WO2019100456A1 - 一种通过对核酸酶系统敲除以调控体外生物合成活性的方法 - Google Patents

一种通过对核酸酶系统敲除以调控体外生物合成活性的方法 Download PDF

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WO2019100456A1
WO2019100456A1 PCT/CN2017/115966 CN2017115966W WO2019100456A1 WO 2019100456 A1 WO2019100456 A1 WO 2019100456A1 CN 2017115966 W CN2017115966 W CN 2017115966W WO 2019100456 A1 WO2019100456 A1 WO 2019100456A1
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protein
protein synthesis
gene
exn53
synthesis system
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PCT/CN2017/115966
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English (en)
French (fr)
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郭敏
薛银鸽
代田纯
姜灵轩
郑竹霞
柴智
刘帅龙
于雪
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康码（上海）生物科技有限公司
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Priority to JP2020545834A priority Critical patent/JP7093417B2/ja
Priority to EP17932666.5A priority patent/EP3715462A4/en
Priority to KR1020207018328A priority patent/KR102345759B1/ko
Priority to US16/766,845 priority patent/US20230192781A1/en
Publication of WO2019100456A1 publication Critical patent/WO2019100456A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a method for modulating in vitro biosynthetic activity by knocking out a nuclease system.
  • Protein is an important molecule in cells and is involved in the execution of all functions of cells. The sequence and structure of the protein are different, which determines the difference in function. In cells, proteins can act as enzymes to catalyze various biochemical reactions. They can act as signal molecules to coordinate various activities of organisms, support biological forms, store energy, transport molecules, and make organisms move. In the field of biomedicine, protein antibodies are important drugs for treating diseases such as cancer as a targeted drug [1][2] .
  • the four processes of protein translation include translation initiation, translation extension, translation termination, and ribosome recycling, where translation initiation is the most regulated process [3] .
  • translation initiation is the most regulated process [3] .
  • the ribosomal small subunit (40S) binds (tRNA) i Met and recognizes the 5' end of the mRNA under the action of a translation initiation factor.
  • the small subunit moves downstream and binds to the ribosomal large subunit (60S) at the initiation codon (ATG) to form a complete ribosome and enters the translational extension phase [4] .
  • In vitro biosynthesis system refers to the completion of specific chemical molecules or organisms by adding exogenously encoded nucleic acid DNA, RNA, substrate and energy source in a lysing system of bacteria, fungi, plant cells or animal cells. High-efficiency synthesis of macromolecules (DNA, RNA, protein) in vitro.
  • a common in vitro biosynthesis system is an in vitro protein synthesis system that performs rapid and efficient translation of exogenous recombinant proteins by exogenous mRNA or DNA template and using cell lysates [5] .
  • the commercially available in vitro protein synthesis system is an in vitro transcription-translation system (IVTT), which transcribes mRNA intermediates via RNA polymerase, and then uses amino acids and ATP. Divide, complete one-step efficient translation of foreign proteins.
  • IVTT in vitro transcription-translation system
  • common commercial in vitro protein expression systems include Escherichia coli extract (ECE), Rabbit reticu Locyte lysate (RRL), Wheat germ extract (WGE), and insect (Insect cell extract, ICE) and human source systems.
  • Nucleic acid mRNA and DNA proteins encode substrates in in vitro synthesis systems whose stability affects protein yield.
  • a nuclease is a protein that hydrolyzes a phosphodiester bond between nucleotides in the first step of nucleic acid decomposition. Some nucleases can only act on RNA, called RNase. Some nucleases can only act on DNA, called deoxyribonuclease (DNase). The nuclease can be further divided into an exonuclease and an endonuclease depending on the position of the action of the nuclease.
  • RNA in cells The degradation of most RNA in cells is mainly through the 5'to 3' exonuclease localized to the cytoplasm and the 5'to 3' exonuclease localized to the nucleus, or by localization to the cytoplasm and nucleus, with 3' To 5' exonuclease and endonuclease activity of the protein subunit complex exosome degradation.
  • CRISPR/Cas Clustered ReguLatory Interspaced Short Palindromic Repeats/CRISPR associated
  • gRNA guide RNA
  • PAM protospacer adjacent motif
  • CRISPR/Cas9 duplex the gene cleaved by CRISPR/Cas9 duplex can be recombined into a new sequence in HDR for genetic modification purposes.
  • donor DNA donor DNA
  • CRISPR/Cas9 duplex can be recombined into a new sequence in HDR for genetic modification purposes.
  • Saccharomyces. Cerevisiae there are many examples of genomic engineering using the CRISPR/Cas9 system, including gene point mutations, gene knockouts, and gene insertions.
  • Nucleases may be retained in current in vitro synthesis systems. These nucleases affect the stability of nucleic acids in in vitro synthesis systems by degrading mRNA and DNA in in vitro synthesis systems.
  • a first aspect of the invention provides an in vitro cell-free protein synthesis system, the cell-free protein synthesis system comprising:
  • the content of the EXN53 protein in the yeast cell extract is ⁇ 10%, preferably ⁇ 5%, more preferably ⁇ 2%.
  • the content of EXN53 in the yeast cell extract is zero.
  • the EXN53 is derived from a yeast selected from one or more of the following groups: Pichia pastoris, Kluyveromyces, preferably, derived from Kluyveromyces.
  • the Kluyveromyces cerevisiae comprises Kluyveromyces cerevisiae, and/or Kluyveromyces lactis.
  • nucleotide sequence of the EXN53 is set forth in SEQ ID NO.: 1.
  • the protein sequence of the EXN53 is set forth in SEQ ID NO.: 6.
  • the cell-free protein synthesis system further comprises one or more components selected from the group consisting of:
  • the cell-free protein synthesis system further comprises one or more components selected from the group consisting of:
  • the yeast cell is selected from the group consisting of yeast of one or more sources: Pichia pastoris, Kluyveromyces, or a combination thereof; preferably, the yeast cell comprises: g Luwei yeast, more preferably Kluyveromyces marxianus, and/or Kluyveromyces lactis.
  • the yeast cell extract is an aqueous extract of yeast cells.
  • the yeast cell extract is free of yeast endogenous long chain nucleic acid molecules.
  • the yeast cell extract is prepared by a method comprising the steps of:
  • the solid-liquid separation comprises centrifugation.
  • centrifugation is carried out in a liquid state.
  • the centrifugation conditions are from 5,000 to 100,000 x g, preferably from 8,000 to 30,000 x g.
  • the centrifugation time is from 0.5 to 2 h, preferably from 20 min to 50 min.
  • the centrifugation is carried out at 1-10 ° C, preferably at 2-6 ° C.
  • the washing treatment is carried out using a washing liquid at a pH of 7-8 (preferably, 7.4).
  • the washing liquid is selected from the group consisting of potassium 4-hydroxyethylpiperazine ethanesulfonate, potassium acetate, magnesium acetate, or a combination thereof.
  • the cell disruption treatment comprises high pressure disruption, freeze-thaw (eg, liquid nitrogen cryolysis) disruption.
  • the substrate for the synthetic RNA comprises: a nucleoside monophosphate, a nucleoside triphosphate, or a combination thereof.
  • the substrate of the synthetic protein comprises: 1-20 natural amino acids, and unnatural amino acids.
  • the magnesium ion is derived from a source of magnesium ions selected from the group consisting of magnesium acetate, magnesium glutamate, or a combination thereof.
  • the potassium ion is derived from a source of potassium ions selected from the group consisting of potassium acetate, potassium glutamate, or a combination thereof.
  • the energy regeneration system is selected from the group consisting of a phosphocreatine/phosphocreatase system, a glycolysis pathway and its intermediate energy system, or a combination thereof.
  • the cell-free protein synthesis system further comprises (f1) a synthetic tRNA.
  • the buffering agent is selected from the group consisting of 4-hydroxyethylpiperazineethanesulfonic acid, trishydroxymethylaminomethane, or a combination thereof.
  • the cell-free protein synthesis system further comprises (g1) a foreign DNA molecule for directing protein synthesis.
  • the DNA molecule is linear.
  • the DNA molecule is cyclic.
  • the DNA molecule contains a sequence encoding a foreign protein.
  • the sequence encoding the foreign protein comprises a genomic sequence, a cDNA sequence.
  • sequence encoding the foreign protein further comprises a promoter sequence, a 5' untranslated sequence, and a 3' untranslated sequence.
  • the cell-free protein synthesis system comprises a component selected from the group consisting of 4-hydroxyethylpiperazineethanesulfonic acid, potassium acetate, magnesium acetate, nucleoside triphosphate, amino acid, creatine phosphate Dithiothreitol (DTT), phosphocreatine kinase, RNA polymerase, or a combination thereof.
  • the polyethylene glycol is selected from the group consisting of PEG3000, PEG 8000, PEG 6000, PEG 3350, or a combination thereof.
  • the polyethylene glycol comprises polyethylene glycol having a molecular weight (Da) of from 200 to 10,000, preferably polyethylene glycol having a molecular weight of from 3,000 to 10,000.
  • the concentration (v/v) of the component (a) in the protein synthesis system is from 20% to 70%, preferably from 30% to 60%, more preferably from 40% to 50%. %, based on the total volume of the protein synthesis system.
  • the concentration (w/v, for example, g/mL) of the component (b) in the protein synthesis system is 0.1 to 8%, preferably 0.5 to 4%, more preferably, 1-2%.
  • the concentration of component (c) in the protein synthesis system is 0.2 to 4%, preferably 0.5 to 4%, more preferably 0.5 to 1%, to synthesize the protein.
  • the total volume of the system is 0.2 to 4%, preferably 0.5 to 4%, more preferably 0.5 to 1%, to synthesize the protein.
  • the nucleoside triphosphate is selected from the group consisting of adenosine triphosphate, guanosine triphosphate, cytidine triphosphate, uridine nucleoside triphosphate, or a combination thereof.
  • the concentration of the component (e1) in the protein synthesis system is from 0.1 to 5 mM, preferably from 0.5 to 3 mM, more preferably from 1 to 1.5 mM.
  • the amino acid is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, phenylalanine, valine, tryptophan, serine, Tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, or a combination thereof.
  • the amino acid comprises a D-form amino acid and/or an L-form amino acid.
  • the concentration of the component (e2) in the protein synthesis system is 0.01 to 0.48 mM, preferably 0.04 to 0.24 mM, more preferably 0.04 to 0.2 mM, optimally , 0.08 mM.
  • the concentration of the component (e3) in the protein synthesis system is 1-10 mM, preferably 1-5 mM, more preferably 2-4 mM.
  • the concentration of the component (e4) in the protein synthesis system is 30-210 mM, preferably 30-150 mM, more preferably 30-60 mM.
  • the concentration of the component (e6) in the protein synthesis system is 0.01 to 0.3 mg/mL, preferably 0.02 to 0.1 mg/mL, more preferably 0.027 to 0.054 mg. /mL.
  • the concentration of 4-hydroxyethylpiperazineethanesulfonic acid in the protein synthesis system is 5 to 50 mM, preferably 10 to 50 mM, preferably 15 to 30 mM, more preferably , 20-25 mM.
  • the concentration of the potassium acetate in the protein synthesis system is 20-210 mM, preferably 30-210 mM, preferably 30-150 mM, more preferably 30-60 mM.
  • the magnesium acetate has a concentration of 1-10 mM, preferably 1-5 mM, more preferably 2-4 mM.
  • the concentration of creatine phosphate is 10-50 mM, preferably 20-30 mM, more preferably 25 mM.
  • the concentration of the heme in the protein synthesis system is 0.01 to 0.1 mM, preferably 0.02 to 0.08 mM, more preferably 0.03 to 0.05 mM, most preferably 0.04 mM. .
  • the spermidine concentration in the protein synthesis system is 0.05-1 mM, preferably 0.1-0.8 mM, more preferably, more preferably 0.2-0.5 mM, more preferably Ground, 0.3-0.4 mM, optimally, 0.4 mM.
  • the concentration of the dithiothreitol (DTT) in the protein synthesis system is from 0.2 to 15 mM, preferably from 0.2 to 7 mM, more preferably from 1 to 2 mM.
  • the concentration of the phosphocreatine kinase in the protein synthesis system is 0.1 to 1 mg/mL, preferably 0.2 to 0.5 mg/mL, more preferably 0.27 mg/mL.
  • the concentration of the T7 RNA polymerase in the protein synthesis system is 0.01-0.3 mg/mL, preferably 0.02-0.1 mg/mL, more preferably 0.027-0.054 mg/mL. .
  • the cell-free in vitro synthesis system has the following properties:
  • composition of the cell-free protein synthesis system comprises:
  • composition of the cell-free protein synthesis system further comprises:
  • the PEG is selected from the group consisting of PEG 3350, PEG 3000, and/or PEG 8000.
  • the RNA polymerase is T7 RNA polymerase.
  • a second aspect of the present invention provides a yeast cell extract having an EXN53 protein content of ⁇ 10%, preferably ⁇ 5%, more preferably ⁇ 2%.
  • a third aspect of the present invention provides a method for producing an in vitro cell-free protein synthesis system according to the first aspect of the present invention, comprising the steps of:
  • the concentration (v/v) of the component (a) in the protein synthesis system is from 20% to 70%, preferably from 30% to 60%, more preferably from 40% to 50%. %, based on the total volume of the protein synthesis system.
  • the concentration (w/v, for example, g/mL) of the component (b) in the protein synthesis system is 0.1 to 8%, preferably 0.5 to 4%, more preferably, 1-2%.
  • a fourth aspect of the invention provides a method of synthesizing a protein in vitro comprising the steps of:
  • step (ii) incubating the protein synthesis system of step (i) for a period of time T1 under suitable conditions to synthesize the protein encoded by the exogenous DNA.
  • the method further comprises: (iii) isolating or detecting the protein encoded by the exogenous DNA, optionally from the protein synthesis system.
  • the exogenous DNA is from a prokaryote, a eukaryote.
  • the exogenous DNA is from an animal, a plant, or a pathogen.
  • the exogenous DNA is from a mammal, preferably a primate, a rodent, including a human, a mouse, a rat.
  • the coding sequence of the foreign protein encodes a foreign protein selected from the group consisting of luciferin, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, Aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, variable region of antibody, luciferase mutant, alpha-amylase, enterobacterin A, C Hepatitis B virus E2 glycoprotein, insulin precursor, interferon alpha A, interleukin-1 beta, lysozyme, serum albumin, single-chain antibody fragment (scFV), thyroxine transporter, tyrosinase, xylan Enzyme, or a combination thereof.
  • luciferin or luciferase (such as firefly luciferase)
  • green fluorescent protein yellow fluorescent protein
  • Aminoacyl tRNA synthetase
  • the exogenous protein is selected from the group consisting of luciferin, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, glyceraldehyde- 3-phosphate dehydrogenase, catalase, actin, variable region of antibody, luciferase mutation, alpha-amylase, enteromycin A, hepatitis C virus E2 glycoprotein, insulin precursor Interferon alpha A, interleukin-1 beta, lysozyme, serum albumin, single chain antibody fragment (scFV), thyroxine transporter, tyrosinase, xylanase, or a combination thereof.
  • luciferin or luciferase (such as firefly luciferase)
  • green fluorescent protein yellow fluorescent protein
  • aminoacyl tRNA synthetase aminoacyl tRNA synthetase
  • the exogenous DNA encodes a foreign protein selected from the group consisting of luciferin, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA Synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, variable region of antibody, luciferase mutant, alpha-amylase, enteromycin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon alpha A, interleukin-1 beta, lysozyme, serum albumin, single-chain antibody fragment (scFV), thyroxine transporter, tyrosinase, xylanase, or Its combination.
  • luciferin or luciferase (such as firefly luciferase)
  • green fluorescent protein yellow fluorescent protein
  • aminoacyl tRNA Synthetase glyceraldehyde
  • the protein encoded by the exogenous DNA is selected from the group consisting of luciferin, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, Glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, variable region of antibody, luciferase mutation, alpha-amylase, enteromycin A, hepatitis C virus E2 glycoprotein, Insulin precursor, interferon alpha A, interleukin-1 beta, lysozyme, serum albumin, single chain antibody fragment (scFV), thyroxine transporter, tyrosinase, xylanase, or a combination thereof.
  • luciferin or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, Glyceraldehyde-3-
  • the reaction temperature is 20 to 37 ° C, preferably 20 to 25 ° C.
  • the reaction time is from 1 to 6 h, preferably from 2 to 4 h.
  • a fifth aspect of the present invention provides an engineered strain which is a Kluyveromyces strain, and wherein expression or activity of an EXN53 gene (nuclease gene) in the strain is decreased.
  • the "reduction" means that the expression level of the EXN53 gene is ⁇ 10%, preferably ⁇ 5%, more preferably ⁇ 2%.
  • the "reduction" means that the expression or activity of the EXN53 gene is decreased to satisfy the following conditions:
  • the ratio of A1/A0 is ⁇ 30%, preferably ⁇ 10%, more preferably ⁇ 5%, more preferably ⁇ 2%, most preferably 0-2%;
  • A1 is the expression or activity of the EXN53 gene
  • A0 is the expression or activity of the wild-type EXN53 gene.
  • the expression or activity of the EXN53 gene (nuclease gene) in the strain is reduced by a method selected from the group consisting of gene mutation, gene knockout, gene disruption, RNA interference technology, Crispr technology Or a combination thereof.
  • a sixth aspect of the invention provides the use of the engineered strain of the fifth aspect of the invention for improving the efficiency of protein synthesis in vitro.
  • Figure 1 shows a design roadmap for improving the stability of nucleic acids by reducing nucleases in in vitro biosynthetic systems and utilizing the analysis and specific modification of nuclease genes in the K. lactis genome.
  • A K. lactis wild type cells.
  • B K. lactis nuclease engineered cells.
  • C The K. lactis cell lysate after the nuclease was engineered.
  • D The yeast strain prepared by modifying the specific gene was prepared into a yeast cell solution, which has more stable nucleic acid characteristics and prepared an enhanced in vitro biosynthesis system.
  • Figure 2 shows a distribution and functional analysis of all 61 nuclease genes in the K. lactis genome.
  • AF is a six-chromosome of K.lactis, and the nucleases on the A chromosome are: KlSEN54, KlDNA2, KlTRM2, KlFCF1, KlDOM34, KlRAD2, KlRNH70, KlDIS3, KlNPP1; nucleases on the B chromosome are: KlOGG1; nucleases located on the C chromosome There are: KlPOL2, KlRAD50, KlYSH1, KlRCL1, KlNGL2, KlMRE11, KlPOP3, KlMKT1, KlAPN1, KlRPP1, KlPOP2; nucleases on the D chromosome are: KlNUC1, KlRPP6, KlDBR1, KlRPS3, KlRPM2, KlSUV3, KlRAD1, KlIRE1, KlPOP1;
  • Figure 3 shows the sequence of the five nucleases genetically engineered in the K. lactis genome and the design of the CRISPR system.
  • Donor DNA two homologous arms homologous arm 1 and homologous arm 2 are the ORFs of the nuclease gene to be knocked out, and the gene sequence of about 1000 bp downstream, construct two guide gRNAs, and recognize the nuclease gene ORF to be knocked out.
  • the 5' and 3' PAM sequences direct the Cas9 nuclease to cleave the DNA, and homologous recombination occurs after DSB (double strand break), thereby achieving gene knockout.
  • Figure 4 shows a schematic representation of five nuclease protein domains in the K. lactis genome.
  • Figure 5 shows the KLEXN53 protein and the homologous protein ScEXN53 (KEGG No.: YGL173C) in Saccharomyces cerevisiae, and the homologous protein SpEXO2 (KEGG No.: SPAC17A5.14) in Schizosaccharomyces pombe, and the homologous protein HsEXN53 (KEGG) in Homo sapiens No.:54464) partial amino acid sequence alignment map, active site marked with #.
  • Figure 6 shows the homologous protein ScDIS3 (KEGG No.: YOL021C) of KlDIS3 protein and Saccharomyces cerevisiae, and the homologous protein SpDIS3 (KEGG No.: SPBC26H8.10) in Schizosaccharomyces pombe, and homologous protein HsDIS3 (KEGG) in Homo sapiens No.: 22894) partial amino acid sequence alignment map, active site marked with #.
  • Figure 7 shows the KlRAT1 protein and the homologous protein ScRAT1 (KEGG No.: YOR048C) in Saccharomyces cerevisiae, the homologous protein SpRAT1 (KEGG No.: SPAC26A3.12c) in Schizosaccharomyces pombe, and the homologous protein HsRAT1 (KEGG) in Homo sapiens No.: 22803) partial amino acid sequence alignment map, active site marked with #.
  • Figure 8 shows the KlRRP6 protein and the homologous protein ScRRP6 (KEGG No.: YOR001W) in Saccharomyces cerevisiae, the homologous protein SpRRP6 (KEGG No.: SPAC1F3.01) in Schizosaccharomyces pombe, and the homologous protein HsRRP6 (KEGG) in Homo sapiens No.: 5394) Partial amino acid sequence alignment map, the active site is marked with #.
  • Figure 9 shows the KlNGL2 protein and the homologous protein ScNGL3 (KEGG No.: YML118W) in Saccharomyces cerevisiae, the homologous protein SpNGL2 (KEGG No.: SPBC9B6.11c) in Schizosaccharomyces pombe, and the homologous protein HsANGEL2 (KEGG) in Homo sapiens No.: 90806) Amino acid sequence alignment map.
  • Figure 10 shows the plasmid map of pHoCas9_SE_Kana_tRNA_ScRNR2_KlEXN53-1&2.
  • the gRNA1 and gRNA2 of KLEXN53 are the two gRNAs at the 5' and 3' ends of the KLEXN53 gene ORF, respectively, with the tRNA-Tyr promoter and the SNR52 terminator, PMZ374-cas9 is the optimized cas9, and the plasmid carries the kana screening marker.
  • Figure 11 shows the KLEXN53-DD1-pMD18 plasmid map.
  • the homologous arm 1 and the homologous arm 2 are the gene sequences of about 1000 bp upstream and downstream of the ORF of the KLEXN53 gene, and the plasmid carries the Amp screening marker.
  • Figure 12 shows the plasmid map of pHoCas9_SE_Kana_tRNA_ScRNR2_KlDIS3-1&2.
  • the gRNA1 and gRNA2 of KlDis3 are the two gRNAs at the 5' and 3' ends of the KlDis3 gene ORF, respectively, with the tRNA-Tyr promoter and the SNR52 terminator, PMZ374-cas9 is the optimized cas9, and the plasmid carries the kana screening marker.
  • Figure 13 shows the KlDIS3-DD1-pMD18 plasmid map.
  • the homologous arm 1 and the homologous arm 2 are the gene sequences of about 1000 bp upstream and downstream of the ORF of the KlDis3 gene, and the plasmid carries the Amp screening marker.
  • Figure 14 shows the plasmid map of pHoCas9_SE_Kana_tRNA_ScRNR2_KlRat1-1&2.
  • the gRNA1 and gRNA2 of KlRat1 are the two gRNAs at the 5' and 3' ends of the KlRat1 gene ORF, respectively, with the tRNA-Tyr promoter and the SNR52 terminator, PMZ374-cas9 is the optimized cas9, and the plasmid carries the kana screening marker.
  • Figure 15 shows the KlRat1-DD1-pMD18 plasmid map.
  • the homologous arm 1 and the homologous arm 2 are the gene sequences of about 1000 bp upstream and downstream of the ORF of the KlRat1 gene, and the plasmid carries the Amp screening marker.
  • Figure 16 shows the plasmid map of pHoCas9_SE_Kana_tRNA_ScRNR2_KlRrp6-1&2.
  • the gRNA1 and gRNA2 of KlRrp6 are the two gRNAs at the 5' and 3' ends of the KlRrp6 gene ORF, respectively, with the tRNA-Tyr promoter and the SNR52 terminator, PMZ374-cas9 is the optimized cas9, and the plasmid carries the kana screening marker.
  • Figure 17 shows the KlRrp6-DD1-pMD18 plasmid map.
  • the homologous arm 1 and the homologous arm 2 are the gene sequences of about 1000 bp upstream and downstream of the ORF of the KlRrp6 gene, and the plasmid carries the Amp screening marker.
  • Figure 18 shows the plasmid map of pHoCas9_SE_kana_tRNA_ScRNR2_KlNGL2-1&2.
  • the gRNA1 and gRNA2 of KlNGL3 are the two gRNAs at the 5' and 3' ends of the K1NGL3 gene ORF, respectively, with the tRNA-Tyr promoter and the SNR52 terminator, PMZ374-cas9 is the optimized cas9, and the plasmid carries the kana screening marker.
  • Figure 19 shows the Kl NGL2-DD1-pMD18 plasmid map.
  • the homologous arm 1 and the homologous arm 2 are the gene sequences of about 1000 bp upstream and downstream of the ORF of the KlNGL3 gene, and the plasmid carries the Amp screening marker.
  • Figure 20 shows a graph of in vitro translational activity assay data for engineered strains.
  • the fluorescence intensity of firefly fluorescent protein (Fluc) is used to indicate the ability of the recombinant protein to synthesize in vitro biosynthesis systems.
  • Fluc firefly fluorescent protein
  • wt represents wild type Kluyveromyces
  • ⁇ KLEXN53EXN53 represents a KLEXN53EXN53 gene knockout Kluyveromyces strain.
  • nucleases After extensive and in-depth research, through the extensive screening and exploration, five nucleases are screened out from a variety of nucleases, one of which is down-regulated or knocked out, and unexpected findings can be improved.
  • the stability of nucleic acids and the efficiency of protein production in in vitro protein synthesis systems Specifically, after down-regulating or knocking out EXN53, the luciferase activity of the ⁇ klexn53 yeast strain in IVTT was ⁇ 2 times that of the wild type. On the basis of this, the inventors completed the present invention.
  • Yeast combines the advantages of simple, efficient protein folding, and post-translational modification. Among them, Saccharomyces cerevisiae and Pichia pastoris are model organisms that express complex eukaryotic proteins and membrane proteins. Yeast can also be used as a raw material for the preparation of in vitro translation systems.
  • Kluyveromyces is an ascomycete, in which Kluyveromyces marxianus and Kluyveromyces lactis are industrially widely used yeasts.
  • Kluyveromyces cerevisiae has many advantages over other yeasts, such as superior secretion capacity, better large-scale fermentation characteristics, food safety levels, and the ability to simultaneously modify post-translational proteins.
  • the yeast in vitro protein synthesis system is not particularly limited, and a preferred yeast in vitro protein synthesis system is the Kluyveromyces expression system (more preferably, the K. lactis expression system).
  • the yeast in vitro protein synthesis system comprises:
  • the EXN53 is derived from a yeast selected from one or more of the following groups: Pichia pastoris, Kluyveromyces, preferably, derived from Kluyveromyces (eg, Markscroft) S. cerevisiae, Kluyveromyces lactis).
  • Kluyveromyces cerevisiae e.g., Kluyveromyces lactis
  • Kluyveromyces lactis is not particularly limited, and includes any Kluvi (e.g., Kluyveromyces lactis) strain capable of improving the efficiency of synthetic proteins.
  • the protein sequence of the EXN53 is set forth in SEQ ID NO.: 6, and the nucleotide sequence of the EXN53 is set forth in SEQ ID NO.: 1.
  • the in vitro protein synthesis system comprises: yeast 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, RNase inhibitor, fluorescein, luciferase DNA, RNA polymerase.
  • yeast cell extract 4-hydroxyethylpiperazineethanesulfonic acid
  • potassium acetate magnesium acetate
  • adenosine triphosphate (ATP) adenosine triphosphate
  • GTP guanosine triphosphate
  • CTP cytosine triphosphate
  • TTP thymidine triphosphate
  • amino acid mixture amino acid mixture
  • creatine phosphate dithio
  • the RNA polymerase is not particularly limited and may be selected from one or more RNA polymerases, and a typical RNA polymerase is T7 RNA polymerase.
  • the ratio of the yeast cell extract in the in vitro protein synthesis system is not particularly limited, and generally the yeast cell extract accounts for 20-70% of the in vitro protein synthesis protein synthesis system, preferably Ground, 30-60%, more preferably, 40-50%.
  • the yeast cell extract does not contain intact cells, and typical yeast cell extracts include ribosomes for protein translation, transfer RNA, aminoacyl tRNA synthetase, initiation factors required for protein synthesis, and The elongation factor and the termination release factor.
  • the yeast extract contains some other proteins in the cytoplasm derived from yeast cells, especially soluble proteins.
  • the yeast cell extract contains a protein content of 20 to 100 mg/mL, preferably 50 to 100 mg/mL.
  • the method for determining protein content is a Coomassie Brilliant Blue assay.
  • the preparation method of the yeast cell extract is not limited, and a preferred preparation method comprises the following steps:
  • the solid-liquid separation method is not particularly limited, and a preferred mode is centrifugation.
  • the centrifugation is carried out in a liquid state.
  • the centrifugation conditions are not particularly limited, and a preferred centrifugation condition is 5,000 to 100,000 x g, preferably 8,000 to 30,000 x g.
  • the centrifugation time is not particularly limited, and a preferred centrifugation time is from 0.5 min to 2 h, preferably from 20 min to 50 min.
  • the temperature of the centrifugation is not particularly limited.
  • the centrifugation is carried out at 1-10 ° C, preferably at 2-6 ° C.
  • the washing treatment method is not particularly limited, and a preferred washing treatment method is treatment with a washing liquid at a pH of 7-8 (preferably, 7.4), and the washing liquid is not particularly Typically, the wash liquor is typically selected from the group consisting of potassium 4-hydroxyethylpiperazine ethanesulfonate, potassium acetate, magnesium acetate, or combinations thereof.
  • the manner of the cell disruption treatment is not particularly limited, and a preferred cell disruption treatment includes high pressure disruption, freeze-thaw (e.g., liquid nitrogen low temperature) disruption.
  • the mixture of nucleoside triphosphates in the in vitro protein synthesis system is adenine nucleoside triphosphate, guanosine triphosphate, cytidine triphosphate, and uridine nucleoside triphosphate.
  • the concentration of each of the single nucleotides is not particularly limited, and usually the concentration of each single nucleotide is from 0.5 to 5 mM, preferably from 1.0 to 2.0 mM.
  • the mixture of amino acids in the in vitro protein synthesis system can include natural or unnatural amino acids, and can include D-form or L-form amino acids.
  • Representative amino acids include, but are not limited to, 20 natural amino acids: glycine, alanine, valine, leucine, isoleucine, phenylalanine, valine, tryptophan, serine, Tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine.
  • the concentration of each amino acid is usually from 0.01 to 0.5 mM, preferably from 0.02 to 0.2 mM, such as 0.05, 0.06, 0.07, 0.08 mM.
  • the in vitro protein synthesis system further comprises polyethylene glycol or an analog thereof.
  • concentration of polyethylene glycol or the like is not particularly limited, and usually, the concentration (w/v) of polyethylene glycol or the like is from 0.1 to 8%, preferably from 0.5 to 4%, more preferably, 1-2%, based on the total weight of the protein synthesis system.
  • Representative examples of PEG include, but are not limited to, PEG3000, PEG 8000, PEG 6000, and PEG 3350. It should be understood that the system of the present invention may also include other various molecular weight polyethylene glycols (e.g., PEG 200, 400, 1500, 2000, 4000, 6000, 8000, 10000, etc.).
  • the in vitro protein synthesis system further comprises sucrose.
  • concentration of sucrose is not particularly limited, and usually, the concentration of sucrose is from 0.03 to 40% by weight, preferably from 0.08 to 10% by weight, more preferably from 0.1 to 5% by weight, based on the total weight of the protein synthesis system.
  • a particularly preferred in vitro protein synthesis system in addition to the yeast extract, contains the following components: 22 mM, 4-hydroxyethylpiperazineethanesulfonic acid having a pH of 7.4, 30-150 mM potassium acetate, 1.0-5.0 mM Magnesium acetate, 1.5-4 mM nucleoside triphosphate mixture, 0.08-0.24 mM amino acid mixture, 25 mM phosphocreatine, 1.7 mM dithiothreitol, 0.27 mg/mL phosphocreatine kinase, 1%-4% polyethylene Alcohol, 0.5% to 2% sucrose, 8-20 ng/ ⁇ l of firefly luciferase DNA, 0.027-0.054 mg/mL T7 RNA polymerase.
  • coding sequence of a foreign protein is used interchangeably with “foreign DNA” and refers to a foreign DNA molecule for directing protein synthesis.
  • the DNA molecule is linear or circular.
  • the DNA molecule contains a sequence encoding a foreign protein.
  • examples of the sequence encoding the foreign protein include, but are not limited to, a genomic sequence, a cDNA sequence.
  • the sequence encoding the foreign protein further comprises a promoter sequence, a 5' untranslated sequence, and a 3' untranslated sequence.
  • the selection of the exogenous DNA is not particularly limited.
  • the exogenous DNA is selected from the group consisting of a luciferin protein, or a luciferase (such as firefly luciferase), a green fluorescent protein, and a yellow fluorescent protein. , aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, exogenous DNA of a variable region of an antibody, DNA of a luciferase mutant, or a combination thereof.
  • the exogenous DNA may also be selected from the group consisting of alpha-amylase, enteromycin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon alpha A, interleukin-1 beta, lysozyme, serum white. Protein, single-chain antibody fragment (scFV), thyroxine transporter, tyrosinase, exogenous DNA of xylanase, or a combination thereof.
  • alpha-amylase enteromycin A
  • hepatitis C virus E2 glycoprotein insulin precursor
  • interferon alpha A interleukin-1 beta
  • lysozyme serum white.
  • Protein single-chain antibody fragment (scFV), thyroxine transporter, tyrosinase, exogenous DNA of xylanase, or a combination thereof.
  • the exogenous DNA encodes a protein selected from the group consisting of: green fluorescent protein (eGFP), yellow fluorescent protein (YFP), and Escherichia coli beta-galactosidase ( ⁇ -galactosidase, LacZ), human lysine-tRNA synthetase, human leucine-tRNA synthetase, Arabidopsis glyceraldehyde 3-phosphate dehydrogenase (Glyceraldehyde-3-phosphate) Dehydrogenase), murine catalase (Catalase), or a combination thereof.
  • eGFP green fluorescent protein
  • YFP yellow fluorescent protein
  • Escherichia coli beta-galactosidase ⁇ -galactosidase, LacZ
  • human lysine-tRNA synthetase human leucine-tRNA synthetase
  • the invention provides an in vitro high-throughput protein synthesis method, comprising the steps of:
  • step (ii) incubating the protein synthesis system of step (i) for a period of time T1 under suitable conditions to synthesize the protein encoded by the exogenous DNA.
  • the present invention provides a design and method for knocking out a nuclease gene in a yeast genome to increase the efficiency of protein translation in vitro, comprising the steps of:
  • the monoclonal cells are screened for expansion culture, the yeast genome is extracted, and the primers are designed to amplify the knockout sites.
  • the amplified PCR products can be verified by sequencing to obtain a strain that knocks out a specific gene;
  • the yeast cell strain prepared by knocking out the specific gene is prepared into a yeast cell solution, added to the protein in vitro translation system, and allowed to stand for 2-6 hours in an environment of 20-30 ° C, and read by a multi-function microplate reader (Perkin Elmer). Firefly luciferase activity was detected.
  • the present invention is designed for the first time to improve the stability of nucleic acids by reducing nucleases in the system, and to systematically analyze and specifically modify the nuclease gene in the K. lactis genome, thereby providing a universal regulation.
  • Technical routes and methods for in vitro biosynthetic activity are designed for the first time to improve the stability of nucleic acids by reducing nucleases in the system, and to systematically analyze and specifically modify the nuclease gene in the K. lactis genome, thereby providing a universal regulation.
  • the present invention screens a large number of nucleases, and for the first time, screens out five nucleases from a wide variety of nucleases, wherein one of the nucleases (such as EXN53) is down-regulated or knocked out, and unexpected findings can actually be improved.
  • one of the nucleases such as EXN53
  • the stability of nucleic acids and the efficiency of protein production in in vitro protein synthesis systems is ⁇ 2 times (e.g., 2.46-fold) that of the wild type.
  • the present invention firstly found a K. lactis strain which can significantly increase the activity of protein synthesis in vitro.
  • Example 1 improves the stability of nucleic acids by reducing nucleases in in vitro biological systems, and analyzes and specifically modifies the nuclease gene in the K. lactis genome.
  • In vitro biosynthesis systems use cell lysates by disrupting different types of cells, including microorganisms, animals, and plants, for translation of foreign proteins.
  • cell lysates In order to achieve functions such as transcription, translation, protein folding and energy metabolism, cell lysates must contain elements for energy regeneration and protein synthesis, including ribosomes, aminoacyl-tRNA synthetases, translation initiation and elongation factors. , ribosome releasing factor, nucleotide cyclic enzyme, metabolic enzyme, molecular chaperone and folding enzyme.
  • Substances that require exogenous addition include amino acids, nucleotides, DNA templates, energy substrates, cofactors, and salt molecules.
  • the stability of the different components in the system has an important effect on the duration of the reaction and the final protein yield, and the depletion of certain substrates (such as ATP and cysteine) is an important reason for the termination of the reaction.
  • the stability of the DNA template and the mRNA generated by its transcription has an important influence on the duration and yield of the in vitro translation system.
  • the cells are disrupted, cell lysates are collected and used to construct an in vitro translation system containing, in addition to the various components necessary for protein synthesis, nucleases, proteases, and the like which are detrimental to the reaction.
  • the efficiency of protein translation is significantly improved by the absence of inhibitory factors.
  • nuclease gene knockout and the addition of stabilizing factors, etc. can effectively improve the stability of nucleic acids in the in vitro translation system, thereby improving the translation efficiency of the protein.
  • the stability of nucleic acids is improved by reducing nucleases in in vitro biosynthetic systems, and a design roadmap for the analysis and specific modification of the nuclease gene in the K. lactis genome (shown in Figure 1) is utilized.
  • Nucleases also known as nuclear polymerases or polynucleotide enzymes are enzymes capable of hydrolyzing phosphodiester bonds between nucleotides in the first step of nucleic acid decomposition.
  • the nuclease can be classified into an exonuclease and an endonuclease depending on the position of the action of the nuclease.
  • Exonuclease hydrolyzes nucleotides one by one from the 3' end, called 3'to 5' exonuclease.
  • the exonuclease hydrolyzes nucleotides one by one from the 5' end, which is called 5'to 3' exo.
  • Enzymes, endonucleases catalyze the hydrolysis of phosphodiester bonds within the polynucleotide. Nucleases are further divided into deoxyribonuclease (DNase) and ribonuclease (RNase), the former acting on DNA and the latter acting on RNA. The exonuclease is further divided into a 5' to 3' exonuclease and a 3' to 5' exonuclease according to the direction of action of the nuclease.
  • DNase deoxyribonuclease
  • RNase ribonuclease
  • the exonuclease is further divided into a 5' to 3' exonuclease and a 3' to 5' exonuclease according to the direction of action of the nuclease.
  • K. lactis contains 61 nucleases distributed on six chromosomes of K.lactisA-F (Fig. 2).
  • the nucleases located on chromosome A are: KlSEN54, KlDNA2, KlTRM2, KlFCF1, KlDOM34, KlRAD2, KlRNH70, KlDIS3, KlNPP1;
  • KlSEN54 is encoded in the KEGG database: KlLA0A00803g
  • KlDNA2 is encoded in the KEGG database: KlLA0A05324g
  • KlTRM2 is encoded in the KEGG database: KlLA0A05665g
  • KlFCF1 is encoded in the KEGG database
  • KlLA0A07018g the code of KlDOM34 in KEGG database is: KlLA0A08646g
  • the code of KlRAD2 in KEGG database is: KlLA0A09427g
  • the nuclease located on the B chromosome is: KlOGG1; the coding of KlOGG1 in the KEGG database is: KlLA0B05159g.
  • the nucleases located on the C chromosome are: KlPOL2, KlRAD50, KlYSH1, KlRCL1, KlNGL2, KlMRE11, KlPOP3, KlMKT1, KlAPN1, KlRPP1, KlPOP2; KlPOL2 is encoded in the KEGG database: KlLA0C02585g, and the code of KlRAD50 in the KEGG database is: KlLA0C02915g, KlYSH1 in the KEGG database is encoded as: KlLA0C04598g, KlRCL1 is encoded in the KEGG database: KlLA0C05984g, KlNGL2 is encoded in the KEGG database: KlLA0C06248g, KlMRE11 is encoded in the KEGG database: KlLA0C06930g, KlPOP3 in the KEGG database The code in the code is: KlLA0C12199g, the code of
  • Nucleases on the D chromosome are: KlNUC1, KlRPP6, KlDBR1, KlRPS3, KlRPM2, KlSUV3, KlRAD1, KlIRE1, KlPOP1;
  • KlNUC1 is encoded in the KEGG database: KlLA0D00440g,
  • KlRPP6 is encoded in the KEGG database: KlLA0D01309g, KlDBR1
  • the code in the KEGG database is: KlLA0D04466g
  • the code of KlRPS3 in the KEGG database is: KlLA0D08305g
  • the code of KlRPM2 in the KEGG database is: KlLA0D10483g
  • the code of KlSUV3 in the KEGG database is: KlLA0D12034g
  • the code of KlRAD1 in the KEGG database is KlLA0D12210g
  • the code of KlIRE1 in the KEGG database is
  • Nucleases located on the E chromosome are: KlPOL3, KlDXO1, KlMUS81, KlPAN3, KlAPN2, KlRAI1, KlEXO1, KlREX4, KlPAN2, KlLCL3; KlPOL3 is encoded in the KEGG database: KlLA0E01607g, KlDXO1 is encoded in the KEGG database: KlLA0E02245g, The encoding of KlMUS81 in the KEGG database is: KlLA0E03015g, the encoding of KlPAN3 in the KEGG database is: KlLA0E08097g, the encoding of KlAPN2 in the KEGG database is: KlLA0E18877g, the encoding of KlRAI1 in the KEGG database is: KlLA0E12893g, KlEXO1 in the KEGG database The code is: KlLA0E16743g
  • the nucleases located on the F chromosome are: KlRAD17, KlPOP4, KlPOP5, KlRAD27, KlRNH201, KlVMA1, KlRAT1, KlNGL1, KlREX2, KlTRL1, KlPOL31, KlCCR4, KlTRZ1, KlSEN2, KlREX3, KlSWT1, KLEXN53, KlRNT1, KlSEN15, KlNTG1, KlNOB1, KlDDP1.
  • the encoding of KlRAD17 in the KEGG database is: KlLA0F00330g
  • the encoding of KlPOP4 in the KEGG database is: KlLA0F02211g
  • the encoding of KlPOP5 in the KEGG database is: KlLA0F02453g
  • the encoding of KlRAD27 in the KEGG database is: KlLA0F02992g
  • the code is: KlLA0F04774g
  • the code of KlVMA1 in the KEGG database is: KlLA0F05401g
  • the code of KlRAT1 in the KEGG database is: KlLA0F07469g
  • the code of KlNGL1 in the KEGG database is: KlLA0F07733g
  • the code of KlREX2 in the KEGG database is: KlLA0F08998g
  • KlPOL31 The code in the
  • K.lactis nuclease can be divided into two types, among which there are 18 DNases, 41 RNases, and 2 of no functional classifications.
  • KlAPN1 is a 3'to 5' function in DNase
  • KlRAD27 is a 5'to 3' function
  • KlEXO1 is a 5'to 3' function
  • KlRNH70, KlDIS3, KlNGL2, KlRPP6, 5 have a 3'to 5' function in RNase.
  • the 'to 3' function is KlDXO1, KlRAT1, KLEXN53.
  • RNA and DNA proteins encode substrates in in vitro synthesis systems whose stability affects protein yield.
  • the 5' end of RNA will instantaneously complete the processing of the hat structure, and the 5' hat structure of RNA plays an important role in the stability of RNA and the efficiency of translation.
  • RNA degradation In eukaryotes, there is a mechanism of RNA degradation that relies on adenosylation and adenosylation-independent, and in the de-adenosylated RNA degradation mechanism, the 5' end mature hat structure of RNA can be removed by recruiting a capping complex or In the immature hat structure, RNA also undergoes 5' to 3' degradation under the action of nucleases. In the mechanism of mRNA degradation independent of adenosine, under the action of the endonuclease, the nucleic acid molecule generates a chain scission from the middle, and the exonuclease degrades the RNA from 3' to 5'.
  • exogenous linear or circular DNA is used as a template to transcribe mRNA with a tail of polyA 3', depending on the promoter and RNA transcriptase, possibly With or without a 5' end mature hat structure. Therefore, DNase, RNase, exonuclease and endonuclease will affect the stability of the template in the in vitro protein synthesis system. The modification of these enzymes may enhance the biosynthesis activity in vitro.
  • the 5' to 3' exonuclease in K. lactis includes EXN53 and Rat1, wherein EXN53 is localized in the cytoplasm and Rat1 is localized in the nucleus.
  • a protein subunit complex exosome having 3'to 5' exonuclease and endonuclease activity which can be localized in the cytoplasm and nucleus after de-adenosylation in a mechanism dependent on de-adenosylated RNA degradation. Degraded by action.
  • lactis includes Rrp6, Dis3 (also known as Rrp44) and NGL2, etc., wherein Rrp6 and Dis3 are both exonuclease and endonuclease activity protein subunit complex exosome component.
  • Rrp6 is a 3'to 5' exonuclease, a member of the RNase D family, which is located in the nucleus and is a key catalytic subunit of the exosome in the nucleus. It is responsible for the processing, degradation and quality control of various RNAs in the cell.
  • Dis3 (also known as Rrp44) is a key catalytic subunit of exosome, a highly conserved member of the RNaseII/RNB family, localized in the nucleus and cytoplasm, with 3'to 5' exonuclease and endonuclease activity, responsible for the nucleus And in the cytoplasmic RNA metabolism and processing of various types of RNA, NGL2 is an enzyme that specifically recognizes Poly A, which hydrolyzes RNA from the 3' to 5' direction.
  • the present invention selects 5 genes of EXN53, Rat1, Rrp6, Dis3 and NGL2 for transformation, and the method includes, but is not limited to, gene knockout or gene mutation.
  • the stability of nucleic acids is improved by reducing nucleases in in vitro biosynthesis systems, and the design and technical details of in vitro protein synthesis activities are regulated by analysis and specific modification of the nuclease gene in the K. lactis genome.
  • the present invention modifies the K. lactis genome by the CRISPR-Cas9 gene editing technology.
  • CRISPR is a widely used gene editing technology. It has a simple structure, convenient operation, high editing efficiency, no need to introduce antibiotics, and can achieve simultaneous knockout of multiple sites of target genes. It has formed a mature in yeast. Conversion system.
  • the CRISPR system used in the present invention is an optimized system for K. lactis strain, and optimizes parameters and conditions such as Cas9 protein coding sequence, gRNA promoter, donor DNA homology arm length and yeast competent preparation and transformation. Efficient editing in K. lactis yeast strains is achieved.
  • the two homologous arms of the donor DNA are the gene sequences of about 1000 bp upstream and downstream of the ORF of the nuclease gene to be knocked out, and two gRNAs are constructed, and 5' and 3 of the ORF of the nuclease gene to be knocked out are simultaneously recognized.
  • the 'end of the PAM sequence which guides the Cas9 nuclease to cleave the DNA, and the homologous recombination occurs after DSB (double strand break), thereby achieving nuclease gene knockout.
  • knockout of the five genes of EXN53, Rat1, Rrp6, Dis3, and NGL3 is performed by designing a gRNA in the vicinity of the start codon and the stop codon.
  • the design of the five nuclease genetically modified CRISPR system in the K. lactis genome is shown in Figure 3.
  • the EXN53 gene sequence (SEQ ID NO.1) in K. lactis was determined by BLAST alignment analysis with EXN53 gene in KEGG database.
  • the KEGG database encodes KlLA0F22385g, and the sequence homology of EXN53 gene in S. cerevisiae yeast is 60.93. %, the protein sequence homology is 57.65%, the homology with the EXN53 gene sequence in S. pombe yeast is 48.38%, the homology is 38.29%, and the homology of EXN53 gene in H. sapiens yeast is 43.81%, the protein sequence is the same.
  • the source is 33.48% (Fig. 5) and contains the characteristic EXN535'to 3' exonuclease domain, Amelogenin domain (Fig. 4).
  • the gene was named KLEXN53 (located at 2091235...2095596 of chromosome F).
  • the PAM sequence (NGG) was searched for at the KLEXN53 gene start codon and stop codon, and the gRNA sequence was determined.
  • the principle of gRNA selection is: GC content is moderate, the standard of the invention is GC content of 40%-60%; avoid the existence of poly T structure.
  • the KLEXN53gRNA-1 sequence determined by the present invention is AGAGTTCGACAATTTGTACT, and the KLEXN53gRNA-2 sequence is CGTCGTGGCCGTAGTAATCG.
  • the plasmid construction and transformation methods were as follows: using the primers pCas9-KLEXN53-F1: AGAGTTCGACAATTTGTACTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTC
  • pHoCas9_SE_Kana_tRNA_ScRNR2_KLEXN53-1 primers pCas9-F1:TAGGTCTAGAGATCTGTTTAGCTTGCCTCG (SEQ ID NO.:11) and pCas9-R1:TATCCACTAGACAGAAGTTTGCGTTCC (SEQ ID NO.:12), using pHoCas9_SE_Kana_tRNA_ScRNR2_KLEXN533-2 as a template, using primers pCas9-F2: TATGGAACGCAAACTTCTGTCTAGTGGATAGTATATGTGTTATGTAGTATACTCTTTCTTCAACAATTAAATACTCTCGG (SEQ ID NO.: 13) and pCas9-F2: CGAGGCAAGCTAAACAGATCTCTAGACCTATATCCACTAGACAGAAGTTTGCGTTCC (SEQ ID NO.: 14) were subjected to PCR
  • the present invention first inserts donor DNA into a pMD18 plasmid, and then obtains a linear donor DNA sequence by PCR amplification.
  • PCR amplification was carried out using K. lactis genomic DNA as a template, primers KLEXN53-F1:GTACCCGGGGATCCTCTAGAGATCCAGTTGCAGAGCCTCCGAA (SEQ ID NO.: 15) and KLEXN53-R1:CATGCCTGCAGGTCGACGATGCGAAACCTTAGCTCTTTATCGAAC (SEQ ID NO.: 16); pMD18 plasmid as template PCR amplification was carried out with primers pMD18-F: ATCGTCGACCTGCAGGCATG (SEQ ID NO.: 17) and pMD18-R: ATCTCTAGAGGATCCCCGGG (SEQ ID NO.: 18).
  • KLEXN53-pMD18 plasmid was used as a template, and the primer KLEXN53-F2: ATGTTGGTTTGAATGGACTATTAACAGTAAATATTATATCACTTC (SEQ ID NO.: 19)
  • KLEXN53-R2 GAAGTGATATAATATTTACTGTTAATAGTCCATTCAAACCAACATTTATTTTAGTTAAGCCACAAACCGTAATTAATTGAACAC
  • KLEXN53-DD-pMD18 shown in Figure 11 was constructed. The specific steps were as follows: 8.5 ⁇ L of each of the two PCR products were mixed, and 1 ⁇ L of Dpn I, 2 ⁇ L of 10 ⁇ digestion buffer was added, and the mixture was incubated at 37° C. for 3 hours.
  • the competent cells were thawed at 37 ° C for 15-30 s, centrifuged at 13,000 g for 2 min and the supernatant was removed.
  • the DIS3 gene sequence (SEQ ID NO. 2) in the Kluyveromyces cerevisiae was determined by BLAST alignment analysis of the DIS3 gene in the KEGG database.
  • the KEGG database encodes KlLA0A10835g, and the sequence homology of the DIS3 gene in S. cerevisiae yeast is 70.54. %, protein sequence homology 77.59%, 56.86% homology with DIS3 gene sequence in S. pombe yeast, 51.82% protein sequence homology, and 48.83% homology with DIS3 gene sequence in H. sapiens yeast, protein sequence The homology was 40.19% (Fig. 6), containing the characteristic VacB domain, PIN_Rrp44domain (Fig. 4).
  • the gene was named KlDIS3 (located at position 938712..941738 of chromosome A).
  • the PAM sequence was searched for near the KlDIS3 gene start codon and stop codon, and the gRNA sequence was determined.
  • the principle of gRNA selection is: GC content is moderate, the standard of the invention is GC content of 40%-60%; avoid the existence of poly T structure.
  • the KlDIS3gRNA-1 sequence determined by the present invention is TTCAGCAGCTAAGAAGGAAG (SEQ ID NO.: 23), and the KlDIS3gRNA-2 sequence is AGAGGTCAGTGTCTTTGATA (SEQ ID NO.: 24).
  • Plasmid construction and transformation methods are as follows: using primers pCas9-KlDIS3-F1: ATGGGACTTTTTCAGCAGCTAAGAAGGAAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCC (SEQ ID NO.:25) and pCas9-KlDIS3-R1: AGCTCTAAAACCTTCCTTCTTAGCTGCTGAAAAAGTCCCATTCGCCACCCG (SEQ ID NO.:26),pCas9-KlDIS3-F2:ATGGGACTTTAGAGGTCAGTGTCTTTGATAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCC(SEQ ID NO.: 27) and pCas9-KlDIS3-R2: AGCTCTAAAACTATCAAAGACACTGACCTCTAAAGTCCCATTCGCCACCCG (SEQ ID NO.: 28) were all PCR amplified using the pCAS plasmid as a template.
  • the present invention first inserts donor DNA into a pMD18 plasmid, and then obtains a linear donor DNA sequence by PCR amplification.
  • PCR amplification was carried out using K. lactis genomic DNA as a template, primers KlDIS3-F1: CCCGGGGATCCTCTAGAGATGCTGCTAGGTGACAGAAGGTTGTCC (SEQ ID NO.: 33) and KlDIS3-R1:CATGCCTGCAGGTCGACGATCCAAAGAAGAACGTCGTAAGACCGC (SEQ ID NO.: 34); pMD18 plasmid as template PCR amplification was carried out with primers pMD18-F: ATCGTCGACCTGCAGGCATG (SEQ ID NO.: 35) and pMD18-R: ATCTCTAGAGGATCCCCGGG (SEQ ID NO.: 36).
  • KlDIS3-pMD18 plasmid as a template, PCR amplification was performed with primers KlDIS3-F2: CTCTTCTGTTTAGCACCCGGTTATAGCTTAATTTATTAATTATGTACATTATATAAAAACTATTGTC (SEQ ID NO.: 37) and KlDIS3-R2: AAGCTATAACCGGGTGCTAAACAGAAGAGTATGACGTTTTATACTTCTCCAG (SEQ ID NO.: 38); construct KlDIS3-DD-pMD18 (eg Figure 13)).
  • the specific steps were as follows: 8.5 ⁇ L of each of the two PCR products were mixed, and 1 ⁇ L of Dpn I, 2 ⁇ L of 10 ⁇ digestion buffer was added, and the mixture was incubated at 37° C. for 3 hours. 10 ⁇ L of Dpn I-treated product was added to 100 ⁇ L of DH5 ⁇ competent cells, placed on ice for 30 min, and heat-shocked at 42 ° C for 45 s. Then, 1 mL of LB liquid medium was added and shaken at 37 ° C for 1 h, and applied to Amp-resistant LB solid culture, 37 Incubate in °C until the monoclonal grows out. Five monoclonal clones were picked and shaken in LB liquid medium. After PCR detection was positive and sequenced, the plasmid was extracted and stored.
  • the competent cells were thawed at 37 ° C for 15-30 s, centrifuged at 13,000 g for 2 min and the supernatant was removed.
  • the Rat1 gene sequence (SEQ ID NO. 3) in K. lactis was determined by BLAST alignment analysis with the Rat1 gene in the KEGG database.
  • the KEGG database encodes KlLA0A10835g, and the sequence homology with the Rat1 gene sequence in S. cerevisiae yeast is 60.38. %, protein sequence homology 62.30%, 50.53% homology with Rat1 gene sequence in S. pombe yeast, 39.73% homology of protein sequence, 50.15% homology with Rat1 gene sequence in H. sapiens yeast, protein sequence The homology was 41.41% (Fig. 7) and contained the characteristic EXN535 'to 3' exonuclease domain (Fig. 4).
  • the gene was named KlRat1 (located on chromosome F: 703955..706933).
  • the PAM sequence was searched for near the start codon and stop codon of the Rat1 gene, and the gRNA sequence was determined.
  • the principle of gRNA selection is: GC content is moderate, the standard of the invention is GC content of 40%-60%; avoid the existence of poly T structure.
  • the K1Rat1gRNA-1 sequence determined by the present invention is GTAAGGCCAGGTACTCACAA (SEQ ID NO.: 41), and the KlRat1gRNA-2 sequence is CTCGCAACAGAGACAGCCAC (SEQ ID NO.: 42).
  • Plasmid and transformation methods are as follows: using primers pCas9-KlRat1-F1: ATGGGACTTTGTAAGGCCAGGTACTCACAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCC (SEQ ID NO.:43) and pCas9-KlRat1-R1: AGCTCTAAAACTTGTGAGTACCTGGCCTTACAAAGTCCCATTCGCCACCCG (SEQ ID NO.:44),pCas9-KlRat1-F2:ATGGGACTTTCTCGCAACAGAGACAGCCACGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCC(SEQ ID NO.: 45) and pCas9-KlRat1-R2: AGCTCTAAAACGTGGCTGTCTCTGTTGCGAGAAAGTCCCATTCGCCACCCG (SEQ ID NO.: 46), both of which were PCR amplified using the pCAS plasmid as
  • pHoCas9_SE_Kana_tRNA_ScRNR2_KlRat1-1 primers pCas9-F1:TAGGTCTAGAGATCTGTTTAGCTTGCCTCG (SEQ ID NO.:47) and pCas9-R1:TATCCACTAGACAGAAGTTTGCGTTCC (SEQ ID NO.:48) were used, and pHoCas9_SE_Kana_tRNA_ScRNR2_KlDIS3-2 was used as a template, using primers pCas9-F2: TATGGAACGCAAACTTCTGTCTAGTGGATAGTATATGTGTTATGTAGTATACTCTTTCTTCAACAATTAAATACTCTCGG (SEQ ID NO.: 49) and pCas9-R2: CGAGGCAAGCTAAACAGATCTCTAGACCTATATCCACTAGACAGAAGTTTGCGTTCC (SEQ ID NO.: 50) were subjected to
  • the present invention first inserts donor DNA into a pMD18 plasmid, and then obtains a linear donor DNA sequence by PCR amplification.
  • PCR amplification was carried out using K. lactis genomic DNA as a template, primers KlRat1-F1: CCCGGGGATCCTCTAGAGATGCTGCATGGTCACAGGAGATGC (SEQ ID NO.: 51) and KlRat1-R1:CATGCCTGCAGGTCGACGATGGTACGTGAGGCGACAATATGGTCC (SEQ ID NO.: 52); pMD18 plasmid as template PCR amplification was carried out with primers pMD18-F: ATCGTCGACCTGCAGGCATG (SEQ ID NO.: 53) and pMD18-R: ATCTCTAGAGGATCCCCGGG (SEQ ID NO.: 54).
  • KlRat1-pMD18 plasmid Using KlRat1-pMD18 plasmid as template, PCR amplification was performed with primers K1Rat1-F2:CCAGGTACTCACATGAACTGTGGACAATTTTATACCCGTTTATATCAGCAC (SEQ ID NO.:55) and KlRat1-R2:TGTCCACAGTTCATGTGAGTACCTGGCCTTACTTCTCGC (SEQ ID NO.:56); KlRat1-DD-pMD18 was constructed (eg Figure 15). The specific steps were as follows: 8.5 ⁇ L of each of the two PCR products were mixed, and 1 ⁇ L of Dpn I, 2 ⁇ L of 10 ⁇ digestion buffer was added, and the mixture was incubated at 37° C. for 3 hours.
  • the competent cells were thawed at 37 ° C for 15-30 s, centrifuged at 13,000 g for 2 min and the supernatant was removed.
  • the Rrp6 gene sequence (SEQ ID NO. 4) in Kluyveromyces cerevisiae was determined by BLAST alignment analysis of Rrp6 gene in KEGG database.
  • the KEGG database encodes KlLA0D01309g, the name hypothetical protein, and the Rrp6 gene sequence in S. cerevisiae yeast.
  • the homology is 55.04%
  • the protein sequence homology is 46.93%
  • the homology with the Rrp6 gene sequence in S. pombe yeast is 44.04%
  • the protein sequence homology is 29.85%
  • the Rrp6 gene sequence homology in H. sapiens yeast is 40.44. %
  • protein sequence homology is 23.53% (Fig.
  • the PAM sequence (NGG) was searched for near the start codon and stop codon of the Rrp6 gene, and the gRNA sequence was determined.
  • the principle of gRNA selection is: GC content is moderate, the standard of the invention is GC content of 40%-60%; avoid the existence of poly T structure.
  • the KlRrp6gRNA-1 sequence determined by the present invention is CACCATGTCTTCAGAGGATA, and the KlRrp6g RNA-2 sequence is CCGACATGTTCAACAGAGTA.
  • Plasmid and transformation methods are as follows: using primers pCas9-KlRrp6-F1: ATGGGACTTTCACCATGTCTTCAGAGGATAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCC (SEQ ID NO.:59) and pCas9-KlRrp6-R1: AGCTCTAAAACTATCCTCTGAAGACATGGTGAAAGTCCCATTCGCCACCCG (SEQ ID NO.:60),pCas9-KlRrp6-F2:ATGGGACTTTCCGACATGTTCAACAGAGTAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCC(SEQ ID NO.: 61) and pCas9-Kl Rrp6-R2: AGCTCTAAAACTACTCTGTTGAACATGTCGGAAAGTCCCATTCGCCACCCG (SEQ ID NO.: 62) were all PCR amplified using the pCAS plasmi
  • pHoCas9_SE_Kana_tRNA_ScRNR2_KlRrp6-1 primers pCas9-F1:TAGGTCTAGAGATCTGTTTAGCTTGCCTCG (SEQ ID NO.:63) and pCas9-R1:TATCCACTAGACAGAAGTTTGCGTTCC (SEQ ID NO.:64), using pHoCas9_SE_Kana_tRNA_ScRNR2_KlRrp6-2 as a template, using primers pCas9-F2: TATGGAACGCAAACTTCTGTCTAGTGGATAGTATATGTGTTATGTAGTATACTCTTTCTTCAACAATTAAATACTCTCGG (SEQ ID NO.: 65) and pCas9-R2: CGAGGCAAGCTAAACAGATCTCTAGACCTATATCCACTAGACAGAAGTTTGCGTTCC (SEQ ID NO.: 66) were subjected to
  • the present invention first inserts donor DNA into a pMD18 plasmid, and then obtains a linear donor DNA sequence by PCR amplification.
  • PCR amplification was carried out using K. lactis genomic DNA as a template, primers KlRrp6-F1: CCCGGGGATCCTCTAGAGATGCGATAGCTTTAATCTGAGTGAACACCG (SEQ ID NO.: 67) and KlRrp6-R1:CATGCCTGCAGGTCGACGATGGGTACTCGTTGATAACATGATGCGTAG (SEQ ID NO.: 68); pMD18 plasmid as template PCR amplification was carried out with primers pMD18-F: ATCGTCGACCTGCAGGCATG (SEQ ID NO.: 69) and pMD18-R: ATCTCTAGAGGATCCCCGGG (SEQ ID NO.: 70).
  • KlRrp6-pMD18 plasmid Using KlRrp6-pMD18 plasmid as a template, PCR amplification was performed with primers K1Rrp6-F2: CTGACTCTAATCCACCAGCATCTTGAGCAGCTCTAATGGTATAAATATCG (SEQ ID NO.: 71) and KlRrp6-R2: GCTCAAGATGCTGGTGGATTAGAGTCAGCTGGTAGTCTAC (SEQ ID NO.: 72); construct KlRrp6-DD-pMD18 (eg Figure 17).
  • the specific steps were as follows: 8.5 ⁇ L of each of the two PCR products were mixed, and 1 ⁇ L of Dpn I, 2 ⁇ L of 10 ⁇ digestion buffer was added, and the mixture was incubated at 37° C. for 3 hours. 10 ⁇ L of Dpn I-treated product was added to 100 ⁇ L of DH5 ⁇ competent cells, placed on ice for 30 min, and heat-shocked at 42 ° C for 45 s. Then, 1 mL of LB liquid medium was added and shaken at 37 ° C for 1 h, and applied to Amp-resistant LB solid culture, 37 Incubate in °C until the monoclonal grows out. Five monoclonal clones were picked and shaken in LB liquid medium. After PCR detection was positive and sequenced, the plasmid was extracted and stored.
  • the competent cells were thawed at 37 ° C for 15-30 s, centrifuged at 13,000 g for 2 min and the supernatant was removed.
  • the NLAG gene sequence (SEQ ID NO. 5) in the Kluyveromyces cerevisiae was determined by BLAST alignment analysis with the NGL3 gene in the KEGG database.
  • the KEGG database encodes KlLA0C06248g, and the NGL3 gene sequence homology with S. cerevisiae yeast 46.26 %, protein sequence homology 46.34%, 45.42% homology with NGL2 gene sequence in S. pombe yeast, 29.07% homology of protein sequence, 38.72% homology with NGL3 gene sequence in H. sapiens yeast, protein sequence The homology was 19.89% (Fig.
  • the PAM sequence was searched for near the KlNGL2 gene start codon and stop codon, and the gRNA sequence was determined.
  • the principle of gRNA selection is: GC content is moderate, the standard of the invention is GC content of 40%-60%; avoid the existence of poly T structure.
  • the K1NGL2gRNA-1 sequence determined by the present invention is GCTGGTAGTACGCAAGACAC (SEQ ID NO.: 75), and the K1NGL2gRNA-2 sequence is TTGTGCATGATTGTTAAACT (SEQ ID NO.: 76).
  • the plasmid construction and transformation methods were as follows: using primers pCas9-KlNGL2-F1:TATCCAGACACCAAAGTCAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTC (SEQ ID NO.: 77) and pCas9-Kl NGL3-R1:GCTCTAAAACCTGACTTTGGTGTCTGGATAAAAGTCCCATTCGCCACCCG (SEQ ID NO.:78), pCas9-KlNGL2-F2:TTGTGCATGATTGTTAAACTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGT ( SEQ ID NO.: 79) and pCas9-K1 NGL2-R2CGGGTGGCGAATGGGACTTTTTGTGCATGATTGTTAAACTGTTTTAGAGC (SEQ ID NO.: 80): PCR amplification was carried out using the pCAS plasmid as a template.
  • pHoCas9_SE_Kana_tRNA_ScRNR2_KlNGL2-1 primers pCas9-F1:TAGGTCTAGAGATCTGTTTAGCTTGCCTCG (SEQ ID NO.:81) and pCas9-R1:TATCCACTAGACAGAAGTTTGCGTTCC (SEQ ID NO.:82), using pHoCas9_SE_Kana_tRNA_ScRNR2_KlNGL2-2 as a template, using primers pCas9-F2: TATGGAACGCAAACTTCTGTCTAGTGGATAGTATATGTGTTATGTAGTATACTCTTTCTTCAACAATTAAATACTCTCGG (SEQ ID NO.: 83) and pCas9-R2: CGAGGCAAGCTAAACAGATCTCTAGACCTATATCCACTAGACAGAAGTTTGCGTTCC (SEQ ID NO.: 84) were subjected to PCR
  • the present invention first inserts donor DNA into a pMD18 plasmid, and then obtains a linear donor DNA sequence by PCR amplification.
  • PCR amplification was carried out using K. lactis genomic DNA as a template, primers K1NGL2-F1:GAGCTCGGTACCCGGGGATCCTCTAGAGATCGAATACGTGAAACAGCCTAGGAA (SEQ ID NO.:85) and KlNGL2-R1:GCCAAGCTTGCATGCCTGCAGGTCGACGATCACGGCCCTAGTACTAATCCCAT (SEQ ID NO.:86); pMD18 plasmid as template PCR amplification was carried out with primers pMD18-F: ATCGTCGACCTGCAGGCATG (SEQ ID NO.: 87) and pMD18-R: ATCTCTAGAGGATCCCCGGG (SEQ ID NO.: 88).
  • KlNGL3-pMD18 plasmid as a template, PCR amplification was performed with primers K1NGL2-F2:GAAGTAATAATTTGAGCCAATATATTCATAAACTGTTTAACTATGGACTACACTACAG (SEQ ID NO.:89) and KlNGL2-R2:CCATAGTTAAACAGTTTATGAATATATTGGCTCAAATTATTACTTCTACTTTGCAGTG (SEQ ID NO.:90); construct KlNGL2-DD-pMD18 (eg Figure 19).
  • the specific steps were as follows: 8.5 ⁇ L of each of the two PCR products were mixed, and 1 ⁇ L of Dpn I, 2 ⁇ L of 10 ⁇ digestion buffer was added, and the mixture was incubated at 37° C. for 3 hours. 10 ⁇ L of Dpn I-treated product was added to 100 ⁇ L of DH5 ⁇ competent cells, placed on ice for 30 min, and heat-shocked at 42 ° C for 45 s. Then, 1 mL of LB liquid medium was added and shaken at 37 ° C for 1 h, and applied to Amp-resistant LB solid culture, 37 Incubate in °C until the monoclonal grows out. Five monoclonal clones were picked and shaken in LB liquid medium. After PCR detection was positive and sequenced, the plasmid was extracted and stored.
  • the competent cells were thawed at 37 ° C for 15-30 s, centrifuged at 13,000 g for 2 min and the supernatant was removed.
  • Luciferase activity assay After the reaction is complete, add an equal volume of substrate luciferine to a 96-well white plate or a 384-well white plate and place immediately on the Envision 2120 Multiplate Reader (Perkin Elmer). The firefly luciferase activity was measured, and the relative light unit value (RLU) was used as the activity unit, as shown in FIG.
  • Example 7 of the present invention indicate that among all nuclease knockout mutants, the ⁇ klexn53 strain can significantly enhance the efficiency of protein production by the yeast in vitro protein synthesis system (Table 2), and it can also be seen from Fig. 20 that the wild type is in IVTT.
  • the value of luciferase activity was 2.90 ⁇ 10 8
  • the luciferase activity of ⁇ klexn53 yeast strain in IVTT was 7.16 ⁇ 10 8
  • its activity was 2.46 times that of wild type.

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Abstract

提供了一种通过对核酸酶系统敲除以调控体外生物合成活性的方法，包括从很多种核酸酶中筛出5种核酸酶，并对其中一种核酸酶(如EXN53)进行下调或敲除处理，可以提高核酸的稳定性和体外蛋白质合成体系产生蛋白质的效率。

Description

一种通过对核酸酶系统敲除以调控体外生物合成活性的方法 技术领域
本发明涉及生物技术领域，具体地，涉及一种通过对核酸酶系统敲除以调控体外生物合成活性的方法。
背景技术
蛋白质是细胞中的重要分子，几乎参与了细胞所有功能的执行。蛋白的序列和结构不同，决定了其功能的不同。在细胞内，蛋白可以作为酶类催化各种生化反应，可以作为信号分子协调生物体的各种活动，可以支持生物形态，储存能量，运输分子，并使生物体运动。在生物医学领域，蛋白质抗体作为靶向药物，是治疗癌症等疾病的重要手段 [1][2]。
在细胞中，蛋白质翻译的调节在应对营养缺失等外界压力，细胞发育与分化等很多过程中发挥重要作用。蛋白质翻译的四个过程包括翻译起始、翻译延伸、翻译终止和核糖体再循环，其中翻译起始是受调控最多的一个过程 [3]。在翻译起始阶段，核糖体小亚基(40S)结合(tRNA) i Met，并在翻译起始因子的作用下识别mRNA 5’末端。小亚基向下游移动，并在起始密码子(ATG)位置与核糖体大亚基(60S)结合，形成完整核糖体，并进入翻译延伸阶段 [4]。
体外生物合成系统(in vitro biosynthesis system)是指在细菌、真菌、植物细胞或动物细胞的裂解体系中，通过加入外源编码的核酸DNA、RNA、底物和能量源，完成特定化学分子或生物大分子(DNA,RNA,蛋白质)的体外高效合成。常见的体外生物合成系统是体外蛋白质合成系统(in vitro protein synthesis system)，通过外源mRNA或者DNA模板、利用细胞裂解物，完成外源重组蛋白的快速高效翻译 [5]。
商业上常见的体外蛋白质合成系统是体外转录-翻译偶联的体系(in vitro transcription-translation system，简称IVTT)，通过DNA模板、经RNA聚合酶转录出mRNA中间体，再利用氨基酸和ATP等组分，完成外源蛋白的一步高效翻译。目前，常见的商业化体外蛋白表达系统包括大肠杆菌系统(Escherichia coli extract，ECE)、兔网织红细胞(Rabbit reticuLocyte lysate，RRL)、麦胚(Wheat germ extract,WGE)、昆虫(Insect cell extract,ICE)和人源系统。
核酸mRNA和DNA蛋白质体外合成系统中的编码底物，它们的稳定性影响着 蛋白质的得率。核酸酶是在核酸分解的第一步中，水解核苷酸之间的磷酸二酯键的一种蛋白质。有些核酸酶只能作用于RNA，称为核糖核酸酶(RNase)，有些核酸酶只能作用于DNA，称为脱氧核糖核酸酶(DNase)。根据核酸酶作用的位置不同，又可将核酸酶分为核酸外切酶(exonuclease)和核酸内切酶(endonuclease)。细胞内绝大多数RNA的降解主要通过定位于细胞质的5′to 3′核酸外切酶和定位于细胞核的5′to 3′核酸外切酶，或者通过定位于细胞质和细胞核的，具有3′to 5′核酸外切酶和核酸内切酶活性的蛋白亚基复合体exosome的作用而降解。
CRISPR/Cas(Clustered ReguLatory Interspaced Short Palindromic Repeats/CRISPR associated)是广泛存在于细菌和古细菌中的一种生物防御系统。其中经过改造的Ⅱ型系统，CRISPR/Cas9，成为现在广泛使用的基因组改造工具。在guide RNA(gRNA)的介导下，Cas9蛋白识别基因组上protospacer adjacent motif(PAM)及其上游20bp序列，并在PAM上游3bp位置产生双链切口。在同时提供供体DNA(donor DNA)的情况下，被CRISPR/Cas9双链切开的基因可以HDR的方式重组入新的序列，以达到基因改造的目的。在酿酒酵母(Saccharomyces.cerevisiae)中，利用CRISPR/Cas9系统进行基因组改造的实例很多，包括基因点突变、基因敲除和基因插入。
目前的体外合成系统中可能留存一些核酸酶，这些核酸酶会通过降解体外合成系统中的mRNA和DNA，从而影响体外合成系统中核酸的稳定性。
因此，本领域迫切需要开放一种通过稳定核酸实现外源蛋白稳定表达的方法。
发明内容
本发明的目的在于提供一种通过稳定核酸实现外源蛋白稳定表达的方法。
本发明第一方面提供了一种体外的无细胞的蛋白合成体系，所述无细胞的蛋白合成体系包括：
(a)酵母细胞提取物；
(b)聚乙二醇；
(c)任选的外源蔗糖；和
(d)任选的溶剂，所述溶剂为水或水性溶剂，
其中，所述酵母细胞提取物中的EXN53蛋白的含量≤10％，较佳地，≤5％， 更佳地，≤2％。
在另一优选例中，所述酵母细胞提取物中的EXN53的含量为0。
在另一优选例中，所述EXN53来源于选自下组的一种或多种来源的酵母：毕氏酵母、克鲁维酵母，较佳地，来源于克鲁维酵母。
在另一优选例中，所述克鲁维酵母包括马克斯克鲁维酵母、和/或乳酸克鲁维酵母。
在另一优选例中，所述EXN53的核苷酸序列如SEQ ID NO.:1所示。
在另一优选例中，所述EXN53的蛋白序列如SEQ ID NO.:6所示。
在另一优选例中，所述无细胞的蛋白合成体系还包括选自下组的一种或多种组分：
(e1)用于合成RNA的底物；
(e2)用于合成蛋白的底物；
(e3)镁离子；
(e4)钾离子；
(e5)缓冲剂；
(e6)RNA聚合酶；
(e7)能量再生系统。
在另一优选例中，所述无细胞的蛋白合成体系还包括选自下组的一种或多种组分：
(e8)血红素；
(e9)亚精胺。
在另一优选例中，所述酵母细胞选自下组的一种或多种来源的酵母：毕氏酵母、克鲁维酵母、或其组合；较佳地，所述的酵母细胞包括：克鲁维酵母，更佳地为马克斯克鲁维酵母、和/或乳酸克鲁维酵母。
在另一优选例中，所述的酵母细胞提取物为对酵母细胞的水性提取物。
在另一优选例中，所述酵母细胞提取物不含酵母内源性的长链核酸分子。
在另一优选例中，所述的酵母细胞提取物是用包括以下步骤的方法制备：
(i)提供酵母细胞；
(ii)对酵母细胞进行洗涤处理，获得经洗涤的酵母细胞；
(iii)对经洗涤的酵母细胞进行破细胞处理，从而获得酵母粗提物；和
(iv)对所述酵母粗提物进行固液分离，获得液体部分，即为酵母细胞提取物。
在另一优选例中，所述的固液分离包括离心。
在另一优选例中，在液态下进行离心。
在另一优选例中，所述离心条件为5000-100000×g，较佳地，8000-30000×g。
在另一优选例中，所述离心时间为0.5－2h，较佳地，20min－50min。
在另一优选例中，所述离心在1-10℃下进行，较佳地，在2-6℃下进行。
在另一优选例中，所述的洗涤处理采用洗涤液在pH为7-8(较佳地，7.4)下进行处理。
在另一优选例中，所述洗涤液选自下组：4-羟乙基哌嗪乙磺酸钾、醋酸钾、醋酸镁、或其组合。
在另一优选例中，所述的破细胞处理包括高压破碎、冻融(如液氮低温)破碎。
在另一优选例中，所述的合成RNA的底物包括：核苷单磷酸、核苷三磷酸、或其组合。
在另一优选例中，所述的合成蛋白的底物包括：1-20种天然氨基酸、以及非天然氨基酸。
在另一优选例中，所述镁离子来源于镁离子源，所述镁离子源选自下组：醋酸镁、谷氨酸镁、或其组合。
在另一优选例中，所述钾离子来源于钾离子源，所述钾离子源选自下组：醋酸钾、谷氨酸钾、或其组合。
在另一优选例中，所述能量再生系统选自下组：磷酸肌酸/磷酸肌酸酶系统、糖酵解途径及其中间产物能量系统、或其组合。
在另一优选例中，所述无细胞的蛋白合成体系还包括(f1)人工合成的tRNA。
在另一优选例中，所述缓冲剂选自下组：4-羟乙基哌嗪乙磺酸、三羟甲基氨基甲烷、或其组合。
在另一优选例中，所述无细胞的蛋白合成体系还包括(g1)外源的用于指导蛋白质合成的DNA分子。
在另一优选例中，所述的DNA分子为线性的。
在另一优选例中，所述的DNA分子为环状的。
在另一优选例中，所述的DNA分子含有编码外源蛋白的序列。
在另一优选例中，所述的编码外源蛋白的序列包括基因组序列、cDNA序列。
在另一优选例中，所述的编码外源蛋白的序列还含有启动子序列、5'非翻译序列、3'非翻译序列。
在另一优选例中，所述无细胞的蛋白合成体系包括选自下组的成分：4-羟乙基哌嗪乙磺酸、醋酸钾、醋酸镁、核苷三磷酸、氨基酸、磷酸肌酸，二硫苏糖醇(DTT)、磷酸肌酸激酶、RNA聚合酶、或其组合。
在另一优选例中，所述聚乙二醇选自下组：PEG3000、PEG8000、PEG6000、PEG3350、或其组合。
在另一优选例中，所述聚乙二醇包括分子量(Da)为200-10000的聚乙二醇，较佳地，分子量为3000-10000的聚乙二醇。
在另一优选例中，所述蛋白合成体系中，组分(a)的浓度(v/v)为20％-70％，较佳地，30-60％，更佳地，40％-50％，以所述蛋白合成体系的总体积计。
在另一优选例中，所述蛋白合成体系中，组分(b)的浓度(w/v，例如g/mL)为0.1-8％，较佳地，0.5-4％，更佳地，1-2％。
在另一优选例中，所述蛋白合成体系中，组分(c)的浓度为0.2-4％，较佳地，0.5-4％，更佳地，0.5-1％，以所述蛋白合成体系的总体积计。
在另一优选例中，所述核苷三磷酸选自下组：腺嘌呤核苷三磷酸、鸟嘌呤核苷三磷酸、胞嘧啶核苷三磷酸、尿嘧啶核苷三磷酸、或其组合。
在另一优选例中，所述蛋白合成体系中，组分(e1)的浓度为0.1-5mM，较佳地，0.5-3mM，更佳地，1-1.5mM。
在另一优选例中，所述氨基酸为选自下组：甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸、组氨酸、或其组合。
在另一优选例中，所述氨基酸包括D型氨基酸和/或L型氨基酸。
在另一优选例中，所述蛋白合成体系中，所述组分(e2)的浓度为0.01-0.48mM，较佳地，0.04-0.24mM，更佳地，0.04-0.2mM，最佳地，0.08mM。
在另一优选例中，所述蛋白合成体系中，所述组分(e3)的浓度为1-10mM，较佳地，1-5mM，更佳地，2-4mM。
在另一优选例中，所述蛋白合成体系中，所述组分(e4)的浓度为30-210mM，较佳地，30-150mM，更佳地，30-60mM。
在另一优选例中，所述蛋白合成体系中，所述组分(e6)的浓度为0.01-0.3mg/mL，较佳地，0.02-0.1mg/mL，更佳地，0.027-0.054mg/mL。
在另一优选例中，所述蛋白合成体系中，4-羟乙基哌嗪乙磺酸的浓度为5-50mM，较佳地，10-50mM，较佳地，15-30mM，更佳地，20-25mM。
在另一优选例中，所述蛋白合成体系中，所述醋酸钾的浓度为20-210mM，较佳地，30-210mM，较佳地，30-150mM，更佳地，30-60mM。
在另一优选例中，所述蛋白合成体系中，所述醋酸镁的浓度为1-10mM，较佳地，1-5mM，更佳地，2-4mM。
在另一优选例中，所述蛋白合成体系中，所述磷酸肌酸的浓度为10-50mM，较佳地，20-30mM，更佳地，25mM。
在另一优选例中，所述蛋白合成体系中，所述血红素的浓度为0.01-0.1mM，较佳地，0.02-0.08mM，更佳地，0.03-0.05mM，最佳地，0.04mM。
在另一优选例中，所述蛋白合成体系中，所述亚精胺的浓度为0.05-1mM，较佳地，0.1-0.8mM，更佳地，更佳地，0.2-0.5mM，更佳地，0.3-0.4mM，最佳地，0.4mM。
在另一优选例中，所述蛋白合成体系中，所述二硫苏糖醇(DTT)的浓度为0.2-15mM，较佳地，0.2-7mM，更佳地，1-2mM。
在另一优选例中，所述蛋白合成体系中，所述磷酸肌酸激酶的浓度为0.1-1mg/mL，较佳地，0.2-0.5mg/mL，更佳地，0.27mg/mL。
在另一优选例中，所述蛋白合成体系中，所述T7RNA聚合酶的浓度为0.01-0.3mg/mL，较佳地，0.02-0.1mg/mL，更佳地，0.027-0.054mg/mL。
在另一优选例中，所述的无细胞体外合成体系具有以下性能：
在合成体系里，蛋白合成总量达到3ug蛋白/mL体系。
在另一优选例中，所述的无细胞的蛋白合成体系的组成包括：
Figure PCTCN2017115966-appb-000001
Figure PCTCN2017115966-appb-000002
在另一优选例中，所述无细胞的蛋白合成体系的组成还包括：
亚精胺，            0.2-0.4mM      0.3-0.4mM；
血红素，            0.01-0.04mM    0.03-0.04mM。
在另一优选例中，所述的PEG选自PEG3350、PEG3000、和/或PEG8000。
在另一优选例中，所述RNA聚合酶为T7RNA聚合酶。
本发明第二方面提供了一种酵母细胞提取物，所述酵母细胞提取物中的EXN53蛋白的含量≤10％，较佳地，≤5％，更佳地，≤2％。
本发明第三方面提供了一种本发明第一方面所述的体外的无细胞的蛋白合成体系的生产方法，包括步骤：
将(a)酵母细胞提取物与(b)聚乙二醇；(c)任选的外源蔗糖；和(d)任选的溶剂混合，从而获得本发明第一方面所述的体外的无细胞的蛋白合成体系，其中，所述溶剂为水或水性溶剂，所述酵母细胞提取物中的EXN53蛋白的含量≤10％，较佳地，≤5％，更佳地，≤2％。
在另一优选例中，所述蛋白合成体系中，组分(a)的浓度(v/v)为20％-70％，较佳地，30-60％，更佳地，40％-50％，以所述蛋白合成体系的总体积计。
在另一优选例中，所述蛋白合成体系中，组分(b)的浓度(w/v，例如g/mL)为 0.1-8％，较佳地，0.5-4％，更佳地，1-2％。
本发明第四方面提供了一种体外合成蛋白的方法，包括步骤：
(i)提供本发明第一方面所述的体外的无细胞的蛋白合成体系，并加入外源的用于指导蛋白质合成的DNA分子，其中；所述蛋白合成体系中的EXN53蛋白的含量≤10％，较佳地，≤5％，更佳地，≤2％；
(ii)在适合的条件下，孵育步骤(i)的蛋白合成体系一段时间T1，从而合成由所述外源DNA编码的蛋白质。
在另一优选例中，所述的方法还包括：(iii)任选地从所述蛋白合成体系中，分离或检测所述的由外源DNA编码的蛋白质。
在另一优选例中，所述外源DNA来自原核生物、真核生物。
在另一优选例中，所述外源DNA来自动物、植物、病原体。
在另一优选例中，所述外源DNA来自哺乳动物，较佳地灵长动物，啮齿动物，包括人、小鼠、大鼠。
在另一优选例中，所述的外源蛋白的编码序列编码选自下组的外源蛋白：荧光素蛋白、或荧光素酶(如萤火虫荧光素酶)、绿色荧光蛋白、黄色荧光蛋白、氨酰tRNA合成酶、甘油醛-3-磷酸脱氢酶、过氧化氢酶、肌动蛋白、抗体的可变区域、萤光素酶突变体、α-淀粉酶、肠道菌素A、丙型肝炎病毒E2糖蛋白、胰岛素前体、干扰素αA、白细胞介素-1β、溶菌酶素、血清白蛋白、单链抗体段(scFV)、甲状腺素运载蛋白、酪氨酸酶、木聚糖酶、或其组合。
在另一优选例中，所述外源蛋白选自下组：荧光素蛋白、或荧光素酶(如萤火虫荧光素酶)、绿色荧光蛋白、黄色荧光蛋白、氨酰tRNA合成酶、甘油醛-3-磷酸脱氢酶、过氧化氢酶、肌动蛋白、抗体的可变区域、萤光素酶突变、α-淀粉酶、肠道菌素A、丙型肝炎病毒E2糖蛋白、胰岛素前体、干扰素αA、白细胞介素-1β、溶菌酶素、血清白蛋白、单链抗体段(scFV)、甲状腺素运载蛋白、酪氨酸酶、木聚糖酶、或其组合。
在另一优选例中，所述的外源DNA编码选自下组的外源蛋白：荧光素蛋白、或荧光素酶(如萤火虫荧光素酶)、绿色荧光蛋白、黄色荧光蛋白、氨酰tRNA合成酶、甘油醛-3-磷酸脱氢酶、过氧化氢酶、肌动蛋白、抗体的可变区域、萤光素酶突变体、α-淀粉酶、肠道菌素A、丙型肝炎病毒E2糖蛋白、胰岛素前体、干扰素αA、白细胞介素-1β、溶菌酶素、血清白蛋白、单链抗体段(scFV)、甲状腺素运 载蛋白、酪氨酸酶、木聚糖酶、或其组合。
在另一优选例中，所述外源DNA编码的蛋白质选自下组：荧光素蛋白、或荧光素酶(如萤火虫荧光素酶)、绿色荧光蛋白、黄色荧光蛋白、氨酰tRNA合成酶、甘油醛-3-磷酸脱氢酶、过氧化氢酶、肌动蛋白、抗体的可变区域、萤光素酶突变、α-淀粉酶、肠道菌素A、丙型肝炎病毒E2糖蛋白、胰岛素前体、干扰素αA、白细胞介素-1β、溶菌酶素、血清白蛋白、单链抗体段(scFV)、甲状腺素运载蛋白、酪氨酸酶、木聚糖酶、或其组合。
在另一优选例中，所述步骤(ii)中，反应温度为20-37℃，较佳地，20-25℃。
在另一优选例中，所述步骤(ii)中，反应时间为1-6h，较佳地，2-4h。
本发明第五方面提供了一种工程菌株，所述菌株为克鲁维酵母菌株，并且所述菌株中的EXN53基因(核酸酶基因)的表达或活性被降低。
在另一优选例中，所述“降低”指EXN53基因的表达量≤10％，较佳地，≤5％，更佳地，≤2％。
在另一优选例中，所述“降低”是指将EXN53基因的表达或活性降低满足以下条件：
A1/A0的比值≤30％，较佳地≤10％，更佳地≤5％，更佳地，≤2％，最佳地为0-2％；
其中，A1为EXN53基因的表达或活性；A0为野生型EXN53基因的表达或活性。
在另一优选例中，所述菌株中的EXN53基因(核酸酶基因)的表达或活性被降低通过选自下组的方式实现：基因突变、基因敲除、基因中断、RNA干扰技术、Crispr技术、或其组合。
本发明第六方面提供了一种本发明第五方面所述的工程菌株的用途，用于提高体外蛋白合成效率。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了通过降低体外生物合成体系中的核酸酶来提高核酸的稳定性，并利用对K.lactis基因组中核酸酶基因进行分析和特异性改造的设计路线图。(A)K.lactis野生型细胞。(B)K.lactis核酸酶改造后的细胞。(C)收集改造核酸酶后的K.lactis细胞lysate.(D)利用改造特定基因后的酵母菌株制备成酵母细胞溶液，具有更稳定的核酸特性，制备出增强的体外生物合成体系。
图2显示了K.lactis基因组中全部61种核酸酶基因的分布和功能分析图。A-F为K.lactis六条染色体，位于A染色体的核酸酶有：KlSEN54，KlDNA2，KlTRM2，KlFCF1，KlDOM34，KlRAD2，KlRNH70，KlDIS3，KlNPP1；位于B染色体的核酸酶有：KlOGG1；位于C染色体的核酸酶有：KlPOL2，KlRAD50，KlYSH1，KlRCL1，KlNGL2，KlMRE11，KlPOP3，KlMKT1，KlAPN1，KlRPP1，KlPOP2；位于D染色体的核酸酶有：KlNUC1，KlRPP6，KlDBR1，KlRPS3，KlRPM2，KlSUV3，KlRAD1，KlIRE1，KlPOP1；位于E染色体的核酸酶有：KlPOL3，KlDXO1，KlMUS81，KlPAN3，KlAPN2，KlRAI1，KlEXO1，KlREX4，KlPAN2，KlLCL3；位于F染色体的核酸酶有：KlRAD17，KlPOP4，KlPOP5，KlRAD27，KlRNH201，KlVMA1，KlRAT1，KlNGL1，KlREX2，KlPOL31，KlCCR4，KlTRZ1，KlSEN2，KlREX3，KlSWT1，KLEXN53，KlRNT1，KlSEN15，KlNTG1，KlNOB1，KlDDP1。
图3显示了K.lactis基因组中5种核酸酶基因改造的序列和CRISPR系统设计图。供体donor DNA两个同源臂homologous arm 1和homologous arm 2分别为待敲除核酸酶基因的ORF上、下游1000bp左右的基因序列，构建两个向导gRNA，同时识别待敲除核酸酶基因ORF的5′和3′端的PAM序列，引导Cas9核酸酶对DNA定点切割,出现DSB(double strand break)后发生同源重组，从而实现基因敲除。
图4显示了K.lactis基因组中5种核酸酶蛋白结构域示意图。
图5显示了KLEXN53蛋白与Saccharomyces cerevisiae中同源蛋白ScEXN53(KEGG No.:YGL173C)，与Schizosaccharomyces pombe中同源蛋白SpEXO2(KEGG No.:SPAC17A5.14)，与Homo sapiens中同源蛋白HsEXN53(KEGG No.:54464)部分氨基酸序列比对图，活性位点用#标记。
图6显示了KlDIS3蛋白与Saccharomyces cerevisiae中同源蛋白ScDIS3(KEGG No.:YOL021C)，与Schizosaccharomyces pombe中同源蛋白SpDIS3(KEGG No.:SPBC26H8.10)，与Homo sapiens中同源蛋白HsDIS3(KEGG No.:22894)部分氨基酸序列比对图，活性位点用#标记。
图7显示了KlRAT1蛋白与Saccharomyces cerevisiae中同源蛋白ScRAT1(KEGG No.:YOR048C)，与Schizosaccharomyces pombe中同源蛋白SpRAT1(KEGG No.:SPAC26A3.12c)，与Homo sapiens中同源蛋白HsRAT1(KEGG No.:22803)部分氨基酸序列比对图，活性位点用#标记。
图8显示了KlRRP6蛋白与Saccharomyces cerevisiae中同源蛋白ScRRP6(KEGG No.:YOR001W)，与Schizosaccharomyces pombe中同源蛋白SpRRP6(KEGG No.:SPAC1F3.01)，与Homo sapiens中同源蛋白HsRRP6(KEGG No.:5394)部分氨基酸序列比对图，活性位点用#标记。
图9显示了KlNGL2蛋白与Saccharomyces cerevisiae中同源蛋白ScNGL3(KEGG No.:YML118W)，与Schizosaccharomyces pombe中同源蛋白SpNGL2(KEGG No.:SPBC9B6.11c)，与Homo sapiens中同源蛋白HsANGEL2(KEGG No.:90806)氨基酸序列比对图。
图10显示了pHoCas9_SE_Kana_tRNA_ScRNR2_KlEXN53-1&2质粒图谱。KLEXN53的gRNA1和gRNA2分别为KLEXN53基因ORF的5′和3′端两个gRNA，带有tRNA-Tyr启动子和SNR52终止子，PMZ374-cas9是优化后的cas9，质粒带有kana筛选标记。
图11显示了KLEXN53-DD1-pMD18质粒图谱。homologous arm 1和homologous arm 2分别为KLEXN53基因的ORF上、下游1000bp左右的基因序列，质粒带有Amp筛选标记。
图12显示了pHoCas9_SE_Kana_tRNA_ScRNR2_KlDIS3-1&2质粒图谱。KlDis3的gRNA1和gRNA2分别为KlDis3基因ORF的5′和3′端两个gRNA，带有tRNA-Tyr启动子和SNR52终止子，PMZ374-cas9是优化后的cas9，质粒带有kana筛选标记。
图13显示了KlDIS3-DD1-pMD18质粒图谱。homologous arm 1和homologous arm 2分别为KlDis3基因的ORF上、下游1000bp左右的基因序列，质粒带有Amp筛选标记。
图14显示了pHoCas9_SE_Kana_tRNA_ScRNR2_KlRat1-1&2质粒图谱。KlRat1的gRNA1和gRNA2分别为KlRat1基因ORF的5′和3′端两个gRNA，带有tRNA-Tyr启动子和SNR52终止子，PMZ374-cas9是优化后的cas9，质粒带有kana筛选标记。
图15显示了KlRat1-DD1-pMD18质粒图谱。homologous arm 1和homologous  arm 2分别为KlRat1基因的ORF上、下游1000bp左右的基因序列，质粒带有Amp筛选标记。
图16显示了pHoCas9_SE_Kana_tRNA_ScRNR2_KlRrp6-1&2质粒图谱。KlRrp6的gRNA1和gRNA2分别为KlRrp6基因ORF的5′和3′端两个gRNA，带有tRNA-Tyr启动子和SNR52终止子，PMZ374-cas9是优化后的cas9，质粒带有kana筛选标记。
图17显示了KlRrp6-DD1-pMD18质粒图谱。homologous arm 1和homologous arm 2分别为KlRrp6基因的ORF上、下游1000bp左右的基因序列，质粒带有Amp筛选标记。
图18显示了pHoCas9_SE_kana_tRNA_ScRNR2_KlNGL2-1&2质粒图谱。KlNGL3的gRNA1和gRNA2分别为KlNGL3基因ORF的5′和3′端两个gRNA，带有tRNA-Tyr启动子和SNR52终止子，PMZ374-cas9是优化后的cas9，质粒带有kana筛选标记。
图19显示了Kl NGL2-DD1-pMD18质粒图谱。homologous arm 1和homologous arm 2分别为KlNGL3基因的ORF上、下游1000bp左右的基因序列，质粒带有Amp筛选标记。
图20显示了改造菌株体外翻译活性测定数据图。利用萤火虫荧光蛋白(Fluc)的荧光强度指示体外生物合成系统的重组蛋白的合成能力。其中wt代表野生型克鲁维酵母，ΔKLEXN53EXN53代表KLEXN53EXN53基因敲除的克鲁维酵母菌株。
具体实施方式
经过广泛而深入的研究，通过大量筛选和摸索，从很多种核酸酶中筛出5种核酸酶，其中对其中一种核酸酶(如EXN53)进行下调或敲除处理，意外的发现居然可以提高核酸的稳定性，并显著提高体外蛋白质合成体系产生蛋白质的效率。具体地，下调或敲除EXN53后，Δklexn53酵母菌株在IVTT中荧光素酶活性是野生型的≥2倍。在此基础上，本发明人完成了本发明。
除非另行说明，本发明所有的％均为基于质量的百分比浓度(wt％)。
酵母体外蛋白质合成体系
酵母(yeast)兼具培养简单、高效蛋白质折叠、和翻译后修饰的优势。其中酿酒酵母(Saccharomyces cerevisiae)和毕氏酵母(Pichia pastoris)是表达复杂真核蛋白质和膜蛋白的模式生物，酵母也可作为制备体外翻译系统的原料。
克鲁维酵母(Kluyveromyces)是一种子囊孢子酵母，其中的马克斯克鲁维酵母(Kluyveromyces marxianus)和乳酸克鲁维酵母(Kluyveromyces lactis)是工业上广泛使用的酵母。与其他酵母相比，乳酸克鲁维酵母具有许多优点,如超强的分泌能力,更好的大规模发酵特性、食品安全的级别、以及同时具有蛋白翻译后修饰的能力等。
在本发明中，酵母体外蛋白质合成体系不受特别限制，一种优选的酵母体外蛋白质合成体系为克鲁维酵母表达系统(更佳地，乳酸克鲁维酵母表达系统)。
在本发明中，所述酵母体外蛋白质合成体系包括：
(a)酵母细胞提取物；
(b)聚乙二醇；
(c)任选的外源蔗糖；和
(d)任选的溶剂，所述溶剂为水或水性溶剂，其中，所述酵母细胞提取物中的EXN53蛋白的含量≤10％，较佳地，≤5％，更佳地，≤2％。
在一优选实施方式中，所述EXN53来源于选自下组的一种或多种来源的酵母：毕氏酵母、克鲁维酵母，较佳地，来源于克鲁维酵母(如马克斯克鲁维酵母、乳酸克鲁维酵母)。
在本发明中，克鲁维酵母(如乳酸克鲁维酵母)不受特别限制，包括任何一种能够提高合成蛋白效率的克鲁维(如乳酸克鲁维酵母)菌株。
在一优选实施方式中，所述EXN53的蛋白序列如SEQ ID NO.:6所示，所述EXN53的核苷酸序列如SEQ ID NO.:1所示。
在一特别优选的实施方式中，本发明提供的体外蛋白合成体系包括：酵母细胞提取物，4-羟乙基哌嗪乙磺酸，醋酸钾，醋酸镁，腺嘌呤核苷三磷酸(ATP)，鸟嘌呤核苷三磷酸(GTP)，胞嘧啶核苷三磷酸(CTP)，胸腺嘧啶核苷三磷酸(TTP)，氨基酸混合物，磷酸肌酸，二硫苏糖醇(DTT)，磷酸肌酸激酶，RNA酶抑制剂，荧光素，萤光素酶DNA，RNA聚合酶。
在本发明中，RNA聚合酶没有特别限制，可以选自一种或多种RNA聚合酶，典型的RNA聚合酶为T7RNA聚合酶。
在本发明中，所述酵母细胞提取物在体外蛋白合成体系中的比例不受特别限制，通常所述酵母细胞提取物在体外蛋白质合成蛋白合成体系中所占体系为20-70％，较佳地，30-60％，更佳地，40-50％。
在本发明中，所述的酵母细胞提取物不含完整的细胞，典型的酵母细胞提取物包括用于蛋白翻译的核糖体、转运RNA、氨酰tRNA合成酶、蛋白质合成需要的起始因子和延伸因子以及终止释放因子。此外，酵母提取物中还含有一些源自酵母细胞的细胞质中的其他蛋白，尤其是可溶性蛋白。
在本发明中，所述的酵母细胞提取物所含蛋白含量为20-100mg/mL,较佳为50-100mg/mL。所述的测定蛋白含量方法为考马斯亮蓝测定方法。
在本发明中，所述的酵母细胞提取物的制备方法不受限制，一种优选的制备方法包括以下步骤：
(i)提供酵母细胞；
(ii)对酵母细胞进行洗涤处理，获得经洗涤的酵母细胞；
(iii)对经洗涤的酵母细胞进行破细胞处理，从而获得酵母粗提物；
(iv)对所述酵母粗提物进行固液分离，获得液体部分，即为酵母细胞提取物。
在本发明中，所述的固液分离方式不受特别限制，一种优选的方式为离心。
在一优选实施方式中，所述离心在液态下进行。
在本发明中，所述离心条件不受特别限制，一种优选的离心条件为5000-100000×g，较佳地，8000-30000×g。
在本发明中，所述离心时间不受特别限制，一种优选的离心时间为0.5min-2h，较佳地，20min-50min。
在本发明中，所述离心的温度不受特别限制，优选的，所述离心在1-10℃下进行，较佳地，在2-6℃下进行。
在本发明中，所述的洗涤处理方式不受特别限制，一种优选的洗涤处理方式为采用洗涤液在pH为7-8(较佳地，7.4)下进行处理，所述洗涤液没有特别限制，典型的所述洗涤液选自下组：4-羟乙基哌嗪乙磺酸钾、醋酸钾、醋酸镁、或其组合。
在本发明中，所述破细胞处理的方式不受特别限制，一种优选的所述的破细胞处理包括高压破碎、冻融(如液氮低温)破碎。
所述体外蛋白质合成体系中的核苷三磷酸混合物为腺嘌呤核苷三磷酸、鸟嘌呤核苷三磷酸、胞嘧啶核苷三磷酸和尿嘧啶核苷三磷酸。在本发明中，各种单核苷酸的浓度没有特别限制，通常每种单核苷酸的浓度为0.5-5mM，较佳地为1.0-2.0mM。
所述体外蛋白质合成体系中的氨基酸混合物可包括天然或非天然氨基酸，可包括D型或L型氨基酸。代表性的氨基酸包括(但并不限于)20种天然氨基酸：甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸和组氨酸。每种氨基酸的浓度通常为0.01-0.5mM，较佳地0.02-0.2mM，如0.05、0.06、0.07、0.08mM。
在优选例中，所述体外蛋白质合成体系还含有聚乙二醇或其类似物。聚乙二醇或其类似物的浓度没有特别限制，通常，聚乙二醇或其类似物的浓度(w/v)为0.1-8％，较佳地，0.5-4％，更佳地，1-2％，以所述蛋白合成体系的总重量计。代表性的PEG例子包括(但并不限于)：PEG3000，PEG8000，PEG6000和PEG3350。应理解，本发明的体系还可包括其他各种分子量的聚乙二醇(如PEG200、400、1500、2000、4000、6000、8000、10000等)。
在优选例中，所述体外蛋白质合成体系还含有蔗糖。蔗糖的浓度没有特别限制，通常，蔗糖的浓度为0.03-40wt％，较佳地，0.08-10wt％，更佳地，0.1-5wt％，以所述蛋白合成体系的总重量计。
一种特别优选的体外蛋白质合成体系，除了酵母提取物之外，还含有以下组分：22mM，pH为7.4的4-羟乙基哌嗪乙磺酸，30-150mM醋酸钾，1.0-5.0mM醋酸镁，1.5-4mM核苷三磷酸混合物，0.08-0.24mM的氨基酸混合物，25mM磷酸肌酸，1.7mM二硫苏糖醇，0.27mg/mL磷酸肌酸激酶，1％-4％聚乙二醇，0.5％-2％蔗糖，8-20ng/μl萤火虫荧光素酶的DNA，0.027-0.054mg/mL T7 RNA聚合酶。
外源蛋白的编码序列(外源DNA)
如本文所用，术语“外源蛋白的编码序列”与“外源DNA”可互换使用，均指外源的用于指导蛋白质合成的DNA分子。通常，所述的DNA分子为线性的或环状的。所述的DNA分子含有编码外源蛋白的序列。在本发明中，所述的编码外 源蛋白的序列的例子包括(但并不限于)：基因组序列、cDNA序列。所述的编码外源蛋白的序列还含有启动子序列、5’非翻译序列、3’非翻译序列。
在本发明中，所述外源DNA的选择没有特别限制，通常，外源DNA选自下组：编码荧光素蛋白、或荧光素酶(如萤火虫荧光素酶)、绿色荧光蛋白、黄色荧光蛋白、氨酰tRNA合成酶、甘油醛-3-磷酸脱氢酶、过氧化氢酶、肌动蛋白、抗体的可变区域的外源DNA、萤光素酶突变体的DNA、或其组合。
外源DNA还可以选自下组：编码α-淀粉酶、肠道菌素A、丙型肝炎病毒E2糖蛋白、胰岛素前体、干扰素αA、白细胞介素-1β、溶菌酶素、血清白蛋白、单链抗体段(scFV)、甲状腺素运载蛋白、酪氨酸酶、木聚糖酶的外源DNA、或其组合。
在一优选实施方式中，所述外源DNA编码选自下组的蛋白：绿色荧光蛋白(enhanced GFP,eGFP)、黄色荧光蛋白(YFP)、大肠杆菌β-半乳糖苷酶(β-galactosidase,LacZ)、人赖氨酸-tRNA合成酶(Lysine-tRNA synthetase)、人亮氨酸-tRNA合成酶(Leucine-tRNA synthetase)、拟南芥甘油醛3-磷酸脱氢酶(Glyceraldehyde-3-phosphate dehydrogenase)、鼠过氧化氢酶(Catalase)、或其组合。
体外高通量蛋白合成方法(提高蛋白质体外翻译效率方法)
本发明提供了一种体外高通量蛋白合成方法，包括步骤：
(i)提供本发明第一方面所述的体外的无细胞的蛋白合成体系，并加入外源的用于指导蛋白质合成的DNA分子，其中；所述蛋白合成体系中的EXN53蛋白的含量≤10％，较佳地，≤5％，更佳地，≤2％；
(ii)在适合的条件下，孵育步骤(i)的蛋白合成体系一段时间T1，从而合成由所述外源DNA编码的蛋白质。
在一优选实施方式中，本发明提供了一种对酵母基因组中核酸酶基因进行敲除从而提高蛋白质体外翻译效率的设计和方法，包括下述步骤：
(1)增强体外生物合成活性的设计、分析方法如下：
A、体外生物合成系统中各组分对活性的影响分析；
B、K.lactis基因组中核酸酶基因的功能和分布分析；
C、K.lactis基因组中选定核酸酶基因的改造设计；
(2)靶向敲除基因克隆载体的构建，构建方法如下：
A、针对特定基因的序列，设计引导核酸内切酶切割的sgRNA序列；
B、将上述sgRNA序列重组至含有核酸内切酶的载体中，得到sgRNA和核酸内切酶共表达的第一载体；
(3)构建敲除特定基因的供体DNA，构建方法如下：
A、从基因数据库中下载特定基因核苷酸序列，利用基因上下游序列各1000bp构建第二载体；
B、用引物M13F和M13R对第二载体进行扩增，获得的PCR产物进行乙醇沉淀浓缩，最终获得供体DNA；
(4)将第一载体和供体DNA同时转化到酵母感受态细胞中；
筛选出单克隆细胞进行扩大培养，抽提酵母基因组，设计引物对敲除位点进行扩增，扩增的PCR产物经测序验证后即可获得敲除特定基因的菌株；
(5)无细胞体外蛋白质合成体系
利用敲除特定基因后的酵母菌株制备成酵母细胞溶液，加入到蛋白体外翻译体系中，20-30℃的环境中，静置反应2-6h，利用多功能酶标仪(Perkin Elmer)读数，检测萤火虫荧光素酶活性。
本发明的主要优点包括：
(1)本发明首次设计了通过降低体系中的核酸酶来提高核酸的稳定性，并对K.lactis基因组中核酸酶基因进行系统分析和特异性改造，从而提供了一种通用的用于调控体外生物合成活性的技术路线和方法。
(2)本发明对大量的核酸酶筛选，首次从很多种核酸酶中筛出5种核酸酶，其中对其中一种核酸酶(如EXN53)进行下调或敲除处理，意外的发现居然可以提高核酸的稳定性，并显著提高体外蛋白质合成体系产生蛋白质的效率。具体地，下调或敲除EXN53后，Δklexn53酵母菌株在IVTT中荧光素酶活性是野生型的≥2倍(如2.46倍)。
(3)本发明首次发现一种能显著提高体外蛋白质合成活性的K.lactis菌株。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York: Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
如无特别说明，则本发明实施例中所用的材料和试剂均为市售产品。
实施例1通过降低体外生物体系中的核酸酶来提高核酸的稳定性，并对K.lactis基因组中核酸酶基因进行分析和特异性改造
1.通过K.lactis基因组改造以调控体外生物合成活性的的设计原理
1.1体外生物合成体系分析
1.1.1不同体外生物合成体系中的组成分析
体外生物合成系统通过对不同种类的细胞(包括微生物、动物和植物)进行破碎，提取细胞裂解物，从而用于外源蛋白的翻译。体外生物合成系统为了实现转录、翻译、蛋白折叠和能量代谢等功能，细胞裂解物中必须包含能量再生和蛋白质合成方面的元件，包括核糖体、氨酰-tRNA合成酶、翻译起始和延伸因子、核糖体释放因子、核苷酸循环酶、代谢酶、分子伴侣和折叠酶等。需要外源加入的物质包括氨基酸、核苷酸、DNA模板、能量底物、辅助因子和盐分子等。系统中不同组分的稳定性对反应延续时间及最终蛋白产量有重要影响，某些底物的消耗殆尽(例如ATP和半胱氨酸等)是反应终止的重要原因。
1.1.2体外生物合成体系中的组分对合成产物的影响分析模型；
在体外翻译系统中，能量代谢底物(磷酸肌酸等)、参与mRNA合成的底物(NTP等)、参与蛋白质合成的底物(氨基酸等)以及磷酸盐浓度和pH值等组分，随着反应的进行，量值会产生变化，最终导致反应终止。通过不同技术手段，补充相应组分(NTP和氨基酸等)或稳定相应条件(pH值和磷酸盐浓度等)，均能有效延长体外翻译系统的反应时间，从而提高蛋白产量。
1.1.3体外蛋白质合成体系中的核酸和核酸酶对蛋白质合成的影响分析；
作为蛋白质翻译的重要底物，DNA模板及其转录生成的mRNA的稳定性，对体外翻译系统反应持续时间及产量有重要影响。细胞经过破碎，收取细胞裂解液并用于构建体外翻译体系，其中除了含有蛋白质合成所必需的各种组分外，也包含核酸酶、蛋白酶等不利于反应进行的组分。在由经过纯化的各种组分组成的系统当中，由于不含有抑制因子，蛋白质翻译的效率显著提升。
同时，通过不同的技术手段(包括核酸酶基因敲除和加入稳定因子等)能有效提高体外翻译系统中核酸的稳定性，进而提高蛋白质的翻译效率。通过降低体外生物合成体系中的核酸酶来提高核酸的稳定性，并利用对K.lactis基因组中核酸酶基因进行分析和特异性改造的设计路线图(如图1所示)。
1.2K.lactis基因组中核酸酶基因分析
1.2.1K.lactis基因组中核酸酶基因的分类和分布；
核酸酶(也称为核聚合酶或多核苷酸酶)是核酸分解的第一步中能够水解核苷酸之间的磷酸二酯键的酶。根据核酸酶作用的位置不同，可将核酸酶分为核酸外切酶(exonuclease)和核酸内切酶(endonuclease)。核酸外切酶从3′端开始逐个水解核苷酸，称为3′to 5′外切酶，核酸外切酶从5′端开始逐个水解核苷酸，称为5′to 3′外切酶，核酸内切酶催化水解多核苷酸内部的磷酸二酯键。核酸酶又分为脱氧核糖核酸酶(DNase)和核糖核酸酶(RNase)，前者对DNA起作用，后者对RNA起作用。根据核酸酶的作用方向又将核酸外切酶分为5′to 3′核酸外切酶和3′to 5′核酸外切酶。
通过数据库基因功能比对分析，K.lactis中共含有61个核酸酶，分布在K.lactisA-F六条染色体上(图2)，其中位于A染色体的核酸酶有：KlSEN54，KlDNA2，KlTRM2，KlFCF1，KlDOM34，KlRAD2，KlRNH70，KlDIS3，KlNPP1；KlSEN54在KEGG数据库中的编码为：KlLA0A00803g，KlDNA2在KEGG数据库中的编码为：KlLA0A05324g，KlTRM2在KEGG数据库中的编码为：KlLA0A05665g，KlFCF1在KEGG数据库中的编码为：KlLA0A07018g，KlDOM34在KEGG数据库中的编码为：KlLA0A08646g，KlRAD2在KEGG数据库中的编码为：KlLA0A09427g，KlRNH70在KEGG数据库中的编码为：KlLA0A10065g，KlDIS3在KEGG数据库中的编码为：KlLA0A10835g，KlNPP1在KEGG数据库中的编码为：KlLA0A11374g。
位于B染色体的核酸酶有：KlOGG1；KlOGG1在KEGG数据库中的编码为：KlLA0B05159g。
位于C染色体的核酸酶有：KlPOL2，KlRAD50，KlYSH1，KlRCL1，KlNGL2，KlMRE11，KlPOP3，KlMKT1，KlAPN1，KlRPP1，KlPOP2；KlPOL2在KEGG数据库中的编码为：KlLA0C02585g，KlRAD50在KEGG数据库中的编码为：KlLA0C02915g，KlYSH1在KEGG数据库中的编码为：KlLA0C04598g，KlRCL1在KEGG数据库中的编码为：KlLA0C05984g，KlNGL2在KEGG数据库中的编码为：KlLA0C06248g， KlMRE11在KEGG数据库中的编码为：KlLA0C06930g，KlPOP3在KEGG数据库中的编码为：KlLA0C12199g，KlMKT1在KEGG数据库中的编码为：KlLA0C13926g，KlAPN1在KEGG数据库中的编码为：KlLA0C14256g，KlRPP1在KEGG数据库中的编码为：KlLA0C14718g，KlPOP2在KEGG数据库中的编码为：KlLA0C18821g。
位于D染色体的核酸酶有：KlNUC1，KlRPP6，KlDBR1，KlRPS3，KlRPM2，KlSUV3，KlRAD1，KlIRE1，KlPOP1；KlNUC1在KEGG数据库中的编码为：KlLA0D00440g，KlRPP6在KEGG数据库中的编码为：KlLA0D01309g，KlDBR1在KEGG数据库中的编码为：KlLA0D04466g，KlRPS3在KEGG数据库中的编码为：KlLA0D08305g，KlRPM2在KEGG数据库中的编码为：KlLA0D10483g，KlSUV3在KEGG数据库中的编码为：KlLA0D12034g，KlRAD1在KEGG数据库中的编码为：KlLA0D12210g，KlIRE1在KEGG数据库中的编码为：KlLA0D12210g，KlPOP1在KEGG数据库中的编码为：KlLA0D19448g。
位于E染色体的核酸酶有：KlPOL3，KlDXO1，KlMUS81，KlPAN3，KlAPN2，KlRAI1，KlEXO1，KlREX4，KlPAN2，KlLCL3；KlPOL3在KEGG数据库中的编码为：KlLA0E01607g，KlDXO1在KEGG数据库中的编码为：KlLA0E02245g，KlMUS81在KEGG数据库中的编码为：KlLA0E03015g，KlPAN3在KEGG数据库中的编码为：KlLA0E08097g，KlAPN2在KEGG数据库中的编码为：KlLA0E18877g，KlRAI1在KEGG数据库中的编码为：KlLA0E12893g，KlEXO1在KEGG数据库中的编码为：KlLA0E16743g，KlREX4在KEGG数据库中的编码为：KlLA0E17865g，KlPAN2在KEGG数据库中的编码为：KlLA0E18877g，KlLCL3在KEGG数据库中的编码为：KlLA0E19163g。
位于F染色体的核酸酶有：KlRAD17，KlPOP4，KlPOP5，KlRAD27，KlRNH201，KlVMA1，KlRAT1，KlNGL1，KlREX2，KlTRL1，KlPOL31，KlCCR4，KlTRZ1，KlSEN2，KlREX3，KlSWT1，KLEXN53，KlRNT1，KlSEN15，KlNTG1，KlNOB1，KlDDP1。KlRAD17在KEGG数据库中的编码为：KlLA0F00330g，KlPOP4在KEGG数据库中的编码为：KlLA0F02211g，KlPOP5在KEGG数据库中的编码为：KlLA0F02453g，KlRAD27在KEGG数据库中的编码为：KlLA0F02992g，KlRNH201在KEGG数据库中的编码为：KlLA0F04774g，KlVMA1在KEGG数据库中的编码为：KlLA0F05401g，KlRAT1在KEGG数据库中的编码为：KlLA0F07469g，KlNGL1在KEGG数据库中的编码为：KlLA0F07733g，KlREX2在KEGG数据库中的编码为：KlLA0F08998g，KlPOL31在KEGG数据库中的编码为：KlLA0F14949g,KlCCR4在 KEGG数据库中的编码为：KlLA0F15884g，KlTRZ1在KEGG数据库中的编码为：KlLA0F16665g，KlSEN2在KEGG数据库中的编码为：KlLA0F19866g，KlREX3在KEGG数据库中的编码为：KlLA0F19910g，KlSWT1在KEGG数据库中的编码为：KlLA0F20361g，KLEXN53在KEGG数据库中的编码为：KlLA0F22385g，KlRNT1在KEGG数据库中的编码为：KlLA0F24816g，KlSEN15在KEGG数据库中的编码为：KlLA0F26334g，KlNTG1在KEGG数据库中的编码为：KlLA0F07711g，KlNOB1在KEGG数据库中的编码为：KlLA0F16280g，KlDDP1在KEGG数据库中的编码为：KlLA0F20273g.
按照功能分类，可将K.lactis核酸酶分为两类，其中DNA酶共有18个，RNA酶共有41个，无功能分类的2个见表1。其中在DNA酶中具有3′to 5′功能的是KlAPN1，5′to 3′功能的是KlRAD27，KlEXO1；在RNA酶中具有3′to 5′功能的是KlRNH70，KlDIS3，KlNGL2，KlRPP6，5′to 3′功能的是KlDXO1，KlRAT1，KLEXN53。
表1
Figure PCTCN2017115966-appb-000003
Figure PCTCN2017115966-appb-000004
1.2.2K.lactis基因组中与体外蛋白质合成相关的核酸酶基因的选取分析；
核酸RNA和DNA蛋白质体外合成系统中的编码底物，它们的稳定性影响着蛋白质的得率。在真核生物翻译过程中，RNA的5′端会瞬时完成帽子结构的加工，而RNA的5′帽子结构对于RNA的稳定性和翻译的效率起着举足轻重的作用。
在真核生物中，存在依赖去腺苷化和非依赖腺苷化的RNA降解机制，在依赖去腺苷化RNA降解机制中可以通过招募去帽复合物去除RNA的5′端成熟帽子结构或者非成熟的帽子结构，RNA同时也在核酸酶的作用下发生5′to 3′的降解。在非依赖腺苷化的mRNA降解机制中，在内切核酸酶的作用下，核酸分子从中间产生断链，核酸外切酶从3′to 5′降解RNA。
在K.lactis的体外蛋白质合成体系中，采用外源线性或环状DNA作为模板，转录出带有polyA 3′端尾巴的mRNA,取决于启动子(promotor)和RNA转录酶的不同，可能带有或不带有5′端成熟帽子结构。因此，DNase酶、RNase酶、核酸外切酶、核酸内切酶均会对体外蛋白质合成体系中模板的稳定性产生影响，改造这些酶对于提高体外生物合成活性均有可能有提升作用。
这其中，由于mRNA的特殊不稳定性和体内RNase酶的高活性，mRNA的寿命是制约体外蛋白质合成的主要瓶颈。因此，通过改造以减少体内RNase酶是首选的改造方案。当然，本专利仍然适用于其他核酸酶的设计改造以提高体外蛋白质合成活性。
1.2.3K.lactis基因组中核酸酶基因EXN53，Rat1，Rrp6，Dis3，NGL2的特异性改造设计；
K.lactis内5′to 3′核酸外切酶包括EXN53和Rat1，其中EXN53定位在细胞质内，Rat1定位在细胞核内。在依赖去腺苷化RNA降解机制中也可以经 过脱腺苷化后通过定位于细胞质和细胞核的，具有3′to 5′核酸外切酶和核酸内切酶活性的蛋白亚基复合体exosome的作用而降解。K.lactis内3′to 5′核酸外切酶包括Rrp6，Dis3(又称Rrp44)和NGL2等，其中Rrp6和Dis3都是核酸外切酶和核酸内切酶活性的蛋白亚基复合体exosome的组成部分。Rrp6是一种3′to 5′核酸外切酶，是RNase D家族的成员，定位于细胞核内，是细胞核内exosome关键的催化亚基，负责细胞内各种RNA的加工，降解和质量控制。Dis3(又称Rrp44)是exosome关键的催化亚基，是高度保守的RNaseII/RNB家族成员，定位于细胞核和细胞质内，具有3′to 5′核酸外切酶和核酸内切酶活性，负责细胞核和细胞质内的RNA代谢和各类RNA的加工，NGL2是一种特异性识别Poly A的酶，这种酶从3′到5′的方向水解RNA。
基于以上分析，本发明选取EXN53，Rat1，Rrp6，Dis3，NGL2共5个基因进行改造，方法包括但不限于基因敲除或者基因突变。通过降低体外生物合成体系中的核酸酶来提高核酸的稳定性，并利用对K.lactis基因组中核酸酶基因进行分析和特异性的改造设计，并调控体外蛋白质合成活性的设计和技术细节。
1.3K.lactis基因组中靶向基因改造的方法分析
1.3.1K.lactis基因组改造的方法和选取分析；
本发明通过CRISPR-Cas9基因编辑技术对K.lactis基因组进行改造。CRISPR是现在应用广泛的基因编辑技术，具有结构简单，操作方便，编辑效率高，不需要引入抗生素，可实现对靶基因多个位点同时敲除等优点，在酵母中已经形成了比较成熟的转化体系。
1.3.2针对K.lactis基因组的CRISPR/Cas9的技术设计；
本发明使用的CRISPR体系，是针对K.lactis菌株经过优化的体系，对Cas9蛋白编码序列、gRNA启动子、供体DNA同源臂长度及酵母感受态制备和转化等参数和条件进行了优化，实现了在K.lactis酵母菌株中的高效编辑。
1.3.3针对K.lactis基因组的K.lactis核酸酶基因EXN53,Rat1，Rrp6，Dis3，NGL3敲除的CRISPR/Cas9设计；
在本专利中，供体DNA两个同源臂分别为待敲除核酸酶基因的ORF上下游1000bp左右的基因序列，构建两个gRNA，同时识别待敲除核酸酶基因ORF的5′和3′端的PAM序列，引导Cas9核酸酶对DNA定点切割,出现DSB (double strand break)后发生同源重组，从而实现核酸酶基因敲除。例如EXN53,Rat1，Rrp6，Dis3,NGL3这5个基因的敲除是利用在起始密码子和终止密码子附近各设计一个gRNA进行敲除。K.lactis基因组中5种核酸酶基因改造的CRISPR系统设计如图3所示。
实施例2通过CRISPR/Cas9靶向敲除EXN53基因
2.1KLEXN53序列检索及CRISPR gRNA序列确定
2.1.1靶向敲除基因克隆载体质粒的构建：
在KEGG数据库中以EXN53基因进行BLAST比对分析，确定乳酸克鲁维酵母中EXN53基因序列(SEQ ID NO.1)，KEGG数据库编码为KlLA0F22385g，与S.cerevisiae酵母中EXN53基因序列同源性60.93％，蛋白序列同源性57.65％，与S.pombe酵母中EXN53基因序列同源性47.38％蛋白序列同源性38.29％，与H.sapiens酵母中EXN53基因序列同源性43.81％，蛋白序列同源性33.48％(图5)，含有特征性的EXN535'to 3'核酸外切酶domain，Amelogenin domain(图4)。该基因命名为KLEXN53(位于染色体F的2091235...2095596位)。
在KLEXN53基因起始密码子和终止密码子搜索PAM序列(NGG)，并确定gRNA序列。gRNA选择的原则为：GC含量适中，本发明的标准为GC含量为40％-60％；避免poly T结构的存在。最终，本发明确定的KLEXN53gRNA-1序列为AGAGTTCGACAATTTGTACT，KLEXN53gRNA-2序列为CGTCGTGGCCGTAGTAATCG。
质粒构建及转化方法如下：使用引物pCas9-KLEXN53-F1：AGAGTTCGACAATTTGTACTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTC
(SEQ ID NO.:7)和pCas9-KLEXN53-R1：GCTCTAAAACAGTACAAATTGTCGAACTCTAAAGTCCCATTCGCCACCCG(SEQ ID NO.:8)，pCas9-KLEXN53-F2：CGTCGTGGCCGTAGTAATCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTC(SEQ IDNO.:9)和pCas9-KLEXN53-R2：GCTCTAAAACCGATTACTACGGCCACGACGAAAGTCCCATTCGCCACCCG(SEQ ID NO.:10)，均以pCAS质粒为模板，进行PCR扩增。分别将扩增产物17μL混合，加入1μL Dpn I，2μL 10×digestion buffer，37℃温浴3h。将Dpn I处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Kan抗性LB固体培 养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，分别命名为pHoCas9_SE_Kana_tRNA_ScRNR2_KLEXN53-1和pHoCas9_SE_Kana_tRNA_ScRNR2_KLEXN53-2。以pHoCas9_SE_Kana_tRNA_ScRNR2_KLEXN53-1为模板，使用引物pCas9-F1：TAGGTCTAGAGATCTGTTTAGCTTGCCTCG(SEQ ID NO.:11)和pCas9-R1：TATCCACTAGACAGAAGTTTGCGTTCC(SEQ ID NO.:12)，以pHoCas9_SE_Kana_tRNA_ScRNR2_KLEXN533-2为模板，使用引物pCas9-F2：TATGGAACGCAAACTTCTGTCTAGTGGATAGTATATGTGTTATGTAGTATACTCTTTCTTCAACAATTAAATACTCTCGG(SEQ ID NO.:13)和pCas9-F2：CGAGGCAAGCTAAACAGATCTCTAGACCTATATCCACTAGACAGAAGTTTGCGTTCC(SEQ IDNO.:14)进行PCR扩增，pCas9-F1/pCas9-R1和pCas9-F2/pCas9-R2的PCR扩增产物按照1:5进行混合，加入1μL Dpn I，2μL10×digestion buffer，37℃温浴3h。将Dpn I处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Kan抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，分别命名为pHoCas9_SE_Kana_tRNA_ScRNR2_KLEXN53-1&2(如图10所示)。
2.2供体DNA质粒构建及扩增
为了便于线性供体DNA的保存及扩增，本发明首先将供体DNA插入到pMD18质粒中，然后通过PCR扩增得到线性供体DNA序列。
以乳酸克鲁维酵母基因组DNA为模板，以引物KLEXN53-F1：GTACCCGGGGATCCTCTAGAGATCCAGTTGCAGAGCCTCCGAA(SEQ ID NO.:15)和KLEXN53-R1：CATGCCTGCAGGTCGACGATGCGAAACCTTAGCTCTTTATCGAAC(SEQ ID NO.:16)进行PCR扩增；以pMD18质粒为模板，以引物pMD18-F：ATCGTCGACCTGCAGGCATG(SEQ ID NO.:17)和pMD18-R：ATCTCTAGAGGATCCCCGGG(SEQ ID NO.:18)进行PCR扩增。将两次扩增产物各8.5μL混合，加入1μL Dpn I，2μL 10×digestion buffer，37℃温浴3h。将Dpn I处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Amp抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培 养，PCR检测阳性并测序确认后，提取质粒保存，命名为KLEXN53-pMD18。
以KLEXN53-pMD18质粒为模板，以引物KLEXN53-F2：ATGTTGGTTTGAATGGACTATTAACAGTAAATATTATATCACTTC(SEQ ID NO.:19)
和KLEXN53-R2：GAAGTGATATAATATTTACTGTTAATAGTCCATTCAAACCAACATTTATTTTAGTTAAGCCACAAACCGTAATTAATTGAACAC(SEQ ID NO.:20)进行PCR扩增；构建KLEXN53-DD-pMD18(如图11所示)。具体步骤为：两种PCR产物各8.5μL混合，加入1μL Dpn I，2μL 10×digestion buffer，37℃温浴3h。将Dpn I处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Amp抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存。
2.3乳酸克鲁维酵母转化及阳性鉴定
2.3.1将乳酸克鲁维酵母菌液在YPD固体培养基上划线并挑取单克隆，于25mL 2×YPD液体培养基中振荡培养过夜，取2mL菌液于50mL液体2×YPD培养基中继续振荡培养2-8h。20℃条件下3000g离心5min收集酵母细胞，加入500μL无菌水重悬，同样条件下离心收集细胞。配制感受态细胞溶液(5％v/v甘油，10％v/v DMSO)并将酵母细胞溶解于500μL该溶液中。分装50μL至1.5mL离心管中，-80℃保存。
1.3.2将感受态细胞置于37℃融化15-30s，13000g离心2min并去除上清。配制转化缓冲液：PEG 3350(50％(w/v))260μL，LiAc(1.0M)36μL，carrier DNA(5.0mg/mL)20μL，Cas9/gRNA质粒5μL，供体DNA 10μL，加入无菌水至最终体积360μL。热激后，13000g离心30s去除上清。加入1mL YPD液体培养基，培养2-3h，吸取200μL涂布于固体YPD(200μg/mL G418)培养基，培养2-3天至单菌落出现。
1.3.3在乳酸克鲁维酵母转化后的平板上挑取10-20个单克隆，置于1mL YPD(200μg/mL G418)液体培养基中振荡培养过夜，以菌液为模板，以CRISPRInsertion Check引物KLEXN53-CICF1：TTTGCTGGTTGCCCGTATTCCC(SEQ IDNO.:21)，KLEXN53-CICR1：TAATAGCACAGGGAATGCACCTT(SEQ ID NO.:22)，对样品进行PCR检测。PCR结果阳性并经测序鉴定的菌株，确定为阳性菌株。
实施例3通过CRISPR/Cas9靶向敲除DIS3基因
3.1KlDIS3序列检索及CRISPR gRNA序列确定
3.1.1靶向敲除基因克隆载体质粒的构建：
在KEGG数据库中以DIS3基因进行BLAST比对分析，确定乳酸克鲁维酵母中DIS3基因序列(SEQ ID NO.2)，KEGG数据库编码为KlLA0A10835g，与S.cerevisiae酵母中DIS3基因序列同源性70.54％，蛋白序列同源性77.59％，与S.pombe酵母中DIS3基因序列同源性56.86％，蛋白序列同源性51.82％，与H.sapiens酵母中DIS3基因序列同源性48.83％，蛋白序列同源性40.19％(图6)，含有特征性的VacB domain，PIN_Rrp44domain(图4)。该基因命名为KlDIS3(位于染色体A的938712..941738位置)。
在KlDIS3基因起始密码子和终止密码子附近搜索PAM序列(NGG)，并确定gRNA序列。gRNA选择的原则为：GC含量适中，本发明的标准为GC含量为40％-60％；避免poly T结构的存在。最终，本发明确定的KlDIS3gRNA-1序列为TTCAGCAGCTAAGAAGGAAG(SEQ ID NO.:23)，KlDIS3gRNA-2序列为AGAGGTCAGTGTCTTTGATA(SEQ ID NO.:24)。
质粒构建及转化方法如下：使用引物pCas9-KlDIS3-F1：ATGGGACTTTTTCAGCAGCTAAGAAGGAAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCC(SEQ ID NO.:25)和pCas9-KlDIS3-R1：AGCTCTAAAACCTTCCTTCTTAGCTGCTGAAAAAGTCCCATTCGCCACCCG(SEQ ID NO.:26)，pCas9-KlDIS3-F2：ATGGGACTTTAGAGGTCAGTGTCTTTGATAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCC(SEQ ID NO.:27)和pCas9-KlDIS3-R2：AGCTCTAAAACTATCAAAGACACTGACCTCTAAAGTCCCATTCGCCACCCG(SEQ ID NO.:28)，均以pCAS质粒为模板，进行PCR扩增。分别将扩增产物17μL混合，加入1μL Dpn I，2μL10×digestion buffer，37℃温浴3h。将Dpn I处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Kan抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，分别命名为pHoCas9_SE_Kana_tRNA_ScRNR2_KlDIS3-1和pHoCas9_SE_Kana_tRNA_ScRNR2_KlDIS3-2。以 pHoCas9_SE_Kana_tRNA_ScRNR2_KlDIS3-1为模板，使用引物pCas9-F1：TAGGTCTAGAGATCTGTTTAGCTTGCCTCG(SEQ ID NO.:29)和pCas9-R1：TATCCACTAGACAGAAGTTTGCGTTCC(SEQ ID NO.:30)，以pHoCas9_SE_Kana_tRNA_ScRNR2_KlDIS3-2为模板，使用引物pCas9-F2：TATGGAACGCAAACTTCTGTCTAGTGGATAGTATATGTGTTATGTAGTATACTCTTTCTTCAACAATTAAATACTCTCGG(SEQ ID NO.:31)和pCas9-R2：CGAGGCAAGCTAAACAGATCTCTAGACCTATATCCACTAGACAGAAGTTTGCGTTCC(SEQ ID NO.:32)进行PCR扩增，pCas9-F1/pCas9-R1和pCas9-F2/pCas9-R2的PCR扩增产物按照1:5进行混合，加入1μL Dpn I，2μL10×digestion buffer，37℃温浴3h。将Dpn I处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Kan抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，分别命名为pHoCas9_SE_Kana_tRNA_ScRNR2_KlDIS-1&2(如图12所示)。
3.2供体DNA质粒构建及扩增
为了便于线性供体DNA的保存及扩增，本发明首先将供体DNA插入到pMD18质粒中，然后通过PCR扩增得到线性供体DNA序列。
以乳酸克鲁维酵母基因组DNA为模板，以引物KlDIS3-F1：CCCGGGGATCCTCTAGAGATGCTGCTAGGTGACAGAAGGTTGTCC(SEQ ID NO.:33)和KlDIS3-R1：CATGCCTGCAGGTCGACGATCCAAAGAAGAACGTCGTAAGACCGC(SEQ ID NO.:34)进行PCR扩增；以pMD18质粒为模板，以引物pMD18-F：ATCGTCGACCTGCAGGCATG(SEQ ID NO.:35)和pMD18-R：ATCTCTAGAGGATCCCCGGG(SEQ ID NO.:36)进行PCR扩增。将两次扩增产物各8.5μL混合，加入1μL Dpn I，2μL10×digestion buffer，37℃温浴3h。将Dpn I处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Amp抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，命名为KlDIS3-pMD18。
以KlDIS3-pMD18质粒为模板，以引物KlDIS3-F2：CTCTTCTGTTTAGCACCCGGTTATAGCTTAATTTATTAATTATGTACATTATATAAAAACTATTGTC(SEQ ID NO.:37)和KlDIS3-R2： AAGCTATAACCGGGTGCTAAACAGAAGAGTATGACGTTTTATACTTCTCCAG(SEQ ID NO.:38)进行PCR扩增；构建KlDIS3-DD-pMD18(如图13所示)。具体步骤为：两种PCR产物各8.5μL混合，加入1μL Dpn I，2μL10×digestion buffer，37℃温浴3h。将Dpn I处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Amp抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存。
3.3乳酸克鲁维酵母转化及阳性鉴定
3.3.1将乳酸克鲁维酵母菌液在YPD固体培养基上划线并挑取单克隆，于25mL 2×YPD液体培养基中振荡培养过夜，取2mL菌液于50mL液体2×YPD培养基中继续振荡培养2-8h。20℃条件下3000g离心5min收集酵母细胞，加入500μL无菌水重悬，同样条件下离心收集细胞。配制感受态细胞溶液(5％v/v甘油，10％v/v DMSO)并将酵母细胞溶解于500μL该溶液中。分装50μL至1.5mL离心管中，-80℃保存。
3.3.2将感受态细胞置于37℃融化15-30s，13000g离心2min并去除上清。配制转化缓冲液：PEG 3350(50％(w/v))260μL，LiAc(1.0M)36μL，carrier DNA(5.0mg/mL)20μL，Cas9/gRNA质粒15μL，供体DNA 10μL，加入无菌水至最终体积360μL。热激后，13000g离心30s去除上清。加入1mL YPD液体培养基，培养2-3h，吸取200μL涂布于固体YPD(200μg/mL G418)培养基，培养2-3天至单菌落出现。
3.3.3在乳酸克鲁维酵母转化后的平板上挑取10-20个单克隆，置于1mL YPD(200μg/mL G418)液体培养基中振荡培养过夜，以菌液为模板，以CRISPR Insertion Check引物KlDIS3-CICF1：CACCAACAACAGGAAATCTCATG(SEQ ID NO.:39)，KlDIS3-CICR1：CAGTACAGAA GCTCAGCAAC AACC(SEQ ID NO.:40)，对样品进行PCR检测。PCR结果阳性并经测序鉴定的菌株，确定为阳性菌株。
实施例4通过CRISPR/Cas9靶向敲除Rat1基因
4.1KlRat1序列检索及CRISPR gRNA序列确定
4.1.1靶向敲除基因克隆载体质粒的构建：
在KEGG数据库中以Rat1基因进行BLAST比对分析，确定乳酸克鲁维酵母中 Rat1基因序列(SEQ ID NO.3)，KEGG数据库编码为KlLA0A10835g，与S.cerevisiae酵母中Rat1基因序列同源性60.38％，蛋白序列同源性62.30％，与S.pombe酵母中Rat1基因序列同源性50.53％，蛋白序列同源性39.73％，与H.sapiens酵母中Rat1基因序列同源性50.15％，蛋白序列同源性41.41％(图7)，含有特征性的EXN535'to 3'核酸外切酶domain(图4)。该基因命名为KlRat1(位于染色体F:703955..706933位置)。
在Rat1基因起始密码子和终止密码子附近搜索PAM序列(NGG)，并确定gRNA序列。gRNA选择的原则为：GC含量适中，本发明的标准为GC含量为40％-60％；避免poly T结构的存在。最终，本发明确定的KlRat1gRNA-1序列为GTAAGGCCAGGTACTCACAA(SEQ ID NO.:41)，KlRat1gRNA-2序列为CTCGCAACAGAGACAGCCAC(SEQ ID NO.:42)。
质粒构建及转化方法如下：使用引物pCas9-KlRat1-F1：ATGGGACTTTGTAAGGCCAGGTACTCACAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCC(SEQ ID NO.:43)和pCas9-KlRat1-R1：AGCTCTAAAACTTGTGAGTACCTGGCCTTACAAAGTCCCATTCGCCACCCG(SEQ ID NO.:44)，pCas9-KlRat1-F2：ATGGGACTTTCTCGCAACAGAGACAGCCACGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCC(SEQ ID NO.:45)和pCas9-KlRat1-R2：AGCTCTAAAACGTGGCTGTCTCTGTTGCGAGAAAGTCCCATTCGCCACCCG(SEQ ID NO.:46)，均以pCAS质粒为模板，进行PCR扩增。分别将扩增产物17μL混合，加入1μL Dpn I，2μL10×digestion buffer，37℃温浴3h。将Dpn I处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Kan抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，分别命名为pHoCas9_SE_Kana_tRNA_ScRNR2_KlRat1-1和pHoCas9_SE_Kana_tRNA_ScRNR2_KlRat1-2。以pHoCas9_SE_Kana_tRNA_ScRNR2_KlRat1-1为模板，使用引物pCas9-F1：TAGGTCTAGAGATCTGTTTAGCTTGCCTCG(SEQ ID NO.:47)和pCas9-R1：TATCCACTAGACAGAAGTTTGCGTTCC(SEQ ID NO.:48)，以pHoCas9_SE_Kana_tRNA_ScRNR2_KlDIS3-2为模板，使用引物pCas9-F2： TATGGAACGCAAACTTCTGTCTAGTGGATAGTATATGTGTTATGTAGTATACTCTTTCTTCAACAATTAAATACTCTCGG(SEQ ID NO.:49)和pCas9-R2：CGAGGCAAGCTAAACAGATCTCTAGACCTATATCCACTAGACAGAAGTTTGCGTTCC(SEQ ID NO.:50)进行PCR扩增，pCas9-F1/pCas9-R1和pCas9-F2/pCas9-R2的PCR扩增产物按照1:5进行混合，加入1μL Dpn I，2μL10×digestion buffer，37℃温浴3h。将Dpn I处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Kan抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，分别命名为pHoCas9_SE_Kana_tRNA_ScRNR2_KlRat1-1&2(如图14所示)。
4.2供体DNA质粒构建及扩增
为了便于线性供体DNA的保存及扩增，本发明首先将供体DNA插入到pMD18质粒中，然后通过PCR扩增得到线性供体DNA序列。
以乳酸克鲁维酵母基因组DNA为模板，以引物KlRat1-F1：CCCGGGGATCCTCTAGAGATGCTGCATGGTCACAGGAGATGC(SEQ ID NO.:51)和KlRat1-R1：CATGCCTGCAGGTCGACGATGGTACGTGAGGCGACAATATGGTCC(SEQ ID NO.:52)进行PCR扩增；以pMD18质粒为模板，以引物pMD18-F：ATCGTCGACCTGCAGGCATG(SEQ ID NO.:53)和pMD18-R：ATCTCTAGAGGATCCCCGGG(SEQ ID NO.:54)进行PCR扩增。将两次扩增产物各8.5μL混合，加入1μL Dpn I，2μL10×digestion buffer，37℃温浴3h。将Dpn I处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Amp抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，命名为KlRat1-pMD18。
以KlRat1-pMD18质粒为模板，以引物KlRat1-F2：CCAGGTACTCACATGAACTGTGGACAATTTTATACCCGTTTATATCAGCAC(SEQ ID NO.:55)和KlRat1-R2：TGTCCACAGTTCATGTGAGTACCTGGCCTTACTTCTCGC(SEQ ID NO.:56)进行PCR扩增；构建KlRat1-DD-pMD18(如图15所示)。具体步骤为：两种PCR产物各8.5μL混合，加入1μL Dpn I，2μL10×digestion buffer，37℃温浴3h。将Dpn I处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培 养1h，涂布于Amp抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存。
4.3乳酸克鲁维酵母转化及阳性鉴定
4.3.1将乳酸克鲁维酵母菌液在YPD固体培养基上划线并挑取单克隆，于25mL 2×YPD液体培养基中振荡培养过夜，取2mL菌液于50mL液体2×YPD培养基中继续振荡培养2-8h。20℃条件下3000g离心5min收集酵母细胞，加入500μL无菌水重悬，同样条件下离心收集细胞。配制感受态细胞溶液(5％v/v甘油，10％v/v DMSO)并将酵母细胞溶解于500μL该溶液中。分装50μL至1.5mL离心管中，-80℃保存。
4.3.2将感受态细胞置于37℃融化15-30s，13000g离心2min并去除上清。配制转化缓冲液：PEG 3350(50％(w/v))260μL，LiAc(1.0M)36μL，carrier DNA(5.0mg/mL)20μL，Cas9/gRNA质粒15μL，供体DNA 10μL，加入无菌水至最终体积360μL。热激后，13000g离心30s去除上清。加入1mL YPD液体培养基，培养2-3h，吸取200μL涂布于固体YPD(200μg/mL G418)培养基，培养2-3天至单菌落出现。
4.3.3在乳酸克鲁维酵母转化后的平板上挑取10-20个单克隆，置于1mLYPD(200μg/mL G418)液体培养基中振荡培养过夜，以菌液为模板，以CRISPR Insertion Check引物KlRat1-CICF1：TAAGACAGGTACCCTCACGACG(SEQ ID NO.:57)，KlRat1-CICR1：CTTGGAAGTG GATACATTTCTAGAGG(SEQ ID NO.:58)，对样品进行PCR检测。PCR结果阳性并经测序鉴定的菌株，确定为阳性菌株。
实施例5通过CRISPR/Cas9靶向敲除Rrp6基因
5.1靶向敲除基因克隆载体质粒的构建：
5.1.1KlRrp6序列检索及CRISPR gRNA序列确定
在KEGG数据库中以Rrp6基因进行BLAST比对分析，确定乳酸克鲁维酵母中Rrp6基因序列(SEQ ID NO.4)，KEGG数据库编码为KlLA0D01309g，名称hypothetical protein，与S.cerevisiae酵母中Rrp6基因序列同源性55.04％，蛋白序列同源性46.93％，与S.pombe酵母中Rrp6基因序列同源性44.04％，蛋白序列同源性29.85％，与H.sapiens酵母中Rrp6基因序列同源性40.44％，蛋白序列同源性23.53％(图8)，含有特征性的Rrp6p_like核酸外切酶 domain，PMC2NT domain，Helicase and RNase D C-terminal domain(图4)。该基因命名为KlRrp6(位于染色体D的114713..116947位置)。
在Rrp6基因起始密码子和终止密码子附近搜索PAM序列(NGG)，并确定gRNA序列。gRNA选择的原则为：GC含量适中，本发明的标准为GC含量为40％-60％；避免poly T结构的存在。最终，本发明确定的KlRrp6gRNA-1序列为CACCATGTCTTCAGAGGATA，KlRrp6g RNA-2序列为CCGACATGTTCAACAGAGTA。
质粒构建及转化方法如下：使用引物pCas9-KlRrp6-F1：ATGGGACTTTCACCATGTCTTCAGAGGATAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCC(SEQ ID NO.:59)和pCas9-KlRrp6-R1：AGCTCTAAAACTATCCTCTGAAGACATGGTGAAAGTCCCATTCGCCACCCG(SEQ ID NO.:60)，pCas9-KlRrp6-F2：ATGGGACTTTCCGACATGTTCAACAGAGTAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCC(SEQ ID NO.:61)和pCas9-Kl Rrp6-R2：AGCTCTAAAACTACTCTGTTGAACATGTCGGAAAGTCCCATTCGCCACCCG(SEQ ID NO.:62)，均以pCAS质粒为模板，进行PCR扩增。分别将扩增产物17μL混合，加入1μL Dpn I，2μL10×digestion buffer，37℃温浴3h。将Dpn I处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Kan抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，分别命名为pHoCas9_SE_Kana_tRNA_ScRNR2_KlRrp6-1和pHoCas9_SE_Kana_tRNA_ScRNR2_KlRrp6-2。以pHoCas9_SE_Kana_tRNA_ScRNR2_KlRrp6-1为模板，使用引物pCas9-F1：TAGGTCTAGAGATCTGTTTAGCTTGCCTCG(SEQ ID NO.:63)和pCas9-R1：TATCCACTAGACAGAAGTTTGCGTTCC(SEQ ID NO.:64)，以pHoCas9_SE_Kana_tRNA_ScRNR2_KlRrp6-2为模板，使用引物pCas9-F2：TATGGAACGCAAACTTCTGTCTAGTGGATAGTATATGTGTTATGTAGTATACTCTTTCTTCAACAATTAAATACTCTCGG(SEQ ID NO.:65)和pCas9-R2：CGAGGCAAGCTAAACAGATCTCTAGACCTATATCCACTAGACAGAAGTTTGCGTTCC(SEQ ID NO.:66)进行PCR扩增，pCas9-F1/pCas9-R1和pCas9-F2/pCas9-R2的PCR扩增产物按照1:5进行混合，加入1μL Dpn I，2μL10×digestion buffer， 37℃温浴3h。将Dpn I处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Kan抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，分别命名为pHoCas9_SE_Kana_tRNA_ScRNR2_KlRrp6-1&2(如图16所示)。
5.2供体DNA质粒构建及扩增
为了便于线性供体DNA的保存及扩增，本发明首先将供体DNA插入到pMD18质粒中，然后通过PCR扩增得到线性供体DNA序列。
以乳酸克鲁维酵母基因组DNA为模板，以引物KlRrp6-F1：CCCGGGGATCCTCTAGAGATGCGATAGCTTTAATCTGAGTGAACACCG(SEQ ID NO.:67)和KlRrp6-R1：CATGCCTGCAGGTCGACGATGGGTACTCGTTGATAACATGATGCGTAG(SEQ ID NO.:68)进行PCR扩增；以pMD18质粒为模板，以引物pMD18-F：ATCGTCGACCTGCAGGCATG(SEQ ID NO.:69)和pMD18-R：ATCTCTAGAGGATCCCCGGG(SEQ ID NO.:70)进行PCR扩增。将两次扩增产物各8.5μL混合，加入1μL Dpn I，2μL 10×digestion buffer，37℃温浴3h。将Dpn I处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Amp抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，命名为KlRrp6-pMD18。
以KlRrp6-pMD18质粒为模板，以引物KlRrp6-F2：CTGACTCTAATCCACCAGCATCTTGAGCAGCTCTAATGGTATAAATATCG(SEQ ID NO.:71)和KlRrp6-R2：GCTCAAGATGCTGGTGGATTAGAGTCAGCTGGTAGTCTAC(SEQ ID NO.:72)进行PCR扩增；构建KlRrp6-DD-pMD18(如图17所示)。具体步骤为：两种PCR产物各8.5μL混合，加入1μL Dpn I，2μL 10×digestion buffer，37℃温浴3h。将Dpn I处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Amp抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存。
5.3乳酸克鲁维酵母转化及阳性鉴定
5.3.1将乳酸克鲁维酵母菌液在YPD固体培养基上划线并挑取单克隆，于 25mL 2×YPD液体培养基中振荡培养过夜，取2mL菌液于50mL液体2×YPD培养基中继续振荡培养2-8h。20℃条件下3000g离心5min收集酵母细胞，加入500μL无菌水重悬，同样条件下离心收集细胞。配制感受态细胞溶液(5％v/v甘油，10％v/v DMSO)并将酵母细胞溶解于500μL该溶液中。分装50μL至1.5mL离心管中，-80℃保存。
5.3.2将感受态细胞置于37℃融化15-30s，13000g离心2min并去除上清。配制转化缓冲液：PEG 3350(50％(w/v))260μL，LiAc(1.0M)36μL，carrier DNA(5.0mg/mL)20μL，Cas9/gRNA质粒15μL，供体DNA 10μL，加入无菌水至最终体积360μL。热激后，13000g离心30s去除上清。加入1mL YPD液体培养基，培养2-3h，吸取200μL涂布于固体YPD(200μg/mL G418)培养基，培养2-3天至单菌落出现。
5.3.3在乳酸克鲁维酵母转化后的平板上挑取10-20个单克隆，置于1mL YPD(200μg/mL G418)液体培养基中振荡培养过夜，以菌液为模板，以CRISPR Insertion Check引物KlRrp6-CICF1：TGGAGGCGTACAATGCAGTG(SEQ ID NO.:73)，KlRrp6-CICR1：TGAACCCTCT TCCATGTCTC ATC(SEQ ID NO.:74)，对样品进行PCR检测。PCR结果阳性并经测序鉴定的菌株，确定为阳性菌株。
实施例6通过CRISPR/Cas9靶向敲除NGL2基因
6.1KlNGL2序列检索及CRISPR gRNA序列确定
6.1.1靶向敲除基因克隆载体质粒的构建：
在KEGG数据库中以NGL3基因进行BLAST比对分析，确定乳酸克鲁维酵母中NGL基因序列(SEQ ID NO.5)，KEGG数据库编码为KlLA0C06248g，与S.cerevisiae酵母中NGL3基因序列同源性46.26％，蛋白序列同源性46.34％，与S.pombe酵母中NGL2基因序列同源性45.42％，蛋白序列同源性29.07％，与H.sapiens酵母中NGL3基因序列同源性38.72％，蛋白序列同源性19.89％(图9)，含有特征性的Exonuclease-Endonuclease-Phosphatase(EEP)domain(图4)。该基因命名为KlNGL2(位于染色体C的552493…554043位置)。
在KlNGL2基因起始密码子和终止密码子附近搜索PAM序列(NGG)，并确定gRNA序列。gRNA选择的原则为：GC含量适中，本发明的标准为GC含量为40％-60％；避免poly T结构的存在。最终，本发明确定的KlNGL2gRNA-1序列为GCTGGTAGTACGCAAGACAC(SEQ ID NO.:75)，KlNGL2gRNA-2序列为 TTGTGCATGATTGTTAAACT(SEQ ID NO.:76)。
质粒构建及转化方法如下：使用引物pCas9-KlNGL2-F1：TATCCAGACACCAAAGTCAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTC(SEQ ID NO.:77)和pCas9-Kl NGL3-R1：GCTCTAAAACCTGACTTTGGTGTCTGGATAAAAGTCCCATTCGCCACCCG(SEQ ID NO.:78)，pCas9-KlNGL2-F2：TTGTGCATGATTGTTAAACTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGT(SEQ ID NO.:79)和pCas9-Kl NGL2-R2CGGGTGGCGAATGGGACTTTTTGTGCATGATTGTTAAACTGTTTTAGAGC(SEQ ID NO.:80)：以pCAS质粒为模板，进行PCR扩增。分别将扩增产物17μL混合，加入1μL Dpn I，2μL10×digestion buffer，37℃温浴3h。将Dpn I处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Kan抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，分别命名为pHoCas9_SE_Kana_tRNA_ScRNR2_KlNGL2-1和pHoCas9_SE_Kana_tRNA_ScRNR2_KlNGL3-2。以pHoCas9_SE_Kana_tRNA_ScRNR2_KlNGL2-1为模板，使用引物pCas9-F1：TAGGTCTAGAGATCTGTTTAGCTTGCCTCG(SEQ ID NO.:81)和pCas9-R1：TATCCACTAGACAGAAGTTTGCGTTCC(SEQ ID NO.:82)，以pHoCas9_SE_Kana_tRNA_ScRNR2_KlNGL2-2为模板，使用引物pCas9-F2：TATGGAACGCAAACTTCTGTCTAGTGGATAGTATATGTGTTATGTAGTATACTCTTTCTTCAACAATTAAATACTCTCGG(SEQ ID NO.:83)和pCas9-R2：CGAGGCAAGCTAAACAGATCTCTAGACCTATATCCACTAGACAGAAGTTTGCGTTCC(SEQ ID NO.:84)进行PCR扩增，pCas9-F1/pCas9-R1和pCas9-F2/pCas9-R2的PCR扩增产物按照1:5进行混合，加入1μL Dpn I，2μL10×digestion buffer，37℃温浴3h。将Dpn I处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Kan抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，分别命名为pHoCas9_SE_Kana_tRNA_ScRNR2_KlNGL3-1&2(如图18所示)。
6.2供体DNA质粒构建及扩增
为了便于线性供体DNA的保存及扩增，本发明首先将供体DNA插入到pMD18质粒中，然后通过PCR扩增得到线性供体DNA序列。
以乳酸克鲁维酵母基因组DNA为模板，以引物KlNGL2-F1：GAGCTCGGTACCCGGGGATCCTCTAGAGATCGAATACGTGAAACAGCCTAGGAA(SEQ ID NO.:85)和KlNGL2-R1：GCCAAGCTTGCATGCCTGCAGGTCGACGATCACGGCCCTAGTACTAATCCCAT(SEQ ID NO.:86)进行PCR扩增；以pMD18质粒为模板，以引物pMD18-F：ATCGTCGACCTGCAGGCATG(SEQ ID NO.:87)和pMD18-R：ATCTCTAGAGGATCCCCGGG(SEQ ID NO.:88)进行PCR扩增。将两次扩增产物各8.5μL混合，加入1μL Dpn I，2μL 10×digestion buffer，37℃温浴3h。将Dpn I处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Amp抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，命名为KlNGL2-pMD18。
以KlNGL3-pMD18质粒为模板，以引物KlNGL2-F2：GAAGTAATAATTTGAGCCAATATATTCATAAACTGTTTAACTATGGACTACACTACAG(SEQ ID NO.:89)和KlNGL2-R2：CCATAGTTAAACAGTTTATGAATATATTGGCTCAAATTATTACTTCTACTTTGCAGTG(SEQ ID NO.:90)进行PCR扩增；构建KlNGL2-DD-pMD18(如图19所示)。具体步骤为：两种PCR产物各8.5μL混合，加入1μL Dpn I，2μL10×digestion buffer，37℃温浴3h。将Dpn I处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Amp抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存。
6.3乳酸克鲁维酵母转化及阳性鉴定
6.3.1将乳酸克鲁维酵母菌液在YPD固体培养基上划线并挑取单克隆，于25mL 2×YPD液体培养基中振荡培养过夜，取2mL菌液于50mL液体2×YPD培养基中继续振荡培养2-8h。20℃条件下3000g离心5min收集酵母细胞，加入500μL无菌水重悬，同样条件下离心收集细胞。配制感受态细胞溶液(5％ v/v甘油，10％v/v DMSO)并将酵母细胞溶解于500μL该溶液中。分装50μL至1.5mL离心管中，-80℃保存。
6.3.2将感受态细胞置于37℃融化15-30s，13000g离心2min并去除上清。配制转化缓冲液：PEG 3350(50％(w/v))260μL，LiAc(1.0M)36μL，carrier DNA(5.0mg/mL)20μL，Cas9/gRNA质粒15μL，供体DNA 10μL，加入无菌水至最终体积360μL。热激后，13000g离心30s去除上清。加入1mL YPD液体培养基，培养2-3h，吸取200μL涂布于固体YPD(200μg/mL G418)培养基，培养2-3天至单菌落出现。
6.3.3在乳酸克鲁维酵母转化后的平板上挑取10-20个单克隆，置于1mL YPD(200μg/mL G418)液体培养基中振荡培养过夜，以菌液为模板，以CRISPR Insertion Check引物KlNGL2-CICF1：ATATTGTCTAGCAGCTCATCGCGTA(SEQ ID NO.:91)，KlNGL2-CICR1：AAGCAGATTTATGCACGAATTGCC(SEQ ID NO.:92)，对样品进行PCR检测。PCR结果阳性并经测序鉴定的菌株，确定为阳性菌株。
实施例7无细胞体外蛋白质合成体系的制备和蛋白合成效率的测定
实验方法：
i.体外蛋白质合成体系的储存液配制：1M pH为7.4的4-羟乙基哌嗪乙磺酸，5M醋酸钾，250mM醋酸镁，25mM 4四种核苷三磷酸的混合物，包括腺嘌呤核苷三磷酸、鸟嘌呤核苷三磷酸、胞嘧啶核苷三磷酸和尿嘧啶核苷三磷酸，1mM 2二十种氨基酸的混合物：甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸和组氨酸，20种氨基酸的浓度均为1.0mM，1M磷酸肌酸，1M二硫苏糖醇，6.48mg/mL磷酸肌酸激酶，1.7mg/mL T7RNA聚合酶20％-50％聚乙二醇(polyethylene glycol,PEG)3350或者(polyethylene glycol,PEG)8000,20％-40％蔗糖；
ii.体外蛋白质合成反应体系：终浓度为22mM pH为7.4的4-羟乙基哌嗪乙磺酸，30-150mM醋酸钾，1.0-5.0mM醋酸镁，1.5mM-4mM核苷三磷酸混合物(腺嘌呤核苷三磷酸、鸟嘌呤核苷三磷酸、胞嘧啶核苷三磷酸和尿嘧啶核苷三磷酸)，0.08-0.24mM的氨基酸混合物(甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸和 组氨酸)，25mM磷酸肌酸，1.7mM二硫苏糖醇，0.27mg/mL磷酸肌酸激酶，8-20ng/μL萤火虫荧光素酶DNA，0.027-0.054mg/mL T7RNA聚合酶，1％-4％的聚乙二醇，最后加入50％体积的酵母细胞提取物；
iii.体外蛋白质合成反应：将上述的反应体系放置在20-30℃的环境中，静置反应2-6h；
iv.萤光素酶活性测定：反应结束后，在96孔白板或384孔白板中加入等体积的底物荧光素(luciferine)，立即放置于Envision 2120多功能酶标仪(Perkin Elmer),读数，检测萤火虫荧光素酶活性，相对光单位值(RLU)作为活性单位，如图20所示。
实验结果：
本发明实施例7的结果表明：在所有核酸酶敲除突变株中，Δklexn53菌株能够显著增强酵母体外蛋白质合成体系产生蛋白质的效率(表2)，从图20中也可以看出野生型在IVTT中荧光素酶活性数值为：2.90×10 8，Δklexn53酵母菌株在IVTT中荧光素酶活性数值为：7.16×10 8，其活性是野生型的2.46倍。
表2
Figure PCTCN2017115966-appb-000005
参考文献：
1.Ayyar,B.V.,S.Arora,and R.O'Kennedy,Coming-of-Age of Antibodies in Cancer Therapeutics.Trends Pharmacol Sci,2016.37(12): p.1009-1028.
2.Scott,A.M.,J.D.Wolchok,and L.J.,Antibody therapy of cancer.Nature Reviews Cancer,2012.12(4):p.278.
3.Sonenberg,N.and A.G.Hinnebusch,ReguLation of translation initiation in eukaryotes:mechanisms and biological targets.Cell,2009.136(4):p.731-45.
4.M.,D.J.R.G.,Nucleic Acid.Encyclopedia of Cell Biology,Elsevier,2015.
5.Chong,S.,Overview of Cell-Free Protein Synthesis:Historic Landmarks,Commercial Systems,and Expanding Applications.2014:John Wiley&Sons,Inc.16.30.1-16.30.11.
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种体外的无细胞的蛋白合成体系，其特征在于，所述无细胞的蛋白合成体系包括：
    (a)酵母细胞提取物；
    (b)聚乙二醇；
    (c)任选的外源蔗糖；和
    (d)任选的溶剂，所述溶剂为水或水性溶剂，
    其中，所述酵母细胞提取物中的EXN53蛋白的含量≤10％，较佳地，≤5％，更佳地，≤2％。
  2. 如权利要求1所述的蛋白合成体系，其特征在于，所述EXN53来源于选自下组的一种或多种来源的酵母：毕氏酵母、克鲁维酵母，较佳地，来源于克鲁维酵母。
  3. 如权利要求1所述的蛋白合成体系，其特征在于，所述EXN53的核苷酸序列如SEQ ID NO.:1所示。
  4. 如权利要求1所述的蛋白合成体系，其特征在于，所述EXN53的蛋白序列如SEQ ID NO.:6所示。
  5. 如权利要求1所述的蛋白合成体系，其特征在于，所述酵母细胞提取物中的EXN53的含量为0。
  6. 如权利要求1所述的蛋白合成体系，其特征在于，所述无细胞的蛋白合成体系还包括选自下组的一种或多种组分：
    (e1)用于合成RNA的底物；
    (e2)用于合成蛋白的底物；
    (e3)镁离子；
    (e4)钾离子；
    (e5)缓冲剂；
    (e6)RNA聚合酶；
    (e7)能量再生系统。
  7. 一种酵母细胞提取物，其特征在于，所述酵母细胞提取物中的EXN53 蛋白的含量≤10％，较佳地，≤5％，更佳地，≤2％。
  8. 一种权利要求1所述的体外的无细胞的蛋白合成体系的生产方法，其特征在于，包括步骤：
    将(a)酵母细胞提取物与(b)聚乙二醇；(c)任选的外源蔗糖；和(d)任选的溶剂混合，从而获得权利要求1所述的体外的无细胞的蛋白合成体系，其中，所述溶剂为水或水性溶剂，所述酵母细胞提取物中的EXN53蛋白的含量≤10％，较佳地，≤5％，更佳地，≤2％。
  9. 一种体外合成蛋白的方法，其特征在于，包括步骤：
    (i)提供权利要求1所述的体外的无细胞的蛋白合成体系，并加入外源的用于指导蛋白质合成的DNA分子，其中；所述蛋白合成体系中的EXN53蛋白的含量≤10％，较佳地，≤5％，更佳地，≤2％；
    (ii)在适合的条件下，孵育步骤(i)的蛋白合成体系一段时间T1，从而合成由所述外源DNA编码的蛋白质。
  10. 如权利要求9所述的方法，其特征在于，所述的方法还包括：(iii)任选地从所述蛋白合成体系中，分离或检测所述的由外源DNA编码的蛋白质。
  11. 如权利要求9所述的方法，其特征在于，所述外源DNA编码的蛋白质选自下组：荧光素蛋白、或荧光素酶、绿色荧光蛋白、黄色荧光蛋白、氨酰tRNA合成酶、甘油醛-3-磷酸脱氢酶、过氧化氢酶、肌动蛋白、抗体的可变区域、萤光素酶突变、α-淀粉酶、肠道菌素A、丙型肝炎病毒E2糖蛋白、胰岛素前体、干扰素αA、白细胞介素-1β、溶菌酶素、血清白蛋白、单链抗体段(scFV)、甲状腺素运载蛋白、酪氨酸酶、木聚糖酶、或其组合。
  12. 一种工程菌株，其特征在于，所述菌株为克鲁维酵母菌株，并且所述菌株中的EXN53基因(核酸酶基因)的表达或活性被降低。
  13. 如权利要求12所述的工程菌株，其特征在于，所述“降低”指EXN53基因的表达量≤10％，较佳地，≤5％，更佳地，≤2％。
  14. 如权利要求12所述的工程菌株，其特征在于，所述“降低”是指将EXN53基因的表达或活性降低满足以下条件：
    A1/A0的比值≤30％，较佳地≤10％，更佳地≤5％，更佳地，≤2％，最佳地为0-2％；
    其中，A1为EXN53基因的表达或活性；A0为野生型EXN53基因的表达或活 性。
  15. 一种权利要求12所述的工程菌株的用途，其特征在于，用于提高体外蛋白合成效率。
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