WO2017128509A1 - 大肠杆菌细胞内原位进化目标蛋白质的新方法 - Google Patents

大肠杆菌细胞内原位进化目标蛋白质的新方法 Download PDF

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WO2017128509A1
WO2017128509A1 PCT/CN2016/077246 CN2016077246W WO2017128509A1 WO 2017128509 A1 WO2017128509 A1 WO 2017128509A1 CN 2016077246 W CN2016077246 W CN 2016077246W WO 2017128509 A1 WO2017128509 A1 WO 2017128509A1
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escherichia coli
situ
dxs
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徐志南
濮悦
黄磊
董昌
卢建刚
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Zhejiang University ZJU
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Definitions

  • the present invention relates to a novel method for the in situ evolution of proteins in E. coli cells, and belongs to the field of biotechnology.
  • Directed evolution technology is a simulation of Darwinian evolution in the laboratory, the evolution of proteins that do not exist in nature or have better properties, and can change the metabolic flow by changing the catalytic efficiency of the enzyme, expanding or constructing new metabolic pathways. To weaken or eliminate unnecessary or harmful metabolic pathways, thereby achieving the purpose of increasing the yield of certain metabolic products or degrading harmful substances.
  • Directed evolution technology has two main steps: firstly, using modern molecular biology methods to construct a mutant library of genes, and then coupling screening methods to screen the library. The construction of mutant libraries can provide sufficient diversity for screening and is a key step in directed evolution. Techniques for constructing mutant libraries can be classified into in vitro mutations and in vivo mutations, depending on where the DNA mutation occurs.
  • In vitro mutation techniques include error prone PCR, DNA shuffling, Staggered extension process (StEP), Site directed saturation mutagenesis (SDSM), iterative saturation mutation (Iterative) Saturation mutagenesis, ISM), etc.
  • Error prone PCR is a random introduction of point mutations by changing the polymerase chain reaction conditions;
  • DNA shuffling is an in vitro recombination technique for DNA molecules by using a set of sequence-related DNA sequences.
  • DNase I was randomly spliced into small fragments, followed by PCR remerization to achieve gene rearrangement to obtain a mutant library, and then screened for the gene with the expected trait, which can achieve 70% homology sequence recombination with the parent sequence;
  • Staggered Extension Process (StEP) is a simplified DNA shuffling technique.
  • SDSM Site directed saturation mutagenesis
  • ISM Iterative saturation mutagenesis
  • the mutated gene must be linked to the corresponding vector by digestion.
  • the mutated gene is transcribed and translated into a corresponding protein to exhibit a certain phenotype, and the corresponding screening technique can be coupled to screen the mutant library.
  • the steps of enzymatic cleavage and transformation are complicated and complicated, and cannot be, continuously evolved, cannot spontaneously evolve, and require manual intervention.
  • PACE phage-assisted continuous evolution
  • MAGE multi-site automated genome editing
  • Physical and chemical mutagenesis is the use of physical, chemical and other factors to deal with organisms to induce mutations in genetic material, thus A variation in morphological characteristics.
  • This traditional mutagenesis method has low mutation efficiency and no target.
  • Harvard University David R Liu Lab developed a continuous evolutionary system based on phage growth, which is expressed in E. coli.
  • the mutated DNA polymerase produces mutations and establishes a screening system in combination with M13 phage to automate directed evolution.
  • the mutation efficiency of PACE technology is only 10 -5 , and saturation mutations are achieved at 3 sites randomized to a 1 kb gene. Almost impossible, in addition, PACE technology can only evolve on one gene.
  • MAGE Multiple Automated Genome Transformation Technology
  • MAGE Multiple Automated Genome Transformation Technology
  • It also designs a series of single-stranded oligonucleotide degenerate primers for different regions of the genome, and integrates these degenerate primers into the genome using the ⁇ -Red homologous recombination system to achieve transformation of multiple sites in a single cell genome.
  • Diversity of genome modifications between cell populations They designed an automated operating device that would enable the process for the periodicity and scalability of the technology.
  • TAAGGAGGT classical SD sequence
  • the present invention relates to a novel method for the in situ evolution of proteins in E. coli cells and for the enhancement of the catalytic performance of one or more enzymes to increase the yield of specific target metabolites of the microorganism.
  • nucleic acid library comprising one or more mutation sites, including an in vitro synthesized oligonucleotide single-stranded library or a prepared single-stranded DNA library or a double-stranded DNA library, by ⁇ -Red homology
  • the recombinant technology utilizes the nucleic acid library to recombine the structural gene on the intracellular vector or the genome of the E. coli to achieve the purpose of evolving the molecular structure of the enzyme to improve the catalytic efficiency of the enzyme.
  • Oligonucleotides can be obtained by chemical synthesis; single-stranded nucleotides can be obtained by, but not limited to, T7 reverse transcription, exonuclease, denaturing high performance liquid chromatography, magnetic bead capture, asymmetric PCR , two-step PCR method, etc.; double-stranded nucleotides can be synthesized by PCR.
  • Oligonucleotides and single-stranded nucleotides may be modified at bases 1 to 4 of their 5' ends; double-stranded nucleotides are modified at positions 1 to 4 of the 5' end of one of the strands, Avoid degradation by nucleases after transformation into cells. Modifications include, but are not limited to, 5' bromouracil modification, 5' chlorouracil modification, 5' iodine uracil modification, phosphate modification, thiophosphoric acid modification, and the like.
  • Oligonucleotides, single-stranded nucleotides, and double-stranded nucleotides can be introduced into cells by transformation or transfection. Transformation and transfection as referred to herein refers to some techniques for the mature introduction of exogenous nucleotides into a target cell, including but not limited to calcium phosphate method, calcium chloride method, lipofection, electroporation, light Perforation method, etc. Transfection media include, but are not limited to, water, calcium chloride, liposomes, and the like.
  • E. coli cells were cultured to a concentration at 30 °C prior to transformation or transfection and induced at 42 °C.
  • the ⁇ -Red homologous recombination system on the genome of the cell is under the control of the pL promoter and is regulated by the cI857 repressor protein.
  • the pL promoter was repressed and not expressed at 30 ° C, and induced three proteins required for expression of ⁇ -Red homologous recombination at 42 °C. After 15 min induction, the cells were stored at 4 ° C to prevent induced protein degradation.
  • the medium needs to be replaced with a transfection medium to remove ions from it.
  • the manner in which this process can be employed includes, but is not limited to, centrifugation, membrane filtration, and the like.
  • a nucleic acid library is added, and a resistance-recovering nucleotide fragment is introduced, which can be screened on the resistant plate and the recombination efficiency of the position near the resistance gene is increased.
  • Suitable resistance genes include, but are not limited to, an ampicillin resistance gene, a tetracycline resistance gene, a kanamycin resistance gene, a neomycin resistance gene, a chloramphenicol resistance gene, and the like.
  • Screening markers include, but are not limited to, various enzymes, helper groups, fluorescent labels, luminescent labels, bioluminescent groups, and the like.
  • Fluorescent labels include, but are not limited to, yellow fluorescent protein, green fluorescent protein, cyan fluorescent protein, rhodamine, and the like.
  • Bioluminescent groups include, but are not limited to, luciferase, luminescent proteins, and the like.
  • Some enzymes that produce visual signals include, but are not limited to, galactosidase, phosphatase, peroxidase, cholinesterase, and the like.
  • the present invention provides an in vivo mutation method for achieving a high mutation rate in vivo and mutating one or more genes on the genome, and mutating the entire genome of Escherichia coli by repeated ⁇ -Red homologous recombination strategy.
  • the modification of structural genes achieves the in situ evolutionary purpose of the genes corresponding to functional proteins. This method can evolve a single gene on the genome, or modify multiple genes at the same time, and couple them to find the best effect achieved by the synergy of multiple mutant proteins.
  • the novel method for in situ evolution of proteins in E. coli cells provided by the present invention is a method for efficiently obtaining desired biomolecules or biological substances, which is simpler and more efficient than the conventional methods.
  • the novel method for in situ evolution of proteins in Escherichia coli cells provided by the present invention can improve the thermal stability and catalysis of pyrroloquinoline-dependent glucose dehydrogenase by in situ saturation mutation of key amino acids of E. coli intracellular target genes. Specificity.
  • the present invention provides a novel method for in situ evolution of proteins in Escherichia coli cells, which can be applied to in situ recombinant mutant lycopene synthesis key enzyme 1-deoxy-D-xylulose-5-phosphate synthase Genes that increase the level of solubility and catalytic efficiency of their expressed proteins.
  • 1-deoxy-D-xylulose-5-phosphate synthase (DXS) mutants were obtained by screening.
  • the soluble expressions of the mutants DXSa (S217P, L226H), DXSb (K214E, K227E, G512H), DXSc (E230G) and DXSd (Y115H, E182A, L226R, F435S) were increased by 2 times compared with the wild-type DXS protein. the above.
  • the specific enzyme activities of DXS mutants (DXSa, DXSb, DXSd and DXSe (I170T K213E, N249S)) were significantly improved, thereby increasing the biosynthesis lycopene level of E. coli containing the corresponding mutant gene by 2-5 times.
  • the invention provides a novel method for in situ protein evolution in E. coli cells, which method can simultaneously mutate a plurality of lycopene key enzyme genes to increase the production level of E. coli synthetic lycopene.
  • the present invention also provides Escherichia coli strain EcLYCb, which stores the Escherichia coli strain numbered CCTCC M 2015691, and the soluble expression level and enzyme catalytic efficiency of the strain are significantly improved.
  • the invention also provides Escherichia coli strain EcLYC246, save number CCTCC M 2015692, which improves the production level of lycopene synthesized by Escherichia coli.
  • Figure 1 Schematic diagram of the comparison between the process of the present invention and conventional DNA rearrangement techniques.
  • DNA Shuffling Method DNA rearrangement technology
  • DNase I Digestion DNase I digestion
  • cycles of denaturation, annealing, extension multiple denaturation, ligation, extension
  • Cloning, Transformation cloning, transformation
  • Gene gene
  • Prone PCR error-prone PCR
  • Asymmetric PCR asymmetric PCR
  • Synthetic Oligos synthetic oligonucleotides
  • cycles of Recombination multiple recombination
  • Genome genome
  • E. coli E. coli.
  • Figure 2 Thermal stability analysis of wild-type and GDH mutants at 55 °C.
  • Figure 3 Comparison of soluble expression of wild-type and DXS mutant proteins. Among them, Comparison of Soluble DXS Expression: comparison of soluble DXS protein expression; Marker: protein molecular weight standard size.
  • Figure 4 Comparison of specific enzyme activities of wild-type and DXS mutant proteins. Among them, Retention time: retention time; Specific enzyme activity: than enzyme activity.
  • Figure 5 Comparison of the ability of wild type and E. coli strains containing dxs mutant to produce lycopene; among them, Lycopene Production: lycopene production.
  • Figure 6 Comparison of the ability of wild type and E. coli strains containing dxs mutant to produce lycopene; among them, Lycopene Production: lycopene production.
  • Figure 7 Comparison of the ability of wild-type and E. coli strains containing multiple gene mutations to produce lycopene; among them, Lycopene Production: lycopene production.
  • Figure 8 Comparison of the ability of wild type and E. coli strains containing multiple gene mutations to produce lycopene; among them, Lycopene Production: lycopene production.
  • Fig. 1 The operation flow of the present invention is shown in Fig. 1, and is better illustrated by the following five examples, respectively, to improve the thermal stability and catalytic specificity of pyrroloquinoline-dependent glucose dehydrogenase, and to improve 1-deoxy-D-wood
  • Example 1 in situ evolution of pyrroloquinoline-dependent glucose dehydrogenase in Escherichia coli
  • Escherichia coli EcNR2 (Harris H. Wang et al. 2009 Nature. 460: 894-890) can express the desired protein of ⁇ -Red recombination under induction of temperature conditions, exhibiting high recombination efficiency for 80-100 nt oligonucleotides.
  • a single colony of Escherichia coli EcGDH picked from the plate was placed in a shake flask of 50 mL LB medium, and 25 ⁇ L of kanamycin was added thereto, and cultured overnight at 30 ° C under a shaker;
  • the tube was placed in a 42 ° C water bath and shaken for 15 min to induce the full expression of ⁇ -Red recombinant protein;
  • test tube was placed on ice for 5 min;
  • the supernatant was removed, and the cells were resuspended in 100 ⁇ L of pre-cooled sterile water, and an oligonucleotide mutation library and a resistance-recovering oligonucleotide were added;
  • the above mixture was transferred to a 2 mm electric rotor, and an electric shock was performed under the conditions of a capacitance of 25 ⁇ F, a voltage of 2.5 kV, and a resistance of 200 ⁇ ;
  • the prepared 1 ml SOC medium was quickly added to the electric shock cup, and the cells were suspended by gently blowing.
  • the mixture in the electric shock cup was transferred to a test tube containing 1 ml of the medium, and placed in a shaker at 30 ° C to resuscitate the cells;
  • the substrate specificity of the GDH mutants in the Escherichia coli strains EcGDHa, EcGDHb, EcGDHc, EcGDHd was improved. Relative activity to lactose and maltose was reduced by 28% to 42% relative to GDH in wild-type E. coli.
  • the thermal stability of the GDH mutants in the E. coli strains EcGDHb and EcGDHf was 1.69 and 1.6 times higher, respectively, relative to wild-type GDH.
  • Double-stranded nucleotides have a relatively low efficiency of ⁇ -Red homologous recombination in E. coli, only 0.01%. It was found that E. coli EcNR2 (Harris H. Wang et al. (2009) Nature. 460: 894-890) can express the protein required for ⁇ -Red recombination under temperature conditions, and can be significantly improved by co-transformation with resistant fragments. Reorganization efficiency.
  • 1-Deoxy-D-xylulose-5-phosphate synthase is a key enzyme in the isoprene synthesis pathway in Escherichia coli.
  • 1-Deoxygenation is synthesized using 3-phospho-glyceraldehyde and pyruvate as substrates.
  • -D-xylulose-5-phosphate The exogenous gene crtE, crtB, crtI was introduced into E. coli EcNR2, so that the Escherichia coli can produce lycopene, which is named EcLYC. Since lycopene has a visible red color, it is possible to screen high-throughput strains with increased lycopene production based on the depth of the red color of the colonies.
  • the upper primer dxs-1-for (SEQ ID NO. 4), dxs-2-for (SEQ ID NO. 5), dxs-3-for (SEQ ID NO), respectively, modified with a 5' end 4 base thio group .6) and the lower primer dxs-1-rev (SEQ ID NO. 7), dxs-2-rev (SEQ ID NO. 8), dxs-3-rev (SEQ ID NO. 9), with the addition of Mn 2+ Under the conditions, 94 ° C 45s, 54 ° C 50s, 72 ° C 1min cycle 30 times to do error-prone PCR, tapping purification and recovery of the resulting double-stranded mutant library containing mutations.
  • a single colony of Escherichia coli EcLYC picked from the plate was placed in a shake flask of 50 mL of LB medium, and 25 ⁇ L of kanamycin was added thereto, and cultured at 30 ° C overnight on a shaker;
  • the tube was placed in a 42 ° C water bath and shaken for 15 min to induce the full expression of ⁇ -Red recombinant protein;
  • test tube was placed on ice for 5 min;
  • the supernatant was removed, and the cells were resuspended in 100 ⁇ L of pre-cooled sterile water, and a double-stranded mutant library with mutation and a resistance-recovering oligonucleotide were added;
  • the above mixture was transferred to a 2 mm electric rotor, and an electric shock was performed under the conditions of a capacitance of 25 ⁇ F, a voltage of 2.5 kV, and a resistance of 200 ⁇ ;
  • the prepared 1 ml SOC medium was quickly added to the electric shock cup, and the cells were suspended by gently blowing.
  • the mixture in the electric shock cup was transferred to a test tube containing 1 ml of the medium, and placed in a shaker at 30 ° C to resuscitate the cells;
  • Escherichia coli EcNR2 (Harris H. Wang et al. (2009) Nature. 460: 894-890) can express the protein required for ⁇ -Red recombination under the induction of temperature conditions, and the long-term modification of the 5' end 4 base thio modification
  • the chain also has a higher recombination efficiency because there is less step of converting the double strand into a single strand during the recombination process.
  • 1-Deoxy-D-xylulose-5-phosphate synthase is a key enzyme in the isoprene synthesis pathway in Escherichia coli.
  • 1-Deoxygenation is synthesized using 3-phospho-glyceraldehyde and pyruvate as substrates.
  • -D-xylulose-5-phosphate The foreign gene crtE, crtB, crtI was introduced into Escherichia coli EcNR2, so that Escherichia coli can produce lycopene as a screening marker and named EcLYC.
  • the first primer dxs-for (SEQ ID NO. 16) with a 5' thiol modification at the 5' end and the lower primer dxs-rev (SEQ ID NO. 17) phosphorylated at the 5' end were used as the dxs gene.
  • the PCR product obtained above is treated with Lambda EXO exonuclease, and the dxs double-stranded enzyme is cleaved into a single strand;
  • a single colony of Escherichia coli EcLYC picked from the plate was placed in a shake flask of 50 mL of LB medium, and 25 ⁇ L of kanamycin was added thereto, and cultured at 30 ° C overnight on a shaker;
  • the tube was placed in a 42 ° C water bath and shaken for 15 min to induce the full expression of ⁇ -Red recombinant protein;
  • test tube was placed on ice for 5 min;
  • the supernatant was removed, and the cells were resuspended in 100 ⁇ L of pre-cooled sterile water, and a 5'-end 4 base thio-modified dxs single-strand mutant library and a resistance-recovering oligonucleotide were added;
  • the above mixture was transferred to a 2 mm electric rotor, and an electric shock was performed under the conditions of a capacitance of 25 ⁇ F, a voltage of 2.5 kV, and a resistance of 200 ⁇ ;
  • the prepared 1 ml SOC medium was quickly added to the electric shock cup, and the cells were suspended by gently blowing.
  • the mixture in the electric shock cup was transferred to a test tube containing 1 ml of the medium, and placed in a shaker at 30 ° C to resuscitate the cells;
  • the lycopene accumulation amount of the Escherichia coli strain containing the DXS mutant of each strain was improved relative to the wild type strain, and the EcLYC-d was more than doubled.
  • Escherichia coli EcNR2 which introduced the exogenous gene crtE, crtB, crtI, was able to synthesize lycopene (EcLYC).
  • the synthesis process relied mainly on the isoprene synthesis pathway in E. coli, and both ATP and NADPH were required.
  • the dxs gene was used as an example to illustrate the preparation of a long-chain single-stranded dxs gene with a 5'-end 4 base thio-modified mutation in vitro. the process of.
  • the first primer dxs-for (SEQ ID NO. 16) with a 5' thiol modification at the 5' end and the lower primer dxs-rev (SEQ ID NO. 17) phosphorylated at the 5' end were used as the dxs gene.
  • the PCR product obtained above is treated with Lambda EXO exonuclease, and the dxs double-stranded enzyme is cleaved into a single strand;
  • dux, dxr, idi, ispA, talB and rpos with a 5' end 4 base thio-modified single strand with a mutation.
  • a single colony of Escherichia coli EcLYC picked from the plate was placed in a shake flask of 50 mL of LB medium, and 25 ⁇ L of kanamycin was added thereto, and cultured at 30 ° C overnight on a shaker;
  • the tube was placed in a 42 ° C water bath and shaken for 15 min to induce the full expression of ⁇ -Red recombinant protein;
  • test tube was placed on ice for 5 min;
  • the supernatant was removed, and the cells were resuspended in 100 ⁇ L of pre-cooled sterile water, and a single-stranded DNA mutation library and a resistance-recovering oligonucleotide were added;
  • the above mixture was transferred to a 2 mm electric rotor, and an electric shock was performed under the conditions of a capacitance of 25 ⁇ F, a voltage of 2.5 kV, and a resistance of 200 ⁇ ;
  • the prepared 1 ml SOC medium was quickly added to the electric shock cup, and the cells were suspended by gently blowing.
  • the mixture in the electric shock cup was transferred to a test tube containing 1 ml of the medium, and placed in a shaker at 30 ° C to resuscitate the cells;
  • the lycopene accumulation amount of the Escherichia coli strain containing the DXS mutant of each strain was improved relative to the wild type strain, and EcLYC-5 was 1.5 times or more of the original.
  • Escherichia coli EcNR2 which introduces the foreign genes crtE, crtB, crtI, can synthesize lycopene (EcLYC), and its accumulation is affected by many aspects: 1. The accumulation of lycopene in E. coli depends on the accumulation of precursor substance IPP. Synthetic via the isoprene pathway.
  • knockdown of some of the isoflavone pathway genes of isoprene can increase the yield of lycopene; 4, the energy supply in E. coli also affects the accumulation of lycopene (sucAB, talB, sdhABCD).
  • the dxs gene was used as an example to illustrate the preparation of a long-chain single-stranded dxs gene with a 5'-end 4 base thio-modified mutation in vitro. the process of.
  • the first primer dxs-for (SEQ ID NO. 16) with a 5' thiol modification at the 5' end and the lower primer dxs-rev (SEQ ID NO. 17) phosphorylated at the 5' end were used as the dxs gene.
  • the PCR product obtained above is treated with Lambda EXO exonuclease, and the dxs double-stranded enzyme is cleaved into a single strand;
  • the above-described single-stranded nucleotide library is mixed with a synthetic oligonucleotide having a nonsense mutation for recombination of the gene.
  • a single colony of Escherichia coli EcLYC picked from the plate was placed in a shake flask of 50 mL of LB medium, and 25 ⁇ L of kanamycin was added thereto, and cultured at 30 ° C overnight on a shaker;
  • the tube was placed in a 42 ° C water bath and shaken for 15 min to induce the full expression of ⁇ -Red recombinant protein;
  • test tube was placed on ice for 5 min;
  • the supernatant was removed, and the cells were resuspended in 100 ⁇ L of pre-cooled sterile water, and a single-stranded NDA mutant library and resistance-recovering oligonucleotides were added;
  • the above mixture was transferred to a 2 mm electric rotor, and an electric shock was performed under the conditions of a capacitance of 25 ⁇ F, a voltage of 2.5 kV, and a resistance of 200 ⁇ ;
  • the prepared 1 ml SOC medium was quickly added to the electric shock cup, and the cells were suspended by gently blowing.
  • the mixture in the electric shock cup was transferred to a test tube containing 1 ml of the medium, and placed in a shaker at 30 ° C to resuscitate the cells;
  • the lycopene accumulation amount of the Escherichia coli strain containing the DXS mutant of each strain was improved relative to the wild type strain.
  • EcLYC246 has been applied for the preservation of the patented species, and the strain storage number is CCTCC M 2015692.
  • the lycopene-related gene in this strain of Escherichia coli has been mutated to make the accumulation of lycopene more than 4 times that of the wild-type E. coli strain.
  • SEQ ID NO.3 90nt oligodeoxynucleotide of chloramphenicol resistance gene
  • SEQ ID NO. 16 4' base 4 base thio-modified upper primer dxs-for

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Abstract

提供了一种在大肠杆菌细胞内原位进化蛋白质的方法，所述方法可用于微生物单个基因的原位进化或多个目标基因的同时原位进化，用以提高酶的催化活性，并提高微生物代谢产物的产率。

Description

大肠杆菌细胞内原位进化目标蛋白质的新方法 发明领域
本发明涉及一种在大肠杆菌细胞内原位进化蛋白质的新方法，属于生物技术领域。
背景技术
定向进化技术是一种在实验室中模拟达尔文进化,进化出在自然界并不存在的或是具有更优性质的蛋白质，并可以通过改变酶的催化效率改变代谢流，扩展或构建新的代谢途径，弱化或消除不必要或有害的代谢途径，从而达到提高某种代谢产物产率或降解有害物质的目的。定向进化技术主要有两个步骤：首先利用现代分子生物学方法构建基因的突变文库，然后耦合筛选方法对文库进行筛选。突变文库的构建可以提供足够的多样性以供筛选，是定向进化的关键步骤。按照DNA突变发生的场所，构建突变文库的技术可以分为体外突变和体内突变。
体外突变技术主要有易错PCR(error prone PCR)，DNA改组(DNA shuffling)，交错延伸过程(Staggered extension process,StEP)，定点饱和突变技术(Site directed saturation mutagenesis,SDSM)，迭代饱和突变(Iterative saturation mutagenesis，ISM)等。易错PCR(error prone PCR)是通过改变聚合酶链式反应条件而随机引入点突变的；DNA改组(DNA shuffling)是一种DNA分子的体外重组技术，通过将一组序列相关的DNA序列用DNase I随机切割成小片段，随后进行PCR重聚实现基因重排得到突变文库，再筛选出有预期性状的基因，这种方法可以实现与亲本序列有70％同源性序列的重组；而交错延伸过程(Staggered extension process,StEP)是一种简化了的DNA改组技术。定点饱和突变技术(Site directed saturation mutagenesis,SDSM)是将目的蛋白靶位点的氨基酸用分别其他19种氨基酸替代，分析靶位点改造后蛋白的活性；迭代饱和突变(Iterative saturation mutagenesis，ISM)是一种反复的饱和突变方法，通过选择结构已知蛋白的几个重要区域的1-2个或3个位点同时进行反复饱和突变后获得突变体文库，并进行筛选以获得功能提高的突变体。以上这些体外突变技术虽然突变的效率比较高，但是必须将突变的基因通过酶切连接到相应的载体上转化到生 物体内，突变基因被转录翻译为对应的蛋白表现出一定表型，才可以耦合相应的筛选技术对突变文库进行筛选。酶切连接转化的步骤操作繁琐、过程复杂，不能、连续进化，不能自发进化，需要人工干预。
体内突变技术有物理化学诱变，PACE(噬菌体辅助持续进化,MAGE(多位点自动化基因组编辑)。物理、化学诱变是利用物理、化学等因素处理生物体，以诱发遗传物质的突变，从而引起形态特征的变异。这一传统的诱变方法的突变效率低而且没有目标性。最近，哈佛大学David R Liu实验室开发了一种基于噬菌体生长的连续进化系统，通过在大肠杆菌体内表达易突变的DNA聚合酶产生突变，并结合M13噬菌体建立了筛选体系实现了定向进化的自动化。但是PACE技术的突变效率比较低只有10-5，对一个1kb的基因随机的3个位点实现饱和突变几乎没有可能，另外PACE技术只可以对一个基因进行进化。
George Church实验室发明了MAGE(多重自动基因组改造技术)。它同时针对基因组的不同区域设计一系列的单链寡核苷酸简并引物，利用λ-Red同源重组系统将这些简并引物整合到基因组上，实现单个细胞基因组多个位点的改造或细胞群体间基因组改造的多样性。他们针对该技术的周期性和可扩展性设计了可以实现该过程的自动化操作装置。利用该技术定向改造大肠杆菌中番茄红素合成过程中的20个基因的RBS区，设计不同的简并引物，使他们定向进化到认为可以提高表达量的经典的SD序列(TAAGGAGGT)模式，最终筛选得到高产菌株，并对这些高产菌株的基因序列分析得出参与番茄红素合成起始以及末端的基因的RBS序列趋于相似。MAGE技术虽然在体内实现了对代谢通路中多基因RBS序列的突变文库的构建，但是并没有实现对代谢途径中单个或多个目标蛋白质的结构基因进行原位定向进化。但在大肠杆菌细胞内，一个目标基因所编码的酶蛋白在微生物代谢网络中所起到的代谢调控作用，往往取决于该基因的体内表达水平和编码蛋白质的酶活性。已报道，有很多方法被发明如何调控目标基因的表达，但还没有出现在大肠杆菌细胞内进行原位同源重组进化酶蛋白基因的方法。
发明内容
本发明涉及一种在大肠杆菌细胞内原位进化蛋白质的新方法，并应用于一种或多种酶催化性能的提高，从而提高微生物特定目标代谢产物的产率。
在本发明中，包括构建一个包含一个或多个突变位点的核酸库，包括体外合成的寡核苷酸单链库或制备的单链DNA库或双链DNA库，通过λ-Red同源重组技术，利用所述核酸库对大肠杆菌胞内载体上或基因组上的结构基因进行原位重组，达到进化酶分子结构提高酶催化效率的目的。寡核苷酸可以以化学合成的方式获取；单链核苷酸的获取方式包括但不限于T7逆转录法，核酸外切酶法，变性高效液相色谱法，磁珠捕获法，不对称PCR，两步PCR法等；双链核苷酸可以通过PCR法合成获得。
寡核苷酸和单链核苷酸可以在其5’端的1至4位碱基处进行修饰；双链核苷酸在其中一条链的5’端的1至4位碱基处进行修饰，以避免在转化入细胞后被核酸酶所降解。修饰的方式包括但不限于5’端溴尿嘧啶修饰，5’端氯尿嘧啶修饰，5’端碘代尿嘧啶修饰，磷酸酯修饰，硫代磷酸修饰等。
寡核苷酸、单链核苷酸和双链核苷酸可以通过转化或者转染的方式进入细胞内。这里提到的转化和转染指的是一些成熟地把外源核苷酸导入目标细胞的技术，包括但不限于磷酸钙法，氯化钙法，脂质体转染，电穿孔法，光穿孔法等。转染介质包括但不限于水，氯化钙，脂质体等。
在转化或转染之前，大肠杆菌细胞在30℃培养至一定浓度，并于42℃进行诱导。在细胞基因组上的λ-Red同源重组系统在pL启动子控制下，并受到cI857阻遏蛋白的调控。在30℃时pL启动子受阻遏不表达，42℃时诱导表达λ-Red同源重组所需的三个蛋白。经过15min诱导之后的细胞放于4℃保存，防止诱导的蛋白降解。
培养基需要置换成转染介质，去除其中的离子。此过程可以采用的方式包括但不限于离心法，膜过滤法等。
在转化或转染过程中，加入核酸库的同时，引入抗性回复核苷酸片段，可以在抗性平板上进行筛选，并提高抗性基因附近位置的重组效率。合适的抗性基因包括但不限于氨苄抗性基因，四环素抗性基因，卡那霉素抗性基因，新霉素抗性基因，氯霉素抗性基因等。
合适的筛选标记可以被应运于平板筛选，流式细胞仪筛选，微流控等。筛选标记包括但不限于各种酶，辅助基团，荧光标记，发光标记，生物发光基团等。荧光标记包括但不限于黄色荧光蛋白，绿色荧光蛋白，青色荧光蛋白，罗丹明等。 生物发光基团包括但不限于萤光素酶，发光蛋白质等。一些能产生视觉信号的酶包括但不限于半乳糖苷酶，磷酸酶，过氧化物酶，胆碱酯酶等。
本发明为了在体内实现较高的突变率并对基因组上的一个或多个基因进行突变，提供了一种体内突变方法，通过反复λ-Red同源重组的策略对大肠杆菌的整个基因组进行突变，尤其是对结构基因的修饰，达到对功能蛋白所对应基因的原位进化目的。此方法可以对基因组上单一基因进行进化，也可以同时对多个基因进行修饰，并耦合筛选，寻找出多个突变蛋白协同作用所达到的最佳效果。
本发明所提供的在大肠杆菌细胞内原位进化蛋白质的新方法是一种有效得获得所需生物分子或生物物质的方法，比以往的方法更加简便、高效。
本发明所提供的在大肠杆菌细胞内原位进化蛋白质的新方法，可以通过大肠杆菌胞内目标基因关键氨基酸的原位饱和突变，提高吡咯喹啉依赖型葡萄糖脱氢酶的热稳定性和催化特异性。
本发明所提供的在大肠杆菌细胞内原位进化蛋白质的新方法，所述方法可应用于体内原位重组突变番茄红素合成关键酶1-脱氧-D-木酮糖-5-磷酸合成酶基因，提高其表达蛋白的可溶性水平和催化效率。通过筛选得到的5个1-脱氧-D-木酮糖-5-磷酸合成酶(DXS)突变体。其中，突变体DXSa(S217P，L226H)，DXSb(K214E，K227E，G512H)，DXSc(E230G)和DXSd(Y115H，E182A，L226R，F435S)的可溶表达相对于野生型DXS蛋白都提高了2倍以上。DXS突变体(DXSa，DXSb，DXSd和DXSe(I170T K213E，N249S))的比酶活都得到了明显提高，从而提高了含相应突变基因的大肠杆菌生物合成番茄红素水平2-5倍。
本发明所提供的在大肠杆菌细胞内原位进化蛋白质的新方法，所述方法可采用同时突变多个番茄红素关键酶基因从而提高大肠杆菌合成番茄红素的生产水平。
本发明还提供了大肠杆菌菌株EcLYCb，保存编号为CCTCC M 2015691的大肠杆菌菌株，该菌株的可溶性表达水平和酶催化效率都得到明显提高。
本发明还提供了大肠杆菌菌株EcLYC246，保存编号为CCTCC M 2015692，提高了大肠杆菌合成番茄红素的生产水平。
附图说明
图1：本发明流程与传统DNA重排技术对比示意图。其中，DNA Shuffling Method：DNA重排技术；DNase I Digestion：DNase I酶切；cycles of denaturation，annealing，extension：多次变性，连接，延伸；Cloning，Transformation：克隆，转化；Gene：基因；Error-Prone PCR：易错PCR；Asymmetric PCR：不对称PCR；Synthetic Oligos：合成寡聚核苷酸；cycles of Recombination：多次重组；Genome：基因组；E.coli：大肠杆菌。
图2：野生型及GDH突变体在55℃的热稳定性分析图。
图3：野生型及DXS突变蛋白的可溶表达情况对比图。其中，Comparation of Soluble DXS Expression：可溶DXS蛋白表达情况对比；Marker：蛋白分子量标准大小。
图4：野生型及DXS突变蛋白的比酶活对比图。其中，Retention time：保留时间；Specific enzyme activity：比酶活。
图5：野生型及含dxs突变体的大肠杆菌菌株产番茄红素能力对比图；其中，Lycopene Production：番茄红素产量。
图6：野生型及含dxs突变体的大肠杆菌菌株产番茄红素能力对比图；其中，Lycopene Production：番茄红素产量。
图7：野生型及含多个基因突变的大肠杆菌菌株产番茄红素能力对比图；其中，Lycopene Production：番茄红素产量。
图8：野生型及含多个基因突变的大肠杆菌菌株产番茄红素能力对比图；其中，Lycopene Production：番茄红素产量。
具体实施方式
本发明的操作流程如图1所示，并通过以下五个例子更好得阐述，分别为提高吡咯喹啉依赖型葡萄糖脱氢酶热稳定性和催化特异性，提高1-脱氧-D-木酮糖-5-磷酸合成酶的可溶表达和催化活性，以及提高大肠杆菌中番茄红素合成途径的多个关键酶的催化活性。
实施例1，大肠杆菌体内原位进化吡咯喹啉依赖型葡萄糖脱氢酶
大肠杆菌EcNR2(Harris H.Wang等.2009Nature.460:894-890)在温度条件诱导下可以表达λ-Red重组所需蛋白，表现出对80-100nt寡核苷酸的高重组效率。
把吡咯喹啉依赖型葡萄糖脱氢酶基因(gdh)通过λ-Red同源重组插入到上 述菌株基因组上，命名为EcGDH。设计两条90nt含兼并碱基的寡核苷酸分别对应gdh基因的两个活性位点：热稳定性(S231，SEQ ID NO.1)和催化特异性(N452，SEQ ID NO.2)，构成一个寡核苷酸突变库，并用该突变库在基因组上进行点饱和突变。同时合成一条用于恢复原本基因组上被沉默的氯霉素抗性基因的90nt寡核苷酸(SEQ ID NO.3)。
在50mL LB培养基的摇瓶中接入从平板上挑取的大肠杆菌EcGDH单菌落，并加入25μL卡那霉素，30℃下摇床过夜培养；
转接1％的菌液至2mL LB培养基的试管中，置于30℃下摇床培养2.5-3h至OD达到0.7左右；
将试管置于42℃水浴中振荡培养15min，诱导λ-Red重组蛋白充分表达；
将试管置于冰上5min；
取1mL菌液移入1.5mL预冷离心管中，4℃下以13000r/min离心30s，弃去上清液，加入预冷的无菌水1ml重悬洗涤，弃去上清液，并用预冷的无菌水1ml重悬洗涤2次；
去除上清液，并用100μL预冷无菌水重悬菌体，加入寡核苷酸突变库和抗性回复寡核苷酸；
将上述混合物转移到2mm电转杯中，在电容25μF，电压2.5kV，电阻200Ω条件下进行电击；
迅速将准备好的1ml SOC培养基加入到电击杯中，轻轻吹打使细胞悬浮。将电击杯中的混合液转移到装有1ml培养基的试管中，放入30℃摇床培养，使细胞复苏；
当细胞复苏到OD达到0.7左右时，重复上述步骤，进行第二轮转化。该转化过程一共进行3-5轮；
最后一轮电转后，复苏12h。在氯霉素抗生素平板上进行筛选，通过与一系列底物的显色反应来筛选热稳定性提高或催化特异性提高的突变子。野生型EcGDH及GDH突变体(EcGDHa，EcGDHb，EcGDHc，EcGDHd，EcGDHe，EcGDHf)的催化特异性对比如表1所示，其在55℃的热稳定性分析如图2所示。
表1：各种GDH突变体的氨基酸突变及催化特性总结
Figure PCTCN2016077246-appb-000001
Figure PCTCN2016077246-appb-000002
从表中可以看出，大肠杆菌菌株EcGDHa，EcGDHb，EcGDHc，EcGDHd中的GDH突变体的底物特异性得到了提高。相对于野生型大肠杆菌中的GDH，其对于乳糖和麦芽糖的相对活性降低了28％至42％。大肠杆菌菌株EcGDHb和EcGDHf中的GDH突变体的热稳定相对于野生型GDH分别提高了1.69和1.6倍。
实施例2用双链核苷酸在大肠杆菌体内原位进化1-脱氧-D-木酮糖-5-磷酸合成酶
双链核苷酸在大肠杆菌中通过λ-Red同源重组的效率比较低，只有0.01％。研究发现，大肠杆菌EcNR2(Harris H.Wang等.(2009)Nature.460:894-890)在温度条件诱导下可以表达λ-Red重组所需蛋白，通过与抗性回复片段共同转化可以显著提高重组效率。
1-脱氧-D-木酮糖-5-磷酸合成酶(dxs)是大肠杆菌中异戊二烯合成途径中一个关键酶，以3-磷酸-甘油醛和丙酮酸为底物合成1-脱氧-D-木酮糖-5-磷酸。在大肠杆菌EcNR2中引入外源基因crtE，crtB，crtI，使得该大肠杆菌可以产番茄红素，命名为EcLYC。由于番茄红素具有可见的红色，可以根据菌落红色的深浅，高通量的筛选番茄红素产量提高的菌株。
体外制备带突变的双链dxs基因：
根据NCBI上的CCD工具分析结果发现，DXS蛋白有三个保守区域，所以制备了3条带突变的双链核苷酸；
分别用5’端4个碱基硫代修饰的上引物dxs-1-for(SEQ ID NO.4),dxs-2-for(SEQ ID NO.5),dxs-3-for(SEQ ID NO.6)和下引物dxs-1-rev(SEQ ID NO.7),dxs-2-rev(SEQ ID NO.8),dxs-3-rev(SEQ ID NO.9)，在添加Mn2+的条件下，94℃45s，54℃50s，72℃1min循环30次做易错PCR，割胶纯化回收得到的含有突变的双链突变库。
在50mL LB培养基的摇瓶中接入从平板上挑取的大肠杆菌EcLYC单菌落，并加入25μL卡那霉素，30℃下摇床过夜培养；
转接1％的菌液至2mL LB培养基的试管中，置于30℃下摇床培养2.5-3h至OD达到0.7左右；
将试管置于42℃水浴中振荡培养15min，诱导λ-Red重组蛋白充分表达；
将试管置于冰上5min；
取1mL菌液移入1.5mL预冷离心管中，4℃下以13000r/min离心30s，弃去上清液，加入预冷的无菌水1ml重悬洗涤，弃去上清液，并用预冷的无菌水1ml重悬洗涤2次；
去除上清液，并用100μL预冷无菌水重悬菌体，加入带突变的双链突变库和抗性回复寡核苷酸；
将上述混合物转移到2mm电转杯中，在电容25μF，电压2.5kV，电阻200Ω条件下进行电击；
迅速将准备好的1ml SOC培养基加入到电击杯中，轻轻吹打使细胞悬浮。将电击杯中的混合液转移到装有1ml培养基的试管中，放入30℃摇床培养，使细胞复苏；
当细胞复苏到OD达到0.7左右时，重复上述步骤，进行第二轮转化。该转化过程一共进行8轮；
最后一轮电转后，复苏12h。在平板上筛选出红色较深的菌落。DXS蛋白的氨基酸突变情况如表2所示，其可溶性表达情况如图3所示，其比酶活情况如图4所示，以及影响产番茄红素能力对比如图5所示(其中EcLYCb已申请专利菌种保存，菌种保藏号为CCTCC M 2015691)。
表2 DXS突变蛋白的氨基酸突变情况
Figure PCTCN2016077246-appb-000003
从图3中可以看出，DXS突变体DXSa(SEQ ID NO.10)，DXSb(SEQ ID NO.11)，DXSc(SEQ ID NO.12)和DXSd(SEQ ID NO.13)的可溶表达相对于野 生型DXS蛋白(SEQ ID NO.14)都提高了2倍以上。可溶表达的提高使得其在胞内的有效蛋白量得到提高。通过体外酶反应可知，DXS突变体DXSa，DXSb，DXSd和DXSe(SEQ ID NO.15)的比酶活都提到了提高，并最终使得各株大肠杆菌的番茄红素积累量相对于野生型菌株提高了4倍以上。其中，EcLYCb已申请专利菌种保存，菌种保藏号为CCTCC M 2015691，此株大肠杆菌中的DXS蛋白突变体可溶表达量高，比酶活高，且番茄红素的积累量是野生型大肠杆菌菌株的5倍以上。
实施例3用单链核苷酸在大肠杆菌体内原位进化1-脱氧-D-木酮糖-5-磷酸合成酶
大肠杆菌EcNR2(Harris H.Wang等.(2009)Nature.460:894-890)在温度条件诱导下可以表达λ-Red重组所需蛋白，对5’端4个碱基硫代修饰的长单链也有较高的重组效率，因为在重组过程中少了把双链变单链的步骤。
1-脱氧-D-木酮糖-5-磷酸合成酶(dxs)是大肠杆菌中异戊二烯合成途径中一个关键酶，以3-磷酸-甘油醛和丙酮酸为底物合成1-脱氧-D-木酮糖-5-磷酸。在大肠杆菌EcNR2中引入外源基因crtE，crtB，crtI，使得该大肠杆菌可以产番茄红素，作为一种筛选标志物，命名为EcLYC。
体外制备带突变的5’端4个碱基硫代修饰的dxs基因长单链：
用5’端4个碱基硫代修饰的上引物dxs-for(SEQ ID NO.16)和5’端磷酸化的下引物dxs-rev(SEQ ID NO.17)两个引物以dxs基因为模板，在添加Mn2+的条件下，94℃45s，54℃50s，72℃2min循环30次做易错PCR，割胶纯化回收得到的双链；
以上述含有突变的双链为模板，在PCR体系中只添加5’端4个碱基硫代修饰的上引物dxs-for(SEQ ID NO.16)，94℃30s，53℃30s，72℃4min循环15次，制备得到dxs单链；
用Lambda EXO外切酶处理上述得到的PCR产物，把其中的dxs双链酶切成单链；
乙醇沉淀得到5’端4个碱基硫代修饰的dxs单链突变库。
在50mL LB培养基的摇瓶中接入从平板上挑取的大肠杆菌EcLYC单菌落，并加入25μL卡那霉素，30℃下摇床过夜培养；
转接1％的菌液至2mL LB培养基的试管中，置于30℃下摇床培养2.5-3h至OD达到0.7左右；
将试管置于42℃水浴中振荡培养15min，诱导λ-Red重组蛋白充分表达；
将试管置于冰上5min；
取1mL菌液移入1.5mL预冷离心管中，4℃下以13000r/min离心30s，弃去上清液，加入预冷的无菌水1ml重悬洗涤，弃去上清液，并用预冷的无菌水1ml重悬洗涤2次；
去除上清液，并用100μL预冷无菌水重悬菌体，加入5’端4个碱基硫代修饰的dxs单链突变库和抗性回复寡核苷酸；
将上述混合物转移到2mm电转杯中，在电容25μF，电压2.5kV，电阻200Ω条件下进行电击；
迅速将准备好的1ml SOC培养基加入到电击杯中，轻轻吹打使细胞悬浮。将电击杯中的混合液转移到装有1ml培养基的试管中，放入30℃摇床培养，使细胞复苏；
当细胞复苏到OD达到0.7左右时，重复上述步骤，进行第二轮转化。该转化过程一共进行4-6轮；
最后一轮电转后，复苏12h。在平板上筛选出红色较深的菌落。野生型EcLYC及含DXS突变体的大肠杆菌菌株(EcLYC-a，EcLYC-b，EcLYC-c，EcLYC-d，EcLYC-e)产番茄红素能力对比如图6所示。
从图中可以看出，各株含有DXS突变体的大肠杆菌菌株的番茄红素积累量相对于野生型菌株都得到了提高，其中EcLYC-d提高了1倍以上。
实施例4大肠杆菌胞内原位同时进化多个番茄红素合成关键基因
引入外源基因crtE，crtB，crtI的大肠杆菌EcNR2能够合成番茄红素(EcLYC)，该合成过程主要依赖大肠杆菌中的异戊二烯合成途径，同时对ATP和NADPH都有需求。在此过程中，存在多个关键酶可以提升异戊二烯代谢流，并提高NADPH的供应量，如dxs，dxr，idi，ispA，talB等。
采用两步PCR和易错PCR获得上述基因同源带突变的单链核苷酸，以dxs基因为例，说明体外制备带突变的5’端4个碱基硫代修饰的dxs基因长单链的过程。
用5’端4个碱基硫代修饰的上引物dxs-for(SEQ ID NO.16)和5’端磷酸化的下引物dxs-rev(SEQ ID NO.17)两个引物以dxs基因为模板，在添加Mn2+的条件下，94℃45s，54℃50s，72℃2min循环30次做易错PCR，割胶纯化回收得到的双链；
以上述易错双链为模板，在PCR体系中只添加5’端4个碱基硫代修饰的上引物dxs-for(SEQ ID NO.16)，94℃30s，53℃30s，72℃4min循环15次，制备得到dxs单链；
用Lambda EXO外切酶处理上述得到的PCR产物，把其中的dxs双链酶切成单链；
乙醇沉淀得到带突变的5’端4个碱基硫代修饰的dxs单链。
重复上述方法，获得dxs，dxr，idi，ispA，talB和rpos带突变的5’端4个碱基硫代修饰的单链。
这些单链混合在一起成为一个单链DNA突变库。
在50mL LB培养基的摇瓶中接入从平板上挑取的大肠杆菌EcLYC单菌落，并加入25μL卡那霉素，30℃下摇床过夜培养；
转接1％的菌液至2mL LB培养基的试管中，置于30℃下摇床培养2.5-3h至OD达到0.7左右；
将试管置于42℃水浴中振荡培养15min，诱导λ-Red重组蛋白充分表达；
将试管置于冰上5min；
取1mL菌液移入1.5mL预冷离心管中，4℃下以13000r/min离心30s，弃去上清液，加入预冷的无菌水1ml重悬洗涤，弃去上清液，并用预冷的无菌水1ml重悬洗涤2次；
去除上清液，并用100μL预冷无菌水重悬菌体，加入单链DNA突变库和抗性回复寡核苷酸；
将上述混合物转移到2mm电转杯中，在电容25μF，电压2.5kV，电阻200Ω条件下进行电击；
迅速将准备好的1ml SOC培养基加入到电击杯中，轻轻吹打使细胞悬浮。将电击杯中的混合液转移到装有1ml培养基的试管中，放入30℃摇床培养，使细胞复苏；
当细胞复苏到OD达到0.7左右时，重复上述步骤，进行第二轮转化。该转化过程一共进行10-15轮；
最后一轮电转后，复苏12h。在平板上筛选红色较红的菌落。野生型(EcLYC)及含多个基因突变的大肠杆菌菌株(EcLYC-1，EcLYC-2，EcLYC-3，EcLYC-4，EcLYC-5)产番茄红素能力对比如图7所示。
从图中可以看出，各株含有DXS突变体的大肠杆菌菌株的番茄红素积累量相对于野生型菌株都得到了提高，其中EcLYC-5是原来的1.5倍以上。
实施例5基因组多位点进化大肠杆菌番茄红素代谢相关基因
引入外源基因crtE，crtB，crtI的大肠杆菌EcNR2能够合成番茄红素(EcLYC)，其积累量受到多方面的影响：1、大肠杆菌中番茄红素的积累依赖前体物质IPP的积累，其通过异戊二烯途径合成。过量表达该途径上的多个基因(dxs，dxr，ispD，ispE，ispG，ispH，idi，ispA)都会提高前体物质IPP的积累；2、鸟枪法研究发现多个功能未明确的基因也会影响番茄红素的积累(appY，rpoS，crl，elbA，elbB，yjiD，purH，rnlA，yggT，ycgZ，ymgA，ariR)；3、敲除异戊二烯的一些旁路代谢途径基因(ytjC，fdhF，aceE，gdhA)可以提高番茄红素的产量；4、大肠杆菌体内的能量供应也会影响番茄红素的积累(sucAB，talB，sdhABCD)。
对从上述基因中挑选的二十个基因(dxs，dxr，idi，ispA，appY，rpoS，crl，elbA，elbB，yjiD，purH，rnlA，yggT，sucA，sucB，talB，sdhA，sdhB，sdhC，sdhD)在基因组上进行原位进化，并同时用合成的含两个无义突变的寡核苷酸对四个基因(ytjC，fdhF，aceE，gdhA)进行沉默。
采用两步PCR和易错PCR获得上述基因同源带突变的单链核苷酸，以dxs基因为例，说明体外制备带突变的5’端4个碱基硫代修饰的dxs基因长单链的过程。
用5’端4个碱基硫代修饰的上引物dxs-for(SEQ ID NO.16)和5’端磷酸化的下引物dxs-rev(SEQ ID NO.17)两个引物以dxs基因为模板，在添加Mn2+的条件下，94℃45s，54℃50s，72℃2min循环30次做易错PCR，割胶纯化回收得到的双链；
以上述易错双链为模板，在PCR体系中只添加5’端4个碱基硫代修饰的上 引物dxs-for(SEQ ID NO.10)，94℃30s，53℃30s，72℃4min循环15次，制备得到dxs单链；
用Lambda EXO外切酶处理上述得到的PCR产物，把其中的dxs双链酶切成单链；
乙醇沉淀得到带突变的5’端4个碱基硫代修饰的dxs单链。
重复上述方法，获得dxs，dxr，idi，ispA，appY，rpoS，crl，elbA，elbB，yjiD，purH，rnlA，yggT，sucA，sucB，talB，sdhA，sdhB，sdhC和sdhD带突变的5’端4个碱基硫代修饰的单链。
这些单链混合在一起成为一个单链DNA突变库。
把上述单链核苷酸库与合成的带无义突变的寡核苷酸混合在一起，用于对基因的重组。
在50mL LB培养基的摇瓶中接入从平板上挑取的大肠杆菌EcLYC单菌落，并加入25μL卡那霉素，30℃下摇床过夜培养；
转接1％的菌液至2mL LB培养基的试管中，置于30℃下摇床培养2.5-3h至OD达到0.7左右；
将试管置于42℃水浴中振荡培养15min，诱导λ-Red重组蛋白充分表达；
将试管置于冰上5min；
取1mL菌液移入1.5mL预冷离心管中，4℃下以13000r/min离心30s，弃去上清液，加入预冷的无菌水1ml重悬洗涤，弃去上清液，并用预冷的无菌水1ml重悬洗涤2次；
去除上清液，并用100μL预冷无菌水重悬菌体，加入单链NDA突变库和抗性回复寡核苷酸；
将上述混合物转移到2mm电转杯中，在电容25μF，电压2.5kV，电阻200Ω条件下进行电击；
迅速将准备好的1ml SOC培养基加入到电击杯中，轻轻吹打使细胞悬浮。将电击杯中的混合液转移到装有1ml培养基的试管中，放入30℃摇床培养，使细胞复苏；
当细胞复苏到OD达到0.7左右时，重复上述步骤，进行第二轮转化。该转化过程一共进行10-15轮；
最后一轮电转后，复苏12h。在平板上筛选红色较红的菌落。野生型(EcLYC)及含多个基因突变的大肠杆菌菌株(EcLYC20，EcLYC45，EcLYC60，EcLYC139，EcLYC246)产番茄红素能力对比如图8所示(其中EcLYC246已申请专利菌种保存，菌种保藏号为CCTCC M 2015692)。
从图中可以看出，各株含有DXS突变体的大肠杆菌菌株的番茄红素积累量相对于野生型菌株都得到了提高。其中，EcLYC246已申请专利菌种保存，菌种保藏号为CCTCC M 2015692，此株大肠杆菌中产番茄红素相关基因经过突变使其番茄红素的积累量是野生型大肠杆菌菌株的4倍以上。
文中序列
SEQ ID NO.1 热稳定性饱和突变寡核苷酸
5’-*T*C*A*CACCTATATGGGTAAAGTACTACGCTTAAATCTTGATGGANNNATTCCAAAGGATAATCCAAGTTTTAACGGGGTGGTTAGCCATA-3’(*为硫代修饰，下同)
SEQ ID NO.2 催化特异性饱和突变寡核苷酸
5’-TCCAGATGGGAATGTCTTATATGTATTAACTGATACTGCCGGANNNGTCCAAAAAGATGATGGCTCAGTAACAAATACATTAGAAAACC-3’
SEQ ID NO.3 氯霉素抗性基因的90nt寡核苷酸
5’-*G*C*A*TCGTAAAGAACATTTTGAGGCATTTCAGTCAGT-3’
SEQ ID NO.4 5’端4个碱基硫代修饰的上引物dxs-1-for
5’-*G*A*G*TTTTGATATTGCCAAATACCCGACCCTGGC-3’
SEQ ID NO.5 5’端4个碱基硫代修饰的上引物dxs-2-for
5’-*T*T*G*CCGAGCTATTCAAAAATCTTTGGCGAC-3’
SEQ ID NO.6 5’端4个碱基硫代修饰的上引物dxs-3-for
5’-*G*G*C*AAAGGCATTGTGAAGCGTCGTG-3’
SEQ ID NO.7 下引物dxs-1-rev
5’-GGTCATGATATGCAGGAACTGCGGG-3’
SEQ ID NO.8 下引物dxs-2-rev
5’-CAGTTCCACGCCGACCGCGTTGCCACGCGGGTA-3’
SEQ ID NO.9 下引物dxs-3-rev
5’-TATGCCAGCCAGGCCTTGATTTTGGC-3’
SEQ ID NO.10 DXSa氨基酸序列
Figure PCTCN2016077246-appb-000004
SEQ ID NO.11 DXSb氨基酸序列
Figure PCTCN2016077246-appb-000005
SEQ ID NO.12 DXSc氨基酸序列
Figure PCTCN2016077246-appb-000006
Figure PCTCN2016077246-appb-000007
SEQ ID NO.13 DXSd氨基酸序列
Figure PCTCN2016077246-appb-000008
SEQ ID NO.14 野生型DXS氨基酸序列
Figure PCTCN2016077246-appb-000009
Figure PCTCN2016077246-appb-000010
SEQ ID NO.15 DXSe氨基酸序列
Figure PCTCN2016077246-appb-000011
SEQ ID NO.16 5’端4个碱基硫代修饰的上引物dxs-for
5’-*G*A*G*TTTTGATATTGCCAAATACCCGACCCTGGC-3’
SEQ ID NO.17 5’端磷酸化的下引物dxs-rev
5’-TTATGCCAGCCAGGCCTTGATTTTG-3’(5’端磷酸化)

Claims (7)

  1. 一种在大肠杆菌细胞内原位进化蛋白质的新方法，其特征在于所述方法包括构建包含一个或多个突变位点的核酸库，包括体外合成的寡核苷酸单链库或制备的单链DNA库或双链DNA库，通过λ-Red同源重组技术，利用所述核酸库对大肠杆菌胞内载体上或基因组上的一个或多个结构基因进行原位重组，以较高的突变率达到进化酶分子结构提高酶催化效率的目的。
  2. 如权利要求1所述的一种在大肠杆菌细胞内原位进化蛋白质的新方法，其特征在于所述方法对大肠杆菌基因组上多个基因同时进行重组突变，应用于代谢途径中多个关键酶的进化，从而达到优化代谢途径提高目标产物合成产率的目的。
  3. 如权利要求1所述的一种在大肠杆菌细胞内原位进化蛋白质的新方法，其特征在于所述方法通过大肠杆菌胞内目标基因关键氨基酸的原位饱和突变，提高吡咯喹啉依赖型葡萄糖脱氢酶的热稳定性和催化特异性。
  4. 如权利要求1所述的一种在大肠杆菌细胞内原位进化蛋白质的新方法，其特征在于所述方法应用于体内原位重组突变番茄红素合成关键酶1-脱氧-D-木酮糖-5-磷酸合成酶基因，提高其表达蛋白的可溶性水平和催化效率。
  5. 如权利要求1所述的一种在大肠杆菌细胞内原位进化蛋白质的新方法，其特征在于所述方法同时突变多个番茄红素关键酶基因从而提高大肠杆菌合成番茄红素的生产水平。
  6. 大肠杆菌菌株EcLYCb，保存编号为CCTCC M 2015691。
  7. 大肠杆菌菌株EcLYC246，保存编号为CCTCC M 2015692。
PCT/CN2016/077246 2016-01-27 2016-03-24 大肠杆菌细胞内原位进化目标蛋白质的新方法 Ceased WO2017128509A1 (zh)

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