WO2025140227A1 - Modified aminoacyl-trna synthetase, nucleic acid construct and genetically engineered strain - Google Patents

Abstract

Provided are a modified aminoacyl-tRNA synthetase, a nucleic acid construct and a genetically engineered strain. The provided recombinant aminoacyl-tRNA synthetase can significantly improve the introduction efficiency of non-natural amino acids in IVTT regardless of exogenous addition or integration into cells.

Description

A modified aminoacyl-tRNA synthetase, nucleic acid construct and genetic engineering strain Technical Field

The present invention relates to the field of biotechnology, and preferably to an aminoacyl-tRNA synthetase and a nucleic acid construct thereof, a genetically engineered strain, and an application thereof in cell-free synthesis of non-natural amino acid proteins.

Background Art

Whether it is protein structure and function research or the production of antibody-drug conjugates, more and more cases require the introduction of new functional groups into protein polypeptide chains. This can be achieved by using the protein orthogonal translation system to encode non-natural amino acids with special chemical functional groups into the peptide chain according to the preset genetic codon. Although the introduction of non-natural amino acids can be achieved in cells, due to competition with the classical protein translation system and the special chemically active side chain groups of non-natural amino acids, they often cannot freely pass through the cell membrane. To achieve large-scale production of proteins containing non-natural amino acids, the cell-free in vitro protein translation system has irreplaceable advantages.

Because the cell-free in vitro protein translation system is not bound by the cell membrane, the reaction can be theoretically promoted by increasing the concentration of the main components in the reaction system. The unnatural amino acid is transported to the ribosome by the aminoacyl-tRNA formed by the catalysis of a specific aminoacyl-tRNA synthetase, and the unnatural amino acid is assembled in the peptide chain under the catalysis of the ribosome by pairing with the codon. Among them, the binding ability of the unnatural aminoacyl-tRNA to the transport protein and the matching degree with the ribosome are weaker than those of the classical aminoacyl-tRNA, and the reaction is not carried out. It is necessary to promote the reaction by increasing the substrate concentration. The orthogonal aminoacyl-tRNA synthetase used for the introduction of unnatural amino acids is usually obtained by mutating the substrate binding site of the natural aminoacyl-tRNA synthetase, and the change in substrate specificity often requires the price of decreased catalytic activity. In order to compensate for this decreased activity, a high concentration of enzyme or tRNA or unnatural amino acid is required to make the reaction proceed in a direction that is conducive to peptide chain synthesis.

The commonly used orthogonal translation system pylrs-tRNA _CUA ^pyl comes from the archaea Methanosarcina mazei and Methanosarcina barkeri PylRS (MmPylRS, MbPylRS). Due to its special structure, the recognition of tRNA by pylrs is independent of the anticodon loop and has substrate recognition plasticity. Through directed evolution, the use of pylrs-tRNA _CUA ^pyl has enabled the introduction of more than 200 unnatural amino acids.

However, both MmPylRS and MbPylRS have an N-terminal domain with low solubility, which leads to their poor solubility. Even after codon optimization, the expression level of MmPylRS and MbPylRS in E. coli is difficult to increase, and the protein concentration after concentration still cannot exceed 8 mg/ml. The advantages of the cell-free in vitro protein translation system cannot be fully utilized, and the introduction efficiency of non-natural amino acids is very low, which is not suitable for actual production applications.

Methanomethylophilus alvus PylRS (PylRS) is an aminoacyl-tRNA synthetase. This synthetase belongs to the nucleotide class and its full name is pyrrolysyl-tRNA synthetase, commonly referred to as PylRS. The main function of PylRS is to help prokaryotes synthesize a special amino acid - pyrrolysine (Pyl), which does not exist in any known eukaryotic organisms ^[1] .

Methanomethylophilus alvus is a methanogenic archaeon originally isolated from the stomach of bovine ruminants. PylRS was discovered by sequence analysis of the M. alvus genome and can be used to modify foreign proteins and express new biological activities, so it has important application value in the field of synthetic biology.

Recently, Chin et al. discovered a pylrs (MaPylRS) from Methanomethylophilus alvus, which has similar catalytic domains and tRNA binding domains as MmPylRS, MbPylRS and mapylrs, but lacks the N-terminal domain.

In current scientific research, MaPylRS is mainly used to modify exogenous proteins in prokaryotes such as Escherichia coli ^[2] , and there are also reports of successful applications in eukaryotic organisms ^[3] . According to the latest research results, when MaPylRS is expressed in Saccharomyces cerevisiae ^[4] , it can achieve efficient site-specific insertion of target non-natural amino acids into target proteins. However, whether in Escherichia coli or yeast, MaPylRS exists in the cell in the form of an exogenous plasmid, and there is no eukaryotic cell system that can directly recombinantly express MaPylRS in its genome stably and efficiently ^[5] .

Protein Expression Systems in vitro refers to the use of cell lysate and other non-in vivo conditions to synthesize proteins. It is only necessary to add DNA or RNA templates, RNA polymerase and necessary amino acids, ATP and auxiliary factors to the reaction system to complete the synthesis of the target protein. Since the complex metabolic steps of the entire cell are not required, the system can achieve rapid and efficient large-scale protein production. Due to its high flexibility and simplified process, it has become one of the widely used tools in the biomedical field and scientific research ^[6] . At present, the commercial in vitro protein expression systems that are frequently used include Escherichia coli system (E. coli extract, ECE) ^[7] , rabbit reticulocyte lysate (Rabbit reticulocyte lysate, RRL) ^[8] , wheat germ extract (WGE) ^[9] , insect cell extract (ICE) ^[10] and human system ^[11] .

Because of the lack of the low-solubility N-terminal domain, mapylrs is easier to express in E. coli, and the final expression product can be concentrated to 40 mg/ml without precipitation. It is suitable for cell-free non-natural amino acid introduction system.

CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated) is an immune system widely found in bacteria and archaea. As part of the organism's defense mechanism, it can identify and eliminate foreign DNA or RNA that invades host cells, and has been developed into an efficient and accurate gene editing tool ^[12] . This technology can precisely cut DNA, allowing researchers to increase or decrease specific parts of genes to study their functions, and can also be used to treat certain genetic diseases ^[13] . When using the CRISPR/Cas9 system for gene editing, a specific gRNA sequence needs to be paired with the Cas9 nuclease protein and delivered to the target cell. When the gRNA binds to Cas9, it will be carried to the target genome. During the formation of the double-stranded DNA nick, the gRNA controls the Cas9 enzyme to find the PAM sequence (i.e. "protospacer adjacent motif") on the genome and recognize the sequence 20bp upstream of it. The Cas9 enzyme then creates a double-stranded nick 3 bases upstream of the PAM. At the same time, if donor DNA is provided, the broken DNA chains can be connected through the mechanism of homologous recombination (HDR), thereby achieving the purpose of genetic modification ^[14] . There are many examples of using the CRISPR/Cas9 system to modify the genome of S. cerevisiae, including gene point mutation, gene knockout and gene insertion ^[15,16] . For example, the pCAS plasmid widely used in the prior art has both the Cas9 gene sequence and the gRNA element ^[17] , which can achieve genome modification of S. cerevisiae through a single transformation. Kluyveromyces is a type of yeast fungus, belonging to S. cerevisiae. It has a wide range of industrial applications, especially in the food and beverage industry. Kluyveromyces is widely used in the fermentation process of food and beverages such as dairy products, wine, and beer. It can decompose sugars and produce a variety of enzymes, promote the fermentation process and give the food a good taste and flavor. Compared with other brewing yeasts, Kluyveromyces has some unique characteristics, such as higher temperature adaptability and acid resistance. This makes it more suitable for certain industrial applications under specific conditions, such as high-temperature fermentation and acidic environments. In addition, Kluyveromyces has many advantages as a host system for expressing pharmaceutical proteins. First, through the design of appropriate gene expression vectors and promoter sequences, Kluyveromyces can achieve high-level expression of exogenous genes. Second, Kluyveromyces has a complete protein folding and modification system that can correctly fold complex pharmaceutical proteins and perform necessary glycosylation modifications. Third, Kluyveromyces is easy to culture and can be expressed soluble intracellularly, which facilitates the extraction and purification of target proteins, and the cost of engineered production is low. Overall, Kluyveromyces is a host system with great potential that can be used to efficiently express pharmaceutical proteins.

References

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Summary of the invention

In order to overcome the defects of the prior art, the present invention designs a histidine tag at the N-terminus and C-terminus of the MaPylRS gene, and a thrombin restriction site for convenient tag removal. It is found that adding a sequence at the N-terminus or C-terminus not only promotes the stability of the enzyme, makes it easier to purify, but also further improves the catalytic activity of the enzyme.

In addition, in existing literature reports and commercial kits, MaPylRS protein is manually added exogenously, or a plasmid containing its expression structure is introduced by transformation/transfection, etc. This has a negative impact on the judgment of experimental results, the complexity and stability of experimental results, and the production cost of the target protein. The present invention integrates the MaPylRS gene into the Kluyveromyces genome through the CRISPR/Cas9 efficient gene editing system, achieves stable and efficient expression of the MaPylRS gene in yeast, does not require antibiotic screening and maintenance, and achieves the insertion of non-natural amino acids into the target protein on this basis.

The first invention of the present invention provides a recombinant aminoacyl-tRNA synthetase having the structure described in Formula I:

A1-A2-A3-A4(I):

In Formula I,

A1 is absent or a histidine tag;

A2 is absent or is a thrombin cleavage site;

A3 is absent or a tagged protein;

A4 is an aminoacyl-tRNA synthetase;

"-" is independently a bond or an amino acid linking sequence,

And at least one of A1～A3 exists,

The connection between A1 to A4 can be from the N-terminus to the C-terminus or from the C-terminus to the N-terminus.

Further preferably, the aminoacyl-tRNA synthetase is selected from natural or mutant Pyl-tRNA synthetase (PylRS), Leu-tRNA synthetase (LeuRS), Tyr-tRNA synthetase (TyrRS), Phe-tRNA synthetase (PheRS) or TrP-tRNA synthetase (TrpRS).

Further preferably, the aminoacyl-tRNA synthetase is selected from natural or mutant MaPylRS, MmPylRS, MbPylRS, EcTyrRS, MjTyrRS, EcLeuRS, ScPheRS, ScTrpRS or BsTrpRS.

Further preferably, the aminoacyl-tRNA synthetase is selected from natural or mutated MaPylRS.

Further preferably, the sequence of the aminoacyl-tRNA synthetase is SEQ ID NO:60.

Further preferably, the aminoacyl-tRNA synthetase comprises the sequence shown in SEQ ID NO:60 or an active fragment thereof, or is a polypeptide having ≥85% homology (preferably, ≥90% homology; preferably ≥95% homology; most preferably, ≥97% homology, such as 98% or more, 99% or more) with the amino acid sequence shown in SEQ ID NO:60 and having the same activity as the sequence of SEQ ID NO:60.

More preferably, the structure of the histidine tag is n×His, wherein 1≦n≦50; preferably 2≦n≦30; more preferably, 5≦n≦20; and more preferably 6≦n≦10.

Further preferably, the tag protein is selected from T7 tag, CBP tag, CMyc tag, FLAG tag, Spot tag, C tag, Avi tag, Streg tag, SUMO tag, GST tag, MBP tag or a combination thereof; preferably T7 tag.

Further preferably, the N-terminus or C-terminus of A4 is connected to A1-A2-A3:

More preferably, a his tag is connected to the N-terminus or C-terminus of the aminoacyl-tRNA synthetase.

More preferably, a thrombin cleavage site and a tag protein are connected to the N-terminus of the aminoacyl-tRNA synthetase in order from the N-terminus to the C-terminus.

Further preferably, a his tag, a thrombin cleavage site, and a tag protein are connected to the N-terminus of the aminoacyl-tRNA synthetase in order from the N-terminus to the C-terminus.

Further preferably, the amino acid sequence of the recombinant aminoacyl-tRNA synthetase is selected from any one or more of the following: SEQ ID NO: 1 to SEQ ID NO: 3 or SEQ ID NO: 64.

Further preferably, the recombinant aminoacyl-tRNA synthetase comprises the sequence described in any one of SEQ ID NO:1-3, SEQ ID NO:64 or an active fragment thereof, or is a polypeptide having ≥85% homology (preferably, ≥90% homology; preferably ≥95% homology; most preferably, ≥97% homology, such as 98% or more, 99% or more) with the amino acid sequence described in any one of SEQ ID NO:1-3, SEQ ID NO:64 and having the same activity as the sequence described in any one of SEQ ID NO:1-3, SEQ ID NO:64.

Further preferably, the coding sequence of the recombinant aminoacyl-tRNA synthetase is selected from any one or more of the following: SEQ ID NO: 4, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 63.

Further preferably, the coding sequence of the recombinant aminoacyl-tRNA synthetase comprises any one of SEQ ID NO:4, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63 or an active fragment thereof, or each of which has ≥85% homology (preferably, ≥90% homology; preferably ≥95% homology; most preferably, ≥97% homology, such as 98% or more, 99% or more) with the nucleotide sequence shown above and each of which has the same activity as any one of SEQ ID NO:4, SEQ ID NO:61, SEQ ID NO:62 or SEQ ID NO:63.

The second aspect of the present invention provides a nucleic acid construct encoding the recombinant aminoacyl-tRNA synthetase described in the first aspect of the present invention.

Further preferably, the nucleic acid construct contains at least a nucleic acid sequence as described in Formula II: Z1-Z2-Z3-Z4, wherein Z1 to Z4 are respectively elements used to constitute the construct; "-" is independently a bond or a nucleotide connection sequence; Z1 is absent or is a coding sequence for a histidine tag, Z2 is absent or is a coding sequence for a thrombin cleavage site, Z3 is absent or is a coding sequence for a tag protein, and Z4 is a coding sequence for an aminoacyl-tRNA synthetase; wherein at least one of Z1 to Z3 is present.

The structure of formula II can be from 5' to 3' or from 3' to 5':

That is, the encoding product of Z1-Z2-Z3 is connected to the N-terminus or C-terminus of the encoding product of Z4.

More preferably, the encoding product of Z1-Z2-Z3 is connected to the N-terminus of the encoding product of Z4.

Further preferably, the amino acid sequence encoded by Z1 is HHHHHH; more preferably, Z1 encodes a sequence comprising HHHHHH or an active fragment thereof, or a nucleotide having ≥85% homology with the nucleotide sequence shown above (preferably, ≥90% homology; preferably ≥95% homology; most preferably, ≥97% homology, such as above 98%, above 99%) and having the same activity as the above sequence.

Further preferably, the amino acid sequence encoded by Z2 is LVPRGS; more preferably, Z2 encodes a sequence containing LVPRGS or an active fragment thereof, or a nucleotide having ≥85% homology with the nucleotide sequence shown above (preferably, ≥90% homology; preferably ≥95% homology; most preferably, ≥97% homology, such as above 98%, above 99%) and having the same activity as the above sequence.

Further preferably, the amino acid sequence encoded by the Z3 is SEQ ID NO:59; more preferably, the Z code contains the sequence shown in SEQ ID NO:59 or an active fragment thereof, or is a nucleotide having ≥85% homology with the nucleotide sequence shown above (preferably, ≥90% homology; preferably ≥95% homology; most preferably, ≥97% homology, such as above 98%, above 99%) and having the same activity as the above sequence.

More preferably, the nucleic acid construct further comprises a promoter.

Further preferably, the nucleic acid structure comprises the structure described in Formula III:

Z5-Z1-Z2-Z3-Z4.

Further preferably, the promoter is selected from PGK1, GAP1, ADH1, HXK1, GAPDH1, TEF1 or TIF11.

The third aspect of the present invention provides a vector, wherein the vector contains the nucleic acid construct provided by the second aspect of the present invention.

The fourth aspect of the present invention provides a genetically engineered strain, wherein one or more sites of the genome of the genetically engineered strain are integrated with the nucleic acid construct described in the second aspect of the present invention.

Further preferably, the site is selected from Lys1-5, glpA or UPF1.

More preferably, the nucleic acid construct further comprises a promoter and a terminator.

Further preferably, the promoter is selected from PGK1, GAP1, ADH1, HXK1, GAPDH1, TEF1, TIF11, GAL1, GAL7 or GAL10.

Further preferably, the terminator is selected from CYC1, GPM1, TDH2 or ACT1.

Further preferably, the genetically engineered strain contains the recombinant aminoacyl-tRNA synthetase provided by the first aspect of the present invention.

Further preferably, the genetically engineered strain contains the vector provided in the third aspect of the present invention.

Further preferably, the strain is derived from mammalian cells, plant cells, yeast cells, insect cells, prokaryotic cells or any combination thereof.

The fifth aspect of the present invention provides a method for synthesizing proteins incorporated with non-natural amino acids, using the recombinant aminoacyl-tRNA synthetase described in the first aspect of the present invention, or using the genetically engineered strain described in the fourth aspect of the present invention to provide the recombinant aminoacyl-tRNA synthetase.

The sixth aspect of the present invention provides a cell-free system for synthesizing proteins containing unnatural amino acids, characterized in that the cell-free system at least comprises: (a) a cell extract, and (b) one or more of the recombinant aminoacyl-tRNA synthetase provided by the first aspect of the present invention, the nucleic acid construct provided by the second aspect of the present invention, or the vector provided by the third aspect of the present invention; the cell extract is derived from one of mammalian cells, plant cells, yeast cells, insect cells, prokaryotic cells, or any combination thereof.

The seventh aspect of the present invention provides a cell-free system for synthesizing proteins containing non-natural amino acids, characterized in that the cell-free system at least includes a cell extract, and the cell extract is derived from the genetically engineered strain provided in the fourth aspect of the present invention.

Further preferably, the cell extract is selected from any one or combination of the following sources: Escherichia coli, Kluyveromyces lactis, wheat germ cells, insect cells, rabbit reticulocytes, CHO cells, COS cells, VERO cells, BHK cells, human fibrosarcoma HT1080 cells, or a combination thereof.

Further preferably, the cell extract is derived from yeast cells.

Furthermore, the yeast cell is selected from Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membraneaefaciens, Pichia minuta, Ogataeaminuta, Pichia lindneri, Pichia opuntiae, Pichia thermotolerans ), Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces cerevisiae, Saccharomyces cerevisiae, Saccharomyces molasses, Saccharomyces sp., Hansenula polymorpha, Candida utilis, Kluyveromyces, or a combination thereof.

Furthermore, more preferably, the Kluyveromyces further includes: Kluyveromyces lactis (Kluyveromyces, K.lactis), Kluyveromyces marxianus, Kluyveromyces dobzhanskii, Kluyveromyces aestuarii, Kluyveromyces nonfermentans, Kluyveromyces wickerhamii, Kluyveromyces thermotolerant The yeast cell is one of Kluyveromyces thermotolerans, Kluyveromyces fragilis, Kluyveromyces hubeiensis, Kluyveromyces polysporus, Kluyveromyces siamensis and Kluyveromyces yarrowii, or a combination thereof; preferably, the yeast cell is a Kluyveromyces cell, more preferably a Kluyveromyces lactis cell.

Further preferably, the cell-free system further comprises: a non-natural amino acid, an orthogonal tRNA and a template comprising a target protein gene sequence, wherein the codon encoding the amino acid in the target protein gene sequence is mutated.

More preferably, the non-natural amino acid has a structural formula of the compound of formula (2) or a salt thereof.

wherein n is selected from a natural number of 1-20, _R1 is selected from substituted or unsubstituted C5-C60 aryl or heteroaryl, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl or substituted or unsubstituted C2-C20 alkynyl, and A is selected from O or _-CH2- .

In another preferred embodiment, n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

In another preferred embodiment, n is a natural number selected from 1-10.

In another preferred embodiment, n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In another preferred embodiment, n is a natural number selected from 1-6.

In another preferred embodiment, n is selected from 1, 2, 3, 4, 5 or 6.

In another preferred embodiment, the R ₁ is selected from substituted or unsubstituted C5-C30 aryl or heteroaryl.

In another preferred embodiment, the R ₁ is selected from substituted or unsubstituted phenyl.

In another preferred embodiment, the R ₁ is selected from substituted or unsubstituted C2-C20 alkenyl.

In another preferred embodiment, the R ₁ is selected from substituted or unsubstituted C2-C10 alkenyl.

In another preferred embodiment, the R ₁ is selected from substituted or unsubstituted C2-C6 alkenyl.

In another preferred embodiment, the R ₁ is selected from substituted or unsubstituted C2-C20 alkynyl.

In another preferred embodiment, the R ₁ is selected from substituted or unsubstituted C2-C10 alkynyl.

In another preferred embodiment, the R ₁ is selected from substituted or unsubstituted C2-C6 alkynyl.

In another preferred embodiment, the A is selected from O.

In another preferred embodiment, the A is selected from -CH ₂ -.

In another preferred embodiment, the substituents are common substituents in the art, such as aryl, heteroaryl, alkyl, cycloalkyl, aryloxy, heteroaryloxy, alkyloxy, cycloalkyloxy, hydroxyl, thiol, ester, carboxyl, cyano, halogen, nitro, sulfonic acid, azido, alkenyl, alkynyl, phosphate, etc.

In another preferred embodiment, the structural formula of the non-natural amino acid is selected from one or a combination of the following, or a salt form thereof:

Further preferably, the target protein is selected from: luciferin, luciferase (such as firefly luciferase), fluorescent protein (such as green fluorescent protein, yellow fluorescent protein), aminoacyl-tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, variable region of antibody, luciferase mutation, α-amylase, enterobactin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon αA, cytokine, interferon α2b, interleukin-1β, lysozyme, serum albumin, single-chain antibody fragment (scFV), thyroxine transporter, tyrosinase, xylanase or a combination thereof.

Further preferably, the target protein includes a wild-type protein, a mutant protein or a recombinant protein.

The eighth aspect of the present invention provides a method for preparing the genetically engineered strain described in the fourth aspect of the present invention, characterized in that the nucleic acid construct described in the second aspect of the present invention is transferred or integrated into a cell through transformation, transfection or gene editing technology.

Further preferably, the nucleic acid construct described in the second aspect of the present invention is integrated into the genome of the cell by gene editing technology.

Further preferably, the nucleic acid construct is integrated into the genome of the cell via the active site.

More preferably, the nucleic acid construct further comprises a promoter and a terminator.

Further preferably, the promoter is selected from PGK1, GAP1, ADH1, HXK1, GAPDH1, TEF1 or TIF11.

Further preferably, the terminator is selected from CYC1, GPM1, TDH2 or ACT1.

Further preferably, the site is selected from Lys1-5, glpA or UPF1.

The ninth aspect of the present invention provides a kit, characterized in that the kit contains the reaction system described in the sixth aspect or the seventh aspect of the present invention.

The tenth aspect of the present invention provides a method for in vitro synthesis of proteins containing non-natural amino acids, characterized in that the method is prepared using the cell-free system described in the sixth or seventh aspect of the present invention or the kit described in the ninth aspect of the present invention.

The advantages of the present invention are:

(1) The His tag structures were designed at the N-terminus and C-terminus of the natural or modified MaPylRS synthetase, which further promoted the stability of the enzyme, made it easier to purify it, and increased the catalytic activity of the enzyme, which was beneficial to improving the efficiency of the introduction of non-natural amino acids.

(2) Connecting a His tag, T7 tag, and thrombin restriction site to the N-terminus of the natural or modified MaPylRS synthetase will further promote the stability of the enzyme, make it easier to purify, and increase the catalytic activity of the enzyme, which is beneficial to improving the efficiency of the introduction of non-natural amino acids;

(3) The N-terminal T7 tag and thrombin cleavage site of the natural or modified MaPylRS synthetase will further promote the stability of the enzyme, make it easier to purify, and increase the catalytic activity of the enzyme, which is beneficial to improve the efficiency of the introduction of non-natural amino acids.

(4) The present invention integrates the aforementioned designed MaPylRS recombinant expression structure into the cell genome through CRISPR/Cas9 combined with efficient transformation technology, thereby achieving the stable existence of MaPylRS in the cell genome and the continuous expression of MaPylRS protein.

(5) The Kluyveromyces strain inserted with the recombinant MaPylRS was prepared into an in vitro expression system, which achieved the site-specific insertion of non-natural amino acids into the exogenous target protein, greatly simplified the preparation steps, saved costs, and increased the stability of the synthesized protein with the non-natural amino acid insertion.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the construction diagram of long N-terminal mapylrs

Figure 2 shows the N-his mapylrs construction diagram

Figure 3 shows the construction diagram of C-his mapylrs

Figure 4 shows the electrophoresis of the lysis supernatant of the three proteins after induction, where 1 represents long N-terminal mapylrs, 2 represents N-his mapylrs, and 3 represents C-his mapylrs. The mapylrs with longer N-terminal have higher expression levels and higher protein solubility. The highest protein concentration can reach 120 mg/mL (3.4 mM).

Figure 5 shows the comparison of the activities of proteins constructed and expressed in three forms when unnatural amino acids were introduced at the same concentration. All three forms of MAPYRS can catalyze the expression of proteins containing unnatural amino acids. The inverted triangle in the figure represents the absence of unnatural amino acids, and the dot represents the addition of unnatural amino acids. Nhis represents N-his MAPYRS, chis represents C-his MAPYRS; N-terminal represents long N-terminal MAPYRS.

Figure 6 shows the comparison of the activities of original mapylrs (referring to unrecombinant mapylrs) and recombinant mapylrs (long N-terminal mapylrs and N-his mapylrs) in introducing non-natural amino acids. In the figure, nhis represents N-his mapylrs, long terminal represents long N-terminal mapylrs, and the left part of each comparison bar represents the addition of non-natural amino acids, and the right part represents the addition of non-natural amino acids.

Figure 7 shows the RFP/GFP comparison between the original mapylrs and the recombinant mapylrs (long N-terminal mapylrs and N-his mapylrs) after the introduction of non-natural amino acids. In the figure, nhis represents N-his mapylrs, long terminal represents long N-terminal mapylrs, and no tag represents the original mapylrs.

FIG8 shows a schematic diagram of the structure of the pKM-CAS1.0-K1Lys1-5 plasmid.

FIG. 9 shows a schematic diagram of the structure of the pKM-MaPylRS plasmid.

FIG. 10 shows a schematic diagram of the structure of the pKM-CAS1.0-KlglpA plasmid.

FIG. 11 shows a schematic diagram of the structure of the pKM-CAS1.0-K1UPF1 plasmid.

FIG. 12 is a graph showing the RFP activity measured in Example 9.

FIG. 13 is a graph showing the GFP activity measured in Example 9.

FIG. 14 is a graph showing the RFP/GFP activity measured in Example 9.

FIG. 15 is a graph showing the GFP activity measured in Example 10.

FIG. 16 is a graph showing the RFP activity measured in Example 10.

FIG. 17 is a graph showing the RFP/GFP activity measured in Example 10.

In the figures herein, the words “non” or “control” refer to the addition of non-natural amino acids in the reaction.

DETAILED DESCRIPTION

It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application may be combined with each other.

The present invention is further described below in conjunction with specific embodiments and examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are preferably followed or referred to the conditions indicated in the specific embodiments described above, and then can be followed according to conventional conditions or according to the conditions recommended by the manufacturer.

Unless otherwise specified, the percentages and parts mentioned in the present invention are percentages and parts by weight.

Unless otherwise specified, the materials and reagents used in the examples of the present invention are all commercially available products.

Unless otherwise specified, the temperature units in this application are all degrees Celsius (°C).

Nouns and terms

The following is an explanation or description of the meaning of some relevant "nouns" and "terms" used in the present invention, so as to better understand the present invention. The corresponding explanation or description applies to the full text of the present invention, both below and above. When the present invention involves references, the definitions of relevant terms, nouns, and phrases in the references are also quoted, but when they conflict with the definitions in the present invention, the definitions in the present invention shall prevail. When the definitions in the references conflict with the definitions in the present invention, it does not affect the cited components, substances, compositions, materials, systems, formulations, types, methods, equipment, etc., which shall be subject to the contents determined in the references.

In the present invention, preferred embodiments such as “preferred”, “better”, “more preferred”, “better”, “most preferred” and “further preferred” do not constitute any limitation on the scope of coverage and protection scope of the invention, and are not used to limit the scope and embodiments of the present invention, but are only used to provide some embodiments as examples.

In the description of the present invention, preferred modes such as "one of the preferred embodiments", "one of the preferred modes", "one of the preferred embodiments", "one of the preferred examples", "preferred examples", "in a preferred embodiment", "some preferred examples", "some preferred modes", "preferably", "preferably", "preferably", "more preferably", "more preferably", "further preferably", "most preferably", and illustrative enumeration modes such as "one of the embodiments", "one of the modes", "example", "specific example", "for example", "as an example", "for example", "such as", "such as", etc., do not constitute any limitation on the scope of coverage and protection scope of the invention, and the specific features described in each mode are included in at least one specific embodiment of the present invention. In the present invention, the specific features described in each mode may be combined in a suitable manner in any one or more specific embodiments. In the present invention, the technical features or technical solutions corresponding to each preferred mode may also be combined in any suitable manner.

In the present invention, "any combination thereof" means "greater than 1" in terms of quantity, and means a group consisting of the following situations in terms of coverage: "select any one of them, or select a group consisting of at least two of them".

In the present invention, the descriptions of "one or more", "one or more" and "one or more" have the same meaning as "at least one", "at least one", "a combination thereof", "or a combination thereof", "and combinations thereof", "or any combination thereof", "and any combination thereof", etc. and can be used interchangeably to indicate that the quantity is equal to "1" or "greater than 1".

In the present invention, "or/and" and "and/or" are used to represent "optionally one of them or optionally a combination of them", and also represent at least one of them.

The term "about" can refer to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Sequence identity (or homology) is determined by comparing two aligned sequences along a predetermined comparison window (which can be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of the reference nucleotide sequence or protein) and determining the number of positions at which identical residues occur. Typically, this is expressed as a percentage. The measurement of sequence identity of nucleotide sequences is a method well known to those skilled in the art.

The prior art means described in the present invention in such ways as “usually”, “conventional”, “general”, “frequently” and “often” are also cited as references to the contents of the present invention. Unless otherwise specified, they can be regarded as one of the preferred ways of some technical features of the present invention. It should be noted that they do not constitute any limitation on the scope of coverage and protection scope of the invention.

All documents mentioned in the present invention and documents directly or indirectly cited by these documents are cited in this application as references, just as if each document was cited individually as a reference.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (including but not limited to the embodiments) can be combined with each other to form a new or preferred technical solution, as long as it can be used to implement the present invention. Due to space limitations, they will not be described one by one.

In vitro protein synthesis reaction refers to the reaction of synthesizing protein in an in vitro cell-free synthesis system, including at least the translation process. Including but not limited to IVT reaction (in vitro translation reaction), IVTT reaction (in vitro transcription and translation reaction), IVDTT reaction (in vitro replication transcription and translation reaction). In the present invention, IVTT reaction is preferred. IVTT reaction, corresponding to IVTT system, is the process of transcribing and translating DNA into protein (Protein) in vitro. Therefore, we also refer to this type of in vitro protein synthesis system as D2P system, D-to-P system, D_to_P system, DNA-to-Protein system; the corresponding in vitro protein synthesis method is also called D2P method, D-to-P method, D_to_P method, DNA-to-Protein method.

"Cell-free system" refers to a method of in vitro protein synthesis that is not secretory expression through intact cells. It should be noted that in the in vitro cell-free protein synthesis system of the present invention, it is also allowed to add cell components to promote the reaction, but the added cells are not primarily intended to secrete and express exogenous target proteins. In addition, in the intact cell-free D2P system constructed under the guidance of the present invention, a small amount of intact cells are intentionally added (for example, the protein content provided by them is not more than 30wt% compared to the protein content provided by the cell extract). Such a "circumvention" method is also included in the scope of protection of the present invention.

Target protein: The target expression product of the in vitro protein synthesis system of the present invention is not synthesized by secretion from the host cell, but is synthesized in vitro based on an exogenous nucleic acid template, and can also be called an exogenous protein. The exogenous protein can be a protein, a fusion protein, a mixture of protein molecules or fusion protein molecules; it also broadly includes polypeptides. The product obtained after an in vitro protein synthesis reaction based on a nucleic acid template encoding the target protein can be a single substance or a combination of two or more substances. "Exogenous protein", "target protein", "target protein", "target translation product" have the same meaning and can be translated as "objective protein", "interested protein", "objective translated product", "interested protein product", etc., and can be used interchangeably in the present invention.

D2P, DNA-to-Protein, from DNA template to protein product. For example, D2P technology, D2P system, D2P method, D2P kit, etc.

"The expression system of the present invention", "the in vitro expression system of the present invention", "the in vitro cell-free expression system", "the in vitro cell-free expression system" can be used interchangeably, all referring to the in vitro protein expression system of the present invention, and other descriptions can also be used, such as: protein in vitro synthesis system, in vitro protein synthesis system, cell-free system, cell-free system, cell-free protein synthesis system, cell-free in vitro protein synthesis system, in vitro cell-free protein synthesis system, in vitro cell-free synthesis system, CFS system (cell-free system), CFPS system (cell-free protein synthesis system) and other descriptions. According to the reaction mechanism, it can include an in vitro translation system (which can be abbreviated as IVT system, a mR2P system), an in vitro transcription translation system (which can be abbreviated as IVTT system, a D2P system), an in vitro replication transcription translation system (which can be abbreviated as IVDTT system, a D2P system), etc. In the present invention, the IVTT system is preferred. We also call the in vitro protein synthesis system a "protein synthesis factory" ("Protein Factory" or "proteinfactory" or "Proteinfactory"). The in vitro protein synthesis system provided by the present invention adopts an open description method for its components. The cell-free protein synthesis system of the present invention uses exogenous DNA, mRNA or a combination thereof as a nucleic acid template for protein synthesis, and achieves in vitro synthesis of the target protein by artificially controlling the addition of substrates and transcription, translation-related protein factors and other substances required for protein synthesis.

In the present invention, "protein" and "protein" have the same meaning, are both translated as protein, and can be used interchangeably.

In the present invention, "system" and "system" are both translated as system and can be used interchangeably.

In the present invention, "protein synthesis amount", "protein expression amount" and "protein expression yield" have the same meaning and can be used interchangeably.

In the present invention, cell extract, cell extract, cell lysate, cell disruption and cell lysis product have the same meaning and can be used interchangeably. In English, cell extract, cell lysate and the like can be used as descriptions.

In the present invention, energy system, energy system, and energy supply system have the same meaning and can be used interchangeably. Energy regeneration system and energy regeneration system have the same meaning and can be used interchangeably. The energy regeneration system is a preferred embodiment or component of the energy system.

Furthermore, the present invention provides a cell-free protein synthesis system, which at least includes a cell extract or a cell lysate.

Further preferably, the cell-free protein synthesis system further comprises one or more components selected from the following group: a substrate for synthesizing RNA, a substrate for synthesizing protein, polyethylene glycol or its analogues, magnesium ions, potassium ions, a buffer, RNA polymerase, an energy regeneration system, dithiothreitol, and an optional aqueous solvent.

Further preferably, the substrate for synthesizing RNA includes: nucleoside monophosphate, nucleoside triphosphate or a combination thereof.

Further preferably, the substrate for synthesizing protein includes: 20 natural amino acids and unnatural amino acids.

Further preferably, the magnesium ions are derived from a magnesium ion source, and the magnesium ion source is selected from the following group: magnesium acetate, magnesium glutamate, or a combination thereof.

Further preferably, the potassium ions are derived from a potassium ion source, and the potassium ion source is selected from the following group: potassium acetate, potassium glutamate, or a combination thereof.

Further preferably, the energy regeneration system is selected from the following group: one or a combination of creatine phosphate/creatine phosphate enzyme system, glycolysis pathway and its intermediate energy system.

Further preferably, the energy regeneration system comprises a glucose/phosphate system, and the phosphate is selected from the following group: tripotassium phosphate, triammonium phosphate, trisodium phosphate, dipotassium hydrogen phosphate, diammonium hydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, ammonium dihydrogen phosphate, sodium dihydrogen phosphate, or a combination thereof.

Further preferably, the buffer is selected from the following group: 4-hydroxyethylpiperazineethanesulfonic acid, tris(hydroxymethyl)aminomethane, or a combination thereof.

Further preferably, the in vitro protein synthesis system contains polyethylene glycol (PEG) or its analogs. The concentration of polyethylene glycol or its analogs is not particularly limited. Generally, the concentration (w/v) of polyethylene glycol or its analogs is 0.1-8%, preferably 0.5-4%, more preferably 1-2%, based on the total weight of the protein synthesis system. Representative PEG is selected from the following group: one of PEG3000, PEG3350, PEG6000, PEG8000 or a combination thereof.

Further preferably, the polyethylene glycol includes polyethylene glycol with a molecular weight (Da) of 200-10000, such as PEG200, 400, 1500, 2000, 4000, 6000, 8000, 10000, etc., preferably, polyethylene glycol with a molecular weight of 3000-10000.

In the present invention, the RNA polymerase is not particularly limited and can be selected from one or more RNA polymerases. A typical RNA polymerase is T7 RNA polymerase.

An optional scheme is that the in vitro protein synthesis system provided by the present invention includes: cell extract, 4-hydroxyethylpiperazineethanesulfonic acid, potassium acetate, magnesium acetate, adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytosine triphosphate (CTP), thymidine triphosphate (TTP), amino acid mixture, creatine phosphate, dithiothreitol (DTT), creatine phosphate kinase, and RNA polymerase.

In the present invention, the cell extract does not contain intact cells, and a typical cell extract includes ribosomes, aminoacyl-tRNA synthetases, initiation factors and elongation factors required for protein synthesis, and termination release factors for protein translation. In addition, the cell extract also contains some other proteins derived from the cytoplasm of the cell, especially soluble proteins.

In the present invention, the proportion of the cell extract in the in vitro cell-free protein synthesis system is not particularly limited. Usually, the cell extract accounts for 20-70% of the in vitro cell-free protein synthesis system, preferably 30-60%, and more preferably 40-50%.

In the present invention, the protein content of the cell extract is 20-100 mg/mL, preferably 50-100 mg/mL. The method for determining the protein content is the Coomassie Brilliant Blue determination method.

The present invention also provides a vector or vector combination, wherein the vector contains the nucleic acid construct of the present invention. Preferably, the vector is selected from: bacterial plasmid, bacteriophage, yeast plasmid, animal cell vector, shuttle vector; the vector is a transposon vector. Methods for preparing recombinant vectors are well known to those of ordinary skill in the art. As long as they can replicate and be stable in the host, any plasmid and vector can be used.

Those skilled in the art can use well-known methods to construct expression vectors containing the promoter and/or target gene sequence of the present invention, including in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc.

Template DNA

The template DNA is a nucleotide sequence encoding any target protein to be synthesized, which may be an original sequence, an artificially synthesized sequence, or an artificially modified sequence. The template DNA may be used to synthesize the corresponding RNA and/or protein.

In the present invention, the preparation method of the cell extract is not limited. A preferred preparation method is

The following steps are involved:

(i) providing cells;

(ii) washing the cells to obtain washed cells;

(iii) disrupting the washed cells to obtain a crude cell extract;

(iv) subjecting the crude cell extract to solid-liquid separation to obtain a liquid portion, namely the cell extract.

In the present invention, the solid-liquid separation method is not particularly limited, and a preferred method is centrifugation.

In a preferred embodiment, the centrifugation is performed in a liquid state.

In the present invention, the centrifugation conditions are not particularly limited, and a preferred centrifugation condition is 5000-100000 g, preferably 8000-30000 g.

In the present invention, the centrifugation time is not particularly limited, and a preferred centrifugation time is 0.5 min-2 h, preferably 20 min-50 min.

In the present invention, the centrifugation temperature is not particularly limited. Preferably, the centrifugation is performed at 1-10°C, more preferably, at 2-6°C.

In the present invention, the washing treatment method is not particularly limited. A preferred washing treatment method is to use a washing liquid at a pH of 7-8 (preferably 7.4). The washing liquid is not particularly limited. Typically, the washing liquid is selected from the following group: potassium 4-hydroxyethylpiperazineethanesulfonate, potassium acetate, magnesium acetate, or a combination thereof.

In the present invention, the cell disruption treatment method is not particularly limited. A preferred cell disruption treatment includes high-pressure disruption and freeze-thaw (such as liquid nitrogen cryogenic) disruption.

The nucleoside triphosphate mixture in the in vitro cell-free protein synthesis system is adenosine triphosphate, guanosine triphosphate, cytosine triphosphate and uridine triphosphate. In the present invention, the concentration of various mononucleotides is not particularly limited, and usually the concentration of each mononucleotide is 0.5-5mM, preferably 1.0-2.0mM.

The amino acid mixture in the in vitro cell-free protein synthesis system may include natural or non-natural amino acids, and may include D-type or L-type amino acids. Representative amino acids include (but are not limited to) 20 natural amino acids: glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine. The concentration of each amino acid is generally 0.01-0.5 mM, preferably 0.02-0.2 mM, such as 0.05, 0.06, 0.07, 0.08 mM.

In a preferred embodiment, the in vitro cell-free protein synthesis system further contains polyethylene glycol or its analogs. The concentration of polyethylene glycol or its analogs is not particularly limited. Generally, the concentration (w/v) of polyethylene glycol or its analogs is 0.1-8%, preferably 0.5-4%, more preferably 1-2%, based on the total weight of the biosynthesis system. Representative examples of PEG include (but are not limited to): PEG3000, PEG8000, PEG6000 and PEG3350. It should be understood that the system of the present invention may also include polyethylene glycols of various other molecular weights (such as PEG200, 400, 1500, 2000, 4000, 6000, 8000, 10000, etc.).

In a preferred embodiment, the in vitro cell-free protein synthesis system further contains sucrose. The concentration of sucrose is not particularly limited, and generally, the concentration of sucrose is 0.03-40wt%, preferably 0.08-10wt%, and more preferably 0.1-5wt%, based on the total weight of the protein synthesis system.

A particularly preferred in vitro cell-free protein synthesis system, in addition to yeast cell extract, also contains the following components: 22mM 4-hydroxyethylpiperazineethanesulfonic acid with a pH of 7.4, 30-150mM potassium acetate, 1.0-5.0mM magnesium acetate, 1.5-4mM nucleoside triphosphate mixture, 0.08-0.24mM amino acid mixture, 25mM creatine phosphate, 1.7mM dithiothreitol, 0.27mg/mL creatine phosphate kinase, 1%-4% polyethylene glycol, 0.5%-2% sucrose, and 0.027-0.054mg/mL T7 RNA polymerase.

The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention.

It is not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions, such as the conditions described in Sam Brook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts. The present invention takes Kluyveromyces lactis (K. lactis or kl for short) as an example, but the same design, analysis and experimental methods are also applicable to other eukaryotic cells such as yeast and animal cells, as well as prokaryotic cells.

The present invention uses Kluyveromyces lactis (K. lactis) as an example, but the same design, analysis and experimental methods are also applicable to other yeasts and other lower eukaryotic cells and higher animal cells. The gene modification method in the present invention is CRISPR-Cas9 technology, but it is not limited to this, and can be any known and existing gene modification method.

The in vitro protein synthesis reaction mixture system, also described as an in vitro protein synthesis reaction mixture, a reaction mixture system, or a reaction mixture, refers to a mixed system including an in vitro protein synthesis system and a nucleic acid template encoding a target protein; it may be homogeneous or heterogeneous, and may be a liquid system such as a solution, an emulsion, or a suspension.

Protein of the present invention The final concentrations of the components in Factory are: 80% (v/v) Kluyveromyces lactis extract, 15 mM glucose, 320 mM maltodextrin (measured in molar concentration of glucose monomers), 24 mM tripotassium phosphate, 1.8 mM nucleoside triphosphate mixture (a mixture of adenosine triphosphate, guanosine triphosphate, cytosine triphosphate and uridine triphosphate, with the final concentration of each nucleoside triphosphate being 1.8 mM), 0.7 mM amino acid mixture (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine, with the final concentration of each amino acid being 0.7 mM), magnesium L-aspartate, 80 mM potassium acetate, 2% (w/v) polyethylene glycol 8000, 9.78 mM pH 8.0 Tris·HCl buffer, 6% (w/v) trehalose. The preparation process of Kluyveromyces lactis cell extract adopts conventional technical means, and is prepared by referring to the method described in CN109593656A. In summary, the preparation steps include: providing an appropriate amount of raw materials of Kluyveromyces lactis cells that have been fermented and cultured, freezing the cells with liquid nitrogen, breaking the cells, and collecting the supernatant by centrifugation to obtain a cell extract. The protein concentration in the obtained Kluyveromyces lactis cell extract is 20-40 mg/mL. In the following examples, (i.e., prock, N-E-propargyloxycarbonyl-L-lysine hydrochloride) is a representative of non-natural amino acids (referred to as NCAA), but the NCAA in this application is not limited to only referring to prock.

Example 1: Three methods for constructing Mapylrs proteins:

The first type, Long N-terminal mapylrs:

The N-terminus of the Mapylrs protein contains a 31-amino acid leader peptide, which contains a 6*His affinity purification tag, a T7 tag, and a thrombin restriction site (Figure 1):

Long N-terminal mapylrs amino acid sequence:

Theoretical molecular weight: 34349.98Da

The second type, N-his mapylrs:

The Mapylrs protein has only a 6*His affinity purification tag at the N-terminus, as well as a flexible interface (Figure 2):

Theoretical molecular weight of N-his MAPYRS: 32091.35Da

The third type, C-his mapylrs:

The N-terminus of the Mapylrs protein has one more Gly than the reported sequence, and the C-terminus has an affinity purification tag 6*His (Figure 3):

C-His MAPYLRs theoretical molecular weight: 31685.98

The fourth type, compared to the Long N-terminal mapylrs, removes the his tag:

Example 2: Construction of expression plasmid

Long-N-terminal MAPYLRs expression plasmid:

We commissioned Sangon Biotech to synthesize the full mapylrs gene (WP_015505008), in which the codons were optimized for E. coli expression. The synthesized gene sequence is:

N

Xho

Insert into pET28a vector through NdeI/Xho I restriction site.

The N-his maplys and C-his maplys expression vectors were modified based on pET28a-long-terminal mapylrs by PCR. The primers used were:

N-his mapylrs

C-his mapylrs:

C-his vector R:

The amplified product was digested with DnpI, ligated and transformed into DH5α competent cells, and the plasmid was extracted and sequenced.

Example 3

1) Pick a single bacterial colony and inoculate 100 ml LB (containing 100 mg/L kanamycin) at 37°C overnight.

2) The next day, the cells were inoculated into fresh LB medium (containing 100 mg/L kanamycin) at a ratio of 1:100, cultured at 37°C until OD600≈0.6, and IPTG was added at a final concentration of 0.1 mM. Expression was induced at 16°C for 20 hr.

3) Collect the cells by centrifugation at 5000 rpm for 20 min at 4°C, and resuspend 20 g of wet cells in 100 ml of lysis buffer (25 mM Tris-HCl, 500 mM NaCl, 25 mM imidazole, 5 mM β-mercaptoethanol, 1 mM PMSF, 0.1% Triton X-100). Lyse the cells using a high-pressure homogenizer.

4) Centrifuge twice at 4°C, 20,000 rpm for 20 min, remove bacterial debris repeatedly, and separate and purify the target protein using a HisTrap affinity chromatography column. Buffer A: 25 mM TrisHCl pH 7.8, 500 mM NaCl, 25 mM imidazole; Buffer B: 25 mM TrisHCl pH 7.8, 150 mM NaCl, 500 mM imidazole;

5) After the cell lysis supernatant flows through the affinity chromatography column, the affinity column is repeatedly washed with 10 cv of buffer A, and then the target protein is eluted with 10 cv of buffer B in a gradient from 0% to 100%.

6) Collect and combine the fractions containing the target protein and concentrate the sample using an ultrafiltration centrifuge tube;

7) After the concentrated sample is dialyzed against the dialysis buffer: 50% glycerol, 25mM Hepes pH7.5, the protein concentration is determined and the sample is aliquoted. It can be stored at -80°C for at least 1 year. As shown in Figure 4, the first three forms of recombinant mapylrs are lysed after induction of expression. The mapylrs protein with a longer N-terminal has a higher expression level and a higher solubility. The highest protein concentration can reach 120mg/mL (3.4mM).

Example 4 Comparison of the activity of proteins purified by three construction methods

The concentrations of the three proteins were measured using nanodrop, and then adjusted to the same level using ddH2O that did not contain DNAase RNase.

Establishment of expression system containing unnatural amino acids:

Protein Factory 100ul

Prock (unnatural amino acid) 500mM 1ul

matRNA _CUA ^pyl in vitro transcription product (unpurified) 10ul

GFP-TAG-RFP dual fluorescence reporter gene PCR product 3ul

The final concentration of purified proteins of different forms of Mapylrs was 5uM

Ex485nm/Em535 detects the GFP fluorescence intensity, Ex535nm/Em595nm detects the RFP fluorescence intensity, and the introduction efficiency of the unnatural amino acid is determined based on the RFP fluorescence intensity and the RFP/GFP ratio (see Figure 5 for details).

As shown in Figure 5, according to RFP/GFP, all three forms of Mapylrs can achieve efficient introduction of unnatural amino acids and have high activity. Among them, the N-terminal tag stabilizes the protein structure (Long-N-terminal mapylrs) to make it more active.

Example 5 detected the effects of the original Mapylrs (ie, non-recombinant Mapylrs) and the purified products of N-terminal tagged Mapylrs on the activity of the introduction of non-natural amino acids.

The activities of original Mapylrs, Long-N-terminal mapylrs and N-his maplys were compared. The activity measurement method was the same as in Example 4, using the dual fluorescent protein expression method.

As can be seen from Figures 6 and 7, compared with the original Mapylrs (no tag in the figure), Long-N-terminal mapylrs and N-his maplys have improved activities for the introduction of non-natural amino acids, indicating that after the modification of the present invention, the recombinant Mapylrs obtained have obvious progress in the introduction of non-natural amino acids compared with the original Mapylrs.

In order to overcome the defects of the existing non-natural amino acid insertion system that requires the exogenous manual addition of MaPylRS protein, or the need to introduce a plasmid containing its expression structure by transformation/transfection, the present invention further discloses the integration of MaPylRS protein into the cell genome through gene editing technology, creating a strain that can stably express MaPylRS protein in an appropriate amount, thereby forming a simple and efficient non-natural amino acid insertion system without the need for exogenous addition of MaPylRS.

In the following, only the Long-N-terminal maplyrs with the his tag removed is taken as an example (i.e., the fourth structure is used) to verify the integration of MaPylRS protein into the cell genome, which does not limit other MaPylRS of the present invention.

Example 6 Inserting the MaPylRS expression cassette (i.e., the expression cassette of the fourth structure, the same below) near KlLys1-5 by CRISPR-Cas9 technology

(1) KlLys1-5 sequence retrieval and CRISPR gRNA sequence determination

In order not to affect the normal expression of other genes of Kluyveromyces lactis, the present invention inserts the MaPylRS expression structure near the tDNA KlLys1-5 of Kluyveromyces lactis, and after expression, it combines with non-natural amino acids to perform site-specific insertion.

i. tRNA-Lys-CTT-1-5 was retrieved from http://gtrnadb.ucsc.edu/GtRNAdb2/genomes/eukaryota/Kluy_lact_NRRL_Y_1140/Kluy_lact_NRRL_Y_1140-gene-list.html, and the Lys1-5 gene sequence in K. lactis yeast was obtained. In the present invention, this sequence is named K1Lys1-5 (located at 856274-856346 of chromosome E).

ii. Search for the PAM sequence (NGG) within 500bp upstream and downstream of KlLys1-5, and finally select the PAM located downstream of the gene (sites 856876...856878 of chromosome E), and determine the KlLys1-5 gRNA sequence (GTTCCCATTGATCCCATATC (SEQ ID NO: 11), located at sites 856856...856875 of chromosome E).

(2) Construction of KlLys1-5 CRISPR-Cas9 plasmid

According to the designed gRNA sequence, design two 24nt primers required for vector construction. gRNA-F1: AATCGTTCCCATTGATCCCATATC (SEQ ID NO: 12); gRNA-R1: AAACGATATGGGATCAATGGGAAC (SEQ ID NO: 13) Dilute the primers to 10μM, add 10μL of gRNA-F1 and gRNA-R2 to the PCR tube, mix and centrifuge to the bottom of the tube, and anneal according to the following procedure:

95℃, 3min; 72℃, 30s; 65℃, 2min; 60℃, 2min; 55℃, 2min; 50℃, 2min; 16℃, 2min, then carry out the connection reaction

A. Reaction system: 10×Buffer 1μL, plasmid 20-50ng, annealing product 1μL, enzyme 0.2μL, add water to 10μL.

B. Reaction procedure: 16°C, 60 min.

Take 50 μL of commercial E. coli DH5α competent cells, add all the ligation products and mix well, and complete the transformation process according to the instructions. Screen on LB plates containing 50 mg/L kanamycin and culture overnight. Pick 5 single clones and shake culture in LB liquid culture medium. After sequencing to confirm the positive, extract the plasmid and save it, named pKM-CAS1.0-KlLys1-5 (Figure 8).

(3) Donor DNA construction and amplification

First, the donor Donor, i.e. the homologous recombination sequence of KlLys1-5 sites and the MaPylRS expression structure, was constructed. The promoter of MaPylRS was KlPGK1 promoter, and the terminator was ScCYC1 terminator. The donor DNA construction and transformation method were as follows:

iii. Gene synthesize a plasmid containing the MaPylRS expression cassette (named pKM-MaPylRS, see Figure 9), and use it as a template to perform PCR amplification with primers PF1: AATGTTCCATTGATCCCATATCCTTCGAGCGTCCCAAAACC (SEQ ID NO: 14) and primer PR1: TTCAGTTCAAAAACGCCCCGTTCCTCATCACTAGAAG (SEQ ID NO: 15) to obtain the MaPylRS expression cassette fragment.

iv. Using the lactic acid Kluyveromyces free plasmid as a template, primers PF2: GTTATTAATGTCGTGTGCCATAGGT (SEQ ID NO: 16) and primer PR2: AGGTTTTGGGACGCTCGAAGGATATGGGATCAATGGGAA (SEQ ID NO: 17) were used for PCR amplification to obtain the homology arm 1 fragment of the KlLys1-5 site; using the lactic acid Kluyveromyces free plasmid as a template, primers PF3: TTCTAGTGATGAGGAACGGGGCGTTTTTGAACTGAATTTCG (SEQ ID NO: 18) and primer PR3: CAGCATAGCATTTGAGTATTGTG (SEQ ID NO: 19) were used for PCR amplification to obtain the homology arm 2 fragment of the KlLys1-5 site.

v. Mix the three PCR product fragments obtained above, dilute them 100 times and use them as templates, and perform PCR amplification again with primer PF4: ATAGGTCAATTAATAATATGCCAGCAAT (SEQ ID NO: 20) and primer PR4: GGGGAGCATAGCATTCAAAAACTTC (SEQ ID NO: 21); the three fragments mentioned above can be connected by overlapping extension PCR to form a linear donor DNA Donor. After sequencing confirmation, store it in a -20℃ refrigerator.

(4) Preparation and transformation of efficient Kluyveromyces lactis competent cells

Preparation of competent yeast

Streak the Kluyveromyces lactis liquid on YPD solid medium and pick a single clone, shake culture overnight in 25mL 2×YPD liquid medium, take 2mL of the liquid and continue shaking culture in 50mL liquid 2×YPD medium for 2-8h. Collect yeast cells by centrifugation at 3000g for 5min at 20℃, add 500μL sterile water to resuspend, and collect cells by centrifugation under the same conditions. Prepare competent cell solution (5% v/v glycerol, 10% v/v DMSO) and dissolve yeast cells in 500μL of the solution. Aliquot 50μL into 1.5mL centrifuge tubes and store at -80℃.

Yeast DNA transformation

Thaw the competent cells on ice for 30 seconds, add 200ng of pKM-CAS1.0-KlLys1-5 plasmid and 2000ng of donor DNA. Immediately add 1mL YPD liquid medium after electric shock at 1.5kV for 5mS, culture for 2-3h, and apply 200μL on solid YPD (200μg/mL G418) medium, and culture for 2-3 days until single colonies appear.

(5) Positive identification of gene editing

50-60 single clones were picked from the plate after transformation of Kluyveromyces lactis, and each single clone was placed in 5 μL yeast lysis buffer (Takara Mighty Prep Reagent for DNA). The bacterial lysate was used as a template and the primers Ma R1 (primer within the MaPylRS sequence): ATCTCTTACTTGAACGGTGCTA (SEQ ID NO: 22); 1-5F1 (primer outside the 5' of KlLys1-5 donor DNA): GGTTATCCATTCAGGCAA PCR amplification was performed using primers Ma F1 (primer within the MaPylRS sequence): CCATGTGAGAACCTCTTGG (SEQ ID NO: 24); 1-5R1 (primer outside the 5’ of KlLys1-5 donor DNA): CAGCATAGCATTTGAGTATTGTG (SEQ ID NO: 25) to detect CRISPR insertion at the KlLys1-5 site. The presence of a positive band indicated that the MaPylRS sequence was successfully inserted into the target site.

Example 7 Insertion of the MaPylRS expression cassette near KlglpA by CRISPR-Cas9 technology

(1) KlglpA sequence retrieval and CRISPR gRNA sequence determination

In order not to affect the normal expression of other genes of Kluyveromyces lactis, the present invention inserts the MaPylRS expression structure near the tDNA KlglpA of Kluyveromyces lactis, and after expression, it combines with non-natural amino acids to perform site-specific insertion.

i. Search for glycerol-3-phosphate dehydrogenase in https://www.genome.jp/kegg/kegg2.html and obtain the glpA gene sequence in K. lactis yeast. In the present invention, this sequence is named KlglpA (located at 33084-35012 of chromosome A).

ii. Search for the PAM sequence (NGG) in the range of 1000-2000bp upstream of KlglpA, and finally select the PAM located downstream of the gene (sites 31956...31958 of chromosome A), and determine the KlglpA gRNA sequence (GAAGTAACTCTAGCCATCGG (SEQ ID NO: 26), located at sites 31936...31955 of chromosome A).

(2) Construction of KlglpA CRISPR-Cas9 plasmid

According to the designed gRNA sequence, design two 24nt primers required for vector construction. gRNA-F2: AATCGAAGTAACTCTAGCCATCGG (SEQ ID NO: 27); gRNA-R2: AAACCCGATGGCTAGAGTTACTTC (SEQ ID NO: 28) Dilute the primers to 10μM, add 10μL of gRNA-F2 and gRNA-R2 to the PCR tube, centrifuge to the bottom of the tube after mixing, and anneal according to the following procedure:

95℃, 3min; 72℃, 30s; 65℃, 2min; 60℃, 2min; 55℃, 2min; 50℃, 2min; 16℃, 2min

Then the ligation reaction

A. Reaction system: 10×Buffer 1μL, plasmid 20-50ng, annealing product 1μL, enzyme 0.2μL, add water to 10μL.

B. Reaction procedure: 16°C, 60 min.

Take 50 μL of commercial E. coli DH5α competent cells, add all the ligation products and mix well, and complete the transformation process according to the instructions. Screen on LB plates containing 50 mg/L kanamycin and culture overnight. Pick 5 single clones and shake culture in LB liquid culture medium. After sequencing to confirm the positive, extract the plasmid and save it, named pKM-CAS1.0-KlglpA (Figure 10).

(3) Donor DNA construction and amplification

First, the donor Donor, i.e. the homologous recombination sequence of the KlglpA site and the MaPylRS expression structure, was constructed. The promoter of MaPylRS was KlPGK1 promoter and the terminator was ScCYC1 terminator. The donor DNA construction and transformation method are as follows

iii. Genetically synthesize a plasmid containing the MaPylRS expression cassette, and use it as a template to perform PCR amplification with primer PF5: CCATCAGTTACGGTAGATTCTCCAGTGCCTACGTTCCTCATCACTAGAAG (SEQ ID NO: 29) and primer PR5: TGTTTTGCGCTTGGTTTTCTTTGTGGAGAAATTTCTTCGAGCGTCCCAAA (SEQ ID NO: 30) to obtain the MaPylRS expression cassette fragment.

iv. Using the lactic acid Kluyveromyces free plasmid as a template, primers PF6:AAATTAAGGCAAACATACAGG (SEQ ID NO: 31) and primer PR6:CAACAGTTCGGCTTCTAGTGATGAGGAACGTAGGCACTGGAGAATCTACC (SEQ ID NO: 32) were used for PCR amplification to obtain the homology arm 1 fragment of the KlglpA site; using the lactic acid Kluyveromyces free plasmid as a template, primers PF7:GCTTGAGAAGGTTTTGGGACGCTCGAAGAAATTTCTCCACAAAGAAAACC (SEQ ID NO: 33) and primer PR7:GACCTTTTATTTTGTCACCG (SEQ ID NO: 34) were used for PCR amplification to obtain the homology arm 2 fragment of the KlglpA site.

v. Mix the three PCR product fragments obtained above, dilute them 100 times and use them as templates, and perform PCR amplification again with primer PF8: ATATCGGATGACATGCAGCAA (SEQ ID NO: 35) and primer PR8: TTGTGTACCAAAACTTTCACGG (SEQ ID NO: 36); then the three fragments can be connected by overlapping extension PCR to form a linear donor DNA Donor. After sequencing confirmation, store it in a -20 degrees Celsius refrigerator.

(4) Preparation and transformation of efficient Kluyveromyces lactis competent cells

Preparation of competent yeast

Streak the Kluyveromyces lactis liquid on YPD solid medium and pick a single clone, shake culture overnight in 25mL 2×YPD liquid medium, take 2mL of the liquid and continue shaking culture in 50mL liquid 2×YPD medium for 2-8h. Collect yeast cells by centrifugation at 3000g for 5min at 20℃, add 500μL sterile water to resuspend, and collect cells by centrifugation under the same conditions. Prepare competent cell solution (5% v/v glycerol, 10% v/v DMSO) and dissolve yeast cells in 500μL of the solution. Aliquot 50μL into 1.5mL centrifuge tubes and store at -80℃.

Yeast DNA transformation

Thaw the competent cells on ice for 30 seconds, add 200ng of pKM-CAS1.0-KlglpA plasmid and 2000ng of donor DNA. Immediately add 1mL YPD liquid medium after electric shock at 1.5kV for 5mS, culture for 2-3h, and apply 200μL on solid YPD (200μg/mL G418) medium, and culture for 2-3 days until single colonies appear.

(5) Positive identification of gene editing

50-60 single clones were picked from the plate after transformation of Kluyveromyces lactis, and each single clone was placed in 5 μL yeast lysis buffer (Takara Mighty Prep Reagent for DNA). The bacterial lysate was used as a template and the primers Ma R2 (primer within the MaPylRS sequence): CCATGTGAGAACCTCTTGG (SEQ ID NO: 37); glpA F1 (primer outside the 5' of KlglpA donor DNA): AAATTAAGGCAAACATA CAGG (SEQ ID NO: 38) and primer Ma F2 (primer within the MaPylRS sequence): ATCTCTTACTTGAACGGTGCTA (SEQ ID NO: 39); glpA R1 (primer outside the 5' of KlglpA donor DNA): GACCTTTTATTTTGTCACCG (SEQ ID NO: 40) were used for PCR amplification to detect the CRISPR insertion at the KlglpA site. The presence of a positive band indicated that the MaPylRS sequence was successfully inserted into the target site.

Example 8 KlUPF1 was knocked out and replaced with MaPylRS expression cassette by CRISPR-Cas9 technology

(1) KlUPF1 sequence retrieval and CRISPR gRNA sequence determination

According to the literature, yeast UPF1 protein has been shown to be involved in the degradation of mRNA, and deleting this gene can slow down the degradation rate of immature mRNA transcripts. Therefore, the present invention completely knocks out the K1UPF1 gene and replaces it with the MaPylRS sequence to aggregate the unnatural amino acid transcripts, thereby improving the efficiency of unnatural amino acid insertion.

i. Search for "UPF1" in http://www.yeastgenome.org/ to obtain the ScUPF1 gene sequence in Saccharomyces cerevisiae. Perform BLAST comparison analysis on the UPF1 gene in the NCBI database to determine the UPF1 homologous gene sequence KlUPF1 in Kluyveromyces lactis (located at 567908...570817 on chromosome B).

ii. Search for PAM sequences (NGG) at both ends of the KlUPF1 gene and determine the gRNA sequence. The principles for gRNA selection are: moderate GC content, the standard of the present invention is 40%-60% GC content; avoid the presence of poly T structure. Finally, the KlUPF1 gRNA1 sequence determined by the present invention is TTGGCAAACGCATCGTCATA (SEQ ID NO: 41) and the gRNA1 sequence is CTTAAGGAAGTACAATGGAG (SEQ ID NO: 42).

(2) Construction of KlUPF1 CRISPR-Cas9 plasmid

According to the designed gRNA1 and gRNA2 sequences, design two 24nt primers required for vector construction. gRNA-F3: AATCTTGGCAAACGCATCGTCATA (SEQ ID NO: 43); gRNA-R3: AAACTATGACGATGCGTTTGCCAA (SEQ ID NO: 44); gRNA-F4: AATCCTTTAAGGAAGTACAATGGAG (SEQ ID NO: 45); gRNA-R4: AAACCTCCATTGTACTTCCTTAAG (SEQ ID NO: 46) Dilute the primers to 10μM, add 10μL of gRNA-F and gRNA-R to the PCR tube, centrifuge to the bottom of the tube after mixing, and anneal according to the following procedure:

95℃, 3min; 72℃, 30s; 65℃, 2min; 60℃, 2min; 55℃, 2min; 50℃, 2min; 16℃, 2min

Then the ligation reaction is carried out.

A. Reaction system: 10×Buffer 1μL, plasmid 20-50ng, annealing product 1μL, enzyme 0.2μL, add water to 10μL.

B. Reaction procedure: 16°C, 60 min.

Take 50 μL of commercial E. coli DH5α competent cells, add all the ligation products and mix well, and complete the transformation process according to the instructions. Screen on LB plates containing 50 mg/L kanamycin and culture overnight. Pick 5 single clones and shake culture in LB liquid culture medium. After sequencing to confirm the positive, extract the plasmid and save it, named pKM-CAS1.0-KlUPF1 (Figure 11).

(3) Donor DNA construction and amplification

The present invention first constructs a donor Donor, and replaces the UPF1 coding sequence with the MaPylRS coding sequence. That is, the inserted MaPylRS uses the promoter and terminator of UPF1. The donor DNA construction and transformation method are as follows:

iii. Gene synthesize a plasmid containing the MaPylRS expression cassette, and use it as a template to perform PCR amplification with primer PF9: AGTACAATTAGAATCAAGTTTCCTTATGGGTTCTTCTTCTTCTGG (SEQ ID NO: 47) and primer PR9: TAATATTATTTAATTAATGGATTGATACGCGTTCATGTTTAGTTGATCTTAGCACCGTTC (SEQ ID NO: 48) to obtain the coding sequence of MaPylRS.

iv. Using Kluyveromyces lactis free plasmid as template, primer PF10:CAATGGATACAGTTTCTCGCTA (SEQ ID NO: 49) and primer PR10:ACCAGAAGAAGAAGAACCCATAAGGAAACTTGATTCTAATTGT (SEQ ID NO: 50) were used for PCR amplification to obtain the homology arm 1 fragment of the KlUPF1 site; using Kluyveromyces lactis free plasmid as template, primer PF11:TTGAACGGTGCTAAGATCAACTAAACATGAACGCGTATCAATC (SEQ ID NO: 51) and primer PR11:CTTCGAGACTTCCAATGATCTC (SEQ ID NO: 52) were used for PCR amplification to obtain the homology arm 2 fragment of the KlUPF1 site.

v. Mix the three PCR product fragments obtained above, dilute them 100 times and use them as templates, and perform PCR amplification again with primer PF12: GATCGTCCATTAGCTTATCTACAAATGCC (SEQ ID NO: 53) and primer PR12: GTGAGAATGCCAGACGAT (SEQ ID NO: 54); then the three fragments can be connected by overlapping extension PCR to form a linear donor DNA Donor. After sequencing confirmation, store it in a -20 degrees Celsius refrigerator.

(4) Kluyveromyces lactis transformation and positive identification

Preparation of competent yeast

Streak the Kluyveromyces lactis liquid on YPD solid medium and pick a single clone, shake culture overnight in 25mL 2×YPD liquid medium, take 2mL of the liquid and continue shaking culture in 50mL liquid 2×YPD medium for 2-8h. Collect yeast cells by centrifugation at 3000g for 5min at 20℃, add 500μL sterile water to resuspend, and collect cells by centrifugation under the same conditions. Prepare competent cell solution (5% v/v glycerol, 10% v/v DMSO) and dissolve yeast cells in 500μL of the solution. Aliquot 50μL into 1.5mL centrifuge tubes and store at -80℃.

Yeast DNA transformation

Thaw the competent cells on ice for 30 seconds, add 200ng of pKM-CAS1.0-KlUPF1 plasmid and 2000ng of donor DNA. Immediately add 1mL YPD liquid medium after electric shock at 1.5kV for 5mS, culture for 2-3h, and apply 200μL on solid YPD (200μg/mL G418) medium, and culture for 2-3 days until single colonies appear.

(5) Positive identification of gene editing

50-60 single clones were picked from the plate after transformation of Kluyveromyces lactis, and each single clone was placed in 5 μL yeast lysis buffer (Takara Mighty Prep Reagent for DNA). The bacterial lysate was used as a template and the primers Ma R3 (primer within the MaPylRS sequence): GTCTCTAGAAGCCAAGTCTTC (SEQ ID NO: 55); UPF1 F1 (primer outside the 5' of KlUPF1 donor DNA): GAACTGCCAC PCR amplification was performed using primers Ma F3 (primer within the MaPylRS sequence): GCTGCTCACGACGTTCA (SEQ ID NO: 57); UPF1 R1 (primer outside the 5’ of KlUPF1 donor DNA): GCACTGTAATCAGGCAACT (SEQ ID NO: 58) to detect CRISPR insertion at the KlUPF1 site. The presence of a positive band indicated that the MaPylRS sequence was successfully inserted into the target site.

Example 9 Activity Assay

The genetically modified Kluyveromyces lactis strain was prepared into an in vitro protein synthesis system (IVTT), and a plasmid containing dual reporter genes of green fluorescent protein GFP and red fluorescent protein RFP (green fluorescent protein, GFP; red fluorescent protein, RFP) was added to determine the ability of the modified strain to insert non-natural amino acids into specific sites of specific proteins. GFP is used to detect the overall expression of the protein, while RFP is used to detect whether the non-natural amino acid is successfully inserted at a specific position, that is, the stop codon TAG of GFP. If a red fluorescent signal appears, it means that the translation-related protein reads through the stop codon TAG, which means that the non-natural amino acid is successfully inserted, while the lack of a red fluorescent signal means that the translation-related protein stops at the stop codon TAG and the insertion of the non-natural amino acid fails.

Taking the Kluyveromyces lactis strain obtained in Example 6 as an example, the promoter in the MaPylRS expression cassette was adjusted to obtain different Kluyveromyces lactis strains and prepare them into a Protein Factory.

Establishment of expression system containing unnatural amino acids:

Protein Factory 100ul

Prock (unnatural amino acid) 500mM 1ul

matRNA _CUA ^pyl in vitro transcription product (unpurified) 10ul

GFP-TAG-RFP dual fluorescence reporter gene PCR product 3ul

By detecting the fluorescence intensity of RFP, the efficiency of the introduction of unnatural amino acids can be determined based on the fluorescence intensity of RFP and the ratio of RFP/GFP (see Figures 12 to 14 for details).

In Figures 12 to 14, DW14-1 and DW14-2 are two sets of parallel experiments, and the promoter is TIF11; the promoter of DW14-3 is TEF1; DW14-4 and DW14-5 are two sets of parallel experiments, and the promoter is ADH1; DW14-6 and DW14-7 are two sets of parallel experiments, and the promoter is GAP1; DW14-8 and DW14-9 are two sets of parallel experiments, and the promoter is HXK4; the promoter of DW14-10 is PGK1.

As shown in Figure 12, the modified strains all achieved the read-through of the stop codon TAG, which proves that the modified strains of the present invention can achieve the introduction of non-natural amino acids. As shown in Figure 14, the modified strains of the present invention all have a high non-natural amino acid introduction efficiency, especially DW14-4 to DW14-7 and DW14-10, which have a very high RFP/GFP value, indicating that they all have a very high non-natural amino acid introduction efficiency.

Example 10

The reaction systems obtained after the modified MaPylRS in Example 6 and the original MaPylRS (Example 5) were integrated into yeast to compare the activity effects of ncaa introduction. For specific measurement conditions, see Example 9, and for specific results, see Figures 15 to 17, where sl-3 represents the reaction system of yeast integrated with the original MaPylRS, and sl-9 represents the reaction system of yeast integrated with the modified MaPylRS of the present application. Orthogonal tRNA and Prock were added to the systems, and a system without adding ncaa (i.e., non) was used as a control. It can be seen from Figures 15 to 17 that the reaction systems obtained by integrating the wild-type and modified MaPylRS into yeast cells can both achieve ncaa introduction, but the reaction system obtained after the modified MaPylRS of the present invention is integrated into yeast cells can significantly improve the efficiency of ncaa introduction.

The sequences used in the present invention are shown in Table 1 below

Table 1

Based on the above ideal embodiments of this application, the relevant staff can make various changes and modifications without deviating from the technical concept of this application through the above description. The technical scope of this application is not limited to the content in the specification, and its technical scope must be determined according to the scope of the claims.

Claims (25)

A recombinant aminoacyl-tRNA synthetase having the structure described in Formula I:
A1-A2-A3-A4(I): In Formula I, "-" is independently a bond or an amino acid linking sequence, A1 is absent or is a histidine tag, A2 is absent or is a thrombin cleavage site. A3 is absent or a tagged protein. A4 is an aminoacyl-tRNA synthetase, and at least one of A1 to A3 is present; the connection between A1 to A4 can be from N-terminus to C-terminus, or from C-terminus to N-terminus. The recombinant aminoacyl-tRNA synthetase according to claim 1, characterized in that the aminoacyl-tRNA synthetase is selected from natural or mutant Pyl-tRNA synthetase (PylRS), Leu-tRNA synthetase (LeuRS), Tyr-tRNA synthetase (TyrRS), Phe-tRNA synthetase (PheRS) or TrP-tRNA synthetase (TrpRS). The recombinant aminoacyl-tRNA synthetase according to claim 1 or 2 is characterized in that: the aminoacyl-tRNA synthetase is selected from natural or mutated MaPylRS, MmPylRS, MbPylRS, EcTyrRS, MjTyrRS, EcLeuRS, ScPheRS, ScTrpRS or BsTrpRS; preferably, the aminoacyl-tRNA synthetase comprises the sequence shown in SEQ ID NO:60 or an active fragment thereof, and further preferably, the sequence of the aminoacyl-tRNA synthetase is SEQ ID NO:62; or it is a polypeptide having ≥85%, ≥90%, ≥95%, ≥97%, ≥98% or ≥99% homology with the amino acid sequence shown in SEQ ID NO:60, and having the same activity as the sequence of SEQ ID NO:60. The recombinant aminoacyl-tRNA synthetase according to any one of claims 1 to 3, characterized in that the structure of the histidine tag is n×His, wherein 1≦n≦50; preferably 2≦n≦30; further preferably, 5≦n≦20; more preferably 6≦n≦10. The recombinant aminoacyl-tRNA synthetase according to any one of claims 1 to 4, characterized in that: a his tag is connected to the N-terminus or C-terminus of the aminoacyl-tRNA synthetase; or a thrombin cleavage site and a tag protein are connected to the N-terminus of the aminoacyl-tRNA synthetase in the order from the N-terminus to the C-terminus; or a his tag, a thrombin cleavage site, and a tag protein are connected to the N-terminus of the aminoacyl-tRNA synthetase in the order from the N-terminus to the C-terminus. The recombinant aminoacyl-tRNA synthetase according to any one of claims 1 to 5, characterized in that: the recombinant aminoacyl-tRNA synthetase comprises a sequence or an active fragment thereof of any one of SEQ ID NOs: 1 to 3 and SEQ ID NO: 64, and further preferably, the amino acid sequence of the recombinant aminoacyl-tRNA synthetase is selected from any one or more of the following: SEQ ID NOs: 1 to 3 and SEQ ID NO: 64; or a polypeptide having ≥85% homology, ≥90% homology, ≥95% homology, ≥97% homology, ≥98% homology or ≥99% homology with any one of the amino acid sequences in SEQ ID NOs: 1 to 3 and SEQ ID NO: 64, and having the same activity as any one of the sequences corresponding to the homology in SEQ ID NOs: 1 to 3 and SEQ ID NO: 64; or the recombinant aminoacyl-tRNA synthetase comprises a sequence or an active fragment thereof of any one of SEQ ID NOs: 1 to 3 and SEQ ID NO: 64, and further preferably, the amino acid sequence of the recombinant aminoacyl-tRNA synthetase is selected from any one or more of the following: SEQ ID NOs: 1 to 3 and SEQ ID NO: 64; The coding sequence of RNA synthetase is selected from any one or more of the following: SEQ ID NO:4, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63; or contains any one of the sequences described in SEQ ID NO:4, SEQ ID NO:61, SEQ ID NO:62 or SEQ ID NO:63 or its active fragment, or is a nucleotide having ≥85% homology, ≥90%, ≥95% homology, ≥97% homology, ≥98% homology or ≥99% homology to any one of the sequences described in SEQ ID NO:4, SEQ ID NO:61, SEQ ID NO:62 or SEQ ID NO:63, and has the same activity as any one of the sequences described in SEQ ID NO:4, SEQ ID NO:61, SEQ ID NO:62 or SEQ ID NO:63 with corresponding homology. A nucleic acid construct encoding the recombinant aminoacyl-tRNA synthetase according to any one of claims 1 to 6. The nucleic acid construct according to claim 7, characterized in that: the nucleic acid construct contains at least a structure as described in Formula II: Z1-Z2-Z3-Z4, wherein Z1 to Z4 are respectively elements used to constitute the construct; "-" is independently a bond or a nucleotide linking sequence; Z1 is absent or is a coding sequence for a histidine tag, Z2 is absent or is a coding sequence for a thrombin cleavage site, Z3 is absent or is a coding sequence for a tag protein, and Z4 is a coding sequence for an aminoacyl-tRNA synthetase; wherein at least one of Z1 to Z3 is present. The nucleic acid construct according to claim 8 is characterized in that: the amino acid sequence encoded by Z1 is HHHHHH; the amino acid sequence encoded by Z2 is LVPRGS; the amino acid sequence encoded by Z3 is SEQ ID NO:59; or Z1, Z2 and Z3 each encode a sequence comprising HHHHHH, LVPRGS and SEQ ID NO:59 or an active fragment thereof, or each has ≥85% homology, ≥90% homology, ≥95% homology, ≥97% homology, ≥98% homology or ≥99% homology with the nucleotide sequence corresponding to the nucleotide sequence encoding HHHHHH, LVPRGS and SEQ ID NO:59 and each has the same activity as the nucleotide sequence encoding HHHHHH, LVPRGS and SEQ ID NO:59. The nucleic acid construct according to claim 8 or 9, further comprising a promoter element, Preferably, the nucleic acid structure comprises the structure described in Formula III:
Z5-Z1-Z2-Z3-Z4, In the formula, Z5 is the promoter element, More preferably, the promoter is selected from PGK1, GAP1, ADH1, HXK1, GAPDH1, TEF1 or TIF11. A vector, characterized in that the vector contains the nucleic acid construct according to any one of claims 7 to 10. A genetically engineered strain, characterized in that the nucleic acid construct according to any one of claims 7 to 10 is integrated into one or more sites of the genome of the genetically engineered strain, or the genetically engineered strain contains the recombinant aminoacyl-tRNA synthetase according to any one of claims 1 to 6, or the genetically engineered strain contains the vector according to claim 11. The genetically engineered strain according to claim 12, characterized in that the strain is derived from one of mammalian cells, plant cells, yeast cells, insect cells, prokaryotic cells or any combination thereof. The genetically engineered strain according to claim 12 or 13, characterized in that the site is selected from Lys1-5, glpA or UPF1. The genetically engineered strain according to any one of claims 12 to 14, characterized in that: the nucleic acid construct further comprises a terminator; preferably, the terminator is selected from CYC1, GPM1, TDH2 or ACT1. A method for synthesizing proteins incorporated with non-natural amino acids, characterized in that the recombinant aminoacyl-tRNA synthetase is provided by using the recombinant aminoacyl-tRNA synthetase described in any one of claims 1 to 6, or by using the genetically engineered strain described in any one of claims 12 to 15. A cell-free system for synthesizing proteins containing unnatural amino acids, characterized in that the cell-free system comprises at least: (a) a cell extract, and (b) one or more of the recombinant aminoacyl-tRNA synthetase according to any one of claims 1 to 6, the nucleic acid construct according to any one of claims 7 to 10, or the vector according to claim 11; the cell extract is derived from one of mammalian cells, plant cells, yeast cells, insect cells, prokaryotic cells, or any combination thereof. A cell-free system for synthesizing proteins containing unnatural amino acids, characterized in that the cell-free system at least comprises a cell extract, and the cell extract is derived from the genetically engineered strain according to any one of claims 12-15. A cell-free system for synthesizing proteins containing non-natural amino acids according to claim 17 or 18, characterized in that the cell-free system further comprises: non-natural amino acids, orthogonal tRNAs and a template comprising a target protein gene sequence, wherein the codons encoding amino acids in the target protein gene sequence are mutated. A method for preparing a genetically engineered strain according to any one of claims 12 to 15, characterized in that the nucleic acid construct according to any one of claims 7 to 10 is transferred or integrated into a cell by transformation, transfection or gene editing technology. The method for preparing a genetically engineered strain according to claim 20, characterized in that the nucleic acid construct according to any one of claims 7 to 10 is integrated into the genome of the cell through an active site. The method for preparing a genetically engineered strain according to claim 20 or 21, characterized in that the nucleic acid construct further comprises a terminator; preferably, the terminator is selected from CYC1, GPM1, TDH2 or ACT1. The method for preparing a genetically engineered strain according to claim 21 or 22, characterized in that the site is selected from Lys1-5, glpA or UPF1. A kit, characterized in that it contains the reaction system according to any one of claims 17 to 19. A method for synthesizing a protein containing unnatural amino acids in vitro, characterized in that the protein is prepared using the cell-free system described in any one of claims 17 to 19 or the kit described in claim 24.