WO2022183706A1 - 一种体内连续定向进化系统及其应用 - Google Patents

一种体内连续定向进化系统及其应用 Download PDF

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WO2022183706A1
WO2022183706A1 PCT/CN2021/117642 CN2021117642W WO2022183706A1 WO 2022183706 A1 WO2022183706 A1 WO 2022183706A1 CN 2021117642 W CN2021117642 W CN 2021117642W WO 2022183706 A1 WO2022183706 A1 WO 2022183706A1
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module
genes
gene
protein
promoter
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吴敬
刘展志
张昕昱
许滢
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江南大学
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    • C12N15/1034—Isolating an individual clone by screening libraries
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Definitions

  • the invention relates to an in vivo continuous directional evolution system and its application, belonging to the fields of genetic engineering and enzyme engineering.
  • Directed evolution also known as laboratory evolution
  • laboratory evolution is a method of simulating the evolutionary process at the molecular level in a laboratory environment, resulting in a protein with desired characteristics.
  • this evolutionary process has problems such as low single-round mutation efficiency, huge screening workload, and too complicated screening methods. It usually takes several or even dozens of rounds of evolution to obtain ideal protein properties.
  • David Liu's laboratory at Harvard University proposed a phage-assisted continuous evolution system (PACE), which combines directed evolution with the life cycle of M13 phage and uses mutagenic plasmids to generate target genes. Random mutation enables continuous evolution of target genes and targeted screening.
  • PACE phage-assisted continuous evolution system
  • the gene of interest replaced the gene pIII encoding a key phage coat protein and was integrated into the M13 phage genome.
  • the David Liu lab uses a turbidimeter-lagoon system or a chemostat-lagoon system to continuously supply phages with fresh E. coli host cells to prevent the accumulation of host genome mutations.
  • the expression vector for pill is contained in the host cell, and the expression of pill is coupled with some expected function of the relevant gene. Phage that cannot induce pill expression cannot reproduce and are flushed out of the system by the constant influx of phage. Phages have the advantage of short passage times.
  • a single cell can produce nearly 1,000 progeny phages within 1 hour of infecting the host bacteria, and new phage particles appear 10 minutes after infection. Each generation of phages represents a round of evolution.
  • the system can complete dozens of rounds of evolution in a day, including duplication, mutation and directional selection of the gene of interest. Compared with traditional methods, the system requires minimal manual intervention, and can generate new activities or improve existing activities of the target protein in a shorter time.
  • the PACE system has now been successfully applied to the directed evolution of T7 RNAp polymerase, using PACE's evolutionary system T7 RNAp polymerase to generate recognition activity for the T3 promoter within a few days.
  • the PACE system is a phage-based directed evolution system, it requires high standards for equipment, laboratory researchers' operations, and laboratory specifications.
  • the targeting of the target gene is poor, and the mutation cannot be accurately positioned on the target gene, and random mutations in the non-target gene region may adversely affect the physiological metabolism of the host, reducing the universality of the PACE system. sex. Therefore, it is of high theoretical and practical significance to develop a novel and efficient in vivo continuous directed evolution system and improve the targeting of target genes.
  • the first objective of the present invention is to provide an in vivo continuous directed evolution system, which can randomly mutate the target protein in vivo and conduct directed screening.
  • system host cells include but are not limited to Escherichia coli.
  • the host bacteria in the system comprises two or more functional modules.
  • the functional modules include a random mutation module MP, a programmed death module TAS and a target gene expression module TP; the random mutation module MP is coupled with the target gene expression module TP, and the target gene expression module TP Conjugated to the programmed death module TAS.
  • the coupling of the target gene expression module TP and the programmed death module TAS means that the target gene on the TP module can induce the expression of anti-toxic proteins on the TAS module after random mutation; it is not limited to the following Several: (1) DNA binding proteins: such as coupling the evolution of antitoxin transcription factors with the expression of antitoxin proteins, etc.; (2) proteins acting on small molecules: such as partial carbohydrase, cutinase or other The reaction products of enzymes involved in metabolism or non-metabolite biosynthesis are coupled with the expression of antitoxin proteins, etc.; (3) Fluorescent proteins: such as the use of light-sensitive promoters to couple fluorescent proteins with the expression of antitoxin proteins, etc.; (4) Nucleases, such as Cas9: Coupling the Cas9 function with the expression of antitoxic proteins through corresponding gene regulation.
  • DNA binding proteins such as coupling the evolution of antitoxin transcription factors with the expression of antitoxin proteins, etc.
  • proteins acting on small molecules such as partial carbo
  • the random mutation module MP contains mutagenic genes and auxiliary genes;
  • the target gene expression module TP contains target genes and elements that can be recognized or combined by the random mutation module MP auxiliary genes;
  • the The programmed death module TAS contains a gene encoding a toxin protein and a corresponding gene encoding an antitoxin protein.
  • the mutagenic gene is selected from, but not limited to, the low-fidelity DNA polymerase I encoding gene PolA, the cytosine deaminase encoding gene AID, the cytosine deaminase encoding gene APOBEC, At least one of the genes encoding adenine deaminase, such as TadA.
  • the auxiliary gene is selected from, but not limited to, the coding gene of T7 RNA polymerase, the coding gene of nCas9 lacking the activity of cutting non-complementary strands, the coding gene of dCas9 with only DNA binding ability and other genes. one or more.
  • the target gene is one or more protein-coding genes and/or non-coding genes.
  • the target genes include but are not limited to genes encoding T7 RNA polymerase, antibiotic resistance genes, genes encoding enzymes in catabolic pathways, genes encoding enzymes in anabolic pathways, DNA One or more of the genes encoding binding proteins, nucleases, carbohydrases, and proteases.
  • the elements on the target gene expression module TP that are recognized or bound by auxiliary genes include but are not limited to tac promoter, pac promoter, Sp6 promoter, lac promoter, T7 promoter, pBAD promoter , trc promoter, npr promoter and sgRNA.
  • the promoter for inducing the expression of the gene encoding the toxin protein on the programmed death module TAS is an inducible promoter, including but not limited to pBAD operating system, Lac operating system, Tac operating system and Tet operating system ;
  • the coding gene of the anti-toxic protein is identified and expressed by the target gene after directional evolution; according to the requirements of different target proteins, TAS also contains the expression-assisted recognition or binding protein.
  • the genes encoding the toxin proteins on the programmed death module TAS include, but are not limited to, YdfD, which can cause cell rupture, etc.; PezT, SezT, zeta toxin, etc., which inhibit the formation of cell membranes; and inhibit DNA replication.
  • FicT, CcdB, etc.; TacT, etc. which have inhibitory effects on translation;
  • the encoding gene of anti-toxin protein is selected from DicB/SulA, PezA, SezA, epsilon antitoxin, FicA, CcdA, TacA, etc. corresponding to the toxin protein.
  • the auxiliary recognition or binding proteins include but are not limited to activating and repressing transcription factors, such as lacI, psiR, Lrp, LysG, PcaR, CadR, PadR, NanR, PcaU, BmoR, TgtR , EmrR, FdeR, FrmR, DmpR, BenR, FadR, SoxR, Alks, PobR.
  • activating and repressing transcription factors such as lacI, psiR, Lrp, LysG, PcaR, CadR, PadR, NanR, PcaU, BmoR, TgtR , EmrR, FdeR, FrmR, DmpR, BenR, FadR, SoxR, Alks, PobR.
  • the expression vectors of the random mutation module MP, the target gene expression module TP and the programmed death module TAS include but are not limited to pET series, or pSB1C3, or pRSFDuet, or pCDFDuet plasmids.
  • the expression vectors of the random mutation module MP, the target gene expression module TP and the programmed death module TAS are different.
  • the second object of the present invention is to provide a method for continuous directed evolution of genes, by transforming the continuous directed evolution system into microbial cells.
  • the microbial cells include, but are not limited to, E. coli.
  • the method is to induce microbial cells by an inducer, respectively inducing the random mutation module MP to express the mutagenized protein, the target gene expression module TP to express the target protein, and the programmed death module TAS to express the auxiliary recognition or binding protein. and toxic proteins; for different target proteins, corresponding substrates need to be added.
  • the induced microbial cells are transferred or continuously cultured in a medium containing an inducer.
  • the inducer of the method includes, but is not limited to, inducers IPTG, L-ara.
  • the present invention also protects the use of the continuous targeting system or the method described above in protein engineering.
  • the in vivo continuous directional evolution system of the invention has the targeting ability to the target gene, the mutagenized protein can accurately identify and mediate random mutation of the target gene, and the mutation is accurately located on the target gene. And the system is suitable for screening in Escherichia coli, and the selected target is relatively wide, which is convenient for most laboratories to promote and use.
  • Figure 1 Schematic flow chart of continuous directed evolution in vivo.
  • Figure 6 is a comparison diagram of the number of single colonies formed by the evolution screening product and the control group grown on the screening plate.
  • the continuous directed evolution system in Escherichia coli includes a random mutation module MP, a target gene expression module TP, and a programmed death module TAS.
  • the expression of the antitoxin protein in the programmed death module TAS is coupled with the function of the mutated target protein in the target gene expression module TP. That is, the successfully evolved target protein can directly initiate or regulate the expression of the antitoxin protein in TAS; or the successfully evolved target protein can decompose or synthesize a certain substance, thereby initiating or regulating the expression of the antitoxin protein in TAS.
  • the obtained T7 RNAp mutant can specifically recognize the T7(R13) promoter of the antitoxin protein, and initiate the transcription of the antitoxin protein to antagonize the effect of the antitoxin protein on the host bacteria; no mutation occurs Or the non-dominant T7 RNAp mutant could not recognize the T7(R13) promoter, resulting in the inability to express the antitoxin protein normally, unable to antagonize the toxin protein and cause the cells to be cleaved by the toxin protein.
  • T7 RNAp encoding gene As the example of directed evolution, and any other similar technical solutions are applicable to the present invention.
  • target genes such as carbohydrase gene, cutinase gene, esterase gene, fluorescent protein gene, nuclease gene, etc.
  • their functions can be related to the antitoxin protein in the programmed death module TAS through different principles. expression coupled.
  • the present invention will enumerate several examples of target genes that can be applied to continuous directed evolution. It should be clear that there are many target genes available for selection in the present invention, and are not limited to the following: (1) DNA binding proteins: For example, the evolution of antitoxin transcription factors can be coupled with the expression of antitoxin proteins, etc.; (2) proteins acting on small molecules: such as partial carbohydrase, cutinase or other proteins involved in metabolism or non-metabolite biosynthesis The reaction product of the enzyme is coupled with the expression of anti-toxic protein, etc.; (3) Fluorescent protein: such as using a photosensitive promoter to couple the expression of fluorescent protein and anti-toxic protein, etc.; (4) Nuclease, such as Cas9: Coupling Cas9 function with the expression of antitoxin proteins through corresponding gene regulation, etc.
  • DNA binding proteins For example, the evolution of antitoxin transcription factors can be coupled with the expression of antitoxin proteins, etc.
  • proteins acting on small molecules such as partial carbohydras
  • the programmed death module TAS in FIG. 1 is only an example. According to the different toxicity mechanisms of the TAS poisoning proteins, there are various options for the poison protein-antivirus protein system applicable to the present invention and are not limited to The following: (1) Toxic proteins that can inhibit DNA replication, transcription and other functions: such as FicT/FicA derived from P.
  • FicT modifies DNA gyrase and topoisomerase IV by adenylation, making cellular DNA
  • Toxic proteins that inhibit translation such as TacT/TacA from Salmonella, TacT is an acetyltransferase that blocks charged tRNA molecules The primary amino group of amino acids, thereby inhibiting translation and promoting the formation of surviving cells
  • Toxic proteins that affect cell division such as YdfD derived from Escherichia coli, YdfD can dissolve 99.9% of cells within 2 hours of induction, while cell division SulA, an inhibitor, can eliminate YdfD-induced cell lysis
  • toxic proteins that affect peptidoglycan synthesis such as SezT/SezA from Streptococcus Suis Serotype, PezA/T from Streptococcus Pneumoniae, which can inhibit peptidoglycan synthesis. Synthesized, eventually causing bacterial autolysis.
  • SezT/SezA from Strept
  • the mutagenic protein responsible for the mutation is cytidine deaminase (AID), which can act on single-stranded DNA and can deaminate cytosine into uracil and finally into thymine; T7
  • AID cytidine deaminase
  • T7 The fusion expression of RNAp and AID is responsible for the targeted binding of AID to the downstream of the T7 promoter on the target gene expression module TP, so that the mutagenized protein can be targeted to the target gene.
  • other targeted mutagenic proteins, mutagenic protein mutants or mutagenic fusion proteins can also be used as the spontaneous random mutation module MP in the present invention.
  • DNA polymerase Pol I (D355A/E357A or D424A/I709N/A759R), which is a low-fidelity DNA polymerase mutant that specifically recognizes plasmids with ColE1 Ori and enables the initiation of transcription A nucleotide sequence of a certain length after the site is mutated.
  • the directed evolution method provided by the present invention includes: under the action of an inducer, the random mutation module MP in the system expresses a mutagenized protein, which will recognize and mediate the random mutation of the target gene in the target gene expression module TP; , and then add an inducer that induces the expression of toxic proteins into the system to start the system for directional screening; the successfully evolved target protein mutants can mediate the expression of anti-toxic proteins on the programmed death module TAS, so that the toxic proteins cannot act on host cells. Ultimately only host cells containing the successfully evolved gene of interest will survive.
  • Plasmids pET20b, pRSFDuet and pCDFDuet were purchased from TaKaRa (Dalian) Bioengineering Co., Ltd.; Escherichia coli BL21 (DE3) was preserved by our laboratory.
  • PrimeSTAR HS DNA polymerase, DNA Marker, Dpn I restriction enzyme, etc. involved in the following examples were purchased from TaKaRa (Dalian) Bioengineering Co., Ltd.
  • LB medium 0.5% (W/V) yeast extract powder, 1% (W/V) tryptone, 1% (W/V) sodium chloride, add 2% (W/V) agar powder to be LB solid culture medium.
  • TB medium 1.2% (w/v) tryptone, 2.4% (w/v) yeast extract, 0.4% (v/v) glycerol, phosphate buffer (17 mM KH 2 PO 4 , 72 mM K 2 HPO 4 ) ).
  • the fusion protein whose synthetic nucleotide sequence is shown in SEQ ID NO.1 encodes AID-T7 RNAp(T3) (the amino acid sequence is shown in SEQ ID NO.2), wherein T7 RNAp(T3) represents the T3-type mutation of T7 RNAp body.
  • the AID-T7 RNAp(T3) was ligated into the E. coli expression vector pCDFDuet to construct a recombinant vector pCDFDuet-pT7-AID-T7 RNAp(T3) by means of restriction enzyme ligation.
  • the ligation product was transformed into E.
  • coli JM109 and the transformed product was spread on LB solid medium (containing a final concentration of 50 ⁇ g ⁇ mL -1 streptomycin). After inverting culture at 37°C for 10-12 h, the transformants on the plate were picked and plated in LB liquid medium (containing a final concentration of 50 ⁇ g ⁇ mL -1 streptomycin). After culturing at 37°C and 200rpm for 12-14h, the plasmid was extracted for sequencing and verified as a random mutation module MP, and named pCDFuet-1-AID-T7 RNAp(T3) (as shown in Figure 2).
  • Toxic protein-anti-toxic protein system (TAS) YdfD/SulA was selected, and its sequence was synthesized.
  • the amino acid sequence is shown in SEQ ID NO.3 and SEQ ID NO.4.
  • the nucleotide sequences were introduced into the two multiple cloning sites of pRSFDuet respectively, and the T7 promoter expressing the toxin protein in the vector was replaced with the pBAD operator by MEGAWHOP cloning method.
  • the PCR product was digested with Dpn I, it was transformed into E. coli JM109, and the transformed product was spread on LB solid medium (containing a final concentration of 50 ⁇ g ⁇ mL -1 kanamycin).
  • the transformants on the plate were picked and placed in LB liquid medium (containing a final concentration of 50 ⁇ g ⁇ mL -1 kanamycin). After culturing at 37°C and 200rpm for 12-14h, the plasmids were extracted and sent to a sequencing company for sequencing.
  • the plasmid with correct sequencing result is the recombinant vector pRSFDuet–pT7–antitoxin–araC–pBAD–toxin.
  • Example 3 Identification of programmed death module TAS in E. coli
  • the toxin-antitoxin expression vector pRSFDuet–pT7–antitoxin–araC–pBAD–toxin was transferred into E. coli BL21 (DE3), and then inserted into LB medium to induce fermentation.
  • glycerol bacteria stored in a -80°C refrigerator were added to 10 mL of LB seed solution (containing a final concentration of 50 ⁇ g ⁇ mL -1 kanamycin), and cultured at 37° C. and 200 rpm overnight.
  • the seed liquid was transferred to 50 mL of LB fermentation broth (containing a final concentration of 50 ⁇ g ⁇ mL -1 kanamycin) at a ratio of 1:20.
  • inducers IPTG and L-ara were added to induce fermentation. (Another group was only added with IPTG, one group was only added with L-ara, and one group was not added with inducer as a control). After adding the inducer, the growth of the recombinant bacteria was measured. The results are shown in Figure 4. When only the inducer L-ara is present, the expression of toxin proteins can be induced, which can effectively mediate the death of the strain; when the inducers IPTG and L-ara are present at the same time, because IPTG relieves lac I on T7 class initiation The inhibition of the T7 promoter induces the expression of antitoxin proteins, and the growth of the strain is hardly affected.
  • T7 promoter expressing antitoxin protein was mutated into a T7(R13) promoter by point mutation.
  • Example 4 Construction of target gene expression module TP in Escherichia coli
  • E. coli expression vector pET20b Taking the genome of E. coli BL21 (DE3) as the template, using primers T7RNApRF-F and T7RNApRF-R to amplify the coding gene of T7 RNAp as the target gene, and using the method of MEGAWHOP cloning to connect it to the E. coli expression vector pET20b.
  • the recombinant expression vector pET20b containing the gene encoding T7 RNAp was digested with Dpn I, then transformed into E. coli JM109, and the transformed product was spread on LB solid medium (containing a final concentration of 100 ⁇ g ⁇ mL -1 ampicillin).
  • the transformants on the plate were picked and placed in LB liquid medium (containing a final concentration of 100 ⁇ g ⁇ mL -1 ampicillin). After culturing at 37°C and 200rpm for 12-14h, the plasmids were extracted and verified by sequencing. The plasmid with correct sequencing result is the recombinant vector pET20b-T7 RNAp (as shown in Figure 3).
  • the T7 promoter on the recombinant vector was mutated into a T7(T3) promoter by point mutation, wherein the T7(T3) promoter represents a promoter that can only be recognized by the auxiliary gene T7 RNAp(T3) on the random mutation module MP (
  • the T7 (T3) promoter sequence is shown in the article Meyer A J, Ellefson J W, Ellington A D. Directed Evolution of a Panel of Orthogonal T7 RNA Polymerase Variants for in Vivo or in Vitro Synthetic Circuitry [J].Acs Synthetic Biology, 2015, 4( 10).: Table 1 "P T3 ").
  • the obtained recombinant plasmid pET20b-pT7(T3)-T7 RNAp was the target gene expression module TP.
  • T7RNApRF-F CTTTAAGAAGGAGATATACATATGAACACGATTAACATCGC;
  • T7RNApRF-R TGGTGGTGGTGGTGCTCGAGTTACGCGAACGCGAAGTCC.
  • the random spontaneous mutation module MP, the target gene expression module TP and the programmed death module TAS were sequentially transferred into Escherichia coli BL21 (DE3), and cultured at 37° C. and 200 rpm overnight.
  • the seed liquid was transferred to 50 mL of TB fermentation broth at a ratio of 1:20, and the inducers IPTG and L-ara (final concentrations were 0.4 mM and 0.3%, respectively) were added to induce continuous fermentation after 37 °C, 200 rpm, and 1.5 h.
  • the induced bacterial solution was diluted to a certain number, it was plated (containing 0.3% L-ara), and the number of single colonies was calculated (another group without L-ara was used as a control). The results are shown in FIG.
  • the single colony that can grow on the plate is sequenced for the target gene, and finally the R13 mutant T7 RNAp (R13) of T7 RNAp is obtained.
  • the final mutation efficiency was 4x10 -5 ⁇ -6 /bp, and the screening period was 6 days. The physiological metabolism of the mutant strain was not affected.
  • the random spontaneous mutation module MP, the target gene expression module TP and the programmed death module TAS were sequentially transferred into Escherichia coli BL21 (DE3), and cultured at 37° C. and 200 rpm overnight. Transfer the seed liquid to 50mL TB fermentation broth at a ratio of 1:20, add inducer IPTG (final concentration 0.4mM) for continuous induction fermentation at 37°C, 200rpm for 1.5h, and add inducer L after fermentation for 2-4h -ara (final concentration 0.3%). After the induced bacterial solution was diluted to a certain number, it was plated (containing 0.3% L-ara), and the number of single colonies was calculated (another group without L-ara was used as a control). The results are shown in FIG.
  • the single colony that can grow on the plate is sequenced for the target gene, and finally the R13 mutant T7 RNAp (R13) of T7 RNAp is obtained.
  • the final mutation efficiency was 4x10 -5 ⁇ -6 /bp, and the screening period was 6 days. The physiological metabolism of the mutant strain was not affected.
  • the recombinant bacteria were added with inducer and then subcultured, and the bacterial solution was spread on the plate containing L-ara. No colonies grew after overnight incubation at 37 °C.
  • the inducible gene AID-T7 RNAp(T3) was not contained in the random spontaneous mutation module MP, the recombinant bacteria were added with inducer and then subcultured, and the bacterial solution was spread on the plate containing L-ara, and no colonies were found after overnight incubation at 37°C. grow out.
  • the recombinant bacteria are added with inducer, and after continuous subculture, the bacterial solution is spread on the plate containing L-ara, and no colony grows after overnight incubation at 37°C. out.
  • the above examples illustrate the present invention by taking the evolution of T7 RNAp that recognizes the T7 promoter into the T7 RNAp mutant T7 RNAp(R13) that recognizes the T7(R13) promoter mutant as an example.
  • Replace the T7 RNAp gene in TP with other target genes such as carbohydrase gene, cutinase gene, nuclease gene, fluorescent protein gene, etc.
  • target genes such as carbohydrase gene, cutinase gene, nuclease gene, fluorescent protein gene, etc.
  • the replacement and modification of the mutagenized protein-encoding genes in the random mutation module MP are also included in the description of the present invention.
  • promoter-F-CTTACATTAATTGCGTTGCGCCCGCTTCTAGAGGAGCTGTTGAC promoter-R-GATATTTTTGCCGATCCCCATTGATCTTTTCTCCTCTTTTCCTCC as upstream and downstream primers
  • pET20b-psir-ppsiA the transcription factor-promoter psir-ppsiA gene fragment (nucleotide sequence such as SEQ ID NO.
  • the transcription factor-promoter psir-ppsiA is constructed to pRSFDuet-pT7- by MEGAWHOP Antitoxin–araC–pBAD–toxin obtained the recombinant plasmid pRSFDuet-pT7-psir-ppsiA-antitoxin-araC-pBAD-toxin as the programmed death module TAS;
  • DPE-F-TAAGAAGGAGATATACATCGAGGATGAAACATGGCATCTATT and DPE-R-CCTGGGCATGCCGCTTCAGTGGTGGTGGTGGTGGTG as primers, with pET20b-dpe as template, utilize PCR amplification to obtain the coding gene of DPE as target gene (nucleotide sequence such as SEQ ID NO.6), with example 4.
  • the recombinant plasmid pET20b-pT7(T3)-T7 RNAp was obtained as the template, and the T7 RNAp was replaced by DPE using the method of MEGAWHOP cloning to obtain the plasmid pET20b(+)-pT7(T3)-dpe containing the DPE target gene, as the target gene expression module TP;
  • pCDFuet-1-AID-T7 RNAp(T3) in Example 1 was used as mutant plasmid MP.
  • the MP, TP and TAS were co-transformed into the host cell E.coli BL21 (DE3), and the recombinant strain was obtained as a DPE continuous evolution system in vivo.
  • the recombinant bacteria obtained in Example 6 were inoculated into a shake flask of 10 mL LB (containing 50 ⁇ g/mLamp, 25 ⁇ g/mL Sm, 25 ⁇ g/mL Kana), and the seed liquid was obtained after culturing at 37° C. and 200 r/min for 8-10 h.
  • the seed solution was transferred to 20 mL TB (containing 50 ⁇ g/mL Amp, 25 ⁇ g/mL Sm, 25 ⁇ g/mL Kana) medium at 10% (v/v) inoculum, and incubated for 1 h at 37 °C and 200 r/min.
  • the bacterial solution diluted to a certain gradient was spread on LB (containing 50 ⁇ g/mL Amp, 25 ⁇ g/mL Sm, 25 ⁇ g/mL Kana, 0.5M D-fructose, 0.4mmol/L IPTG, 3mg/mL L-ara) agar plate
  • LB containing 50 ⁇ g/mL Amp, 25 ⁇ g/mL Sm, 25 ⁇ g/mL Kana, 0.5M D-fructose, 0.4mmol/L IPTG, 3mg/mL L-ara

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Abstract

一种体内连续定向进化系统及其应用，属于基因工程和酶工程领域。该系统包括携带随机突变模块MP、程序化死亡模块TAS以及目的基因表达模块TP的大肠杆菌宿主菌。模块间通过相互偶联，通过系统中的随机突变模块MP对目的基因进行多轮连续突变，进一步提高目的基因的突变率，最终实现对宿主菌中目的基因的高效进化以及筛选。该系统使突变准确定位于目的基因上，减少非目的基因区域的随机突变，且具有较好的实用价值，可应用于多种不同功能型蛋白质的定向进化。

Description

一种体内连续定向进化系统及其应用 技术领域
本发明涉及一种体内连续定向进化系统及其应用，属于基因工程和酶工程领域。
背景技术
定向进化也被称为实验室进化，是一种在实验室环境中，从分子水平上模拟进化过程，最终得到具有期望特征的蛋白质的方法。然而，这一进化过程存在着单轮突变效率过低、筛选工作量极大、筛选手段过于复杂等问题，为获得理想的蛋白质特性通常需要几轮进化甚至几十轮进化。基于上述问题，哈佛大学David Liu实验室提出了一种由噬菌体辅助的连续进化系统(PACE)，该系统通过将定向进化与M13噬菌体的生命周期相偶联，并利用诱变质粒使目的基因产生随机突变，能够使目的基因持续进化，并对其进行定向筛选。在PACE中，目的基因取代了编码关键噬菌体外壳蛋白的基因pIII，被整合在M13噬菌体的基因组中。David Liu实验室使用恒浊器-泻湖系统或恒化器-泻湖系统持续向噬菌体提供新鲜大肠杆菌宿主细胞，以防止宿主基因组突变的积累。宿主细胞中包含了pIII的表达载体，并且pIII的表达与相关基因的某些预期的功能偶联在一起。不能诱导pIII表达的噬菌体无法繁殖，并被不断流入的噬菌体冲刷出系统。噬菌体具有传代时间短的优点，单个细胞在感染宿主菌1小时内可产生近1000个子代噬菌体，在感染10分钟后出现新的噬菌体颗粒，每一代噬菌体都代表着一轮进化。该系统可以在一天完成数十轮进化，包括目的基因的复制、突变和定向选择。与传统方法相比，该系统只需要最少的人工干预，可在更短的时间内使目的蛋白产生新的活性或改进目的蛋白已有的活性。PACE系统目前已成功应用于定向进化T7 RNAp聚合酶，利用PACE的进化系统T7 RNAp聚合酶在几天内产生了对T3启动子的识别活性。
尽管具有上述优点，但由于PACE系统是一种基于噬菌体的定向进化系统，因此对于设备、实验室研究人员的操作以及实验室规格标准要求较高。此外，该系统对目的基因的靶向性较差，无法使突变准确定位于目的基因上，而非目的基因区域的随机突变可能会对宿主的生理代谢产生不利影响，降低了PACE系统的普适性。因此，开发新型高效的体内连续定向进化系统并提高对目的基因的靶向性具有较高的理论和应用意义。
发明内容
[技术问题]
现有的定向进化系统对目的基因的靶向性较差，无法使突变准确定位于目的基因上。且现有的定向进化系统，对实验室规格标准以及操作规范要求较高，不利于技术推广应用。
[技术方案]
本发明第一个目的是提供一种体内连续定向进化系统，该系统可以在体内对目的蛋白进行随机突变，并对其进行定向筛选。
作为本发明的进一步改进的方案，所述系统宿主细胞包括但不限于大肠杆菌。
作为本发明的进一步改进的方案，所述系统中宿主菌包含两种及两种以上功能模块。
作为本发明的进一步改进的方案，所述功能模块中包括随机突变模块MP、程序化死亡模块TAS以及目的基因表达模块TP；随机突变模块MP与目的基因表达模块TP偶联，目的基因表达模块TP与程序化死亡模块TAS偶联。
在一种实施方式中，所述目的基因表达模块TP与程序化死亡模块TAS的偶联是指TP模块上目的基因在随机突变后能诱导TAS模块上抗毒蛋白的表达；并不局限于以下几种：(1)DNA结合蛋白：如将抗毒蛋白转录因子的进化与抗毒蛋白的表达相偶联等；(2)作用于小分子的蛋白：如将部分糖酶、角质酶或其他参与代谢或非代谢物生物合成的酶的反应产物与抗毒蛋白的表达相偶联等；(3)荧光蛋白：如利用光敏性启动子将荧光蛋白与抗毒蛋白的表达相偶联等；(4)核酸酶，如Cas9:通过相应的基因调控将Cas9功能与抗毒蛋白的表达相偶联等。
作为本发明的进一步改进的方案，所述随机突变模块MP上含有诱变基因和辅助基因；目的基因表达模块TP上含有目的基因和能被随机突变模块MP辅助基因识别或结合的元件；所述程序化死亡模块TAS上含有编码毒蛋白的基因和相对应的编码抗毒蛋白的基因。
作为本发明的优选方案，所述诱变基因选自但不限于低保真性的DNA聚合酶I的编码基因PolA、胞嘧啶脱氨酶的编码基因AID、胞嘧啶脱氨酶的编码基因APOBEC、腺嘌呤脱氨酶的编码基因TadA等基因中的至少一种。
作为本发明的优选方案，所述辅助基因选自但不限于T7 RNA聚合酶的编码基因、缺少切割非互补链活性的nCas9的编码基因、仅具有DNA结合能力的dCas9的编码基因等基因中的一种或多种。
作为本发明的进一步改进的方案，所述目的基因为一种或多种蛋白的编码基因和/或非编码基因。
作为本发明的优选方案，所述目的基因包括但不限于T7 RNA聚合酶的编码基因、抗生素的抗性基因、分解代谢途径中的酶的编码基因、合成代谢途径中的酶的编码基因、DNA结合蛋白的编码基因、核酸酶的编码基因、糖酶的编码基因、蛋白酶的编码基因等基因中的一种或多种。
作为本发明的优选方案，所述目的基因表达模块TP上被辅助基因识别或结合的元件包括但不限于tac启动子、pac启动子、Sp6启动子、lac启动子、T7启动子、pBAD启动子、trc启动子、npr启动子以及sgRNA。
作为本发明的优选方案，所述诱导程序化死亡模块TAS上毒蛋白的编码基因表达的启动子为诱导型启动子，包括但不限于pBAD操纵系统、Lac操纵系统、Tac操纵系统和Tet操纵系统；抗毒蛋白的编码基因由目的基因定向进化后识别并启动表达；根据不同目的蛋白要求，TAS上还含有表达辅助识别或结合的蛋白。
作为本发明的优选方案，所述程序化死亡模块TAS上毒蛋白的编码基因包括但不限于可引起细胞破裂的YdfD等；阻遏细胞膜生成的PezT、SezT、zeta toxin等；对DNA的复制有抑制作用的FicT、CcdB等；对翻译有抑制作用的TacT等；抗毒蛋白的编码基因选自与毒蛋白对应的DicB/SulA、PezA、SezA、epsilon antitoxin、FicA、CcdA、TacA等。
在一种实施方式中，所述辅助识别或结合的蛋白包括但不限于激活型及抑制型的转录因子，如lacI，psiR，Lrp,LysG,PcaR,CadR,PadR,NanR,PcaU,BmoR,TgtR,EmrR,FdeR,FrmR,DmpR,BenR,FadR,SoxR,Alks,PobR。
在一种实施方式中，所述随机突变模块MP、目的基因表达模块TP和程序化死亡模块TAS的表达载体包括但不限于pET系列，或pSB1C3，或pRSFDuet，或pCDFDuet质粒。
在一种实施方式中，所述随机突变模块MP、目的基因表达模块TP和程序化死亡模块TAS的表达载体各不相同。
本发明第二个目的是提供一种基因连续定向进化方法，将所述连续定向进化系统转化至微生物细胞。
在一种实施方式中，所述微生物细胞包括但不限于大肠杆菌。
在一种实施方式中，所述方法为通过诱导剂诱导微生物细胞，分别诱导随机突变模块MP表达诱变蛋白、目的基因表达模块TP表达目的蛋白和程序化死亡模块TAS表达辅助识别或结合的蛋白和毒蛋白；对于不同的目的蛋白，还需加入相应的底物。
在一种实施方式中，将诱导后的微生物细胞在含有诱导剂的培养基中转接或连续培养。
在一种实施方式中，所述方法的诱导剂是包括但不限于诱导剂IPTG、L-ara。
本发明还保护所述连续定向化系统或上述方法在蛋白质改造中的作用。
有益效果：
本发明的体内连续定向进化系统对目的基因具有靶向性，诱变蛋白能准确识别并介导目的基因产生随机突变，突变准确定位于目的基因上。且该系统适用于在大肠杆菌体内进行筛 选，所选作用对象较为广泛，便于大部分实验室推广使用。
附图说明
图1体内连续定向进化的流程示意图。
图2随机突变模块MP的质粒图谱。
图3目的基因表达模块TP的质粒图谱。
图4程序化死亡模块TAS的致死效果的生长曲线对比图。
图5程序化死亡模块TAS的致死效果的细胞染色对比图。
图6进化筛选产物与对照组在筛选平板上生长形成单菌落数的对比图。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。
如图1所示，本发明一个实施方案的大肠杆菌中的连续定向进化系统包括随机突变模块MP、目的基因表达模块TP、程序化死亡模块TAS。其中，程序化死亡模块TAS中抗毒蛋白的表达与目的基因表达模块TP中突变后的目的蛋白质功能相偶联。即成功进化后的目的蛋白可以直接启动或调控TAS中抗毒蛋白的表达；或者成功进化后的目的蛋白可以分解或者合成某种物质，以此启动或调控TAS中抗毒蛋白的表达。在图1所举实例中，获得的T7 RNAp突变体可以特异性识别抗毒蛋白的T7(R13)启动子，并启动抗毒蛋白的转录，以拮抗毒蛋白对宿主菌的作用；未发生突变或非优势的T7 RNAp突变体则无法识别T7(R13)启动子，导致抗毒蛋白无法正常表达，无法拮抗毒蛋白而导致细胞被毒蛋白裂解。
本领域的技术人员应当理解，本发明并不局限于上述以“T7 RNAp的编码基因”作为定向进化的实施例，其他任何类似的技术方案均适用于本发明。当针对不同的目的基因，如糖酶基因、角质酶基因、酯酶基因、荧光蛋白基因，核酸酶基因等时，可以通过不同的原理将它们的功能与程序化死亡模块TAS中抗毒蛋白的表达偶联起来。
本发明将列举几项可以应用于连续定向进化的目的基因实例，需明确的是，在本发明中可供选择的目的基因有很多，并不局限于以下几种：(1)DNA结合蛋白：如可以将抗毒蛋白转录因子的进化与抗毒蛋白的表达相偶联等；(2)作用于小分子的蛋白：如将部分糖酶、角质酶或其他参与代谢或非代谢物生物合成的酶的反应产物与抗毒蛋白的表达相偶联等；(3)荧光蛋白：如利用光敏性启动子将荧光蛋白与抗毒蛋白的表达相偶联等；(4)核酸酶，如Cas9:通过相应的基因调控将Cas9功能与抗毒蛋白的表达相偶联等。
需说明，图1中的程序化死亡模块TAS仅为一种实施例，根据TAS中毒蛋白的毒性机制不同，可适用于本发明的毒蛋白-抗毒蛋白系统可以有多种选择且不局限于以下几种：(1) 可以抑制DNA复制、转录等功能的毒蛋白：如来源于P.aeruginosa的FicT/FicA，FicT通过腺苷化修饰DNA旋回酶和拓扑异构酶IV，使细胞DNA打结、连接和松弛而导致可逆的细胞生长停止；(2)对翻译有抑制作用的毒蛋白：如来源于Salmonella的TacT/TacA，TacT是一种乙酰转移酶，它可以阻断带电tRNA分子上氨基酸的伯胺基，从而抑制翻译和促进存留细胞的形成；(3)影响细胞分裂的毒蛋白：如来源于大肠杆菌的YdfD，YdfD在诱导2小时内可溶解99.9％的细胞，而细胞分裂抑制剂的SulA可以消除YdfD诱导的细胞裂解；(4)影响肽聚糖合成的毒蛋白：如来源于Streptococcus Suis Serotype的SezT/SezA，Streptococcus Pneumoniae来源的PezA/T，其可以抑制肽聚糖的合成，最终引起细菌自溶。另外，任意可以引起细胞死亡的毒蛋白以及能够拮抗该毒蛋白的抗毒蛋白均可作为本发明中的程序化死亡模块使用。
在本发明的一个实施例中，负责突变的诱变蛋白为胞苷脱氨酶(AID)，AID能作用于单链DNA，可以使胞嘧啶脱氨基转化为尿嘧啶最终转化为胸腺嘧啶；T7 RNAp与AID融合表达，负责使AID可以靶向性地结合在目的基因表达模块TP上T7启动子的下游，使得诱变蛋白对目的基因具有靶向性。除该实施例外，其他具有靶向性的诱变蛋白、诱变蛋白突变体或诱变融合蛋白也可以作为本发明中的自发随机突变模块MP使用。例如但不限于DNA聚合酶PolⅠ(D355A/E357A或D424A/I709N/A759R)，其为一种保真性低的DNA聚合酶突变体，可以特异性识别具有ColE1 Ori的质粒并能使该转录起始位点后一定长度的核苷酸序列发生突变。
本发明提供的定向进化方法，包括：在诱导剂的作用下，系统中随机突变模块MP表达诱变蛋白，其会识别并介导目的基因表达模块TP中目的基因产生随机突变；目的基因突变后，再向系统中加入诱导毒蛋白表达的诱导剂使系统开始定向筛选；成功进化的目的蛋白突变体可以介导程序化死亡模块TAS上抗毒蛋白的表达，使毒蛋白无法作用于宿主细胞，最终只有包含成功进化的目的基因的宿主细胞可以存活。
通过以下实施例说明本发明的可行性，需要说明的是，以下实施例仅是示例性的，并且其目的仅在于说明本发明的可行性，并不意在限制本发明的保护范围。
下述实施例中涉及的质粒和菌株来源如下：
pET20b、pRSFDuet、pCDFDuet质粒购自TaKaRa(大连)生物工程有限公司；大肠杆菌(Escherichia coli)BL21(DE3)由本实验室保藏。
下述实施例中涉及的试剂和培养基如下：
下述实施例中涉及的PrimeSTAR HS DNA聚合酶、DNA Marker、Dpn I限制性内切酶等 购自TaKaRa(大连)生物工程有限公司。
LB培养基：0.5％(W/V)酵母浸粉，1％(W/V)胰蛋白胨，1％(W/V)氯化钠，添加2％(W/V)琼脂粉即为LB固体培养基。
TB培养基：1.2％(W/V)胰蛋白胨、2.4％(W/V)酵母浸粉、0.4％(V/V)甘油、磷酸盐缓冲液(17mM KH 2PO 4、72mM K 2HPO 4)。
实施例1：随机自发突变模块MP的构建
具体步骤如下：
以AID-T7 RNAp介导的随机自发突变模块MP为例。合成核苷酸序列如SEQ ID NO.1所示的融合蛋白编码AID-T7 RNAp(T3)(氨基酸序列如SEQ ID NO.2所示)，其中T7 RNAp(T3)表示T7 RNAp的T3型突变体。通过酶切连接的方式，将AID-T7 RNAp(T3)连接至大肠杆菌表达载体pCDFDuet构建重组载体pCDFDuet-pT7-AID-T7 RNAp(T3)。将连接产物转化至大肠杆菌JM109中，转化产物涂布于LB固体培养基(含终浓度50μg·mL -1链霉素)上。37℃倒置培养10～12h之后，挑取平板上的转化子接至LB液体培养基(含终浓度50μg·mL -1链霉素)中。37℃、200rpm培养12～14h后，提取质粒测序验证，作为随机突变模块MP的一种，并命名为pCDFuet-1-AID-T7 RNAp(T3)(如图2所示)。
实施例2：程序化死亡模块TAS的构建
具体步骤如下：
选取毒蛋白-抗毒蛋白系统(TAS)YdfD/SulA，合成其序列，氨基酸序列如SEQ ID NO.3和SEQ ID NO.4所示。将其核苷酸序列分别导入pRSFDuet的两个多克隆位点中，并使用MEGAWHOP克隆的方法将载体中表达毒蛋白的T7启动子替换为pBAD操纵系统。对PCR产物进行Dpn I消化后，将其转化至大肠杆菌JM109中，转化产物涂布于LB固体培养基(含终浓度50μg·mL -1卡那霉素)上。37℃倒置培养10～12h之后，挑取平板上的转化子接至LB液体培养基(含终浓度50μg·mL -1卡那霉素)中。37℃、200rpm培养12～14h后，提取质粒并将其送至测序公司进行测序。测序结果正确的质粒即为重组载体pRSFDuet–pT7–antitoxin–araC–pBAD–toxin。
实施例3：大肠杆菌中程序化死亡模块TAS的鉴定
具体步骤如下：
将毒蛋白-抗毒蛋白表达载体pRSFDuet–pT7–antitoxin–araC–pBAD–toxin转入大肠杆菌BL21(DE3)中，并将其接入LB培养基中诱导发酵。首先，将保存于-80℃冰箱的甘油菌接入10mL LB种子液(含终浓度50μg·mL -1卡那霉素)中，37℃、200rpm过夜培养。将种子液 以1：20的比例转接至50mL LB发酵液(含终浓度50μg·mL -1卡那霉素)中，37℃、200rpm、1.5h后加入诱导剂IPTG、L-ara诱导发酵(另做一组只加IPTG、一组只加L-ara、一组不加诱导剂的作为对照)。加入诱导剂后，测定重组菌生长情况。结果如图4所示，当只存在诱导剂L-ara时，诱导毒蛋白的表达，能有效介导菌株死亡；当同时存在诱导剂IPTG、L-ara，因为IPTG解除lac I对T7类启动子的抑制诱导T7启动子启动抗毒蛋白的表达，菌株生长几乎不受影响。
诱导6h后，用生理盐水清洗并重悬菌体。在重悬液中加入PI/SYTO9核酸染料染色，用激光共聚焦显微镜观察并鉴定重组菌的死活情况。结果如图5所示，染色实验进一步证明了，当只存在诱导剂L-ara时，pBAD操纵系统诱导毒蛋白的表达，能有效介导菌株死亡；当同时存在诱导剂IPTG、L-ara，因为IPTG解除lac I对T7类启动子的抑制诱导T7启动子启动抗毒蛋白的表达，菌株生长几乎不受影响。
上述实验共同验证了大肠杆菌中程序化死亡模块TAS中毒蛋白和抗毒蛋白的表达是受诱导剂IPTG、L-ara和启动子调控的。
利用实施例3的鉴定方法，通过点突变的方式将表达抗毒蛋白的T7启动子突变为T7(R13)启动子(T7(R13)启动子序列见文章MeyerAJ,Ellefson JW,EllingtonAD.Directed Evolution of a Panel of Orthogonal T7 RNA Polymerase Variants for in Vivo or in Vitro Synthetic Circuitry[J].Acs Synthetic Biology,2015,4(10).：Table 1“P CGTA”)，以进行后续含目的基因定向进化的实施。
实施例4：大肠杆菌中目的基因表达模块TP的构建
具体步骤如下：
以大肠杆菌BL21(DE3)基因组为模板，使用引物T7RNApRF-F和T7RNApRF-R扩增得到T7 RNAp的编码基因作为目的基因，利用MEGAWHOP克隆的方法将其连接至大肠杆菌表达载体pET20b上。将含有T7 RNAp的编码基因的重组表达载体pET20b进行Dpn I消化后，转化至大肠杆菌JM109中，转化产物涂布于LB固体培养基(含终浓度100μg·mL -1氨苄霉素)上。37℃倒置培养10～12h之后，挑取平板上的转化子接至LB液体培养基(含终浓度100μg·mL -1氨苄霉素)中。37℃、200rpm培养12～14h后，提取质粒进行测序验证。测序结果正确的质粒即为重组载体pET20b-T7 RNAp(如图3所示)。以点突变的方式将重组载体上的T7启动子突变为T7(T3)启动子，其中T7(T3)启动子表示仅能被随机突变模块MP上辅助基因T7 RNAp(T3)识别的启动子(T7(T3)启动子序列见文章Meyer A J,Ellefson J W,Ellington A D.Directed Evolution of a Panel of Orthogonal T7 RNA Polymerase Variants for in Vivo or in Vitro Synthetic Circuitry[J].Acs Synthetic Biology,2015,4(10).：Table 1“P T3”)。所得 重组质粒pET20b-pT7(T3)-T7 RNAp为目的基因表达模块TP。
引物T7RNApRF-F:CTTTAAGAAGGAGATATACATATGAACACGATTAACATCGC；
引物T7RNApRF-R：TGGTGGTGGTGGTGCTCGAGTTACGCGAACGCGAAGTCC。
实施例5：大肠杆菌中各个模块的组装与表征
具体步骤如下：
1)将随机自发突变模块MP、目的基因表达模块TP与程序化死亡模块TAS依次转入大肠杆菌BL21(DE3)中，37℃、200rpm过夜培养。将种子液以1：20的比例转接至50mL TB发酵液中，37℃、200rpm、1.5h后加入诱导剂IPTG和L-ara(终浓度分别为0.4mM和0.3％)进行连续诱导发酵。将诱导后的菌液稀释一定倍数后，涂板(含0.3％L-ara)，计算单菌落数(另作一组未加L-ara的作为对照)。结果如图6所示，将平板上能够生长的单菌落进行目的基因测序，最终得到T7 RNAp的R13型突变体T7 RNAp(R13)。最终突变效率为4x10 -5～-6/bp，筛选周期6天，突变型菌株的生理代谢未受到影响。
2)将随机自发突变模块MP、目的基因表达模块TP与程序化死亡模块TAS依次转入大肠杆菌BL21(DE3)中，37℃、200rpm过夜培养。将种子液以1：20的比例转接至50mL TB发酵液中，37℃、200rpm、1.5h后加入诱导剂IPTG(终浓度0.4mM)进行连续诱导发酵，发酵2～4h后加入诱导剂L-ara(终浓度0.3％)。将诱导后的菌液稀释一定倍数后，涂板(含0.3％L-ara)，计算单菌落数(另作一组未加L-ara的作为对照)。结果如图6所示，将平板上能够生长的单菌落进行目的基因测序，最终得到T7 RNAp的R13型突变体T7 RNAp(R13)。最终突变效率为4x10 -5～-6/bp，筛选周期6天，突变型菌株的生理代谢未受到影响。
对比例1
目的基因表达模块TP上不含有目的基因T7 RNAp时，重组菌添加诱导剂经过连续传代培养后将菌液涂布在含有L-ara的平板上，37℃过夜培养后没有菌落长出。
对比例2
随机自发突变模块MP上不含有诱导基因AID-T7 RNAp(T3)时，重组菌添加诱导剂经过连续传代培养后将菌液涂布在含有L-ara的平板上，37℃过夜培养后没有菌落长出。
对比例3
目的基因表达模块TP上不含有被辅助基因识别或结合的序列时，重组菌添加诱导剂经过连续传代培养后将菌液涂布在含有L-ara的平板上，37℃过夜培养后没有菌落长出。
以上实施例是以将识别T7启动子的T7 RNAp进化为识别T7(R13)启动子突变型的T7  RNAp突变体T7 RNAp(R13)为例对本发明进行说明。用其它目的基因(如糖酶基因、角质酶基因、核酸酶基因、荧光蛋白基因等)替换TP中的T7 RNAp基因，并TAS质粒中的抗毒蛋白基因的表达调控和表达后的修饰方式进行相应的调整，使得抗毒蛋白的表达与TP上新目的基因的生物活性偶联在一起，就可以使用本系统对新的目的基因进行定向进化。此外，对随机突变模块MP中的诱变蛋白编码基因进行替换和改造也包括在本发明的说明范畴内。
实施例6 D-阿洛酮糖3-差向异构酶(D-psicose 3-epimerase,DPE)的体内连续进化系统构建
以Promoter-F-CTTACATTAATTGCGTTGCGCCCGCTTCTAGAGGAGCTGTTGAC，promoter-R-GATATTTTTGCCGATCCCCATTGATCTTTTCTCCTCTTTTCCTCC为上下游引物，以pET20b-psir-ppsiA为模板，利用PCR扩增转录因子-启动子psir-ppsiA基因片段(核苷酸序列如SEQ ID NO.5)，对PCR产物进行纯化后作为Mega primer，以实施例2中的pRSFDuet–pT7–antitoxin–araC–pBAD–toxin为模板，通过MEGAWHOP将转录因子-启动子psir-ppsiA构建至pRSFDuet–pT7–antitoxin–araC–pBAD–toxin获得重组质粒pRSFDuet-pT7-psir-ppsiA-antitoxin-araC-pBAD-toxin，作为程序化死亡模块TAS；
以DPE-F-TAAGAAGGAGATATACATCGAGGATGAAACATGGCATCTATT和DPE-R-CCTGGGCATGCCGCTTCAGTGGTGGTGGTGGTGGTG为引物，以pET20b-dpe为模板，利用PCR扩增得到DPE的编码基因作为目的基因(核苷酸序列如SEQ ID NO.6)，以实施例4获得重组质粒pET20b-pT7(T3)-T7 RNAp为模板，利用MEGAWHOP克隆的方法将T7 RNAp替换为DPE，获得含有DPE目的基因的质粒pET20b(+)-pT7(T3)-dpe，作为目的基因表达模块TP；
实施例1中的pCDFuet-1-AID-T7 RNAp(T3)作为突变质粒MP。将MP、TP及TAS共转化到宿主细胞E.coli BL21(DE3)中，得到重组菌作为DPE体内连续进化系统。
实施例7 DPEase体内连续进化筛选
接种实施例6中获得的重组菌至10mL LB(含50μg/mLAmp、25μg/mL Sm、25μg/mL Kana)的摇瓶中，于37℃、200r/min培养8-10h后获得种子液。将种子液按10％(v/v)的接种量转接至20mL TB(含50μg/mLAmp、25μg/mL Sm、25μg/mL Kana)培养基中，于37℃、200r/min培养1h后，加入诱导剂终浓度0.4mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)，于25℃、200r/min培养12h后获得培养液，将培养液按10％(v/v)的接种量转接至新的20mL TB培养基，该过程一共持续8轮诱导发酵。其中，从第6轮开始，在诱导过程中添加终 浓度3mg/mL的L-ara诱导毒蛋白的表达，同时添加0.5M D-果糖作为DPE底物。
取积累突变后的培养液，按5％(v/v)的接种量接种至含有0.5M D-果糖的50mL LB(含50μg/mLAmp、25μg/mL Sm、25μg/mLKana)培养基中，于37℃、200r/min培养1h后，加入诱导剂0.4mmol/L IPTG、3mg/mL L-ara，于25℃、200r/min培养5.5h后，取1mL混匀的菌液，使用1mL无菌PBS洗涤，在4℃、8000r/min下离心1min后，去除上清并重悬于1mLPBS中，使用PBS将菌悬液稀释成一定的梯度。将稀释到一定梯度的菌液涂布于LB(含50μg/mL Amp、25μg/mL Sm、25μg/mL Kana、0.5M D-果糖、0.4mmol/L IPTG、3mg/mL L-ara)琼脂平板上，于37℃下培养12h得到突变体的单克隆。经测序及摇瓶验证后，确定为可溶性表达显著提高的突变体。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (15)

  1. 一种连续定向进化系统，其特征在于，包含随机突变模块MP、程序化死亡模块TAS以及目的基因表达模块TP；所述随机突变模块MP上含有诱变基因和辅助基因；目的基因表达模块TP上含有目的基因和被所述辅助基因识别或结合的元件；所述程序化死亡模块TAS上含有编码毒蛋白的基因和相对应的编码抗毒蛋白的基因。
  2. 根据权利要求1所述的系统，其特征在于，所述随机突变模块MP上的诱变基因包括但不限于低保真性的DNA聚合酶I突变体的编码基因PolA、胞嘧啶脱氨酶的编码基因AID、胞嘧啶脱氨酶的编码基因APOBEC、腺嘌呤脱氨酶的编码基因TadA中的一种或多种。
  3. 根据权利要求1或2所述的系统，其特征在于，所述随机突变模块MP上的辅助基因包括但不限于T7 RNA聚合酶的编码基因、缺少切割非互补链活性的nCas9的编码基因、仅具有DNA结合能力的dCas9的编码基因等基因中的一种或多种。
  4. 根据权利要求1所述的系统，其特征在于，所述目的基因表达模块TP上的目的基因为一种或多种蛋白的编码基因和/或非编码基因。
  5. 根据权利要求1或4所述的系统，其特征在于，所述目的基因包括但不限于T7 RNA聚合酶的编码基因、抗生素的抗性基因、分解代谢途径中的酶的编码基因、合成代谢途径中的酶的编码基因、DNA结合蛋白的编码基因、核酸酶的编码基因、糖酶的编码基因、蛋白酶的编码基因中的一种或多种。
  6. 根据权利要求1所述的系统，其特征在于，所述目的基因表达模块TP上被辅助基因识别或结合的元件包括但不限于tac启动子、pac启动子、Sp6启动子、lac启动子、T7启动子、pBAD启动子、trc启动子、npr启动子以及sgRNA。
  7. 根据权利要求1所述的系统，其特征在于，诱导程序化死亡模块TAS上毒蛋白的编码基因表达的启动子为诱导型启动子，包括但不限于pBAD操纵系统、Lac操纵系统、Tac操纵系统和Tet操纵系统；抗毒蛋白的编码基因由目的基因定向进化后识别并启动表达；根据不同目的蛋白要求，TAS上还含有表达辅助识别或结合的蛋白。
  8. 根据权利要求7所述的系统，其特征在于，所述程序化死亡模块TAS上毒蛋白的编码基因包括但不限于可引起细胞破裂的YdfD；阻遏细胞膜生成的PezT、SezT、zeta toxin；对DNA的复制有抑制作用的FicT、CcdB；对翻译有抑制作用的TacT；抗毒蛋白的编码基因选自与毒蛋白对应的DicB/SulA、PezA、SezA、epsilon antitoxin、FicA、CcdA、TacA基因。
  9. 根据权利要求7所述的系统，其特征在于，所述辅助识别或结合的蛋白包括但不限于激活型及抑制型的转录因子，如lacI，psiR，Lrp,LysG,PcaR,CadR,PadR,NanR,PcaU,BmoR,TgtR,EmrR,FdeR,FrmR,DmpR,BenR,FadR,SoxR,Alks,PobR。
  10. 根据权利要求1所述的系统，其特征在于，所述随机突变模块MP、目的基因表达模 块TP和程序化死亡模块TAS的表达载体包括但不限于pET系列，或pSB1C3，或pRSFDuet，或pCDFDuet质粒。
  11. 含有权利要求1～10任一所述定向进化系统的微生物细胞；所述微生物细胞包括但不限于大肠杆菌。
  12. 一种基因连续定向进化方法，其特征在于，将权利要求1～10任一所述的连续定向进化系统转化至微生物细胞；所述微生物细胞包括但不限于大肠杆菌。
  13. 根据权利要求12所述的一种基因连续定向进化的方法，其特征在于，通过诱导剂诱导微生物细胞，分别诱导随机突变模块MP表达诱变蛋白、目的基因表达模块TP表达目的蛋白和程序化死亡模块TAS表达辅助识别或结合的蛋白和毒蛋白；对于不同的目的蛋白，还需加入相应的底物。
  14. 根据权利要求12所述的一种基因连续定向进化的方法，其特征在于，将诱导后的微生物细胞在含有诱导剂的培养基中转接或连续培养。
  15. 权利要求1～10任一所述连续定向化系统或权利要求12～14任一所述方法在蛋白质改造中的应用。
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