Genetic engineering strain for improving in-vitro cell-free protein synthesis activity and application thereof in D2P

Abstract

The invention provides a genetic engineering strain for improving in-vitro cell-free protein synthesis activity and application of the genetic engineering strain in D2P, specifically, protease encoding protein in the strain is modified to obtain the genetic engineering strain, expression or activity of protease genes in the genetic engineering strain is reduced, and the expression or activity of the protease genes in the genetic engineering strain is reduced. Degradation of target protein can be effectively reduced, IVTT activity of a bacterial strain lysate is improved, and D2P production capacity is remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a genetically engineered strain for improving in vitro cell-free protein synthesis activity, an application thereof in D2P, a preparation method thereof, and an application thereof in D2P. Background Art

[0002] Cell-free protein synthesis (CFPS) is a technology that uses enzymes, amino acid substrates and energy to synthesize proteins in vitro using exogenous mRNA or DNA as templates. Compared with traditional in vivo recombinant expression systems, in vitro cell-free synthesis systems have many advantages, such as the ability to directly use PCR products as templates to simultaneously synthesize multiple proteins in parallel, and to carry out high-throughput peptide or protein drug screening. In addition, in vitro cell-free synthesis systems also have unique advantages in the expression of peptides or proteins that are toxic to cells. Cell-free protein expression systems are an important supplement to cell-based protein expression systems.

[0003] Existing cell-free protein synthesis systems can be mainly divided into two categories: one is to combine and compound the ribosomes, enzymes, tRNA, etc. required for the synthesis of purified proteins in a certain proportion to obtain the ability to express proteins in vitro, that is, the purified cell-free protein expression system (PURE). This system has clear component information and can easily and customizedly adjust the formula to obtain different protein expression characteristics, but the disadvantage is that the preparation process is complicated and costly; the other is to obtain the main active substances from the extracts of living cells with protein expression activity, and appropriately add the required enzymes, substrates, energy substances and other components to obtain the ability to express proteins in vitro, that is, the cell-free protein expression system based on cell extracts. Compared with the PURE system, the latter has the advantages of simple operation process, short cycle and low cost, but the latter also has the disadvantages of complex components and many uncontrollable factors in the system, so it has higher requirements for the characteristics of the source cells of the cell extract. However, there is still huge room for optimization of the supporting technical processes and background strains of the system. Summary of the invention

[0004] The purpose of the present invention is to improve the protein expression level of a cell-free protein expression (DNA to Protein, D2P) system based on Kluyveromyces lactis cell extract through genetic modification.

[0005] The first aspect of the present invention provides a genetically engineered strain for in vitro cell-free protein synthesis, characterized in that the endogenous protease gene of the genetically engineered strain is modified, and the expression or activity of the protease gene in the genetically engineered strain is reduced;

[0006] Preferably, the protease gene is selected from one or more of APE1, APE3, APE4, ATG4, CPS1-1, CPS1-2, CPS1-3, CPS1-4, PEP4, PRB1, and PRC1.

[0007] The genetically engineered strain is a yeast derived from one or more sources selected from the group consisting of: Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces; preferably Kluyveromyces, more preferably Kluyveromyces lactis. In some preferred examples, the Kluyveromyces is one or more types of genetically engineered Kluyveromyces lactis (K. lactis), and the genetic engineering modification is used to improve its in vitro protein expression activity, such that the genetic engineering modification is selected from: EXN53 gene (nuclease gene) knockout, construction of an efficient exogenous gene expression cassette, or K. lactis Y1140 strain.

[0008] The protease gene is derived from Saccharomyces cerevisiae, Pichia pastoris and / or Kluyveromyces.

[0009] The APE1 encoding gene is shown in SEQ ID NO: 1;

[0010] The APE3 encoding gene is shown in SEQ ID NO: 2;

[0011] The coding gene of APE4 is shown in SEQ ID NO: 3;

[0012] The coding gene of ATG4 is shown in SEQ ID NO:4;

[0013] The coding gene of CPS1-1 is shown in SEQ ID NO:5;

[0014] The coding gene of CPS1-2 is shown in SEQ ID NO:6;

[0015] The coding gene of CPS1-3 is shown in SEQ ID NO:7;

[0016] The coding gene of CPS1-4 is shown in SEQ ID NO:8;

[0017] The coding gene of PEP4 is shown in SEQ ID NO:9;

[0018] The coding gene of PRB1 is shown in SEQ ID NO: 10;

[0019] The coding gene of PRC1 is shown in SEQ ID NO:11.

[0020] In a preferred embodiment, the protease gene is selected from one or more of ape4, ape3, atg4, cps1-4, atg4.

[0021] In the present invention, the term "reduction" refers to reducing the expression or activity of the protease gene to meet the following conditions:

[0022] The ratio of B1 / B0 is ≤30%; wherein B1 is the expression or activity of the protease gene in the genetically engineered strain after transformation; B0 is the expression or activity of the protease gene before transformation; preferably, the "reduction" refers to reducing the expression or activity of the protease gene to meet the following conditions: the ratio of B1 / B0 is ≤10%; more preferably, the "reduction" refers to reducing the expression or activity of the protease gene to meet the following conditions: the ratio of B1 / B0 is ≤5%; more preferably, the "reduction" refers to reducing the expression or activity of the protease gene to meet the following conditions: the ratio of B1 / B0 is ≤2%; more preferably, the "reduction" refers to reducing the expression or activity of the protease gene to meet the following conditions: the ratio of B1 / B0 is selected from 0-2%.

[0023] In another preferred embodiment, the expression or activity of the protease gene in the strain is reduced by a method selected from the following group: gene mutation, gene knockout, gene interruption, RNA interference technology, Crispr technology, or a combination thereof.

[0024] In a second aspect of the present invention, the present invention provides a method for preparing the genetically engineered strain for in vitro cell-free protein synthesis according to the first aspect of the present invention, comprising the following steps:

[0025] (1)1. Design of gRNA targeting sequence

[0026] Select the ORF region of the targeted protease gene and use CRISPOR (tefor.net)

[0027] gRNA was designed on the website (http: / / crispor.tefor.net / ), and gRNA with higher scores was selected as the candidate sequence. The corresponding primer pairs were synthesized according to the candidate gRNA targeting sequence;

[0028] (2) Construction of gRNA_Cas9 expression vector

[0029] The primer pairs obtained in step (1) were diluted separately and added to PCR tubes for mixing, and then annealing was completed on a PCR instrument. The pCasMFR vector treated with BsaI and purified was mixed in a T4 ligase system, and the annealed primers were added for ligation reaction; all the ligation products were transformed into Escherichia coli DH5α competent cells, and screened on an LB plate containing kanamycin, and the clones with correct sequencing were selected to extract the gRNA_Cas9 co-expression plasmid and determine the concentration before use in subsequent electroporation experiments;

[0030] (3) Construction of double-stranded DNA donor

[0031] The DNA donor sequence was obtained by Overlap-PCR method. Four primers P1, P2, P3 and P4 were selected from the ORF of the selected protease gene, p1+p2 and p3+p4 were used as forward and reverse primer pairs, respectively, and the genomic DNA of K.lactis Y1140 was used as a template. Phanta Super Fidelity DNA polymerase was used to perform PCR amplification to obtain PCR products H1 and H2; and p1+p4 was used as a template for the second round of PCR, and the double-stranded DNA donor product was obtained. The product was dissolved in D2O and frozen below -20°C for storage for later use after precipitation with 70% ethanol;

[0032] (4) Transformation and screening of yeast strains

[0033] The yeast strain was transformed by electroporation. Before electroporation, 40 μL of the competent cell mixture was fully mixed with the gRNA_Cas9 expression plasmid prepared in step (2) and the DNA Donor prepared in step (3). The electroporation process was completed according to the standard operation procedure. The yeast cells after electroporation were screened on a YPD plate containing 250 μg / mL G418.

[0034] Pick a single colony for PCR verification, select paired primers for verification, and sequence the positive PCR product; streak the single colony with correct sequencing on a plate, and pick a single colony for a second round of PCR verification. Select the strains that have been verified correctly in two rounds to prepare glycerol cryopreservation tubes and store them in a -80℃ refrigerator.

[0035] Furthermore, in step (1), the length of the ORF region is preferably 100 bp-2000 bp;

[0036] Preferably, the gRNA sequence comprises any one of SEQ ID NO: 12 to SEQ ID NO: 33;

[0037] Preferably, the method for synthesizing the corresponding primer pair is: for each gRNA, a 4-base sequence of AATC and AAAC is added to the 5'-end of the forward sequence and the reverse complementary sequence thereof, respectively, to obtain a 24-base primer pair.

[0038] Further, in step (2), preferably, the primer pairs are diluted to a concentration of 1-10 μM;

[0039] Preferably, the annealing process is completed on the PCR instrument according to the following program: 95°C, 3 min; 72°C, 2 min; 65°C, 2 min; 60°C, 2 min; 55°C, 2 min; 50°C, 2 min; 16°C, 2 min.

[0040] Preferably, the temperature of the ligation reaction is 16°C and the time is 1h;

[0041] Preferably, in the ligation reaction, the ratio of the pCasMFR vector treated with BsaI and purified and the annealed primer added is 1-100 ng: 0.1-1 μL.

[0042] Further, in step (3), the primer P1 is selected from the sequence shown in any one of SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:62, SEQ ID NO:66, SEQ ID NO:70, and SEQ ID NO:74; the primer P2 is selected from the sequence shown in any one of SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:43, SEQ ID NO:47, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:59, SEQ ID NO:63, SEQ ID NO:67, SEQ ID NO:71, and SEQ ID NO:75; the primer P3 is selected from the sequence shown in any one of SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:62, SEQ ID NO:66, SEQ ID NO:70, and SEQ ID NO:74. The primer P4 is selected from the sequence shown in any one of SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:63, SEQ ID NO:67, SEQID NO:73, and SEQ ID NO:77.

[0043] Further, in step (4), the yeast strain is a yeast derived from one or more sources selected from the group consisting of: Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces; preferably Kluyveromyces, more preferably Kluyveromyces lactis (Kluyveromy ceslactis, K. lactis);

[0044] In some feasible preferred embodiments, the lactic acid Kluyveromyces strain is a genetically engineered strain in which the expression or activity of the KlEXN53 gene (nuclease gene) is reduced (the specific preparation method is prepared by the method disclosed in CN108949801A), a genetically engineered strain in which the genome is integrated with a first exogenous gene expression cassette of a first nucleic acid construct expressing a fusion protein (such as a fusion protein formed by KlPAB1 and KleIF4G) (the specific preparation method is prepared by the method disclosed in CN111778169A and CN110938649A), and a genetically engineered strain in which the genome is integrated with a first exogenous gene expression cassette of a first nucleic acid construct expressing a fusion protein (such as a fusion protein formed by KlPAB1 and KleIF4G) and the expression or activity of a nuclease (such as KlEXN53) is reduced (the specific preparation method is prepared according to the method disclosed in CN110093284A or CN111778169A). Unless they conflict with the purpose of the present invention, patents CN108949801A, CN111778169A, CN110938649A and CN110093284A and their references are cited in their entirety and for all purposes.

[0045] Preferably, the mixing ratio of the competent cell mixture, gRNA_Cas9 expression plasmid and DNA Donor is 10-100 μL: 300-600 ng: 1-2 μg;

[0046] Preferably, the operation flow of the electroconversion process is:

[0047] Prepare transformation buffer: PEG 3350 (50% (w / v)) 260 μL, LiAc (1.0 M) 36 μL, carrier DNA (5.0 mg / mL) 20 μL, gRNA_Cas9 expression plasmid 5 μL, DNA Donor 10 μL, add sterile water to a final volume of 360 μL. After heat shock, centrifuge at 13000 g for 30 seconds to remove the supernatant. Add 1 mL of YPD liquid medium, culture for 2-3 hours, draw 200 μL and apply it to solid YPD (200 μg / mL G418) medium, and culture for 2-3 days until single colonies appear.

[0048] The third aspect of the present invention provides the use of the genetically engineered strain described in the first aspect or the second aspect, characterized in that it is used to improve the efficiency of in vitro protein synthesis.

[0049] The fourth aspect of the present invention provides an in vitro cell-free protein synthesis system, characterized in that the in vitro cell-free protein synthesis system comprises a lysate or a lysate extract prepared from the strain provided by the first aspect or the second aspect.

[0050] Preferably, the in vitro cell-free protein synthesis system further comprises one or more components selected from the following group:

[0051] (b) polyethylene glycol;

[0052] (c) optionally exogenous sucrose;

[0053] and (d) optionally a solvent, said solvent being water or an aqueous solvent;

[0054] Further preferably, the in vitro cell-free protein synthesis system further comprises one or more components selected from the following group:

[0055] (e1) substrate for RNA synthesis;

[0056] (e2) substrates for protein synthesis;

[0057] (e3) magnesium ion;

[0058] (e4) potassium ion;

[0059] (e5) buffer;

[0060] (e6) RNA polymerase;

[0061] and (e7) an energy regeneration system; more preferably, the energy regeneration system is a creatine phosphate / creatine phosphate enzyme system;

[0062] Further preferably, the in vitro cell-free protein synthesis system further comprises one or more components selected from the following group:

[0063] (e8) heme; and (e9) spermidine.

[0064] The fifth aspect of the present invention provides a method for synthesizing proteins in vitro, characterized in that it comprises the steps of: (i) providing the in vitro cell-free protein synthesis system described in the fourth aspect above, and adding exogenous DNA molecules for guiding protein synthesis; (ii) incubating the in vitro cell-free protein synthesis system of step (i) for a period of time T1 under suitable conditions, thereby synthesizing the protein encoded by the exogenous DNA; preferably, the method for synthesizing proteins in vitro also comprises: (iii) optionally isolating or detecting the exogenous protein encoded by the exogenous DNA from the in vitro cell-free protein synthesis system.

[0065] The in vitro protein synthesis system, template, plasmid, target protein, in vitro protein synthesis reaction (incubation reaction), various preparation methods, various detection methods and other technical elements of the present invention can also be independently selected from the following documents to select appropriate implementation methods or implementation methods, including but not limited to CN111484998A, CN106978349A, CN108535489A, CN108690139A, CN108949801 A, CN108642076A, CN109022478A, CN109423496A, CN109423497A, CN109423509A, CN109837293A, CN109971783A, CN109988801A, CN109971775A, CN110093284A, CN110408635A, CN110408636A, CN110551745A, CN110551700A, CN110551785A, CN110819647A, CN110845622A, CN110938649A, CN110964736A and other documents. Unless otherwise inconsistent with the purpose of the present invention, these documents and the documents cited are cited in their entirety and for all purposes.

[0066] The beneficial effects of the present invention are:

[0067] 1) The present invention systematically analyzes and selectively knocks out genes in the genome of Kluyveromyces lactis strains by means of CRISPR-Cas9-mediated gene editing, thereby providing a general method for regulating in vitro biosynthetic activity.

[0068] 2) The present invention screened a large number of proteases and knocked out some of them, and unexpectedly found that the degradation of the target protein can be effectively reduced, and the efficiency of protein production in the in vitro protein synthesis system can be significantly improved. Specifically, the IVTT activity of down-regulating or knocking out ape3△#2 at 3h and 20h was increased by more than 40% compared with the control strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1The amino acid sequence alignment of KLLA0_A06512g (cps11), KLLA0_E05655g (cps12), KLLA0_E15005g (cps13) and KLLA0_F01903g (cps14) with the Saccharomyces cerevisiae protease ScCPS1. In the K. lactis genome, there are four genes, namely KLLA0_A06512g, KLLA0_E05655g, KLLA0_E15005g and KLLA0_F01903g, which have certain similarities with the amino acid sequence of the Saccharomyces cerevisiae protease ScCPS1.

[0070] Figure 2 Schematic diagram for designing CRISPR-Cas9-mediated protease encoding gene knockout in K. lactis (taking APE3 / KLLA0_D06424g as an example). Both gRNAs were designed inside the ORF, while the H1 and H2 homology arms with a length of 500-1000 bp were designed outside the ORF.

[0071] Figure 3 The IVTT activity test results of the lysates prepared from three ape3 gene knockout mutants (ape3△#1-3). The IVTT activity of the lysates prepared from the knockout mutants was significantly higher than that of the control strain at 3h and 20h.

[0072] Figure 4 The IVTT activity of the lysate prepared by the K. lactis protease gene knockout strain was calibrated with the IVTT activity of the control strain K. lactis. It was confirmed that the knockout of most protease genes was beneficial to improving the IVTT activity of the strain lysate. The protease genes selected by the 3h IVTT activity were ranked in the order of ape4>ape3>atg4>cps14>atg4>cps13~cps11>cps12~prb1~K. lactis>prc1. DETAILED DESCRIPTION

[0073] The advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0074] the term

[0075] Engineered strains

[0076] The "engineered strain" and "genetically engineered strain" described in the present invention are used interchangeably and both refer to the engineered strain for improving the efficiency of in vitro protein synthesis of the present invention, i.e., the strain described in the first aspect of the present invention. The genetically engineered strain is a yeast derived from one or more sources selected from the group consisting of Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces; preferably Kluyveromyces, and more preferably Kluyveromyces lactis.

[0077] Protease gene

[0078] The "protease gene" and "gene encoding protease" described in the present invention are used interchangeably and refer to a general term for a class of enzymes that hydrolyze protein peptide chains. For example, in the K. lactis Y1140 genome, there are dozens of genes encoding proteases. The present invention lists some gene information encoding enzymes that may improve the expression level of D2P after knockout, as shown in Table 1.

[0079] Table 1 K.lactis genes involved in the present invention

[0080]

[0081]

[0082] D2P

[0083] The concept of "D2P" in the present invention includes but is not limited to IVTT reaction (in vitro transcription and translation reaction). In the present invention, IVTT reaction is preferred. IVTT reaction, corresponding to IVTT system, is the process of transcribing and translating DNA into protein in vitro. Therefore, we also refer to this type of in vitro protein synthesis system as D-to-P system, D_to_P system, DNA-to-Protein system; the corresponding in vitro protein synthesis method is also called D2P method, D-to-P method, D_to_P method, DNA-to-Protein method, which has the same meaning as "in vitro cell-free protein synthesis system", "in vitro expression system", "in vitro protein synthesis system", "in vitro protein synthesis reaction system", "cell-free protein synthesis system" and other expressions. Protein in vitro synthesis system, in vitro protein synthesis system, cell-free system, cell-free protein synthesis system, cell-free in vitro protein synthesis system, in vitro cell-free protein synthesis system, in vitro cell-free synthesis system, CFS system (cell-free system), CFPS system (cell-free protein synthesis system) and other descriptions. Including in vitro translation system, in vitro transcription and translation system (IVTT system), etc. We also call the in vitro protein synthesis system a "protein synthesis factory". In vitro protein synthesis reaction refers to the reaction of synthesizing proteins in an in vitro cell-free synthesis system, which at least includes the translation process.

[0084] Foreign DNA

[0085] The exogenous DNA comes from prokaryotes or eukaryotes; in another preferred embodiment, the exogenous DNA comes from animals, plants, or pathogens; in another preferred embodiment, the exogenous DNA comes from mammals, preferably primates, rodents, including humans, mice, and rats.

[0086] Foreign protein

[0087] The exogenous protein is selected from the following group of exogenous proteins: luciferin protein, 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, α-amylase, enterobactin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon αA, interleukin-1β, lysozyme, serum albumin, single-chain antibody fragment (scFV), thyroxine transporter, tyrosinase, xylanase, or a combination thereof. However, the above is only an exemplary introduction to exogenous proteins, and does not only include the above exogenous proteins; it can be any protein that can be expressed exogenously.

[0088] In vitro protein synthesis system

[0089] The in vitro protein synthesis system, template, plasmid, target protein, in vitro protein synthesis reaction (incubation reaction), various preparation methods, various detection methods and other technical elements of the present invention can also be independently selected from the following documents to select appropriate implementation methods or implementation methods, including but not limited to CN111484998A, CN106978349A, CN108535489A, CN108690139A, CN108949801 A, CN108642076A, CN109022478A, CN109423496A, CN109423497A, CN109423509A, CN109837293A, CN109971783A, CN109988801A, CN109971775A, CN110093284A, CN110408635A, CN110408636A, CN110551745A, CN110551700A, CN110551785A, CN110819647A, CN110845622A, CN110938649A, CN110964736A and other documents. Unless otherwise inconsistent with the purpose of the present invention, these documents and the documents cited are cited in their entirety and for all purposes.

[0090] In a feasible embodiment, the in vitro protein synthesis system is not particularly limited. In the present invention, the in vitro protein synthesis system comprises:

[0091] (a) Genetically engineered strain cell extracts;

[0092] (b) polyethylene glycol;

[0093] (c) optionally exogenous sucrose; and

[0094] (d) an optional solvent, wherein the solvent is water or an aqueous solvent.

[0095] In a particularly preferred embodiment, the in vitro protein synthesis system provided by the present invention comprises: genetically engineered strain 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, RNA enzyme inhibitor, luciferin, luciferase DNA, RNA polymerase. In the present invention, RNA polymerase is not particularly limited and can be selected from one or more RNA polymerases, and a typical RNA polymerase is T7 RNA polymerase.

[0096] In the present invention, the proportion of the yeast cell extract in the in vitro protein synthesis system is not particularly limited. Usually, the yeast cell extract accounts for 20-70% of the in vitro protein synthesis system, preferably 30-60%, and more preferably 40-50%.

[0097] Genetically engineered strain cell extracts

[0098] In the present invention, the genetically engineered strain cell extract does not contain intact cells, and a typical cell extract includes ribosomes, transfer RNA, aminoacyl tRNA synthetase, initiation factors and elongation factors required for protein synthesis, and termination release factors for protein translation. In addition, the cell extract also contains some other proteins in the cytoplasm of the genetically engineered strain, especially soluble proteins.

[0099] In the present invention, the preparation method of the genetically engineered cell extract is not limited. A preferred preparation method comprises the following steps:

[0100] (i) providing a genetically engineered strain cell with a protease gene knocked out provided by the first aspect of the present invention;

[0101] (ii) washing the genetically engineered strain cells to obtain washed genetically engineered strain cells;

[0102] (iii) disrupting the washed genetically engineered strain cells to obtain a crude extract of the genetically engineered strain;

[0103] (iv) performing solid-liquid separation on the crude extract of the genetically engineered strain to obtain a liquid portion, which is a cell extract of the genetically engineered strain.

[0104] In the present invention, the solid-liquid separation method is not particularly limited, and a preferred method is centrifugation.

[0105] In a preferred embodiment, the centrifugation is performed in a liquid state.

[0106] In the present invention, the centrifugation conditions are not particularly limited, and a preferred centrifugation condition is 5000-100000×g, preferably 8000-30000×g.

[0107] In the present invention, the centrifugation time is not particularly limited, and a preferred centrifugation time is 0.5 min-2 h, preferably 20 min-50 min.

[0108] In the present invention, the centrifugation temperature is not particularly limited. Preferably, the centrifugation is performed at 1-10°C, more preferably, at 2-6°C.

[0109] In the present invention, the washing treatment method is not particularly limited. A preferred washing treatment method is to use a washing liquid at a pH of 7-8 (preferably 7.4). The washing liquid is not particularly limited. Typically, the washing liquid is selected from the following group: potassium 4-hydroxyethylpiperazineethanesulfonate, potassium acetate, magnesium acetate, or a combination thereof.

[0110] In the present invention, the cell disruption treatment method is not particularly limited. A preferred cell disruption treatment includes high-pressure disruption and freeze-thaw (such as liquid nitrogen cryogenic) disruption.

[0111] The nucleoside triphosphate mixture in the in vitro protein synthesis system is adenosine triphosphate, guanosine triphosphate, cytosine triphosphate and uridine triphosphate. In the present invention, the concentration of various mononucleotides is not particularly limited, and usually the concentration of each mononucleotide is 0.5-5mM, preferably 1.0-2.0mM.

[0112] The amino acid mixture in the in vitro protein synthesis system may include natural or non-natural amino acids, and may include D-type or L-type amino acids. Representative amino acids include (but are not limited to) 20 natural amino acids: glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine. The concentration of each amino acid is generally 0.01-0.5 mM, preferably 0.02-0.2 mM, such as 0.05, 0.06, 0.07, 0.08 mM.

[0113] In a preferred embodiment, the in vitro protein synthesis system further contains polyethylene glycol or its analogs. The concentration of polyethylene glycol or its analogs is not particularly limited. Generally, the concentration (w / v) of polyethylene glycol or its analogs is 0.1-8%, preferably 0.5-4%, more preferably 1-2%, based on the total weight of the protein synthesis system. Representative examples of PEG include (but are not limited to): PEG3000, PEG8000, PEG6000 and PEG3350. It should be understood that the system of the present invention may also include polyethylene glycols of various other molecular weights (such as PEG200, 400, 1500, 2000, 4000, 6000, 8000, 10000, etc.).

[0114] In a preferred embodiment, the in vitro protein synthesis system further contains sucrose. The concentration of sucrose is not particularly limited, and generally, the concentration of sucrose is 0.03-40wt%, preferably 0.08-10wt%, and more preferably 0.1-5wt%, based on the total weight of the protein synthesis system.

[0115] A particularly preferred in vitro protein synthesis system contains the following components in addition to yeast extract: 22 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH 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 creatine phosphate, 1.7 mM dithiothreitol, 0.27 mg / mL creatine phosphate kinase, 1%-4% polyethylene glycol, 0.5%-2% sucrose, 8-20 ng / μl firefly luciferase DNA, and 0.027-0.054 mg / mL T7 RNA polymerase.

[0116] To verify the present invention, there are dozens of genes encoding proteases in the K. lactis Y1140 genome. Figure 1 The amino acid sequence alignment of KLLA0_A06512g (cps11), KLLA0_E05655g (cps12), KLLA0_E15005g (cps13) and KLLA0_F01903g (cps14) with the Saccharomyces cerevisiae protease ScCPS1. In the K. lactis genome, there are four genes, namely KLLA0_A06512g, KLLA0_E05655g, KLLA0_E15005g and KLLA0_F01903g, which have certain similarities with the amino acid sequence of the Saccharomyces cerevisiae protease ScCPS1.

[0117] Through careful analysis and screening, we obtained the information of genes encoding enzymes that may increase the expression level of D2P after knockout, as listed in Table 1. We designed and executed the knockout process of proteases for these genes to obtain artificially modified cells, such as Figure 2 A schematic diagram of the design of CRISPR-Cas9-mediated protease encoding gene knockout in K. lactis (taking APE3 / KLLA0_D06424g as an example). The following is a more specific example.

[0118] The experimental methods for which specific conditions are not indicated in the following examples are usually carried out according to conventional conditions, such as the experimental conditions described in documents such as "Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)", "Cell-free Protein Synthesis Experiment Manual" "Edited by Alexander S. Spirin and James R. Swartz. Cell-free protein synthesis: methods and protocols [M]. 2008", or according to the conditions recommended by the manufacturer, or according to the conditions guided by the specific embodiments described above. Unless otherwise specified, the percentages and parts mentioned in the present invention are weight percentages and weight parts.

[0119] Unless otherwise specified, the reagents and materials in the examples of the present invention are all commercially available products.

[0120] The present invention uses Kluyveromyces lactis (K. lactis) as an example, but the same design, analysis and experimental methods are also applicable to other yeasts and other lower eukaryotic cells and higher animal cells. The gene modification method in the present invention is CRISPR-Cas9 technology, but it is not limited thereto, and can be any known and existing gene modification method.

[0121] Example 1: Design of gRNA targeting sequence

[0122] Select the ORF region (100-2000 bp) of the targeted protease gene and complete the gRNA design on the CRISPOR (tefor.net) (http: / / crispor.tefor.net / ) website, such as Figure 2 The gRNAs shown are all designed inside the ORF, while the H1 and H2 homology arms with a length of 500-1000 bp are designed outside the ORF, and H1 and H2 are assembled by Overlap-PCR. To achieve the successful assembly of H1 and H2, the annealing temperature of primers P1-P4 is 6061°C, and 8-15nt sequences that can be reverse complementary to each other's 5'-ends are added to the 5'-ends of P2 and P3, respectively.

[0123] The gRNAs with higher scores were selected as candidate sequences. For each selected gene, two sequences with the highest comprehensive scores were selected as the final gRNA sequences. The specific information is shown in Table 2. According to the designed gRNA targeting sequence, the corresponding 24-base primer pair was synthesized, that is, for each gRNA, the 4-base sequences of AATC and AAAC were added to the 5'-end of its forward sequence and reverse complementary sequence, respectively, and the final pair of 24-base primers constituted a 24-base primer pair.

[0124] Example 2. Construction of gRNA_Cas9 expression vector

[0125] The paired 24-base primer pairs obtained in Example 1 were diluted to 10 μM, and 10 μL of each was added to the PCR tube and mixed, and the annealing process was completed on the PCR instrument according to the following procedures: 95°C, 3min; 72°C, 2min; 65°C, 2min; 60°C, 2min; 55°C, 2min; 50°C, 2min; 16°C, 2min. 20mg of the pCasM13FR vector (specifically, please refer to the Chinese patent application number CN202211528653.8) treated with BsaI and purified was mixed in 10 μL of T4 ligase system, 0.5 μL of the annealed primer pair, 20n, and the ligation reaction was carried out at 16°C for 1h. All ligation products were transformed into E. coli DH5α competent cells and screened on LB plates containing 50 μg / mL kanamycin. Two single bacteria were randomly selected for sequencing verification. The clones with correct sequencing were selected to extract the gRNA_Cas9 co-expression plasmid and determine the concentration before use for subsequent electroporation.

[0126] Transformation experiments.

[0127]

[0128] Example 3. Construction of double-stranded DNA donor

[0129] like Figure 2As shown, both gRNAs are designed inside the ORF, while the H1 and H2 homology arms with a length of 500-1000bp are designed outside the ORF, and H1 and H2 are assembled by the Overlap-PCR method. The DNA donor sequence used in the present invention is obtained by the Overlap-PCR method. First, the genomic DNA of K.lactis Y1140 is used as a template, and xxxN-p1+xxxN-p2 and xxxN-p3+xxxN-p4 are used as forward and reverse primer pairs (xxxN is the corresponding gene name), respectively. The primer list is shown in Table 3, and Phanta Super Fidelity DNA polymerase is used for PCR amplification to obtain PCR products H1 and H2; and xxxN-p1+xxxN-p4 is used as a template for the second round of PCR, and the products H1 and H2 of the previous step of PCR are used as templates. The products of the second round of PCR are dissolved in 50μL ddH2O after 70% ethanol precipitation and frozen at -20℃ for use.

[0130] To achieve successful assembly of H1 and H2, the annealing temperatures of primers P1-P4 were all 60-61°C, and 8-15 nt sequences were added to the 5'-ends of P2 and P3, respectively, which could be reverse complementary to each other's 5'-ends.

[0131]

[0132]

[0133] Example 4. Transformation and screening of yeast strains

[0134] The transformation of Kluyveromyces lactis strains was completed by electroporation. Competent cells were prepared according to the requirements of the electroporation method. Before electroporation, 40 μL of the competent cell mixture was fully mixed with the corresponding 500 ng gRNA_Cas9 expression plasmid and 1-2 μg DNA Donor. The electroporation process was completed according to the standard operation procedure. The yeast cells after electroporation were screened on a YPD plate containing 250 μg / mL G418. Single colonies were picked for PCR verification. The verification primers are shown in Table 3. Paired primers were selected for verification. The positive PCR products were sent to the sequencing company for sequencing; the single colonies with correct sequencing were streaked on the plate, and single colonies were picked for the second round of PCR verification. The strains selected for the correct two rounds of verification will be prepared into glycerol cryovials and stored in a -80°C refrigerator.

[0135] Example 5. Lysate preparation and IVTT activity detection

[0136] The strain obtained in Example 4, which was frozen in a -80°C refrigerator, was streaked on a YPD plate, and a single colony was picked and inoculated into a 250mL Erlenmeyer flask containing 100mL of seed culture medium, and cultured at 200rpm in a 30°C shaker for 24h; the seed liquid was measured and transferred to a 1000mL Erlenmeyer flask containing 400mL of fermentation culture medium, and cultured at 200rpm in a 30°C shaker until the harvest period, and the cells were collected by high-speed centrifugation, and cell lysate was prepared under liquid nitrogen protection, and then prepared into an in vitro protein synthesis system for subsequent IVTT activity testing. The detection and analysis of the D2P expression activity of the lysate was completed using mEGFP as a reporter protein, and the IVTT activity of the corresponding lysate was judged based on the detected reporter fluorescence intensity (RFU value). The results are as follows: Figure 3 and Figure 4 As shown in the figure, K.lactis refers to Kluyveromyces lactis as an experimental control example; HPC refers to high activity positive control.

[0137] Figure 3 The IVTT activity of the lysate prepared by the knockout mutant was significantly higher than that of the control K. lactis at 3h and 20h, and the IVTT activity at 3h and 20h increased by 6-44% and 23-42%, respectively. The IVTT activity of ape3△#2 at 3h and 20h increased by more than 40% compared with the control strain K. lactis, indicating that the knockout of the ape3 gene has significantly improved the IVTT activity of the K. lactis lysate under the test conditions. It is speculated that the knockout of the ape3 gene effectively reduced the degradation of the target protein.

[0138] Figure 4 The results showed that the knockout of most protease genes was beneficial to improving the IVTT activity of the strain lysate, among which the knockout of ape3, ape4 and atg4 genes had the most obvious effect, among which the knockout of ape4 could increase the IVTT activity by up to 54% at 3h. The protease genes selected by 3h IVTT activity were ranked in the order of ape4>ape3>atg4>cps14>atg4>cps13~cps11>cps12~prb1~K.lactis>prc1. It should be noted that the knockout of cps14 had no positive effect on the improvement of 20h IVTT activity. In addition, the knockout of prc1 was not conducive to the improvement of IVTT activity. In particular, it should be noted that the IVTT activity of the prc1 knockout strain at 20h was significantly reduced compared with the control strain.

[0139] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A genetically engineered strain for improving in vitro cell-free protein synthesis activity, characterized in that: The endogenous protease gene of the genetically engineered strain is modified, and the expression or activity of the protease gene in the genetically engineered strain is reduced compared to before the modification; Preferably, the protease gene is selected from one or more of APE1, APE3, APE4, ATG4, CPS1-1, CPS1-2, CPS1-3, CPS1-4, PEP4, PRB1, and PRC1.

2. The genetically engineered strain according to claim 1, characterized in that The "reduction" refers to reducing the expression or activity of the protease gene to meet the following conditions: the ratio of B1 / B0 ≤30%; wherein B1 is the expression or activity of the protease gene in the genetically engineered strain; B0 is the expression or activity of the protease gene before the modification; preferably, the "reduction" refers to reducing the expression or activity of the protease gene to meet the following conditions: the ratio of B1 / B0 ≤10%; more preferably, the "reduction" refers to reducing the expression or activity of the protease gene to meet the following conditions: the ratio of B1 / B0 ≤5%; more preferably, the "reduction" refers to reducing the expression or activity of the protease gene to meet the following conditions: the ratio of B1 / B0 ≤2%; more preferably, the "reduction" refers to reducing the expression or activity of the protease gene to meet the following conditions: the ratio of B1 / B0 is selected from 0-2%.

3. The genetically engineered strain according to claim 2, characterized in that The genetically engineered strain is derived from one or more yeasts selected from the group consisting of Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces; preferably Kluyveromyces, and more preferably Kluyveromyces lactis.

4. The genetically engineered strain according to claim 3, characterized in that The Kluyveromyces is one or more types of genetically engineered Kluyveromyces lactis, and the genetic engineering is used to improve its in vitro protein expression activity.

5. The genetically engineered strain according to claim 1, characterized in that The protease gene is selected from one or more of ape4, ape3, atg4, cps1-4 and atg4.

6. The genetically engineered strain according to claim 1, characterized in that: The protease gene has the following sequence: The coding gene of APE1 is shown in SEQ ID NO:1; the coding gene of APE3 is shown in SEQ ID NO:2; the coding gene of APE4 is shown in SEQ ID NO:3; the coding gene of ATG4 is shown in SEQ ID NO:4; the coding gene of CPS1-1 is shown in SEQ ID NO:5; the coding gene of CPS1-2 is shown in SEQ ID NO:6; the coding gene of CPS1-3 is shown in SEQ ID NO:7; the coding gene of CPS1-4 is shown in SEQ ID NO:8; the coding gene of PEP4 is shown in SEQ ID NO:9; the coding gene of PRB1 is shown in SEQ ID NO:10; the coding gene of PRC1 is shown in SEQ ID NO:

11.

7. The genetically engineered strain according to claim 1, characterized in that: The expression or activity of the protease gene in the strain is reduced by a method selected from the following group: gene mutation, gene knockout, gene interruption, RNA interference technology, Crispr technology, or a combination thereof.

8. Use of the genetically engineered strain according to any one of claims 1 to 7 for improving the efficiency of in vitro protein synthesis.

9. An in vitro cell-free protein synthesis system, characterized in that: The characteristic is that The in vitro cell-free protein synthesis system comprises preparing a cell extract using the genetically engineered strain according to any one of claims 1 to 7.

10. The synthesis system according to claim 9, characterized in that The in vitro cell-free protein synthesis system further comprises one or more components selected from the group consisting of: (b) polyethylene glycol; (c) optional exogenous sucrose; and (d) optional solvent, wherein the solvent is water or an aqueous solvent; Preferably, the in vitro cell-free protein synthesis system further comprises one or more components selected from the group consisting of: (e1) a substrate for synthesizing RNA; (e2) a substrate for synthesizing protein; (e3) magnesium ions; (e4) potassium ions; (e5) a buffer; (e6) RNA polymerase; and (e7) an energy regeneration system. More preferably, the in vitro cell-free protein synthesis system further comprises one or more components selected from the following group: (e8) heme; and (e9) spermidine.

11. A method for in vitro cell-free protein synthesis, characterized in that: The method comprises the following steps: (i) providing the in vitro cell-free protein synthesis system according to any one of claims 9 to 10, and adding an exogenous DNA molecule for directing protein synthesis; (ii) incubating the in vitro cell-free protein synthesis system of step (i) under suitable conditions for a period of time T1, thereby synthesizing the protein encoded by the exogenous DNA; preferably, the method for in vitro protein synthesis also includes: (iii) optionally isolating or detecting the protein encoded by the exogenous DNA from the in vitro cell-free protein synthesis system; preferably, in step (ii), the reaction temperature is 20-37°C, preferably, 20-25°C; further preferably, in step (ii), the reaction time is 1-72h, preferably, 2-23h.

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