Construction and application of variant tRNA recognizing TAG in yeast

Abstract

The present invention relates to the field of genetic engineering technology, and specifically to the construction and application of variant tRNA for yeast recognition of TAG. The present invention successfully constructs a variant tRNA that can recognize TAG termination codon in Saccharomyces cerevisiae, and the strain containing the variant tRNA can successfully recognize the TAG termination codon inserted in the metabolic pathway, so that the modified metabolic pathway can be artificially controlled to express, thereby preventing the leakage of the artificially modified metabolic pathway.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and in particular to the construction and application of variant tRNA for yeast recognition of TAG. Background Art

[0002] With the widespread application of artificially modified synthetic microorganisms in renewable energy, clinical treatment, industrial production and environmental protection, their unintentional leakage may cause environmental and health problems. At present, a variety of biological escape prevention schemes have been developed for microorganisms containing artificial synthetic metabolic pathways. The most commonly used strategies are the following four: nutritional deficiency, induced switch, orthogonalization of genetic central dogma, and complex gene regulatory network compound control. Among them, the idea of ​​constructing a biological escape prevention system orthogonal to the chassis strain has gradually attracted attention. The construction of orthogonalized metabolic pathways often uses genetic coding mechanisms that are incompatible with natural organisms, such as the use of non-natural coding unit molecules (non-natural amino acids, non-natural nucleotides) or orthogonal design of macromolecular protein machines (enzymes, ribosomes) involved in the process of genetic coding principles. Among them, the introduction of non-natural amino acids to construct non-natural amino acid-dependent orthogonal life forms is a hot area of ​​current research. The introduction of unnatural amino acids often requires the introduction of aminoacyl tRNA synthetase (aaRS) and its corresponding tRNA combination that is orthogonal to the host translation mechanism in the cell. This aminoacyl tRNA synthetase / tRNA pair can encode a specific unnatural amino acid and specifically recognize the stop codon (UAG) to insert the unnatural amino acid with special functions into the target protein. At present, the E. coli C321△A strain has been successfully constructed in which 321 UAG codons are completely replaced with UAA codons. After releasing the UAG stop codon, the insertion rate of unnatural amino acids has been greatly improved. In Salmonella typhimurium, 1557 leucine codons were also successfully replaced by synonymous substitutions to successfully recode the 200kb genome. Escherichia coli and Saccharomyces cerevisiae are the two most commonly used model base bacteria in the field of microbial research. In Escherichia coli, a host strain MT21 tolerant to Tpa has been successfully constructed, and this dependence can be maintained for hundreds of generations without any escape individuals. In Saccharomyces cerevisiae, although the TAG termination codon was replaced with TAA through the Sc 2.0 project and five complete artificial yeast chromosomes were successfully constructed, the corresponding tRNA variants were not developed and applied to the prevention of escape in specific metabolic pathways.

[0003] The existing biological escape prevention strategies for constructing nutrient-deficient strains and developing inducible switches face several difficult-to-avoid problems: 1. They may cause certain survival pressure and metabolic toxicity to the growth and development of the chassis strain itself; 2. They may fail due to gene silencing mechanisms (such as gene mutations and gene recombination); 3. They cannot effectively play a long-term containment role. The present invention focuses on developing an escape prevention strategy based on the orthogonal design principle, which achieves the purpose of escape prevention by constructing metabolic pathways that can only be recognized and expressed by tRNA variants, and the orthogonal design can avoid growth interference with the chassis strain.

[0004] At present, most of the research on the modification of tRNA and its application in the introduction of unnatural amino acids is concentrated in Escherichia coli. As the most commonly used base bacteria in eukaryotic research, there is not much related research on Saccharomyces cerevisiae. Escherichia coli is a prokaryotic organism, while yeast is a eukaryotic organism. There is a species gap between the two. In addition, due to the differences in the structure of tRNA, the structure and type of RNA polymerase, the translation release factor, and the arrangement and metabolism of the genome between prokaryotes and eukaryotes, the tRNA used in Escherichia coli and its modification methods cannot be directly applied to yeast. Therefore, the development of mutant tRNA that acts on yeast and its application as a gene switch in biological escape has great research significance and value. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide the construction and application of a variant tRNA for yeast recognition of TAG.

[0006] The present invention provides a nucleic acid encoding a variant tRNA, which sequentially comprises: an upstream segment, an anticodon CTA and a downstream segment;

[0007] The upstream segment constitutes the 5' terminal loop and arms of the secondary structure of the variant tRNA;

[0008] The downstream segment constitutes the 3'-terminal loop and arm of the secondary structure of the variant tRNA.

[0009] Further, the 5'-terminal loop and arm include: tRNA 5'-terminal amino acid acceptor arm, D stem, D loop, 5'-terminal anticodon stem and anticodon loop anticodon 5'-terminal sequence; the 3'-terminal loop and arm include: tRNA 3'-terminal amino acid acceptor arm, TφC stem, TφC loop, variable arm, 3'-terminal anticodon stem and anticodon loop anticodon 3'-terminal sequence. The tRNA 3'-terminal amino acid acceptor arm is partially reverse complementary to the tRNA 5'-terminal amino acid acceptor arm; the 5'-terminal anticodon stem is reverse complementary to the 3'-terminal anticodon stem.

[0010] In the present invention, the nucleic acid sequence is at least one of SEQ ID NO: 1 to SEQ ID NO: 56.

[0011] Furthermore, the nucleic acid described in the present invention is derived from yeast. Based on the differences in tRNA structure and genome composition between yeast as a eukaryotic microorganism and prokaryotic microorganism, the present invention specifically designs a variant tRNA and a gene switch regulatory system suitable for the TAG termination codon of yeast. The gene switch regulatory system can be applied to the regulation of yeast engineering bacteria genes and metabolic pathway switches, thereby preventing the leakage of artificially modified metabolic pathways.

[0012] Furthermore, the variant tRNA mutates the anticodon of the tRNA corresponding to 20 amino acids in yeast to CTA. The mutated variant tRNA can pair the amino acid carried by the original tRNA with the codon corresponding to the mRNA, so that the yeast does not respond to the TAG termination codon mutation in the structural gene, thereby allowing the structural gene to function normally.

[0013] The present invention provides a variant tRNA vector, which includes the nucleic acid of the present invention and a eukaryotic vector backbone. Further, the screening marker of the eukaryotic vector backbone may include a URA tag, or may include other screening markers adapted to the chassis strain, which is not limited by the present invention. Furthermore, the present invention utilizes the URA tag to screen the constructed yeast expression strain.

[0014] In the present invention, the eukaryotic vector skeleton also includes necessary elements required for gene replication and expression such as promoters and replicons, which are not limited in the present invention. Further, the eukaryotic vector is selected according to different promoter types, and the results show that different promoter types have different effects.

[0015] In the present invention, the promoter includes an inducible promoter and a constitutive promoter. Further, the inducible promoter includes but is not limited to GAL, MET17, CUP1 or AOX1; the constitutive promoter includes but is not limited to TEF1, GPD, ADH1, PRP, ThrP or GAP. Furthermore, the present invention uses constitutive promoters PRP, ThrP and inducible GAL as subjects to express the nucleic acid.

[0016] The present invention provides a yeast strain which is transformed or transfected with the variant tRNA vector of the present invention.

[0017] Furthermore, the yeast strains include but are not limited to Saccharomyces cerevisiae, Pichia pastoris or Yarrowia lipolytica, which is not limited in the present invention.

[0018] Furthermore, the yeast strain of the present invention is based on Saccharomyces cerevisiae; the nucleic acid of the variant tRNA of the present invention is designed according to the genome sequence of Saccharomyces cerevisiae; the vector of the variant tRNA nucleic acid of the present invention is a eukaryotic vector suitable for yeast; the yeast strain of the present invention is a Saccharomyces cerevisiae strain transformed or transfected with the variant tRNA vector of the present invention. The yeast strain, variant tRNA, variant tRNA vector and mutant structural gene of the present invention support each other in function and interact with each other, and they affect the technical effect of the present invention as a whole.

[0019] The present invention provides variant tRNAs produced by the yeast strains of the present invention.

[0020] Furthermore, the variant tRNA described in the present invention, together with the corresponding mutant structural gene, constitutes a yeast gene or metabolic pathway switch regulatory system.

[0021] The present invention provides a method for constructing the variant tRNA, the steps of which include integrating the nucleic acids of the present invention into eukaryotic expression vectors, transforming or transfecting yeast strains to produce variant tRNA. Further, the transformation method includes: chemical transformation and electrotransformation; the transfection method includes calcium phosphate coprecipitation, artificial liposome method, and viral transfection. In some embodiments of the present invention, the method for constructing the variant tRNA includes integrating the nucleic acids of the present invention into eukaryotic expression vectors, and electrotransforming yeast strains to produce variant tRNA.

[0022] Furthermore, the selection of the nucleic acid described in the present invention follows the principle of avoiding essential genes as much as possible. The corresponding tRNA gene sequence and the 20 bp base sequence at both ends of the tRNA gene sequence are selected from the Saccharomyces cerevisiae genome for design, and EcoRI and BamHI restriction sites are inserted at both ends, and the eukaryotic expression vector is integrated by enzyme ligation.

[0023] The present invention provides a characterization system for the response intensity of yeast variant tRNA to TAG stop codon, which includes mutant fluorescent protein and the variant tRNA of the present invention. Furthermore, the mutant fluorescent protein includes but is not limited to red fluorescent protein in which the TAG stop codon is inserted only after the start codon of the nucleic acid encoding it, and the present invention is not limited thereto. The results show that different amino acid variant tRNAs have different promoter types and different response intensities to the TAG stop codon; when Gal is the promoter, trp, glu, tyr, ser, lys, pro, ile, asp, asn and ala all have red fluorescent strains, among which the expression of red fluorescent protein of the three TAG mutant tRNA strains, trp, glu and ile, is relatively stable; when PRP is the promoter, lys, arg, pro, phe, gln, gly, glu, his, cys, leu, Red fluorescence could be detected in all the 12 TAG mutant tRNA strains, including ile and met. Among them, strong red fluorescence could be detected in three parallel groups of 6 TAG mutant tRNA strains, including lys, arg, gln, gly, pro, cys and his, and the recognition response to the TAG termination codon was relatively stable. When ThrP was the promoter, the response intensity of the 12 TAG mutant tRNA strains, including phe, his, lys, met, thr, tyr, trp, leu, gln, glu, asn and ile, was relatively high.

[0024] Furthermore, the characterization system described in the present invention can be used to screen the selection of promoters of tRNA carriers for switch regulation of different genes or metabolic pathways. Selecting a suitable promoter is beneficial to improving the effectiveness of switch regulation.

[0025] The present invention provides a switch control system for yeast engineering strain genes or metabolic pathways, which includes any one of the variant tRNAs described in the present invention or a tRNA designed based on the principle thereof, and the present invention is not limited thereto.

[0026] In the present invention, the mutant structural gene includes inserting TAG in the coding functional region of the structural gene and any position that does not produce frameshift mutation, or replacing any amino acid in its coding functional region with TAG, which is not limited in the present invention.

[0027] Furthermore, the switch regulation system described in the present invention achieves the purpose of regulating the switch of genes or metabolic pathways by combining mutant structural genes with variant tRNA.

[0028] Furthermore, the mutant structural genes of the present invention include but are not limited to genes related to the carotene metabolic pathway. The results show that the yeast strain into which the variant tRNA is transferred can respond to the carotene metabolic pathway.

[0029] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0030] The present invention successfully constructs a variant tRNA that can recognize the TAG stop codon in Saccharomyces cerevisiae. The strain containing the variant tRNA can successfully recognize the TAG stop codon inserted in the metabolic pathway, so that the expression of the modified metabolic pathway can be artificially controlled, thereby preventing the leakage of the artificially modified metabolic pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the rapid construction process of TAG mutant tRNA sequence;

[0032] Figure 2 Shows the expression process of RFP protein with TAG mutation inserted;

[0033] Figure 3 Fluorescence microscope photos of the inducible GAL promoter TAG mutant tRNA strain are shown;

[0034] Figure 4 Shown are the flow cytometry results of the inducible GAL promoter TAG mutant tRNA strain;

[0035] Figure 5 Shown is the fluorescence microscope photo of the PRP promoter TAG mutant tRNA strain;

[0036] Figure 6 The flow cytometry results of the PRP promoter TAG mutant tRNA strain are shown;

[0037] Figure 7 Fluorescence microscope photos of the ThrP promoter TAG mutant tRNA strain are shown;

[0038] Figure 8 The flow cytometry results of the ThrP promoter TAG mutant tRNA strain are shown;

[0039] Fig. 9 The orthogonal carotene metabolic pathway is shown by mutating the three bases of tryptophan into TAG codons;

[0040] Fig.10 The diagram shows the effect of ThrP-TAG mutant tRNA. A is a photo of the strain plate into which the ThrP-TAG mutant tRNA plasmid was transferred; the right side of Figure B is the strain into which the 416 plasmid was transferred. DETAILED DESCRIPTION

[0041] The present invention provides the construction and application of variant tRNA for yeast recognition of TAG, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar replacements and modifications are obvious to those 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 this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0042] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0043] The present invention will be further described below in conjunction with embodiments:

[0044] Example 1

[0045] The present invention firstly constructs a galactose-induced expression plasmid containing TAG mutant tRNA corresponding to 20 amino acids in the Saccharomyces cerevisiae strain by using the long primer self-ligation method, and uses the red fluorescent protein gene as a reporter gene to characterize and screen the recognition efficiency of different TAG mutant tRNAs for TAG stop codons. On this basis, the other two constitutive promoters ThrP and PRP1 were replaced to explore the influence of different promoters on the strength of 20 TAG mutant tRNAs recognizing the stop codon TAG, and finally successfully constructs an orthogonal carotene metabolic pathway that can only be recognized by tRNA variants.

[0046] 1. Construction of TAG mutant-tRNA fragment

[0047] The gene sequences corresponding to 275 tRNAs of Saccharomyces cerevisiae were downloaded from the tRNAdb website (http: / / trna.bioinf.unileipzig.de / DataOutput / Search). The gene maps were used in Snapgene to locate their specific positions on the chromosome. According to the principle of avoiding essential genes as much as possible, a corresponding tRNA sequence was selected, and the three bases at the anticodon position were mutated to TAG. When selecting the tRNA gene sequence on the chromosome of Saccharomyces cerevisiae, the base sequences of 20 bp at both ends were selected, and two restriction sites, EcoRI and BamHI, were introduced at both ends to facilitate the subsequent connection with the vector T4. After obtaining a gene sequence containing tRNA of about 120 bp to 150 bp in length, the tRNA fragment containing the TAG mutation was divided into two parts using four long primers with overlapping front and back primers for annealing and connection. The two primers were annealed to form double strands, and the 5' end was phosphorylated by PNK enzyme to add a phosphate group, and the two tRNA sequences were connected by DNA ligase, thus successfully constructing two tRNA fragments containing EcoRI and BamHI restriction sites. The construction diagram is shown in the figure. Figure 1 shown.

[0048] 2. Obtaining Gal vector.

[0049] Using the URA-tagged plasmid containing the gal-inducible promoter as a template, add EcoRI and BamHI restriction sites to both ends of the vector obtained by PCR using primer loop P. Then perform T4 ligation with the tRNA fragment obtained in the previous step.

[0050] 3. Construction of constitutive TAG mutant tRNA plasmid

[0051] The 20 Gal-TAG mutant tRNA plasmids obtained in the above step are used as templates. It is only necessary to replace the Gal promoter with the constitutive PRP promoter and ThrP promoter. Therefore, 20 Gal-TAG mutant tRNA plasmids are double-digested with XhoI and EcoRI to obtain vector fragments. Among them, the PRP promoter is amplified from the pRS423 plasmid preserved in the laboratory, and the ThrP promoter is amplified from the genome of Saccharomyces cerevisiae BY4742. During amplification, PCR primers are also used to add XhoI and EcoRI restriction sites to both ends of the promoter fragment. The recovered PCR fragment is double-digested with XhoI and EcoRI and then connected to the obtained vector fragment T4 to construct 36 constitutive TAG mutant tRNA plasmids. The present invention has successfully constructed a total of 56 tRNA variants, and the yeast strains containing these variants are stored in a -80°C refrigerator. The strain nomenclature and tRNA mutation sequence are shown in Table 1.

[0052] Table 1

[0053]

[0054]

[0055]

[0056] 4. Characterization of the recognition strength of 56 TAG mutant tRNA variants for TAG stop codon

[0057] In order to facilitate the design of reasonable and effective variant tRNA vectors for target genes in subsequent experiments, we used yeast containing red fluorescent protein (RFP) plasmids to characterize the corresponding fluorescence intensity. The design ideas are as follows Figure 2 . We designed an RFP response plasmid containing a TAG mutation, and inserted the three mutant bases of TAG at the second amino acid position of the RFP gene sequence. There are two main reasons for inserting it at the front end. First, if there is no TAG mutant tRNA that can recognize the TAG codon, the translation of RFP will be directly terminated here, resulting in the inability to synthesize RFP protein normally, thereby ensuring that the final synthesized RFP protein is the RFP protein containing the TAG mutation. Second, inserting an amino acid at the front end of the gene often does not affect the normal folding and expression of the RFP protein. Only when the tRNA variant successfully recognizes the response TAG codon can the red fluorescent Saccharomyces cerevisiae strain be observed under a fluorescence microscope. According to the fluorescence intensity, the tRNA variant with a higher response intensity to the TAG termination codon can be further selected.

[0058] 5.RFP Characterization Results

[0059] like Figure 3 As shown in the figure, red fluorescent strains appeared in trp, glu, tyr, ser, lys, pro, ile, asp, asn, and ala in the Gal promoter plasmid. Among them, the expression of red fluorescent protein in the three TAG mutant tRAN strains of trp, glu, and ile was relatively stable, and the results of fluorescence intensity characterization by flow cytometry were consistent with them, as shown in the figure. Figure 4 As shown. Figure 5 As shown, under the action of the constitutive promoter PRP, red fluorescence can be detected in 12 TAG mutant tRNA strains including lys, arg, pro, phe, gln, gly, glu, his, cys, leu, ile, and met. Among them, strong red fluorescence can be detected in three parallel groups of 6 TAG mutant tRNAs including lys, arg, gln, gly, pro, cys, and his. The recognition response to the TAG stop codon has a strong stability, and the characterization results of the fluorescence intensity by flow cytometry are consistent with it, as shown in Figure 2. Figure 6 As shown. Figure 7 As shown in the figure, under the action of constitutive promoter ThrP, the 12 TAG mutant tRNAs, phe, his, lys, met, thr, tyr, trp, leu, gln, glu, asn, and ile, have higher response intensities. The characterization results of fluorescence intensity by flow cytometry are consistent with this, as shown in the figure. Figure 8 As shown. By comparing the response results under the three promoters, it was found that the TAG mutant tRNA corresponding to the four amino acids lys, glu, pro and ile had a high response intensity to red fluorescent protein under the three promoters and had high robustness. The TAG mutant tRNA corresponding to the eight amino acids trp, tyr, his, cys, leu, gln, gly and asn also had a high red fluorescence expression intensity under the action of the two promoters.

[0060] 6. Construction of orthogonal carotene metabolic pathway using TAG variants

[0061] We selected a tryptophan (trp) in the crtYB gene in the carotene metabolic pathway and replaced the corresponding three bases with the TAG stop codon to construct a tryptophan mutant tRNA, as shown in SEQ ID NO: 54. The recognition principle is similar to that of the TAG-RFP gene designed in the previous section, as shown in Figure 2 As shown. Only when the TAG mutant tRNA can recognize the TAG codon can it be normally translated into the original tryptophan, thus not affecting the normal expression of the carotene metabolic pathway. Therefore, the colonies that only transferred the ThrP-TAG mutant tRNA plasmid and successfully recognized the TAG codon will appear orange. The results showed that after the ThrP-TAG-trp plasmid was transferred, the trp variant successfully recognized the TAG stop codon and translated it into tryptophan, and finally synthesized carotene.

[0062] like Fig.10 As shown, Fig.10 A in the figure is a photo of the strain plate into which the ThrP-TAG mutant tRNA plasmid was transferred. It can be clearly seen that the single colonies that grew are basically orange-red, indicating that the carotene metabolic pathway is normally expressed in these colonies, which means that the ThrP-TAG mutant tRNA plasmid can indeed recognize the TAG stop codon. According to its proportion, it can be clearly seen that ThrP-TAG mutant-tRNA-trp can basically achieve 100% recognition response to the TAG stop codon. Fig.10 Figure B shows that the strains transformed with the 416 plasmid appear white, indicating that the strain cannot recognize the inserted TAG termination codon and translate trypsin, resulting in premature termination of the carotene metabolic pathway.

[0063] 7. Regulation of carotene metabolism in yeast

[0064] The yeast strain after the TAG stop codon mutation of the key base of the carotene metabolic pathway cannot express the carotene metabolic pathway under normal conditions, and the blocking rate of the expression process is 100%. The carotene metabolic pathway can be restored to expression only when and only when the mutant strain cell has the TAGtRNA variant designed by the present invention. Therefore, when the yeast strain after the key base is subjected to the TAG stop codon mutation and the cell does not contain the TAG tRNA variant designed by the present invention escapes to nature, it cannot express carotene, thereby achieving the purpose of preventing the carotene metabolic pathway of the artificially modified yeast strain from escaping, and the anti-escape rate is 100%. Similarly, the key bases of the DNA of other artificially modified yeast metabolic pathways are subjected to TAG mutation to block expression, and the corresponding amino acid TAGtRNA variant designed by the present invention is used to restart the expression of the related metabolic pathway, which can also achieve the purpose of preventing other metabolic pathways from escaping.

[0065] 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 nucleic acid encoding a variant tRNA, the nucleic acid sequence of which is shown in SEQ ID NO:

54.

2. A variant tRNA carrier, characterized in that It comprises the nucleic acid according to claim 1 and a eukaryotic vector backbone.

3. A yeast strain, characterized in that Transform or transfect the variant tRNA vector according to claim 2.

4. A variant tRNA, characterized in that Produced by the yeast strain of claim 3.

5. The method for constructing the variant tRNA according to claim 4, comprising the steps of: After the nucleic acids described in claim 1 are respectively integrated into eukaryotic expression vectors, yeast strains are transformed or transfected to produce variant tRNAs.

6. A characterization system for the response strength of yeast variant tRNA to TAG stop codon, characterized in that: It comprises a mutant fluorescent protein and the variant tRNA according to claim 4.

7. The characterization system according to claim 6, characterized in that The mutant fluorescent protein includes red fluorescent protein.

8. A switch control system for genes or metabolic pathways of yeast engineering strains, comprising the variant tRNA and mutant structural genes according to claim 4; the mutant structural genes include any one of the carotene metabolic pathway genes.

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