, preservatives, etc., which are used to maintain the activity of the recombinant enzyme variant, inversion system or biological material, or to assist them in functioning.

[0046] The present invention provides a method for gene inversion, which comprises performing gene inversion using at least one of the following I) to III):

[0047] I), the recombinase variant of the present invention;

[0048] II), the base inversion system of the present invention;

[0049] III), the biomaterial of the present invention;

[0050] IV), the product described in the present invention.

[0051] The inversion system of the present invention provides a new tool for cell barcoding, genetic circuits and metabolic engineering.

[0052] In the gene inversion method provided by the present invention, the target gene is inserted between the inversion sites with opposite directions, and under the action of the recombinase variant, the conversion of the target gene position and transcription direction between the two inversion sites with opposite directions is completed; specifically, the recombinant fragment obtained between the inversion sites with opposite directions of the target gene insertion and the recombinase variant can be on the same vector or on different vectors, and the present invention is not limited to this; in a specific embodiment of the present invention, the vectors containing the recombinant fragment and the recombinase variant are co-transformed into the host, and the expression of the recombinase variant is induced to function, thereby realizing the conversion of the target gene.

[0053] Specifically, the target gene may be single or multiple; the inversion site may be single or multiple, which is not limited in the present invention.

[0054] The present invention obtains a new tool capable of performing efficient multi-site specific inversion in eukaryotic organisms by directed evolution of recombinase and screening of inversion sites. The experimental results show that the inversion system of the present invention can be used in eukaryotic organisms to achieve efficient multi-site inversion, solve the shortcomings of deletion lethality, make up for the vacancy of multi-site inversion system in eukaryotic organisms, have broad application prospects, and provide new tools for cell barcodes, genetic circuits and metabolic engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 shows the high-throughput screening process;

[0056] Figure 2 shows the single-site and multi-site inversion abilities of mutants;

[0057] Figure 3 The Rci51-5 mutation site is shown;

[0058] Figure 4 The figure shows the comparison of recombination efficiency of Rci51-5 mutant at different inversion sites;

[0059] Figure 5 Taking the carotene synthesis pathway as an example, the composition of the multiple inversion system is explained;

[0060] Figure 6 The following figure shows the difference between this system and the conventional site-specific recombination system Cre / multi-loxP.

[0061] Figure 7 The results showed that the strains (yLJY045, yLJY028) in which the original promoter of crtI gene was replaced by a strong promoter or a medium-strong promoter were associated with higher β-carotene production. DETAILED DESCRIPTION

[0062] The present invention provides a specific inversion system, a method for constructing the system, and an application thereof. A person skilled in the art can refer to the contents of the present invention and appropriately improve the process parameters to achieve the specific inversion system. It should be particularly noted that all similar substitutions and modifications are obvious to a person skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of the present invention without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0063] Amino acid sequence of Rci: MPSPRIRKMSLSRALDKYLKTVSVHKKGHQQEFYRSNVIKRYPIALRNMDEITTVDIATYRDVRLAEINPRTGKPITGNTVRLELALLSSLFNIARVEWGTCRTNPVELVRKPKVSSGRDRRLTSSEERRLSRYFREKNLMLYVIFHLALETAMRQGEILALRWEHIDLRHGVAHLPETKNGHSRDVPL SRRARNFLQMMPVNLHGNVFDYTASGFKNAWRIATQRLRIEDLHFHDLRHEAISRFFELGSLNVMEIAAISGHRSMNMLKRYTHLRAWQLVSKLDARRRQTQKVAAWFVPYPAHITTIDEENGQKAHRIEIGDFDNLHVTATTKEEAVHRASEVLLRTLAIAAQKGERVPSPGALPVNDPDYIMICPLNPGSTPL (SEQ ID NO:1);

[0064] The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below in conjunction with the embodiments:

[0065] Example 1 Construction of multi-site invertase

[0066] 1. Construction of mutants

[0067] The present invention obtains a multi-site reversal enzyme with improved efficiency by directed evolution. The present invention designs a screening system to screen the reversal enzyme, and the screening process is shown in Figure 1. The system comprises three pairs of sfxa101 reversal sites in opposite directions and three auxotrophic genes URA3, hphNT1, and kanMX in the off state. Only when all three auxotrophic genes are reversed can the strain grow on a specific screening medium, and if deletion occurs, the strain cannot grow.

[0068] With the help of this high-throughput screening system, two rounds of progressively stringent directed evolution were performed on the wild-type Rci enzyme from Salmonella typhimurium (SEQ ID NO: 1). The enzyme is regulated by the inducible promoter Gal1. The Rci enzyme mutant library was constructed by introducing random mutations by error-prone PCR. The mutant library was transformed into Saccharomyces cerevisiae carrying the screening system, and after 12 hours of induction, it was plated on different culture media containing two screening tags (SC-URA+HYG-HIS-LEU, SC+HYG+G418-HIS-LEU, SC-URA+G418-HIS-LEU). In the first round, the dominant mutant Rci51 was screened and used as a template for the next round of directed evolution. In the second round of directed evolution, the induction time was reduced to 8 hours, and the culture medium containing three screening tags (SC-URA+G418+HYG-HIS-LEU) was plated to increase the screening pressure, and the best mutant Rci51-5 was screened in the second round.

[0069] The results of two rounds of progressively more stringent directed evolution showed that the multi-site inversion rate of mutant Rci51-5 was increased by 1.6×10 7 , the total reversal efficiency increased by about 1158 times. The strains containing wild-type Rci, Rci51, and Rci51-5 were respectively coated on various culture media to visually display their differences. The results showed that the multi-site reversal ability of the Rci51-5 mutant was greatly improved. See Figure 2 for details.

[0070] Compared with the wild-type Rci enzyme, the mutant Rci51 produced three non-synonymous mutations: F246V, A295S, A330T; the mutant Rci51-5 produced five non-synonymous mutations: Q30K, L109P, F246V, A295S, A330T ( Figure 3 ).

[0071] 2. Verification of mutant site conversion efficiency

[0072] In order to compare the recombination efficiency of the Rci51-5 mutant at different inversion sites, the inversion efficiency of Rci51-5 at different inversion sites (sfxa101, sfxa107, sfxa108, sfxa109, and sfxa112) was tested. The results showed that among the five sfx recombination sites, the sfxa101 site showed the highest efficiency ( Figure 4 ).

[0073] 3. Achieve multi-site conversion in specific target genes

[0074] Based on the mutant Rci51-5 and sfxa101 inversion sites, the present invention constructs a multiple site-specific inversion system Rci51-5 / multi-sfxa101 in eukaryotic organisms for the first time. The system can efficiently achieve DNA inversion between multiple sites, providing a special tool for eukaryotic genome manipulation.

[0075] The present invention characterizes the recombination difference between this system and the conventional site-specific recombination system Cre / multi-loxP, and the results show that in this system, the DNA deletion rate between sites in the same direction is significantly lower than that of Cre / multi-loxP.

[0076] The multiple inversion system constructed in the present invention enriches the types of eukaryotic site-specific inversion systems and provides a new method for metabolic engineering. The experiment takes the β-carotene synthesis pathway of Saccharomyces cerevisiae as an example to illustrate that the multiple inversion system can effectively promote the replacement of promoters in metabolic pathways, creating a paradigm for the application of multiple site-specific inversion systems in metabolic pathway promoter engineering.

[0077] Ten promoters with different expression strengths were selected to form a promoter array, and the sfxa101 inversion site and the terminator of the corresponding promoter were inserted between adjacent promoters. The directions of the three exogenous genes crtE, crtI and crtYB of β-carotene were opposite to the direction of the promoter array. The opposite sfxa101 inversion site, the initial promoter and the corresponding terminator were inserted between the three exogenous genes (specifically, Figure 5 As shown in Figure 1). This design allows the promoters in the promoter array to replace the original promoters of foreign genes through gene inversion between opposite sites under the mediation of recombinase, thereby generating a rich combination of foreign gene transcription. For details, see Figure 6 The mixed bacteria PCR verification results showed that the original promoters of the three exogenous genes could be replaced by any promoter in the array and each replacement could be detected.

[0078] In the experiment, strains with different colors were selected and their promoter replacement was determined by polymerase chain reaction (PCR). The qPCR results showed that the expression level of the exogenous gene was consistent with the change in promoter strength. The carotenoid (β-carotene and lycopene) production of these 12 yeasts with unique structures was tested by high-performance liquid chromatography. The carotenoid production of strains yLJY081, yLJY045, yLJY028, yLJY037, yLJY044, yLJY062, and yLJY055 with orange and dark yellow phenotypes was improved. The strain yLJY081, in which all three initial promoters were replaced, had the largest increase in production. Compared with the control yLJY023, its β-carotene and lycopene production increased by 7.6 times and 12.4 times, respectively. The experiment also found that strains in which the initial promoter of the crtI gene was replaced by a strong promoter or a medium-strong promoter (yLJY045, yLJY028) were associated with higher β-carotene production, which was 6.3 times and 4.9 times higher than the control, respectively. Figure 7 .

[0079] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A recombinase variant, characterized in that The amino acid sequence of the recombinant enzyme is shown in SEQ ID NO: 1, wherein the alanine at position 246 is mutated to valine, the alanine at position 295 is mutated to serine, and the alanine at position 330 is mutated to threonine.

2. The recombinase variant according to claim 1, characterized in that The invention comprises a recombinant enzyme in which the glutamine at position 30 is mutated to lysine, the leucine at position 109 is mutated to proline, the alanine at position 246 is mutated to valine, the alanine at position 295 is mutated to serine, and the alanine at position 330 is mutated to threonine.

3. Inversion system, characterized in that, The invention comprises the recombinase variant and the inversion site according to claim 1 or 2.

4. The reversal system according to claim 3, characterized in that: The inversion site includes at least one of sfxa101, sfxa102, sfxa107, sfxa108, sfxa109 and / or sfxa112.

5. The inversion system according to claim 3 or 4, characterized in that: At least one target gene is also included.

6. The inversion system according to claim 5, characterized in that: The target gene includes a promoter, a terminator, a structural gene, an enhancer, a silencer and / or an inversion site.

7. Biomaterial, characterized in that Including at least one of the following A) to C): A), a nucleic acid encoding the recombinase variant according to claim 1 or 2 and / or the inversion system according to any one of claims 3 to 6; B), a recombinant vector containing the nucleic acid shown in A); C), transforming or transfecting host cells with the recombinant vector shown in B); D) The mixture obtained by culturing the host cells shown in C).

8. Application of at least one of the following a) to c) in target gene conversion: a) the recombinase variant according to claim 1 or 2; b) The inversion system according to any one of claims 3 to 6; c) The biomaterial according to claim 8.

9. A product of target gene conversion, characterized in that: Including auxiliary materials and at least one of the following I) to III): 1), the recombinase variant according to claim 1 or 2; II), the inversion system according to any one of claims 3 to 6; III), the biomaterial according to claim 8.

10. A method for target gene conversion, characterized in that: The method comprises performing gene inversion using at least one of the following I) to IV): 1), the recombinase variant according to claim 1 or 2; II), the base inversion system according to any one of claims 3 to 6; III), the biomaterial according to claim 7; IV), the product according to claim 9.

Images