Method for improving synthesis efficiency of cell-free protein by mutated histone

Abstract

According to the method for improving the synthesis efficiency of the cell-free protein by mutating the histone, provided by the invention, the activity of a cell-free protein synthesis system can be improved and the synthesis efficiency can be improved through a cell extract prepared by culturing a histone site-directed mutagenesis strain.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and specifically relates to a method for improving the efficiency of cell-free protein synthesis. Background Art

[0002] Cell-free protein synthesis (CFPS) is a technology that uses exogenous mRNA or DNA as a template and synthesizes proteins in vitro using enzymes, amino acid substrates, and energy. The cell-free protein expression system is an important supplement to the cell-based protein expression system. Compared with the traditional in vivo recombinant expression system, the in vitro cell-free synthesis system has many advantages, such as being able to directly use PCR products as templates to simultaneously synthesize multiple proteins in parallel, enabling high-throughput screening of polypeptides or protein drugs, with a fast synthesis speed and high efficiency, etc. In addition, the in vitro cell-free synthesis system also has unique advantages in the application of expressing polypeptides or proteins that are toxic to cells.

[0003] The existing cell-free protein synthesis systems can be mainly divided into two categories: One is to combine and reconstitute the purified ribosomes, enzymes, tRNAs, etc. required for protein synthesis in a certain proportion to endow it with the ability to express proteins in vitro, that is, the purified cell-free protein expression system (PURE). This system has clear component information and can conveniently and customarily adjust the formula to obtain different protein expression characteristics, but the disadvantage is that the preparation process is complex and the cost is high. The other category is to obtain the main active substances from the extracts of living cells with protein expression activity, and appropriately add the required enzymes, substrates, energy substances, and other components to endow it with the ability to express proteins in vitro, that is, the cell-free protein expression system based on cell extracts. Compared with the PURE system, the latter has the advantages of simple operation process, short cycle, and low cost, but the latter also has disadvantages such as complex components and many uncontrollable factors in the system, so higher requirements are placed on the cell characteristics of the source of the cell extract.

[0004] Currently, how to further improve the activity of cell-free protein expression by improving cell extracts remains a research topic worthy of study. Summary of the Invention

[0005] The purpose of the present invention is to overcome the problems existing in the above-mentioned prior art, and provide a method for improving the efficiency of cell-free protein synthesis. By genetically modifying to improve the protein expression activity of the cell-free protein expression system of cell extracts, the production efficiency is greatly improved.

[0006] The first aspect of the present invention provides a method for improving the efficiency of cell-free protein synthesis by mutant histone. The cells from which the cell extract used for the cell-free protein synthesis is derived are modified as follows: the first and / or second alleles of the histone H3 gene in the cells are site-directed mutated to obtain mutant strains or cells, and then the cells are cultured and cell extracts are prepared;

[0007] The site-directed mutation refers to mutating lysine or glycine on the first or second allele.

[0008] In another preferred example, the cells are selected from one or any combination of bacteria, mammalian cells, human cells, plant cells, yeast cells, and insect cells, preferably from yeast cells.

[0009] In another preferred example, the yeast cells are selected from one or a combination of Pichia pastoris, Saccharomyces cerevisiae, brewer's yeast, cane molasses yeast, Hansenula, Hansenula polymorpha, Candida utilis, Kluyveromyces, etc.

[0010] Further preferably, the Kluyveromyces is selected from: the Kluyveromyces includes Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces dobzhanskii, Kluyveromyces aestuarii, Kluyveromyces nonfermentans, Kluyveromyces wickerhamii, Kluyveromyces thermotolerans, Kluyveromyces fragilis, Kluyveromyces hubeiensis, Kluyveromyces polysporus, Kluyveromyces siamensis, Kluyveromyces yarrowii, or a combination thereof. In another preferred example, the site-directed mutation is selected from one or more of the following:

[0011] The 27th lysine (K) of the protein encoded by the first or second allele of histone H3 is mutated to methionine (M);

[0012] The glycine (G) at position 34 of the protein encoded by the first or second allele of histone H3 is mutated to arginine (R);

[0013] The glycine (G) at position 34 of the protein encoded by the second allele of histone H3 is mutated to tryptophan (W);

[0014] The lysine (K) at position 36 of the protein encoded by the second allele of histone H3 is mutated to methionine (M).

[0015] In another preferred example, the site-directed mutagenesis is achieved by CRISPR-Cas9-mediated gene editing means.

[0016] In another preferred example, the method of site-directed mutagenesis is as follows:

[0017] Step 1), design the gRNA targeting sequence;

[0018] Step 2), construct the gRNA_Cas9 co-expression vector;

[0019] Step 3), construct the plasmid containing the site-directed mutation of H31 or H32;

[0020] Step 4), construct the double-stranded DNA donor;

[0021] Step 5), cell transformation and screening.

[0022] In another preferred example, the specific operation method of the site-directed mutagenesis is as follows:

[0023] Step 1), design the gRNA for the selected gene and synthesize the corresponding primer pair 1;

[0024] Step 2), perform PCR using the primer pair 1 obtained in Step 1), then react the PCR product with the pCas vector in the T4 ligase system, transform it into Escherichia coli cells, perform plate screening and sequencing to obtain the gRNA_Cas9 co-expression vector;

[0025] Step 3), obtain the genome of the histone, then introduce a synonymous mutation at the position corresponding to the gRNA, and then introduce the mutation in the histone coding region to obtain the vector containing the site-directed mutation of H31 or H32;

[0026] Step 4), use the vector containing the site-directed mutation of H31 or H32 as a template, and obtain the DNA donor by PCR;

[0027] Step 5): Prepare competent cells, add the gRNA_Cas9 expression vector and the DNA donor, perform electrotransformation, screen on plates after transformation and conduct verification. After two rounds of plate screening and verification, a strain containing the site-directed mutation is obtained.

[0028] Further, step 3) includes the following steps:

[0029] 3-1): Using the genomic DNA of the cells as a template, design primer pair 2 for the selected gene, perform high-fidelity PCR amplification, add A to the ends of the PCR products and ligate them into a T-vector to obtain vector 1 containing the selected gene.

[0030] 3-2): Using vector 1 as a template, design primer 3 that introduces a synonymous mutation at the position corresponding to the gRNA, perform high-fidelity PCR to obtain vector 2 with a gene mutation in the gRNA-targeted region.

[0031] 3-3): Using vector 2 as a template, perform high-fidelity PCR with mutant primer pair 4 to introduce a mutation in the histone coding region to obtain mutant vector 3, i.e., a vector containing a site-directed mutation in H31 or H32.

[0032] In another preferred example, the method further includes step 6): After the strain containing the site-directed mutation is obtained, it is expanded in culture, the cells are separated and collected, and after disruption, a cell extract for cell-free protein synthesis is obtained.

[0033] The second aspect of the present invention provides an expression vector or vector combination, which contains nucleic acids encoding the first or second allele of the histone H3 gene as described above after site-directed mutation, and is further prepared by the method described in step 3) as described above. The third aspect of the present invention provides a mutant strain, which is obtained by the transformation described in the first aspect above. Preferably, the strain is selected from one or a combination of Pichia pastoris, Saccharomyces cerevisiae, Hansenula anomala, Candida spp., Kluyveromyces spp., Schizosaccharomyces pombe; preferably one or a combination of Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces spp.; most preferably Kluyveromyces lactis (hereinafter referred to as K. lactis);

[0034] Further, the mutant strain is prepared by the method of the foregoing steps 1)-5).

[0035] The fourth aspect of the present invention provides a cell extract for cell-free protein synthesis, which is obtained by culturing the strain with site-directed mutation prepared by the method of the first aspect above or the mutant strain of the third aspect above, and then centrifuging and lysing.

[0036] The fifth aspect of the present invention provides an in vitro protein synthesis system for expressing exogenous proteins, which comprises the cell extract described in the foregoing third aspect, or the histone contained in the system is obtained by site-directed mutagenesis of the first and / or second alleles of its H3 gene by the mutagenesis described in the first aspect.

[0037] The present invention has the following beneficial effects or advantages compared with the prior art:

[0038] (1) By site-directed mutagenesis of the first or second allele of the histone H3 gene of biological cells, the present invention provides a method to obtain a cell extract with improved expression activity. Using this cell extract for cell-free protein synthesis can significantly improve the expression activity of proteins and enhance the synthesis efficiency.

[0039] (2) The site-directed mutagenesis of the present invention uses CRISPR-Cas9-mediated gene editing means, which has the advantages of high efficiency, precision, easy operation, etc. It can accurately obtain the gene with mutations at the target site, is reproducible, and has practicality and generalizability.

[0040] (3) According to the site of the mutation target, the present invention designs primers for each step and combines with high-fidelity PCR to accurately achieve site-directed mutagenesis, obtain the strain with the target gene mutation, improve the operability and repeatability, and the genetic traits of the transformed cells are stable and can be used for actual production.

[0041] (4) Cells or strains containing the mutant gene are obtained by the method of the present invention. Cell extracts containing the mutant gene or protein are prepared by culturing, collecting, and lysing. The cell extracts are used to enhance the cell-free protein synthesis activity, providing a new idea for the exploration in the field of cell-free protein synthesis.

[0042] (5) The present invention also provides an expression vector or vector combination containing the site-directed mutant gene. The vector is the basis for realizing the site-directed mutagenesis of the cell gene of the present application. The vector is obtained by the foregoing site-directed mutagenesis method and has stable genetic characteristics, and can be used for subsequent further transformation and other related genetic engineering.

[0043] (6) The present invention also provides an in vitro protein synthesis system for expressing proteins. Since it contains the foregoing cell extract with improved expression activity, the protein synthesis efficiency of the system is improved, further enhancing the production efficiency of in vitro protein synthesis.

[0044] 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 (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be repeated one by one here. Description of the Drawings

[0045] Figure 1 Nucleotide sequence alignment of two copies of histone H3 (Query: KLLA0_E08625g and Sbjct: KLLA0_E17623g) in the K. lactis genome.

[0046] Figure 2 gRNA position and primer-mediated nucleotide mutation design scheme. Taking the mutation of H32 (KLLA0_E08625g) as an example, the gRNA was designed in the 5'-UTR of the ORF. Mutations at the gRNA position were introduced by primers H32gF and H32gR. Mutations in the gene coding region were introduced by other primer pairs.

[0047] Figure 3 Growth curve of the mutant strain in YPD liquid medium.

[0048] Figure 4 IVTT activity of the extracts of the mutant strain and the control strain. Detailed implementation manners

[0049] Through extensive and in-depth research, and through a large number of screenings and explorations, the present invention first proposes a method for improving the efficiency of cell-free protein synthesis. This method performs site-directed mutation on the first or second allele of the cellular histone H3 gene through gene editing to obtain a mutant strain or cell, and then cultures and prepares a cell extract, thereby improving the cell-free protein synthesis activity.

[0050] The following further clarifies the present invention in combination with the detailed implementation manners and examples. For the specific methods or materials used in the examples, those skilled in the art can make conventional substitution selections based on the existing technologies on the basis of the technical ideas of the present invention, not limited to the specific records of the embodiments of the present invention.

[0051] For the experimental methods without specific conditions indicated in the following examples, first follow and refer to the conditions guided by the above-mentioned detailed implementation manners, and then follow the conventional conditions, such as those described in "Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)", "Cell-Free Protein Synthesis Experimental Manual" "Edited by Alexander S. Spirin and James R. Swartz. Cell-free protein synthesis: methods and protocols [M]. 2008" and other documents, or follow the conditions recommended by the manufacturer.

[0052] Unless otherwise specified, the percentages and parts mentioned in the present invention are weight percentages and weight parts.

[0053] Unless otherwise specified, the materials and reagents used in the embodiments of the present invention are all commercially available products.

[0054] Unless otherwise specified, the temperature unit in this application is degrees Celsius (°C).

[0055] Term Introduction

[0056] The following are the explanations or descriptions of the meanings of some relevant "nouns" and "terms" adopted in the present invention, so as to better understand the present invention. The corresponding explanations or descriptions apply to the whole text of the present invention, both to the following text and to the above text. When the present invention involves citing documents, the definitions of relevant terms, nouns, and phrases in the cited documents are also cited. However, when there is a conflict with the definitions in the present invention, the definitions in the present invention shall prevail. When there is a conflict between the definition in the cited document and the definition in the present invention, it does not affect the components, substances, compositions, materials, systems, formulations, types, methods, equipment, etc. determined in the cited document to prevail.

[0057] In the present invention, "cell-free protein synthesis", also known as "in vitro protein synthesis", "in vitro cell-free protein synthesis", etc., refers to the reaction of synthesizing proteins in an in vitro cell-free environment synthesis system, which includes at least the translation process. It includes but is not limited to IVT reaction (in vitro translation reaction), IVTT reaction (in vitro transcription-translation reaction), IVDTT reaction (in vitro replication-transcription-translation reaction). In the present invention, the IVTT reaction is preferred. The IVTT reaction, corresponding to the IVTT system, is the process of transcribing and translating DNA into protein (Protein) in vitro. Therefore, we also refer to such in vitro protein synthesis systems as D2P systems, D-to-P systems, D_to_P systems, DNA-to-Protein systems; the corresponding in vitro protein synthesis methods are also called D2P methods, D-to-P methods, D_to_P methods, DNA-to-Protein methods.

[0058] In the present invention, "cell-free", or "cell-free system", means that when performing in vitro protein synthesis, it is not through the way of secretion and expression by 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 do not mainly aim at secreting and expressing exogenous target proteins. In addition, in the D2P system without intact cells 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 does not exceed 30 wt% compared with the protein content provided by the cell extract). Such an "evasion" method is also included in the protection scope of the present invention.

[0059] In the present invention, one of the specific operation modes of the cell-free protein synthesis system further includes, but is not limited to, for example, the cell-free protein synthesis system based on Escherichia coli described in WO2016005982A1. The in vitro cell-free protein synthesis systems including, but not limited to, those based on wheat germ cells, rabbit reticulocytes, Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces marxianus described in other cited documents of the present invention and their directly and indirectly cited documents are also incorporated into the present invention as implementation modes of the in vitro protein synthesis system of the present invention. For example, the in vitro cell-free protein synthesis systems (In vitro cell-free protein synthesis system) described in the cited documents on pages 27-28 of the section "2.1 Systems and Advantages" in the document "Lu, Y. Advances in Cell-Free Biosynthetic Technology. Current Developments in Biotechnology and Bioengineering, 2019, Chapter 2, 23-45" can all be used as the in vitro protein synthesis system for implementing the present invention. For example (unless in conflict with the present invention, the following documents and their cited documents are cited for all purposes and in their entirety), the in vitro cell-free protein synthesis systems, DNA template construction and amplification methods described in documents CN106978349A, CN108535489A, CN108690139A, CN108949801A, CN108642076A, CN109022478A, CN109423496A, CN109423497A, CN109423509A, CN109837293A, CN109971783A, CN109988801A, CN109971775A, CN110093284A, CN110408635A, etc., and their cited documents can all be used as the in vitro protein synthesis system of the present invention and the DNA template construction and amplification method of the present invention.

[0060] In the present invention, "protein" and "protein" have the same meaning and are both translated as "protein" and can be used interchangeably.

[0061] In the present invention, both "system" and "system" are translated as "system" and can be used interchangeably.

[0062] In the present invention, "expression activity", "synthesis activity", "protein synthesis amount", "protein expression amount" and "protein expression yield" have the same meaning and can be used interchangeably, and all represent the ability of the cell-free protein synthesis system to synthesize proteins.

[0063] In the present invention, the meanings of "cell extract", "cell extraction solution", "cell lysate", etc. are the same and can be used interchangeably. They can be described in English as cell extract, cell lysate, etc.

[0064] In the present invention, "site-directed mutagenesis" refers to introducing the required changes (usually changes representing a favorable direction), including base addition, deletion, point mutation, etc. into the target DNA fragment (which can be a genome or a plasmid) by methods such as polymerase chain reaction (PCR), etc., which can quickly and efficiently improve the properties and characteristics of the target protein expressed by the DNA.

[0065] In the present invention, "allele" refers to genes located at the same position on a pair of homologous chromosomes that control different forms of the same trait.

[0066] "CRISPR-Cas9" was originally an adaptive immune defense formed by bacteria and archaea during long-term evolution and can be used to combat invading viruses and foreign DNA. In the present invention, "CRISPR-Cas9" or "CRISPR-Cas9-mediated gene editing" refers to the gene editing technology based on the above principle, that is, the technology for specific DNA modification of the target gene, which is a commonly used and technically mature gene editing means in the field of biotechnology in recent years. Except for the steps described in the present invention, the remaining specific operation methods are all conventional operations recorded in the prior art.

[0067] In the present invention, "vector", "plasmid", "primer", "target gene", "gene", "transformation", "PCR", "high-fidelity PCR", etc. all have their conventional meanings in the prior art and will not be elaborated one by one here.

[0068] In one example, a method for improving the efficiency of cell-free protein synthesis by mutating histone is provided. The strain from which the cell extract used for the cell-free protein synthesis is derived has been modified as follows: the first and / or second alleles of the histone H3 gene in the strain are site-directed mutated to obtain a mutant strain or cell, and then the cell extract is cultured and prepared.

[0069] The site-directed mutagenesis refers to mutating lysine or glycine on the first or second allele.

[0070] Histones are highly conserved in eukaryotes, and there are multiple histone-coding genes in the genomes of eukaryotes. The applicant found that by site-directed mutating lysine or glycine on the first or second allele of the histone H3 gene, then preparing a cell extract using the mutant strain and using it for cell-free protein synthesis, the results showed that the activity of the synthesis system increased.

[0071] In one example, the cells are selected from one or any combination of bacteria, mammalian cells, human cells, plant cells, yeast cells, and insect cells, preferably yeast cells. As is well known, yeast cells are often used as model strains in biotechnological research, especially Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces, etc. In this application, yeast is used as the experimental strain, and site-directed mutagenesis is performed on lysine or glycine on the first or second allele of the H3 gene in its genome, and its effect is verified.

[0072] In one example, the yeast is selected from one or a combination of Pichia pastoris, Saccharomyces cerevisiae, Hansenula anomala, Candida albicans, Kluyveromyces, and Schizosaccharomyces pombe; preferably one or a combination of Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces; most preferably Kluyveromyces lactis.

[0073] More preferably, the Kluyveromyces further includes: the Kluyveromyces includes one or a combination of Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces dobzhanskii, Kluyveromyces aestuarii, Kluyveromyces nonfermentans, Kluyveromyces wickerhamii, Kluyveromyces thermotolerans, Kluyveromyces fragilis, Kluyveromyces hubeiensis, Kluyveromyces polysporus, Kluyveromyces siamensis, and Kluyveromyces yarrowii. Most preferably, Kluyveromyces lactis or Kluyveromyces marxianus is used.

[0074] Kluyveromyces is similar to the model fungus Saccharomyces cerevisiae. In the genome of Kluyveromyces lactis (taking K. lactis Y1140 as an example), there are also 9 histone-coding genes, including two copies each of Histone H2A, H2B, H3, and H4, and one copy of H2A.Z, as shown in Table 1.

[0075] Table 1 Gene List

[0076] Gene source Gene name Gene description KLLA0_C05918g HTZ1 histone H2A.Z KLLA0_E08625g HHT2(H32) histone H3 KLLA0_E08647g HHF2 histone H4 KLLA0_E17337g HTB2 histone H2B KLLA0_E17359g HTA2 histone H2A KLLA0_E17601g HHF1 histone H4 KLLA0_E17623g HHT1(H31) histone H3 KLLA0_F13310g HTB2 histone H2B KLLA0_F13332g HTA2 histone H2A

[0077] In Table 1, the two copies of H3 are H31 and H32 respectively. For convenience of description: H31 is the first allele and H32 is the second allele.

[0078] In one example, the site-directed mutation is selected from one or more of the following:

[0079] Lysine (K) at position 27 of the protein encoded by the first or second allele of histone H3 is mutated to methionine (M);

[0080] Glycine (G) at position 34 of the protein encoded by the first or second allele of histone H3 is mutated to arginine (R);

[0081] Glycine (G) at position 34 of the protein encoded by the second allele of histone H3 is mutated to tryptophan (W);

[0082] Lysine (K) at position 36 of the protein encoded by the second allele of histone H3 is mutated to methionine (M).

[0083] The present invention has constructed a variety of mutation schemes, and the extracts of the mutant strains are used for cell-free synthesis experiments. The results show that the above-mentioned types of site-directed mutations and their combinations can all improve the activity of the cell-free protein synthesis system, producing beneficial effects, that is, achieving technical effects unexpected by those skilled in the art.

[0084] In one example, the site-directed mutation is achieved by CRISPR-Cas9-mediated gene editing means. As mentioned above, CRISPR-Cas9-mediated gene editing means is a mature and precise gene editing method, and the present invention uses this method to perform the above site-directed mutation.

[0085] In one example, in step 1), design the gRNA targeting sequence;

[0086] In step 2), construct the gRNA_Cas9 co-expression vector;

[0087] In step 3), construct a plasmid containing site-directed mutations of H31 and / or H32;

[0088] In step 4), construct the double-stranded DNA donor;

[0089] In step 5), perform cell transformation and screening.

[0090] The above method is designed according to the conventional steps of CRISPR-Cas9 gene editing and the purpose of the present invention. By designing primers corresponding to each step, the construction of the vector and DNA donor is achieved step by step, and finally transformed into cells to achieve site-directed mutagenesis of the genome and obtain the modified strain.

[0091] In one example, the specific operation method for the site-directed mutagenesis is as follows:

[0092] Step 1), design gRNA for the selected gene and synthesize the corresponding primer pair 1.

[0093] Taking the two copies of histone H3 in the K. lactis genome as the modification region, through nucleotide sequence comparison, it shows that more than 95% (391 / 411) of the bases in the ORF region are the same. To ensure the specificity of editing, the gRNA is designed in the non-coding region (5'-UTR) adjacent to the ORF. The sequence alignment is as Figure 1 shown.

[0094] When designing gRNA, according to the selected region (100-2000bp) of the gene targeted by the gRNA, complete the design of gRNA on the CRISPOR (tefor.net) (http: / / crispor.tefor.net / ) website, and select the gRNA with a higher score as the alternative sequence. Then, according to the designed gRNA targeting sequence, synthesize the corresponding base primer pair. Further, add protection bases to the 5'-ends of the forward sequence and reverse complementary sequence of each gRNA respectively.

[0095] Figure 2 Shows the design scheme of gRNA position and primer-mediated nucleotide mutation. Taking the mutation of H32 (KLLA0_E08625g) as an example, the gRNA is designed in the 5'-UTR of the ORF. The mutation at the gRNA position is introduced by the H32gF and H32gR primers. The mutation in the gene coding region is introduced by other primer pairs.

[0096] Step 2), perform PCR using the primer pair 1 obtained in Step 1), then react the PCR product with the pCas vector in the T4 ligase system, transform it into Escherichia coli cells, screen on the plate and sequence to obtain the gRNA_Cas9 co-expression vector. The gRNA_Cas9 co-expression vector can express gRNA and Cas9 simultaneously, thus realizing subsequent gene editing.

[0097] Step 3), obtain the genome of the histone, then introduce synonymous mutations at the positions corresponding to the gRNA, and then introduce mutations in the histone coding region to obtain a vector containing site-directed mutations of H31 or H32. The preferred method for obtaining the genome is PCR amplification, and more preferably high-fidelity PCR to ensure the accuracy of amplification; after adding A to the ends of the PCR products, they are ligated into a T vector to obtain a vector containing the H31 and H32 genes. Considering that the gRNA targeting region needs to be mutated to avoid cleavage of the recombinant sequence, therefore, a vector containing the H31 and H32 genes is used to obtain a vector with synonymous mutations introduced at the positions corresponding to the gRNA by designing mutant primers for PCR. Finally, mutations in the histone coding region are introduced using the aforementioned vector to obtain a vector containing site-directed mutations of H31 or H32.

[0098] Step 4), using the vector containing site-directed mutations of H31 or H32 as a template, obtain the DNA donor by PCR. The PCR is preferably high-fidelity PCR, and the obtained PCR product is precipitated with ethanol and stored frozen for later use.

[0099] Step 5), prepare competent cells, add the gRNA_Cas9 expression vector and the DNA donor, perform electrotransformation, screen and verify on plates after transformation, and obtain the strain containing site-directed mutations after two rounds of plate screening and verification. The gRNA_Cas9 expression vector and the DNA donor are simultaneously introduced into the recipient cells by electrotransformation. The gRNA_Cas9 expression vector expresses gRNA and Cas9 to clip the target gene and achieve site-directed mutation. The electrotransformation can be completed according to the corresponding standard operation procedure adopted. The cells after electrotransformation are cultured on plates, single colonies are picked for PCR and sequencing verification, the single colonies with correct sequencing are re-streaked on plates for the second round of PCR verification, and the mutant strain is obtained after two rounds of verification. When wanting to obtain a strain containing more than two site-directed mutations, it needs to be obtained through two-step transformation and screening, and the specific method is the same.

[0100] Furthermore, step 3) includes the following steps:

[0101] 3-1), using the genomic DNA of cells (such as fungal cells) as a template, design primer pair 2 for the selected gene, perform high-fidelity PCR amplification, add A to the ends of the PCR products and then ligate them into a T vector to obtain vector 1 containing the selected gene;

[0102] 3-2), using vector 1 as a template, design primer 3 for introducing synonymous mutations at the positions corresponding to the gRNA, perform high-fidelity PCR to obtain vector 2 with gene mutations in the gRNA targeting region;

[0103] 3-3), using vector 2 as a template, performing high-fidelity PCR with mutant primer pair 4 to introduce mutations in the histone coding region, and obtaining mutant vector 3, i.e., a vector containing site-directed mutations in H31 or H32.

[0104] In one example, the primer pair 2 sequence used in step 3-1) is selected from one or two groups of SEQ ID NO.3, 4 or SEQ ID NO.17, 18, where SEQ ID NO.3, 4 is one group and SEQ ID NO.17, 18 is one group.

[0105] The primer pair 3 sequence used in step 3-2) is selected from one or two groups of SEQ ID NO.15, 16 or SEQ ID NO.29, 30, where SEQ ID NO.15, 16 is one group and SEQ ID NO.29, 30 is one group.

[0106] The primer pair 4 sequence used in step 3-3) is selected from one or more groups of SEQ ID NO.7-10 or NO.21-28, where SEQ ID NO.7, 8 is one group, SEQ ID NO.9, 10 is one group, SEQ ID NO.21, 22 is one group, and so on.

[0107] In one example, the method further includes step 6), after the obtained strain containing site-directed mutations is expanded in culture, centrifuging to collect cells, and obtaining cell extracts for cell-free protein synthesis after cell disruption. The purpose of the present invention is to use the obtained strain containing site-directed mutations for cell-free protein synthesis. Therefore, after screening and obtaining the mutant strain, it is inoculated and expanded in culture, and then the cells are collected by centrifugation, filtration, etc., and the cells are disrupted by methods such as freeze pulverization and liquid nitrogen pulverization to obtain cell extracts for cell-free protein synthesis reactions. Specifically, the preparation method of the cell extracts or cell lysates is not limited, and a preferred preparation method includes the following steps:

[0108] (i) Culturing the mutant strain and isolating the cells;

[0109] (ii) Washing the cells to obtain washed cells;

[0110] (iii) Performing cell disruption on the washed cells to obtain a crude cell extract;

[0111] (iv) Performing solid-liquid separation on the crude cell extract to obtain the liquid part, which is the cell extract.

[0112] In the present invention, the method for separating and obtaining cells is not particularly limited, and centrifugation, filtration, suction filtration, etc. are preferred; the solid-liquid separation method is not particularly restricted, and one preferred method is centrifugation.

[0113] In one example, an expression vector or vector combination is also provided. The expression vector or vector combination contains nucleic acids obtained by site-directed mutagenesis of the first or second allele of the histone H3 gene as described above, and is further prepared by the method described in step 3) as described above. The vector contains a site-directed mutation in the histone coding region, has a stable shape, and can be used in various genetic engineering, such as constructing double-stranded DNA donors, for further gene editing, etc.

[0114] In one example, a mutant strain is also provided. The strain is obtained by the transformation described in the first aspect above. Preferably, the strain is selected from one or a combination of Pichia pastoris, Saccharomyces cerevisiae, Hansenula anomala, Candida spp., Kluyveromyces spp., Schizosaccharomyces pombe; preferably one or a combination of Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces spp.; most preferably Kluyveromyces lactis;

[0115] Furthermore, the mutant strain is prepared by the method of the foregoing steps 1)-5).

[0116] The mutant strain has stable genetic characteristics, can be expanded in culture and used in actual production, especially for cell-free protein synthesis reaction systems, and has broad application prospects.

[0117] In one example, a cell extract for cell-free protein synthesis is also provided. The cell extract is obtained by centrifuging and lysing a site-directed mutant strain prepared by the method of the foregoing example or the foregoing mutant strain.

[0118] As described above, the cell extract can effectively improve the activity of the cell-free protein synthesis system and improve production efficiency.

[0119] In one example, an in vitro protein synthesis system for expressing proteins is also provided. It contains the foregoing cell extract, or the histone contained in the system is obtained by site-directed mutagenesis of its H3 gene by mutating the first and / or second alleles described in the first aspect. Since the foregoing cell extract containing site-directed mutations is used, the system has the advantages of efficient translation and expression and increased protein yield. The protein is not strictly restricted as long as it is applicable to an in vitro cell-free synthesis system. The cell-free protein synthesis system of the present invention also includes one or more components selected from the following group: substrates for protein synthesis, substrates for RNA synthesis, RNA polymerase, magnesium ions, potassium ions, buffers, energy regeneration systems, polyethylene glycol (PEG) or its analogs, dithiothreitol (DTT), and optionally solvents, and the solvent is water or an aqueous solvent.

[0120] Furthermore, the cell extract does not contain long-chain nucleic acid molecules endogenous to yeast.

[0121] Furthermore, the substrates for synthesizing RNA include: one of nucleoside monophosphates, nucleoside triphosphates, or a combination thereof.

[0122] Furthermore, the substrates for synthesizing proteins include: 20 natural amino acids and unnatural amino acids.

[0123] Furthermore, the magnesium ions are derived from a magnesium ion source, and the magnesium ion source is selected from the group consisting of magnesium acetate, magnesium glutamate, or a combination thereof.

[0124] Furthermore, the potassium ions are derived from a potassium ion source, and the potassium ion source is selected from the group consisting of potassium acetate, potassium glutamate, or a combination thereof.

[0125] Furthermore, the energy regeneration system is selected from the group consisting of creatine phosphate / creatine phosphokinase system, one of the glycolytic pathway intermediate energy systems, sucrose, or a combination thereof.

[0126] Furthermore, the buffer is selected from the group consisting of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, tris(hydroxymethyl)aminomethane, or a combination thereof.

[0127] Furthermore, the protein synthesis system contains polyethylene glycol (PEG) or an analogue thereof. The concentration of polyethylene glycol or its analogue is not particularly limited. Generally, the concentration (w / v) of polyethylene glycol or its analogue is 0.1-8%, preferably 0.5-4%, more preferably 1-2%, based on the total weight of the protein synthesis system. Representative PEGs are selected from the group consisting of PEG3000, PEG3350, PEG6000, PEG8000, or a combination thereof.

[0128] Furthermore, 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.

[0129] An optional solution is that the protein synthesis system provided by the present invention includes: yeast cell extract, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, potassium acetate, magnesium acetate, adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), thymidine triphosphate (TTP), amino acid mixture, creatine phosphate, dithiothreitol (DTT), creatine phosphokinase, RNA polymerase, polyethylene glycol, sucrose.

[0130] The following specific examples are used to explain the detailed method of the present invention.

[0131] Example 1 Design of gRNA targeting sequence

[0132] In this example, two copies of histone H3 in the K. lactis genome were used as the modification region. As described above, through nucleotide sequence comparison, it was shown that more than 95% (391 / 411) of the bases in the ORF region were the same. To ensure the specificity of editing, the gRNA was designed in the non-coding region (5'-UTR) adjacent to the ORF. The sequence alignment is as Figure 1 shown.

[0133] Select the region (100 - 2000 bp) of the gene for which the gRNA needs to be designed, complete the design of the gRNA on the CRISPOR (tefor.net) (http: / / crispor.tefor.net / ) website, and select the gRNAs with higher scores as alternative sequences. For each selected gene, the present invention selects 1 sequence with a relatively high comprehensive score as the final gRNA sequence. The specific information is shown in Table 2. According to the designed gRNA targeting sequence, the corresponding 24-base primer pair was synthesized, that is, 4-base sequences of AATC and AAAC were added to the 5'-ends of the forward sequence and the reverse complementary sequence of each gRNA respectively. The finally obtained pair of 24-base primers constitutes a 24-base primer pair.

[0134] Table 2 gRNA list

[0135]

[0136] Example 2 Construction of gRNA_Cas9 expression vector

[0137] Dilute the 24-base primer pairs synthesized in Example 1 to 10 μM respectively, take 10 μL of each and add them to a PCR tube. After mixing, complete the annealing process on a PCR instrument according to the following program: 95°C, 3 min; 72°C, 2 min; 65°C, 2 min; 60°C, 2 min; 55°C, 2 min; 50°C, 2 min; 16°C, 2 min. Take 0.5 μL of the annealed primer pair and 20 ng of the pCasMFR vector (obtained by the inventors' self-modification, see patent application CN2023116023642 for details) that has been digested with BsaI and purified, mix them in a 10 μL T4 ligase system, and carry out a ligation reaction at 16°C for 1 h. The ligation product was completely transformed into Escherichia coli DH5α competent cells, and screened on an LB plate containing 50 μg / mL kanamycin. Randomly select 2 single colonies for sequencing verification, and select the clone with correct sequencing, extract the gRNA_Cas9 co-expression plasmid, measure its concentration, and use it for subsequent electroporation experiments.

[0138] Example 3 Construction of plasmids (vectors) containing H31 mutations

[0139] 3-1), Using H31-pF + H31-pR as primer pairs, with the genomic DNA of K. lactis Y1140 as the template, PCR amplification was carried out using Phanta Super Fidelity DNA polymerase to obtain PCR products. After adding A to the ends of the PCR products, they were ligated into T vectors to obtain vectors containing H31.

[0140] 3-2), Using the vectors containing H31 as templates, with the H31gF + H31gR primer pair, PCR amplification was carried out using Phanta Super

[0141] Fidelity DNA polymerase to obtain PCR products. After treatment with DpnI, the PCR products were transformed into competent E. coli cells, and monoclonal colonies were picked and verified by sequencing to obtain the mutant vector H31g.

[0142] 3-3), Using H31K27I-pF + H31K27I-pR, with the mutant vector H31g obtained in the previous step as the template, PCR amplification was carried out using Phanta Super Fidelity DNA polymerase to obtain PCR products. After treatment with DpnI, the PCR products were transformed into competent E. coli cells, and monoclonal colonies were picked and verified by sequencing to obtain the H31K27I mutant vector.

[0143] Similarly, in step 3-3), using H31K27M-pF + H31K27M-pR as primers, with the same other operation steps, the H31K27M mutant vector was obtained.

[0144] In step 3-3), using H31G34R-pF + H31G34R-pR as primers, with the same other operation steps, the H31G34R mutant vector was obtained.

[0145] In step 3-3), using H31G34W-pF + H31G34W-pR as primers, with the same other operation steps, the H31G34W mutant vector was obtained.

[0146] In step 3-3), using H31K36M-pF + H31K36M-pR as primers, with the same other operation steps, the H31K36M mutant vector was obtained.

[0147] The specific sequences of the primers used are shown in Table 3.

[0148] Example 4 Construction of plasmids (vectors) containing H32 mutations

[0149] First, using H32-pF + H32-pR as primer pairs and the genomic DNA of K. lactis Y1140 as a template, PCR amplification was performed with Phanta Super Fidelity DNA polymerase to obtain PCR products. After adding A to the ends of the PCR products, they were ligated into T vectors to obtain vectors containing H31.

[0150] Then, using the vectors containing H31 as templates and H32gF + H32gR primer pairs, PCR amplification was performed with Phanta Super Fidelity DNA polymerase to obtain PCR products. After treatment with DpnI, the PCR products were transformed into competent E. coli cells, and monoclonal colonies were picked and verified by sequencing to obtain the mutant vector H31g.

[0151] Subsequently, using H32K27I-pF + H32K27I-pR and the mutant vector H31g obtained in the previous step as a template, PCR amplification was performed with Phanta Super Fidelity DNA polymerase to obtain PCR products. After treatment with DpnI, the PCR products were transformed into competent E. coli cells, and monoclonal colonies were picked and verified by sequencing to obtain the mutant H32K27I vector.

[0152] Similarly, in step 3-3), using H32K27M-pF + H32K27M-pR as primers and with the same other operation steps, the mutant vector H32K27M was obtained.

[0153] In step 3-3), using H32G34R-pF + H32G34R-pR as primers and with the same other operation steps, the mutant vector H32G34R was obtained.

[0154] In step 3-3), using H32G34W-pF + H32G34W-pR as primers and with the same other operation steps, the mutant vector H32G34W was obtained.

[0155] In step 3-3), using H32K36M-pF + H32K36M-pR as primers and with the same other operation steps, the mutant vector H32K36M was obtained.

[0156] The specific sequences of the primers used are shown in Table 3.

[0157] Table 3 Primer List

[0158]

[0159] * The bases represented by the capital characters in the sequence indicate the positions where mutations were introduced.

[0160] Example 5 Construction of double-stranded DNA donor

[0161] In this example, double-stranded DNA was obtained by PCR using the vector containing the mutant sequence obtained in Example 4 as a template. Using the plasmids containing various mutations of H31 and H32 finally obtained in Example 4 as templates respectively, and using H31-pF + H31-pR or H32-pF + H32-pR as primer pairs (see Table 3), PCR amplification was performed using Phanta Super Fidelity DNA polymerase to obtain PCR products. After ethanol precipitation, the PCR products were dissolved in 50 μL ddH 2 O and stored frozen at -20 °C for later use. That is, double-stranded DNA donors containing H31K27I mutation, H31K27M mutation, H31G34R mutation, H31G34W mutation, H31K36M mutation, as well as H32K27I mutation, H32K27M mutation, H32G34R mutation, H32G34W mutation, H32K36M mutation, etc. were obtained respectively.

[0162] Example 6 Transformation and screening of yeast strains

[0163] The transformation of Kluyveromyces lactis strains was completed by electrotransformation. According to the requirements of the electrotransformation method, competent cells were prepared using K. lactis. Before electroporation, 40 μL of the competent cell mixture was fully mixed with the corresponding 500 ng of gRNA_Cas9 expression plasmid and 1 - 2 μg of the DNA Donor constructed in Example 5. The electrotransformation process was completed according to the standard operation procedure to obtain yeasts with different mutations. The electrotransformed yeast cells were screened on YPD plates containing 250 μg / mL G418. Single colonies were picked for PCR verification, and the positive PCR products were sent to a sequencing company for sequencing; the single colonies with correct sequencing were streaked on plates, and single colonies were picked for a second round of PCR verification. Only the strains that passed the two rounds of verification correctly were prepared into glycerol storage tubes and stored in an -80 °C refrigerator. The finally obtained mutant strains are shown in Table 4.

[0164] Table 4 List of strain names.

[0165]

[0166] Example 7 Preparation of lysis solution and verification of IVTT activity

[0167] The single colonies obtained by streaking the test strains cryopreserved in an -80 °C refrigerator in Example 6 on a YPD plate were picked and inoculated into a 250 mL Erlenmeyer flask containing 100 mL of seed medium, and cultured with shaking at 30 °C and 200 rpm for 24 h; an aliquot of the seed culture was transferred to a 1000 mL Erlenmeyer flask containing 400 mL of fermentation medium, and cultured with shaking at 30 °C and 200 rpm until the harvest period. The cells were collected by high-speed centrifugation, and a cell lysate was prepared under liquid nitrogen protection for subsequent IVTT activity testing. A certain amount of the lysate prepared according to the standard procedure was used to complete the detection and analysis of the D2P expression activity of the lysate with mEGFP as the reporter protein according to the operation procedure, and the IVTT activity of the corresponding lysate was judged based on the detected reporter fluorescence intensity (RFU value).

[0168] From Figure 3 the growth curve results, it can be seen from the above experimental results that the growth of most mutant strains in the YPD liquid medium did not show obvious differences from that of the control strain. It is worth noting that although the maximum specific growth rate of H312 did not change significantly compared with the control strain, its lag phase was significantly shortened; while the maximum specific growth rate of H315 and H323 was significantly lower than that of the control strain, indicating that Histone H3K36M would significantly reduce the maximum specific growth rate of the strain.

[0169] From Figure 4 the IVTT activity results, it can be seen that the extracts of the H312 and H324 strains with allelic gene mutations had significantly higher IVTT activities at 3 h and 20 h than those of the control strain. The extracts of the H313 and H321 strains had significantly higher IVTT activities at 3 h and 20 h than those of the control strain, but were slightly lower than those of H324. It shows that both the H3K27M and H3G34R allelic gene mutations have the potential to improve the IVTT activity of yeast cell extracts, and a single copy of the mutation has a significant effect. At the same time, the extracts of the H32G34W and H32K36M mutations also have a certain potential for improvement.

[0170] The above are only partial embodiments of the present invention, and the present invention is not limited to the content of the above embodiments.

[0171] All the documents mentioned in the present invention are cited in this application as references, as if each document was cited separately as a reference. In addition, it should be understood that after reading the above content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A method for improving the efficiency of cell-free protein synthesis by a mutant histone, characterized in that: The cells from which the cell extract used for the cell-free protein synthesis is derived are modified as follows: the first and / or second alleles of the histone H3 gene in the cells are site-directed mutated; The site-directed mutation refers to mutating lysine or glycine on the first or second allele.

2. The method for improving the efficiency of cell-free protein synthesis by a mutant histone according to claim 1, characterized in that, The cells are selected from one or any combination of bacteria, mammalian cells, human cells, plant cells, yeast cells, insect cells, preferably from yeast cells.

3. The method for improving the efficiency of cell-free protein synthesis by a mutant histone according to claim 2, characterized in that, The yeast cell source is selected from one or more of Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Pichia pastoris, Kluyveromyces, Candida utilis, Saccharomyces mellis, methanol-induced yeast, Hansenula; preferably Kluyveromyces; The Kluyveromyces further includes: one or a combination of Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces dobzhanskii, Kluyveromyces aestuarii, Kluyveromyces nonfermentans, Kluyveromyces wickerhamii, Kluyveromyces thermotolerans, Kluyveromyces fragilis, Kluyveromyces hubeiensis, Kluyveromyces polysporus, Kluyveromyces siamensis, Kluyveromyces marxianus var. yarrowii.

4. The method for improving the efficiency of cell-free protein synthesis by a mutant histone according to any one of claims 1-3, characterized in that, The site-directed mutation is selected from one or more of the following: The 27th lysine (K) of the protein encoded by the first or second allele of histone H3 is mutated to methionine (M); The 34th glycine (G) of the protein encoded by the first or second allele of histone H3 is mutated to arginine (R); The 34th glycine (G) of the protein encoded by the second allele of histone H3 is mutated to tryptophan (W); The 36th lysine (K) of the protein encoded by the second allele of histone H3 is mutated to methionine (M).

5. The method for improving the efficiency of cell-free protein synthesis by a mutant histone according to any one of claims 1-4, characterized in that, The site-directed mutation is achieved by CRISPR-Cas9-mediated gene editing means.

6. The method for improving the efficiency of cell-free protein synthesis by a mutant histone according to claim 5, characterized in that, The method of the site-directed mutation is: Step 1), design the gRNA targeting sequence; Step 2), construct the gRNA_Cas9 co-expression vector; Step 3), construct the vector containing the H31 and / or H32 site-directed mutation; Step 4), construct the double-stranded DNA donor; Step 5), cell transformation and screening.

7. The method for improving the efficiency of cell-free protein synthesis by a mutant histone according to claim 6, characterized in that, The specific method of the site-directed mutation is: Step 1), design the gRNA for the selected gene and synthesize the corresponding primer pair 1; Step 2), perform PCR using the primer pair 1 obtained in Step 1), then react the PCR product with the pCas vector in a T4 ligase system, transform it into Escherichia coli cells, screen on a plate and sequence to obtain the gRNA_Cas9 co-expression vector; Step 3), obtain the genome of the histone, then introduce a synonymous mutation at the position corresponding to the gRNA, and then introduce a mutation in the histone coding region to obtain a vector containing a site-directed mutation of H31 or H32; Step 4), use the vector containing the site-directed mutation of H31 or H32 as a template, and obtain the DNA donor by PCR; Step 5), prepare competent cells, add the gRNA_Cas9 expression vector and the DNA donor, perform electrotransformation, screen on a plate after transformation and verify. After two rounds of plate screening and verification, obtain the strain containing the site-directed mutation.

8. The method for improving the efficiency of cell-free protein synthesis by the mutant histone according to claim 7, wherein, Step 3) further includes the following steps: 3-1), using the genomic DNA of the cell as a template, design primer pair 2 for the selected gene, perform high-fidelity PCR amplification, add A to the end of the PCR product and ligate it into the T vector to obtain vector 1 containing the selected gene; 3-2), using vector 1 as a template, design primer 3 for introducing a synonymous mutation at the position corresponding to the gRNA, perform high-fidelity PCR to obtain vector 2 with a gene mutation in the gRNA targeting region; 3-3), using vector 2 as a template, perform high-fidelity PCR with mutant primer pair 4 to introduce a mutation in the histone coding region to obtain mutant vector 3, that is, the vector containing the site-directed mutation of H31 or H32.

9. An expression vector or vector combination, wherein, the expression vector or vector combination contains the nucleic acid after the site-directed mutation of the histone H3 gene as described in claim 1 or 4, and is further prepared by the method described in step 3) of claim 7 or 8.

10. A mutant strain, wherein, the strain is obtained by the transformation as described in claim 1 or 4. Preferably, the strain source is selected from one or a combination of Pichia pastoris, Saccharomyces cerevisiae, Hansenula anomala, Candida spp., Kluyveromyces spp., Schizosaccharomyces pombe; preferably one or a combination of Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces spp.; most preferably Kluyveromyces lactis; Furthermore, the mutant strain is prepared by the method described in any one of claims 6-8.

11. A cell extract for cell-free protein synthesis, wherein, the cell extract is obtained by culturing the site-directed mutant strain prepared by the method described in any one of claims 6-8 or the mutant strain described in claim 10, and then centrifuging and lysing.

12. An in vitro protein synthesis system for expressing proteins, wherein: it contains the cell extract described in claim 11, or the histone contained in the system is obtained by the site-directed mutation of its H3 gene as described in claim 1 or 4.

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