WO2023050552A1 - 蛋白质自动化工程改造优化方法 - Google Patents

蛋白质自动化工程改造优化方法 Download PDF

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WO2023050552A1
WO2023050552A1 PCT/CN2021/133816 CN2021133816W WO2023050552A1 WO 2023050552 A1 WO2023050552 A1 WO 2023050552A1 CN 2021133816 W CN2021133816 W CN 2021133816W WO 2023050552 A1 WO2023050552 A1 WO 2023050552A1
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automated
automatic
pcr
library
workstation
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French (fr)
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司同
付立豪
张智彧
张建志
陈永灿
郭二鹏
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深圳先进技术研究院
中国科学院深圳理工大学(筹)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • the invention relates to a protein automatic engineering transformation optimization method, in particular to protein high-throughput, automatic saturation mutation library construction, high-throughput mixed pool sequencing, mass spectrometry characterization methods and the like.
  • the methods of protein molecular modification are mainly divided into rational design, directed evolution and the combination of the two.
  • Directed evolution is based on non-ideal means of multiple rounds of random mutation and screening to achieve the optimization of target performance without structural and functional information.
  • Deep scanning mutagenesis combines the characteristics of error-prone PCR and saturation mutation methods, and uses degenerate primers to construct a systematic library covering all possible single point mutations of the target protein.
  • Existing deep-scanning mutation methods need to couple protein target properties with cell growth or fluorescent signals, and change the abundance of different mutants in the population through competitive growth or fluorescent sorting.
  • deep mutation scanning methods are mostly limited to experimental models such as fluorescent proteins and protein interactions, and lack a universal method for the sequence-function relationship of enzyme proteins.
  • Random mutation libraries and saturation mutation libraries of enzyme proteins usually have a huge capacity, and high-throughput characterization of mutant performance is required.
  • the establishment of screening methods is usually time-consuming and labor-intensive, and the substrate diversity and product diversity of enzyme proteins make it difficult to couple the subtle differences in molecular structure in the process with signals such as growth rate and fluorescence intensity. Therefore, it is necessary to develop a general mass spectrometry method for qualitative and quantitative screening of reactants at the molecular level. Mass spectrometry analyzes the ions of the target compound.
  • the matrix-assisted laser analysis ionization ion source irradiates the co-crystal film formed by the sample and the matrix with laser, and the matrix absorbs energy from the laser and transfers it to Biomolecules, and the process of ionizing biomolecules by transferring protons to biomolecules or obtaining protons from biomolecules during the ionization process, and finally identifying and quantifying chemicals through time-of-flight detectors.
  • the inventor developed matrix-assisted laser dissociation ionization time-of-flight mass spectrometry imaging technology in the early stage, and realized automatic laser sampling and mass spectrometry analysis (2 seconds/sample) for colonies randomly distributed on the plate.
  • Automated synthetic biology technology completes a large number of engineering trial-and-error experiments at low cost and in multiple cycles, and quickly realizes specific functions.
  • automation technology has developed rapidly.
  • automated synthetic biology technology can greatly accelerate the research process. It involves hardware equipment such as automated pipetting workstations, automated PCR instruments, automated robotic arms, and corresponding integrated control software, which can convert complicated manual operations into automated, high-throughput, and stable experimental data.
  • the high-throughput equipment has a single function, and the serial connection process is complicated, and manual participation is very easy to introduce errors.
  • the existing protein mutation analysis is usually based on the first-generation sequencing method, and each sample is analyzed separately, with low throughput and high cost (30 yuan/sample).
  • Mass spectrometry has high requirements on the quality of samples. Excessive salt ion content will greatly damage the instrument, and reduce detection sensitivity and accuracy. Mass spectrometry sample pretreatment methods are usually cumbersome and costly, requiring and Combined, the analysis throughput is low.
  • An object of the present invention is to provide a protein automatic engineering optimization method.
  • the present invention provides a method for automatic protein engineering transformation optimization, which method includes the process of automatically constructing a protein saturation mutation library, wherein the process of automatically constructing a protein saturation mutation library includes:
  • Design degenerate primers at the mutation site to introduce protein mutations perform automated PCR, automated nucleic acid extraction, automated assembly, and automated transformation, and obtain a saturated mutant library after transformation.
  • the Gibson assembly method or gene synthesis can be used to introduce protein mutations.
  • the automatic PCR is carried out according to the following operations:
  • Mutations are generated by introducing the degenerate codon NNK, and each gene numbering site saturation mutation library is obtained by assembling two fragments amplified using the recombinant plasmid as a template; when the two fragments are assembled, the assembly reagent is placed in the automatic pipetting
  • the workstation mixes the components in the reaction system into the PCR reaction plate according to the edited automated PCR amplification reaction program script, and transfers them to the automated PCR instrument for reaction through the automated pipetting arm; Transported back to the automated pipetting workstation by the automated robotic arm, DpnI enzyme was added to each reaction, and then reacted in an automated PCR instrument to digest the template plasmid; after the reaction was completed, it was placed on the consumable stack, and fragment 1 and fragment 2 were respectively Perform gel electrophoresis.
  • the automatic assembly is performed according to the following operations:
  • the automatic transformation is performed according to the following operations:
  • the protein automatic engineering transformation optimization method of the present invention also includes:
  • the automated high-throughput mixed pool sequencing library was constructed, and the mutant strains with single amino acid mutations in the saturated mutation library were found according to the mixed pool sequencing results.
  • the process of constructing an automated high-throughput mixed pool sequencing library includes:
  • High-throughput selection of saturated mutant libraries place the plate with colonies on the plate position specified by the automatic pick cloner, run the picking clone program, and pick and inoculate the single clones in the plate into deep wells equipped with LB liquid culture Place the deep-well plate in a shaker for culture, and then put it on the consumable stack of the functional island.
  • the automated robotic arm will transfer the deep-well plate to the automatic pipetting workstation, and transform it into competent cells according to the edited automated plasmid.
  • the program script first mixes the bacterial liquid and glycerin solution in a new microplate, and the automatic robotic arm transfers it to the sealing machine for sealing;
  • Automated construction of high-throughput mixed pool sequencing library 2 times of automated PCR: bacterial liquid PCR and index PCR, purification of 2 times of automated PCR products, and DNA quantitative homogenization.
  • the protein automatic engineering transformation optimization method of the present invention also includes:
  • one or more of MALDI ion source and TOF detector mass spectrometer, ESI ion source mass spectrometer, and QQQ detector are used to characterize the catalytic properties of the protein.
  • the catalytic performance of the protein can be analyzed based on the whole cell product, purified protein or cell lysate.
  • the present invention is the automatic engineering of rhamnolipid acyltransferase.
  • the present invention uses an automated functional island, and the instruments and equipment on the island include automated robotic arms, automated liquid transfer workstations, automated PCR instruments, automated centrifuges, consumable stacks, film sealing machines, and film tearing machines.
  • machine, microplate reader, and automated shaking incubator among which Evoware, the operating system of the automated liquid transfer workstation, was provided by the manufacturer, and Momentum, the operating system of the functional island, was provided by Thermo Fisher.
  • the automatic pipetting workstation is equipped with flexible eight-channel pipetting tips, high-throughput 96-channel pipetting tips, multi-functional gripper robotic arm, low temperature control module, heating control module, oscillation module, orifice plate position, tip plate positions and supplies holders.
  • This functional island and other automated equipment such as: automatic clone picking instrument, automatic nucleic acid extraction instrument, etc., are the high-throughput and automatic construction process of the protein saturation mutation library involved in the present invention; the automatic sorting process of the saturation mutation library; high-throughput The construction process of the mixed pool sequencing library; and the automatic mass spectrometry characterization of protein catalytic properties provide strong support, and the relevant process processes are edited into automated program scripts to realize the corresponding automated experimental processes.
  • the automated liquid pipetting workstation may include liquid manipulation instruments using various principles, such as semi-automatic discharge guns, microfluidics, ultrasonic pipetting, and the like.
  • automation devices that are slightly different in instrument configuration and transfer means from the automation platform described in the present invention, such as instruments of different brands, mechanical arms or slide rails of different models, etc., or part of the process is manually operated.
  • PCR and DNA barcoding methods can be used to design and carry out mixed pool NGS sequencing analysis to obtain mutation information, and other alternatives such as methods based on transposon disruption can also be used.
  • the invention is a common technical method for high-throughput and automatic research on enzyme proteins by using multidisciplinary methods such as synthetic biology, analytical chemistry, and automation.
  • the present invention has developed an integrated platform to complete the whole process of protein automation transformation research, the process flow is simple, generalizable, and can meet the optimization of engineering transformation of different proteins.
  • the automated synthetic biology technology involved in the present invention can greatly shorten the construction and characterization time of a systematic single-site saturation mutation library.
  • the method of the present invention can accurately and quantitatively describe the enzyme activity, catalytic specificity, etc. by using a high-throughput mass spectrometry method, and the analysis time for a single sample only needs 2 seconds.
  • the present invention implements parallel analysis of tens of thousands of samples by introducing DNA barcodes to carry out mixed pool sequencing, and greatly reduces costs (no more than 10 yuan/sample); and develops an automated sequencing library construction method , use the automatic pipetting workstation to complete the PCR reaction system configuration, PCR product purification, sequencing library concentration determination and homogenization and other operations during the library construction process, reduce the error in the experimental process, obtain a library with better uniformity, and realize the whole process of unmanned on duty.
  • the automatic mass spectrometry detection method involved in the present invention can quickly realize the automatic pretreatment of mass spectrometry detection by means of a general-purpose pipetting workstation, reduce the pretreatment time and cost of a single sample through parallel operations, and can be directly combined with mass spectrometry without chromatographic separation. It can greatly increase the detection throughput (from 2-5 samples/hour to more than 800 samples/hour).
  • the existing protein single point mutation library is usually based on the error-prone PCR method, which can only construct and characterize some single point mutations (6*number of amino acid residues), while the method of the present invention can systematically characterize all possible single point mutations (19*number of amino acid residues), the coverage rate is more than 3 times higher than that of existing methods, and beneficial mutations that cannot be obtained by existing methods can be obtained.
  • Figure 1 is a schematic diagram of the implementation process for the construction of an enzyme protein saturation mutation library.
  • Fig. 2 is the gel electrophoresis picture of Fragment 1.
  • Fig. 3 is a gel electrophoresis picture of Fragment 2.
  • Figure 4 shows the results of sequencing verification of the saturated mutation library.
  • Figure 5 is a schematic diagram of the implementation process for the automatic construction of a high-throughput mixed pool sequencing library.
  • Figure 6 is a schematic diagram of index PCR primer combinations.
  • Figure 7 is the capillary electrophoresis results of the high-throughput sequencing library.
  • pictures A and B are the results of building a database in the 1-300bp interval
  • pictures C and D are the results of building a database in the 300-600bp interval
  • pictures E and F are the results of building a database in the 601-888bp interval.
  • Figure 8 is a schematic diagram of the implementation path of automatic and high-throughput mass spectrometry characterization of protein catalytic properties.
  • Figure 9 is the structural formula of rhamnolipid homologues.
  • Fig. 10 is a schematic structural diagram of an automation function island adopted in the present invention.
  • the automatic construction of a saturated mutation library for rhamnolipid acyltransferase is carried out, and the mutant library is constructed by designing a degenerate primer (NNK) at the mutation site, as shown in Figure 1.
  • the automated construction process is mainly Including automated PCR, automated nucleic acid extraction, automated assembly, and automated transformation. The route of this process in the automation function and other automated equipment is shown in Figure 1.
  • the consumables stack stores the reagent consumables used in each experimental process, and the automated robotic arm is responsible for Instrument-to-instrument, instrument-to-consumable stack interaction.
  • the rhamnolipid acyltransferase genes rhlA (888bp) and rhlB (1281bp) were constructed into the pRSF-DUET vector to obtain a recombinant plasmid pRSF-rhlA-rhlB with a total length of 5944bp.
  • the mutation is generated by introducing a degenerate codon NNK (N represents any base in A, T, C, and G, K represents any base in T, G), and the saturation mutation primer See Table 1.
  • NNK represents any base in A, T, C, and G
  • K represents any base in T, G
  • Each gene number site saturation mutation library is obtained by Gibson assembly of two fragments amplified using recombinant plasmids as templates.
  • Fragment 1 (PCR-1) is composed of mutation forward primer (S1-F) and reverse primer
  • the forward primer A (S1-R) is amplified through the reaction system 1
  • the fragment 2 (PCR-2) is obtained through the amplification of the forward primer B (S2-F) and the reverse primer (S2-R) through the reaction system 2.
  • the sequence numbered 10-105 in the gene rhlA sequence is used to construct a saturation mutation library.
  • Fragment 1 has 96 groups in total, and fragment 2 is divided into 12 groups based on each 24bp in the rhlA DNA sequence of the target gene.
  • Reaction system 1 is pRSF-rhlA-rhlB plasmid 1ul (concentration 1ng/ul), forward mutation primer 2ul (10mM), reverse primer A 2ul (10mM), PrimeSTAR premixed high-fidelity polymerase 25ul (purchased from Takara), Double distilled water 20ul, the total reaction volume is 50ul; reaction system 2 is pRSF-rhlA-rhlB plasmid 1ul (concentration 1ng/ul), forward primer B 2ul (10mM), reverse primer 2ul (10mM), PrimeSTAR premixed high-density True polymerase 25ul (purchased from Takara), double distilled water 20ul, the total reaction volume is 50ul.
  • the reaction program was pre-denaturation at 98°C for 5 minutes, followed by 30 cycles of reaction, each cycle including: denaturation at 98°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 3 minutes, extension at 72°C for 10 minutes, and finally at 4°C save.
  • Qiagen Automated Nucleic Acid Extractor and Tiangen PCR Product Purification Kit were used to purify the above-mentioned PCR-1 and PCR-2 products, and the adaptability of Tiangen PCR Product Purification Kit to Qiagen instruments was verified.
  • the purification cost is saved, the price of Qiagen’s original PCR purification kit is 16.7 yuan/reaction, and the price of Tiangen PCR purification kit is 2.38 yuan/reaction.
  • the purification operation only needs to put the PCR product and the corresponding reagent consumables in the designated position of the instrument, and the instrument automatically runs the program, which can purify 12 PCR products each time, and store the purified fragments in a 4°C refrigerator. On the consumable stack of the automation function island.
  • an automated pipetting workstation and an automated PCR instrument are used to perform Gibson assembly on the nucleic acid fragments purified in the previous step, and each reaction should assemble a PCR-1 and a PCR-2 into a complete plasmid (that is, a saturated mutant plasmid) , according to the design of PCR primers, every 8 different PCR-1s can be assembled in one PCR-2 respectively, and finally a total of 96 saturated mutant plasmids are assembled, and this process can carry out 96 assembly reactions at the same time.
  • the Gibson assembly reaction solution was pre-configured before the experiment, including 320 ⁇ l 5X ISO buffer, 0.64 ⁇ l T5 exonuclease (10 U/ul, purchased from NEB Company in the United States), 20 ⁇ l Phusion polymerase (2 U/ ⁇ l, purchased from NEB Company in the United States) ), 160 ⁇ l Taq ligase (40U/ul, purchased from NEB Company in the United States), double distilled water to make up to 1.2mL and store at -20°C. Place the components required for the Gibson assembly reaction into the reagent tank of the automated pipetting workstation.
  • the reaction system is: Gibson assembly reaction solution 7.5ul, purified nucleic acid fragment 1 1.25ul, purified nucleic acid fragment 2 1.25ul, the total reaction System 10ul.
  • the automated pipetting workstation mixed the groups in the Gibson assembly reaction according to the edited automated Gibson assembly program script and sent them to the PCR instrument through the automated robotic arm for reaction at 50°C for 1 hour.
  • an automated liquid transfer workstation was used to transform the aforementioned saturated mutant plasmid into Escherichia coli Dh5a competent cells (purchased from Kangti Biology), and this process can carry out 96 transformation experiments simultaneously.
  • 50ul competent cells were added to the saturated mutant plasmid, and incubated for 1h on the 4°C low temperature control module of the automated liquid pipetting workstation, and transferred to Heat shock on the heating control module at 42°C for 45s, transfer to the low temperature control module at 4°C and incubate for 2min, then add 500ul LB medium without antibiotics, transfer to the heating block at 37°C and incubate for 1h, centrifuge to remove 300ul of the supernatant, and leave After resuspending 200ul of the bacterial solution by pipetting, it was evenly added dropwise to the plate containing the antibiotic, and placed in a 37°C microbial incubator to invert the culture
  • the success of the construction is evaluated by sequencing the transformed saturated mutation library.
  • the sequencing results show that there is an NNK peak at the position of the target number, which means that the library construction is successful.
  • Take out the plate cultured overnight in the previous step add 3mL LB medium dropwise on the plate, scrape off all the colonies in the plate with a spreader stick, collect them into a new Ep tube, draw 500ul of bacteria liquid and 500ul of 40% glycerol solution from it After mixing, they were stored in a -80°C refrigerator, and the remaining bacterial liquid was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing.
  • the sequencing results are shown in Figure 4.
  • the constructed 96 rhamnolipid acyltransferase saturation mutation libraries were constructed by automatic high-throughput mixed-pool sequencing library. According to the mixed-pool sequencing results, mutants with single amino acid mutations in the saturation mutation library could be found.
  • the process includes: high-throughput selection of saturated mutant libraries, automatic mutant strain mixing, and automatic construction of high-throughput mixed pool sequencing libraries.
  • the implementation path of this process on the automation function island and other automation equipment is shown in Figure 5.
  • the consumables stack stores the reagent consumables used in each experiment process, and the automated robotic arm is responsible for the connection between instruments and instruments, and between instruments and consumables stacks. interaction.
  • the plasmids of the saturation mutation library were automatically transformed into Escherichia coli BL21(DE3) expression competent cells (purchased from Kangti Life) according to the method in Example 1, and cultured overnight in a 37°C microbial incubator. Place the plate with colonies on the plate position specified by the automatic pick cloner, run the pick clone program, pick and inoculate the single clone in the plate into a 96-well deep-well plate equipped with LB liquid culture, and each saturation mutation 192 single clones were picked from the library (two 96-well deep-well plates).
  • the automated robotic arm will transfer the 96-well deep-well plate to the automatic pipetting workstation.
  • the edited automated plasmid Transform into Escherichia coli competent cell program script First mix 100ul bacterial solution and 100ul 40% glycerol solution in a new 96-well microtiter plate, transfer the automated robotic arm to the sealing machine for sealing, and then transfer to -80°C Store in the refrigerator.
  • the mutant strain bacterial liquids picked out of different numbered saturated mutation libraries were mixed, and the mixing rule was: the mutant strains in the same position of two 96-well deep-well plates picked out of different saturated mutant libraries were mixed (as shown in Figure 6 index PCR primer combination diagram for each site saturation mutation library A1 was mixed, and so on), and finally all the mutants picked from the saturation mutation library were mixed into two new 96-well deep-well plates, and in Store overnight in a -80°C refrigerator.
  • automatic construction of a high-throughput mixed-pool sequencing library is achieved by using an automated liquid pipetting workstation, etc., so that mutant strains with 21 kinds of amino acid (including stop codon) mutations in multiple saturated mutation libraries can be found by one-time sequencing, and can be regenerated. Locate to the 2 picked 96-well plates.
  • the present invention performs high-throughput mixed pool sequencing on 18,432 mutant strains in 96 saturated library libraries, and finally picks out 1,667 non-repetitive mutant strains.
  • the process of automated construction includes 2 times of automated PCR (bacteria liquid PCR and index PCR), 2 times of automated PCR product purification and DNA quantitative normalization.
  • the present invention takes out and thaws the above-mentioned frozen mixed bacterial liquid, and utilizes an automatic pipetting workstation to carry out bacterial liquid PCR to expand the target sequencing sequence. Since the target gene is 888 bp in full length, in order to meet the requirements of next-generation sequencing, 3 pairs of primers are designed for each The 1-300bp, 301-600bp and 601-888bp regions were amplified by PCR, and the primer sequences were shown in Table 2.
  • Bacterial solution PCR reaction system 10ul PrimeSTAR premixed high-fidelity polymerase, 1ul forward primer (10uM), 1ul reverse primer (10uM), 1ul bacterial solution, 7ul double distilled water, total reaction system 20ul, automatic pipetting workstation will The components of the reaction system are mixed and sent to the automatic PCR instrument by the automatic robot arm to run the PCR reaction program of the bacterial liquid: first, pre-denaturation at 98°C for 5 minutes, followed by 18 cycles of denaturation at 98°C for 10 seconds, annealing at 58°C for 10 seconds, and 72°C Extended for 30s, then extended at 72°C for 5min, and finally stored at 4°C.
  • the magnetic bead purification of the PCR product is carried out by the automated liquid transfer workstation, and the PCR product is subjected to magnetic bead adsorption, ethanol elution, removal of the supernatant, and addition of ultra-pure Automated purification steps such as water elution.
  • the purified PCR product was placed on the 4°C cooling control module of the automatic liquid pipetting workstation for the next step of index PCR reaction.
  • the sequence of the index primers is shown in Table 3, N501-N512 is index primer 1, and N701-716 is index primer 2 .
  • index primers are carried out according to Figure 6, and the index primers of A1 well are N501 and N701 respectively.
  • the index PCR reaction system is: 10ul PrimeSTAR premixed high-fidelity polymerase, 2ul index primer 1 (10uM), 2ul index primer 2 (10uM), 6ul product purified by PCR in the previous step, total reaction system 20ul, automatic pipetting workstation
  • the automated robotic arm was transferred to the automated PCR machine to run the index PCR reaction program: first, pre-denaturation at 98°C for 5 minutes, followed by 10 cycles of denaturation at 98°C for 10 seconds, annealing at 58°C for 10 seconds, and extension at 72°C for 30 seconds. Then extend at 72°C for 5 minutes, and finally store at 4°C.
  • the PCR product is purified by magnetic beads on an automated liquid transfer workstation.
  • Example 3 Automated, high-throughput mass spectrometry characterization of protein catalytic properties
  • automated matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF-MS) detection was carried out on the mutant libraries numbered 10-29.
  • the process includes automated cultivation of mutant strains to produce target metabolites, automated sample pretreatment, MALDI automatic detection, etc.
  • 20 saturated mutant libraries were characterized by automated mass spectrometry, and the mass spectrometry data of all mutant strains could be obtained within 4 days.
  • MALDI-ToF-MS could detect 1536 samples within 1 hour, while traditional LC -
  • the MS detection method takes more than 250 hours to complete the detection of a corresponding number of samples.
  • the implementation path of this process between the automation function island and other instruments is shown in Figure 8.
  • the consumables stack stores the reagent consumables used in each experiment process, and the automated robotic arm is responsible for the relationship between instruments and instruments, and between instruments and consumables stacks. interaction between.
  • the mutant strains found in the numbered 10-29 saturated mutation library (20 in total) were automatically cultured to produce target metabolites, and the frozen samples of the 20 saturated mutant libraries stored at -80°C were taken out and placed in On the consumables stack of the automated functional island, after the bacterial liquid is thawed, it is immediately transferred to the automatic pipetting workstation through the automated robotic arm, and 20ul of the mutant strain bacterial liquid is drawn from a specific position of the well plate to the containing 24 wells of LB agar medium were cultured overnight in a microbial incubator at 37°C.
  • the automated liquid pipetting workstation dipped a small amount of bacteria into a 24-well deep-well plate containing LB medium, and transferred it to an automated shaking incubator through an automated robotic arm, and then transferred it to the automated liquid pipetting workstation after shaking and culturing at 37°C for 3-4 hours. Preserve and transfer the bacteria liquid.
  • an automated liquid pipetting workstation is used to perform ethyl acetate extraction on the induced bacterial solution, and the extracted sample is automatically added to the MALDI-ToF-MS metal target plate.
  • Transfer the induced 24-well deep-well plate to an automatic pipetting workstation add an equal volume of ethyl acetate solution, shake it in an automatic shaking incubator for 10 minutes, remove it, and place it in an automatic centrifuge for 2 minutes.
  • Bruker's autoflex MALDI-ToF-MS was used for detection.
  • the metabolite detected in the implementation case was rhamnolipid, and there were four homologues (5a, 5b, 5c, 5d) whose structures were shown in Figure 9.
  • Table 5 The ratio of the four homologues in the mutant product compared to the wild type
  • 013G-Terminator 1.318 0.324 2.977 19.829 013G-A 0.413 1.037 1.398 2.737 013G-C 0.366 1.016 1.587 3.041 013G-D 0.938 0.987 1.089 1.768 013G-E 0.568 1.020 1.317 2.489 013G-F 0.716 0.778 1.898 9.735 013G-G 1.114 0.995 0.900 0.684 013G-H 0.510 1.042 1.376 1.550 013G-I 0.434 1.031 1.379 2.942 013G-K 0.575 0.987 1.372 3.766 013G-L 0.624 0.816 1.764 9.625 013G-M 0.865 0.703 2.065 10.940 013G-N 0.809 1.022 1.133 1.014 013G-P 0.726 0.795 1.917 8.595 013G-Q 0.000 0.000 0.000 0.000 013G-R 0.441
  • 019E-D 0.382 1.100 1.505 1.981 019E-E 0.000 0.000 0.000 0.000 0.000 0.000 019E-F 0.305 1.071 1.901 2.020 019E-G 0.280 1.014 2.134 3.364 019E-H 0.395 1.094 1.626 1.436 019E-I 0.804 1.043 1.095 1.184 019E-K 0.748 1.051 1.202 1.033 019E-L 0.448 1.035 1.677 2.828 019E-M 0.450 1.082 1.518 1.819 019E-N 0.439 1.096 1.519 1.409 019E-P 1.001 0.454 2.838 12.834 019E-Q 0.685 1.066 1.244 0.973 019E-R 0.563 1.063 1.456 1.482 019E-S 0.548 1.075 1.427 1.356 019E-T 0.722 1.076 1.079 0.974 019E-V 0.740 1.048
  • 025P-G 0.823 1.008 1.183 1.195 025P-H 1.229 0.482 2.713 11.583 025P-I 0.996 0.975 1.077 1.693 025P-K 1.151 0.983 0.907 0.911 025P-L 1.025 0.992 0.971 1.296 025P-M 1.018 0.995 0.916 1.593 025P-N 1.221 0.479 2.624 12.344 025P-P 0.444 0.993 1.407 4.067 025P-Q 1.241 0.480 2.568 12.459 025P-R 0.984 1.003 1.016 0.891 025P-S 1.161 0.973 0.903 1.275 025P-T 1.110 0.980 0.979 0.992 025P-V 0.968 1.003 0.992 1.235 025P-W 1.097 0.989 0.895 1.244 025P-Y 0.942 0.991 1.115 1.222 026G-Terminator 0.000
  • 029T-I 0.625 0.923 2.170 0.000 029T-K 1.091 0.769 2.665 0.000 029T-L 1.110 0.654 3.535 0.000 029T-M 0.494 0.976 1.961 0.000 029T-N 0.482 1.023 1.610 0.000 029T-P 1.469 0.434 4.706 0.000 029T-Q 0.636 0.880 2.493 0.000 029T-R 0.757 0.876 2.338 0.000 029T-S 0.435 0.994 1.908 0.000 029T-T 1.183 0.981 0.866 0.000 029T-V 0.487 0.986 1.890 0.000 029T-W 0.960 0.800 2.622 0.000 029T-Y 0.996 0.770 2.801 0.000
  • the above-mentioned experiment on rhamnolipid acyltransferase of the present invention can complete the construction of a systematic single-point mutation library, NGS sequencing analysis and mutation sorting, cell fermentation and product mass spectrometry analysis within 6 weeks, and obtain a systematic mutation sequence- Catalytic performance correspondence, and screening mutants with desired catalytic performance.
  • each of the above-mentioned embodiments of the present invention can use an automated function island, its structure can be seen as shown in Figure 10, and the instruments and equipment in the island include automated manipulators, automated liquid transfer workstations, automated PCR instruments, automated centrifuges, and consumable stacks. , film sealing machine, film tearing machine, microplate reader, and automated shaking incubator, among which Evoware, the operating system of the automated liquid transfer workstation, is provided by the manufacturer, and Momentum, the operating system of the functional island, is provided by Thermo Fisher.
  • the automatic pipetting workstation is equipped with flexible eight-channel pipetting tips, high-throughput 96-channel pipetting tips, multi-functional gripper robotic arm, low temperature control module, heating control module, oscillation module, orifice plate position, tip plate positions and supplies holders.
  • This functional island and other automation equipment such as: automatic clone selection instrument, automatic nucleic acid extraction instrument, etc., are the high-throughput and automatic construction process of the protein saturation mutation library involved in the present invention; the automatic sorting process of the saturation mutation library; high-throughput The construction process of the mixed pool sequencing library; and the automatic mass spectrometry characterization of protein catalytic properties provide strong support, and the relevant process processes are edited into automated program scripts to realize the corresponding automated experimental processes.

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Abstract

一种蛋白质自动化工程改造优化方法，所述方法包括自动化构建蛋白质饱和突变文库的过程，其中，自动化构建蛋白质饱和突变文库的过程包括：在突变位点设计简并引物引入氨基酸突变，进行自动化PCR、自动化核酸提取、自动化组装、自动化转化，转化后获得饱和突变文库。该方法还进一步包括以饱和突变文库为基础，进行自动化高通量混池测序文库的构建，对饱和突变文库进行了自动化质谱表征的过程。

Description

蛋白质自动化工程改造优化方法 技术领域
本发明是关于一种蛋白质自动化工程改造优化方法，具体涉及蛋白质高通量、自动化的饱和突变文库构建、高通量混池测序、质谱表征方法等。
背景技术
蛋白质分子改造的方法主要分为理性设计、定向进化以及二者的结合。定向进化基于多轮随机突变与筛选的非理想手段，在没有结构与功能信息的前提下实现目标性能的优化。深度扫描突变结合了易错PCR和饱和突变方法的特点，利用简并引物构建覆盖目标蛋白所有可能单点突变的系统性文库。现有深度扫描突变方法需要将蛋白目标性质与细胞生长或者荧光信号偶联，通过竞争性生长或荧光分选改变群体中不同突变体丰度。目前深度突变扫描方法多限于荧光蛋白、蛋白质互作等实验模型，缺乏通用的酶蛋白序列-功能关系的通用性方法。
酶蛋白的随机突变文库和饱和突变文库通常容量巨大，需要高通量表征突变株性能。对大多数生化反应而言，需要通过遗传选择、偶联反应、生物传感器等手段，将目标分子的浓度信息转化为易于高通量筛选的信号。但是，建立筛选方法通常耗时耗力，且酶蛋白的底物多样性和产物差异性导致很难将过程中的分子结构的细微差别与生长速率、荧光强度等信号偶联。因此，需要开发分子水平上对反应物的定性定量筛选的通用质谱方法。质谱对目标化合物的离子进行分析，基于分子离子与碎片离子的荷质比可以实现高精度的定性指认，基于离子信号的强度可以实现定量分析。传统方法中，色谱分离过程限制了质谱分析的通量，而基于最新的仪器设计和样品前处理方法，可以利用激光、微流控、超声移液等手段将分析物直接引入质谱提供通量。基质辅助激光解离电离时间飞行质谱近年来发展起来的一种新型的软电离生物质谱,基质辅助激光解析电离离子源用激光照射样品与基质形成的共结晶薄膜，基质从激光中吸收能量传递给生物分子，而电离过程中将质子转移到生物分子或从生物分子得到质子，而使生物分子电离的过程，最后通过飞行时间检测器进行化学物的指认以及定量。发明人前期开发了基质辅助激光解离电离时间飞行质谱成像技术，对平板上随机分布的菌落实现自动化激光取样与质谱分析(2秒/样品)。
由于生物系统高度复杂，目前人工设计的合成生命体很难完全按照预期工作，往往需要长时间的反复调试。自动化合成生物技术通过低成本、多循环地完成海量工程试错 性实验，快速实现特定功能。近年来，在“设计-构建-测试-学习”的各个合成生物研究环节，自动化技术在飞速发展。对于酶蛋白质的深度突变扫描、高通量测序文库构建与自动化质谱检测，自动化合成生物技术可以大大加速研究进程。其中涉及自动化移液工作站、自动化PCR仪、自动化机械臂等硬件设备以及相应的集成控制软件，可以将繁复的手工操作流程转换为自动化、高通量且实验数据稳定的自动化流程。
然而，现有自动化合成生物技术仍存在以下问题：
(1)高通量设备功能单一，串接工艺流程复杂，人工参与极易引入错误。
(2)目前蛋白质饱和突变文库构建过程多为手工操作，工作量极大，文库分选困难，例如对含有300个氨基酸的酶蛋白，需要构建300个单点饱和文库，分选6万个突变株，至少6000个性能检测，无法依靠人力在合理时间内完成。
(3)现有蛋白质突变分析，通常基于第一代测序方法，对每个样品进行单独分析，通量低、成本高(30元/样品)。
(4)质谱检测对样品的质量有较高的要求，盐离子含量过高会极大损害仪器，且降低检测灵敏度和准确度等；质谱样品前处理方法通常繁琐、成本高，需要和色谱方法联用，分析通量低。
发明内容
本发明的一个目的在于提供一种蛋白质自动化工程改造优化方法。
具体而言，本发明提供了一种蛋白质自动化工程改造优化方法，该方法包括自动化构建蛋白质饱和突变文库的过程，其中，自动化构建蛋白质饱和突变文库的过程包括：
在突变位点设计简并引物引入蛋白质突变，进行自动化PCR、自动化核酸提取、自动化组装、自动化转化，转化后获得饱和突变文库。
根据本发明的具体实施方案，本发明的蛋白质自动化工程改造优化方法中，可采用Gibson assembly方法或基因合成引入蛋白质突变。
根据本发明的具体实施方案，本发明的蛋白质自动化工程改造优化方法中，所述自动化PCR按照以下操作进行：
通过引入简并密码子NNK产生突变，每一个基因编号位点饱和突变文库由以重组质粒为模板扩增出来的2个片段进行组装获得；其中两片段组装时，将组装试剂放在自动化移液工作站的试剂槽内，工作站按照编辑好的自动化PCR扩增反应程序脚本将反应体系中的各组分混合到PCR反应板中，通过自动化移液臂转移至自动化PCR仪中反应；反应结束后再由自动化机械臂运回自动化移液工作站，向每一个反应中添加DpnI 酶，再于自动化PCR仪中进行反应，以消化模板质粒；反应结束后放置在耗材堆栈上，分别对片段1和片段2进行凝胶电泳。
根据本发明的具体实施方案，本发明的蛋白质自动化工程改造优化方法中，所述自动化组装按照以下操作进行：
利用自动化移液工作站和自动化PCR仪对纯化过的核酸片段进行Gibson组装；将Gibson组装反应所需组分放置到自动化移液工作站的试剂槽中，自动化移液工作站根据编辑好的自动化Gibson组装程序脚本将Gibson组装反应中的各组混合并通过自动化机械臂送至PCR仪中进行反应。
根据本发明的具体实施方案，本发明的蛋白质自动化工程改造优化方法中，所述自动化转化按照以下操作进行：
使用自动化移液工作站将饱和突变质粒转化至感受态细胞中，根据编辑好的自动化质粒转化至感受态细胞程序脚本将感受态细胞添加到饱和突变质粒中，并在自动化移液工作站的低温控制模块上孵育，通过工作站机械抓手转移到加热控制模块上热激，之后转移至低温控制模块上孵育，后添加无抗生素的培养基，再转移至加热模块上孵育，离心去除上清液，剩下菌液吹打重悬后均匀滴加至含有抗生素的平皿中，放在微生物培养箱中倒置培养过液。
根据本发明的具体实施方案，本发明的蛋白质自动化工程改造优化方法，还包括：
以饱和突变文库为基础，进行自动化高通量混池测序文库的构建，根据混池测序结果找出饱和突变文库中单一氨基酸突变的突变株。
根据本发明的具体实施方案，本发明的蛋白质自动化工程改造优化方法中，自动化高通量混池测序文库的构建的过程包括：
饱和突变文库的高通量挑选：将长有菌落的平皿放置在自动挑克隆仪规定的板位上，运行挑克隆程序，将平皿中的单克隆挑取接种到装有LB液体培养的深孔板中；将深孔板置于摇床中培养，之后放在功能岛的耗材堆栈上，自动化机械臂将深孔板转移至自动化移液工作站中，根据编辑好的自动化质粒转化至感受态细胞程序脚本先将菌液与甘油溶液混合在新的酶标板中，自动化机械臂转移至封膜机上进行封膜处理；
自动化突变株混合：将不同编号饱和突变文库挑取出来的突变株菌液进行混合，混合规则为：不同饱和突变文库挑取出来的2块深孔板的同一位置的突变株进行混合，最终将所有饱和突变文库挑取的突变株混合到新的2块深孔板中；并于-80℃冰箱中过夜静置；
自动化构建高通量混池测序文库：进行2次自动化PCR：菌液PCR和index PCR， 对2次自动化PCR产物进行纯化，进行DNA定量均一化。
根据本发明的具体实施方案，本发明的蛋白质自动化工程改造优化方法还包括：
对饱和突变文库进行了自动化质谱表征。
优选地，使用MALDI离子源和TOF检测器质谱、ESI离子源质谱仪、QQQ检测器中的一种或多种对蛋白质催化性质进行表征。
根据本发明的具体实施方案，本发明的蛋白质自动化工程改造优化方法中，可基于全细胞产物、纯化蛋白或细胞裂解物对蛋白质催化性能进行分析。
根据本发明的一些具体实施方案，本发明是对鼠李糖脂酰基转移酶进行自动化工程改造。
根据本发明的一些具体实施方案，本发明使用了一种自动化功能岛，岛内仪器设备有自动化机械臂、自动化移液工作站、自动化PCR仪、自动化离心机、耗材堆栈、封膜机、撕膜机、酶标仪以及自动化振荡培养箱，其中自动化移液工作站操作系统Evoware由生产商提供，功能岛的操作系统Momentum由赛默飞提供。自动化移液工作站配备了灵活八通道移液吸头、高通量96通道移液吸头、多功能抓手机械臂、低温控制模块、加热控制模块、振荡模块、孔板板位、吸头板位和耗材载架若干。该功能岛与其他自动化设备，如：自动化挑克隆仪、自动化核酸提取仪等，为本发明涉及的蛋白质饱和突变文库高通量、自动化构建流程；饱和突变文库的自动化分选流程；高通量混池测序文库构建流程；以及蛋白质催化性质自动化质谱表征等流程提供了有力支持，将相关工艺流程编辑成自动化程序脚本来实现相应的自动化实验流程。
本发明中，自动化移液工作站可包括使用各种原理的液体操作仪器，如半自动排枪、微流控、超声移液等。
本发明中，使用仪器配置和转运手段与本发明描述的自动化平台稍有区别的自动化装置，如不同品牌仪器、不同型号机械手臂或滑轨等，或部分过程采取人工操作。
本发明中，可使用PCR和DNA条码方法设计和开展混池NGS测序分析获得突变信息，也可使用其他替代方案如基于转座子打断的方法等。
本发明是利用合成生物学、分析化学、自动化等多学科方法进行酶蛋白的高通量、自动化研究的共性技术方法。
综合而言，本发明的技术具有以下技术效果：
(1)本发明开发了一套集成平台完成蛋白自动化改造研究的全过程，工艺流程简洁，具有泛化性，满足不同蛋白的工程改造优化。
(2)本发明涉及的自动化合成生物技术可以大大缩短系统性单位点饱和突变文库的构建和表征时间。本发明方法使用高通量质谱方法可以对酶活、催化特异性等进行精确、定量刻画，单个样品分析时间仅需2秒钟。
(3)本发明基于第二代测序原理，通过引入DNA条码开展混池测序，实现对上万个样品的平行分析，大幅降低成本(不超过10元/样品)；且开发自动化测序文库构建方法，使用自动化移液工作站完成文库构建过程中的PCR反应体系配置、PCR产物纯化、测序文库浓度测定与均一化等操作，减少实验过程的误差，获得均一性更好的文库，可以实现全程无人值守。
(4)本发明涉及的自动化质谱检测方法借助通用的移液工作站可以快速实现质谱检测的自动化前处理，通过平行操作降低单个样品的前处理时间和成本，并且可以不经过色谱分离直接和质谱联用，大幅提高检测通量(从2-5样品/小时提高至800样品/小时以上)。
(5)现有蛋白质单点突变库通常基于易错PCR方法，仅能构建和表征部分单点突变(6*氨基酸残基数)，而本发明方法可以对所有可能单点突变进行系统性表征(19*氨基酸残基数)，比现有方法的覆盖率提高3倍以上，可以获得现有方法无法获得的有益突变。
附图说明
图1为酶蛋白饱和突变文库构建实施流程示意图。
图2为片段1凝胶电泳图。
图3为片段2凝胶电泳图。
图4为饱和突变文库测序验证结果。
图5为高通量混池测序文库自动化构建实施流程示意图。
图6为index PCR引物组合示意图。
图7为高通量测序文库毛细管电泳结果。其中，图片A和图片B为1-300bp区间建库的结果，图片C和图片D为300-600bp区间建库的结果，图片E和图片F为601-888bp区间建库的结果。
图8为蛋白质催化性质自动化、高通量质谱表征实施路径示意图。
图9为鼠李糖脂同系物结构式。
图10为本发明采用的一种自动化功能岛结构示意图。
具体实施方式
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现结合具体实施例及附图对本发明的技术方案进行以下详细说明，应理解这些实施例仅用于说明本发明而不用于限制本发明的范围。除非另外专门定义，本文使用的所有技术和科学术语都与相关领域普通技术人员的通常理解具有相同的含义。实施例中未特别注明的方法操作，按照所属领域现有技术的常规操作或商厂说明书建议的操作进行。
实施例1、蛋白质饱和突变文库的自动化构建
本实施例对鼠李糖脂酰基转移酶进行了饱和突变文库的自动化构建，通过在突变位点设计简并引物(NNK)的方法来构建突变文库，请参见图1所示，自动化构建流程主要包括自动化PCR、自动化核酸提取、自动化组装、自动化转化，该过程在自动化功能及其他自动化设备实施的路线如图1，此外耗材堆材存放每个实验过程中使用到的试剂耗材，自动化机械臂负责仪器与仪器、仪器与耗材堆栈之间的交互。
1.1自动化PCR
本实施例将鼠李糖脂酰基转移酶基因rhlA(888bp)和rhlB(1281bp)构建到pRSF-DUET载体上中，获得总长度为5944bp的重组质粒pRSF-rhlA-rhlB。以重组质粒为模板，通过引入简并密码子NNK(N代表A、T、C、G中的任意一种碱基，K代表T、G中的任意一种碱基)产生突变，饱和突变引物见表1，每一个基因编号位点饱和突变文库由以重组质粒为模板扩增出来的2个片段进行Gibson组装获得，片段1(PCR-1)由突变正向引物(S1-F)和反向引物A(S1-R)经过反应体系1扩增获得，片段2(PCR-2)由正向引物B(S2-F)和反向引物(S2-R)经过反应体系2扩增得到。本实施例对基因rhlA序列中编号10-105的序列进行饱和突变文库的构建，片段1共有96组，片段2以目的基因rhlA DNA序列中每24bp为一组，分为12组，故两片段组装时，每8个不同的片段1分别与同一个片段2进行反应，例如基因编号10-17的片段1需要分别与10-17-S2-R引物扩增出的片段2进行组装。反应体系1为pRSF-rhlA-rhlB质粒1ul(浓度1ng/ul)，正向突变引物2ul(10mM)，反向引物A 2ul(10mM)，PrimeSTAR预混高保真聚合酶25ul(购自Takara)，双蒸水20ul，反应总体积为50ul；反应体系2为pRSF-rhlA-rhlB质粒1ul(浓度1ng/ul)，正向引物B 2ul(10mM)，反向引物2ul(10mM)，PrimeSTAR预混高保真聚合酶25ul(购自Takara)，双蒸水20ul，反应总体积为50ul。将上述试剂放在自动化移液工作站的试剂槽内，工作站按照编辑好的自动化PCR扩增反应程序脚本将反应体系中的各组分混合到PCR反应板中，通过自动化移液臂转移至自动化PCR仪中，反应程序为98℃预变性5min后，进行30个循环反应，每个循环包括：98℃变 性30s，58℃退火30s，72℃延伸3min，之后在于72℃延伸10min，最后于4℃保存。反应结束后再由自动化机械臂运回自动化移液工作站，向每一个反应中添加10U DpnI酶(购自美国NEB公司)，再于自动化PCR仪中进行37℃反应1h，以消化模板质粒。反应结束后放置在耗材堆栈上，分别对片段1和片段2进行凝胶电泳，结果图2与图3所示。
突变引物序列如表1所示。
表1
编号 序列(SEQ ID No.1-SEQ ID No.110) 长度(bp)
10-S1-F agtctgttggtatcgNNKtgcaagggcctgcgggtacatgtcgagcgcgttggg 54
11-S1-F agtctgttggtatcggttNNKaagggcctgcgggtacatgtcgagcgcgttggg 54
12-S1-F agtctgttggtatcggtttgcNNKggcctgcgggtacatgtcgagcgcgttggg 54
13-S1-F agtctgttggtatcggtttgcaagNNKctgcgggtacatgtcgagcgcgttggg 54
14-S1-F agtctgttggtatcggtttgcaagggcNNKcgggtacatgtcgagcgcgttggg 54
15-S1-F agtctgttggtatcggtttgcaagggcctgNNKgtacatgtcgagcgcgttggg 54
16-S1-F agtctgttggtatcggtttgcaagggcctgcggNNKcatgtcgagcgcgttggg 54
17-S1-F agtctgttggtatcggtttgcaagggcctgcgggtaNNKgtcgagcgcgttggg 54
18-S1-F ggcctgcgggtacatNNKgagcgcgttgggcaggatcccgggcgcagcacggtg 54
19-S1-F ggcctgcgggtacatgtcNNKcgcgttgggcaggatcccgggcgcagcacggtg 54
20-S1-F ggcctgcgggtacatgtcgagNNKgttgggcaggatcccgggcgcagcacggtg 54
21-S1-F ggcctgcgggtacatgtcgagcgcNNKgggcaggatcccgggcgcagcacggtg 54
22-S1-F ggcctgcgggtacatgtcgagcgcgttNNKcaggatcccgggcgcagcacggtg 54
23-S1-F ggcctgcgggtacatgtcgagcgcgttgggNNKgatcccgggcgcagcacggtg 54
24-S1-F ggcctgcgggtacatgtcgagcgcgttgggcagNNKcccgggcgcagcacggtg 54
25-S1-F ggcctgcgggtacatgtcgagcgcgttgggcaggatNNKgggcgcagcacggtg 54
26-S1-F gttgggcaggatcccNNKcgcagcacggtgatgctggtcaacggcgcgatggcg 54
27-S1-F gttgggcaggatcccgggNNKagcacggtgatgctggtcaacggcgcgatggcg 54
28-S1-F gttgggcaggatcccgggcgcNNKacggtgatgctggtcaacggcgcgatggcg 54
29-S1-F gttgggcaggatcccgggcgcagcNNKgtgatgctggtcaacggcgcgatggcg 54
30-S1-F gttgggcaggatcccgggcgcagcacgNNKatgctggtcaacggcgcgatggcg 54
31-S1-F gttgggcaggatcccgggcgcagcacggtgNNKctggtcaacggcgcgatggcg 54
32-S1-F gttgggcaggatcccgggcgcagcacggtgatgNNKgtcaacggcgcgatggcg 54
33-S1-F gttgggcaggatcccgggcgcagcacggtgatgctgNNKaacggcgcgatggcg 54
34-S1-F acggtgatgctggtcNNKggcgcgatggcgaccaccgcctcgttcgcccggacc 54
35-S1-F acggtgatgctggtcaacNNKgcgatggcgaccaccgcctcgttcgcccggacc 54
36-S1-F acggtgatgctggtcaacggcNNKatggcgaccaccgcctcgttcgcccggacc 54
37-S1-F acggtgatgctggtcaacggcgcgNNKgcgaccaccgcctcgttcgcccggacc 54
38-S1-F acggtgatgctggtcaacggcgcgatgNNKaccaccgcctcgttcgcccggacc 54
39-S1-F acggtgatgctggtcaacggcgcgatggcgNNKaccgcctcgttcgcccggacc 54
40-S1-F acggtgatgctggtcaacggcgcgatggcgaccNNKgcctcgttcgcccggacc 54
41-S1-F acggtgatgctggtcaacggcgcgatggcgaccaccNNKtcgttcgcccggacc 54
42-S1-F atggcgaccaccgccNNKttcgcccggacctgcaagtgcctggccgaacatttc 54
43-S1-F atggcgaccaccgcctcgNNKgcccggacctgcaagtgcctggccgaacatttc 54
44-S1-F atggcgaccaccgcctcgttcNNKcggacctgcaagtgcctggccgaacatttc 54
45-S1-F atggcgaccaccgcctcgttcgccNNKacctgcaagtgcctggccgaacatttc 54
46-S1-F atggcgaccaccgcctcgttcgcccggNNKtgcaagtgcctggccgaacatttc 54
47-S1-F atggcgaccaccgcctcgttcgcccggaccNNKaagtgcctggccgaacatttc 54
48-S1-F atggcgaccaccgcctcgttcgcccggacctgcNNKtgcctggccgaacatttc 54
49-S1-F atggcgaccaccgcctcgttcgcccggacctgcaagNNKctggccgaacatttc 54
50-S1-F cggacctgcaagtgcNNKgccgaacatttcaacgtggtgctgttcgacctgccc 54
51-S1-F cggacctgcaagtgcctgNNKgaacatttcaacgtggtgctgttcgacctgccc 54
52-S1-F cggacctgcaagtgcctggccNNKcatttcaacgtggtgctgttcgacctgccc 54
53-S1-F cggacctgcaagtgcctggccgaaNNKttcaacgtggtgctgttcgacctgccc 54
54-S1-F cggacctgcaagtgcctggccgaacatNNKaacgtggtgctgttcgacctgccc 54
55-S1-F cggacctgcaagtgcctggccgaacatttcNNKgtggtgctgttcgacctgccc 54
56-S1-F cggacctgcaagtgcctggccgaacatttcaacNNKgtgctgttcgacctgccc 54
57-S1-F cggacctgcaagtgcctggccgaacatttcaacgtgNNKctgttcgacctgccc 54
58-S1-F catttcaacgtggtgNNKttcgacctgcccttcgccgggcagtcgcgtcagcac 54
59-S1-F catttcaacgtggtgctgNNKgacctgcccttcgccgggcagtcgcgtcagcac 54
60-S1-F catttcaacgtggtgctgttcNNKctgcccttcgccgggcagtcgcgtcagcac 54
61-S1-F catttcaacgtggtgctgttcgacNNKcccttcgccgggcagtcgcgtcagcac 54
62-S1-F catttcaacgtggtgctgttcgacctgNNKttcgccgggcagtcgcgtcagcac 54
63-S1-F catttcaacgtggtgctgttcgacctgcccNNKgccgggcagtcgcgtcagcac 54
64-S1-F catttcaacgtggtgctgttcgacctgcccttcNNKgggcagtcgcgtcagcac 54
65-S1-F catttcaacgtggtgctgttcgacctgcccttcgccNNKcagtcgcgtcagcac 54
66-S1-F ctgcccttcgccgggNNKtcgcgtcagcacaacccgcagcgcgggttgatcacc 54
67-S1-F ctgcccttcgccgggcagNNKcgtcagcacaacccgcagcgcgggttgatcacc 54
68-S1-F ctgcccttcgccgggcagtcgNNKcagcacaacccgcagcgcgggttgatcacc 54
69-S1-F ctgcccttcgccgggcagtcgcgtNNKcacaacccgcagcgcgggttgatcacc 54
70-S1-F ctgcccttcgccgggcagtcgcgtcagNNKaacccgcagcgcgggttgatcacc 54
71-S1-F ctgcccttcgccgggcagtcgcgtcagcacNNKccgcagcgcgggttgatcacc 54
72-S1-F ctgcccttcgccgggcagtcgcgtcagcacaacNNKcagcgcgggttgatcacc 54
73-S1-F ctgcccttcgccgggcagtcgcgtcagcacaacccgNNKcgcgggttgatcacc 54
74-S1-F cagcacaacccgcagNNKgggttgatcaccaaggacgacgaggtggaaatcctc 54
75-S1-F cagcacaacccgcagcgcNNKttgatcaccaaggacgacgaggtggaaatcctc 54
76-S1-F cagcacaacccgcagcgcgggNNKatcaccaaggacgacgaggtggaaatcctc 54
77-S1-F cagcacaacccgcagcgcgggttgNNKaccaaggacgacgaggtggaaatcctc 54
78-S1-F cagcacaacccgcagcgcgggttgatcNNKaaggacgacgaggtggaaatcctc 54
79-S1-F cagcacaacccgcagcgcgggttgatcaccNNKgacgacgaggtggaaatcctc 54
80-S1-F cagcacaacccgcagcgcgggttgatcaccaagNNKgacgaggtggaaatcctc 54
81-S1-F cagcacaacccgcagcgcgggttgatcaccaaggacNNKgaggtggaaatcctc 54
82-S1-F atcaccaaggacgacNNKgtggaaatcctcctggcgctgatcgagcgcttcgag 54
83-S1-F atcaccaaggacgacgagNNKgaaatcctcctggcgctgatcgagcgcttcgag 54
84-S1-F atcaccaaggacgacgaggtgNNKatcctcctggcgctgatcgagcgcttcgag 54
85-S1-F atcaccaaggacgacgaggtggaaNNKctcctggcgctgatcgagcgcttcgag 54
86-S1-F atcaccaaggacgacgaggtggaaatcNNKctggcgctgatcgagcgcttcgag 54
87-S1-F atcaccaaggacgacgaggtggaaatcctcNNKgcgctgatcgagcgcttcgag 54
88-S1-F atcaccaaggacgacgaggtggaaatcctcctgNNKctgatcgagcgcttcgag 54
89-S1-F atcaccaaggacgacgaggtggaaatcctcctggcgNNKatcgagcgcttcgag 54
90-S1-F atcctcctggcgctgNNKgagcgcttcgaggtcaatcacctggtctccgcgtcc 54
91-S1-F atcctcctggcgctgatcNNKcgcttcgaggtcaatcacctggtctccgcgtcc 54
92-S1-F atcctcctggcgctgatcgagNNKttcgaggtcaatcacctggtctccgcgtcc 54
93-S1-F atcctcctggcgctgatcgagcgcNNKgaggtcaatcacctggtctccgcgtcc 54
94-S1-F atcctcctggcgctgatcgagcgcttcNNKgtcaatcacctggtctccgcgtcc 54
95-S1-F atcctcctggcgctgatcgagcgcttcgagNNKaatcacctggtctccgcgtcc 54
96-S1-F atcctcctggcgctgatcgagcgcttcgaggtcNNKcacctggtctccgcgtcc 54
97-S1-F atcctcctggcgctgatcgagcgcttcgaggtcaatNNKctggtctccgcgtcc 54
98-S1-F ttcgaggtcaatcacNNKgtctccgcgtcctggggcggtatctccacgctgctg 54
99-S1-F ttcgaggtcaatcacctgNNKtccgcgtcctggggcggtatctccacgctgctg 54
100-S1-F ttcgaggtcaatcacctggtcNNKgcgtcctggggcggtatctccacgctgctg 54
101-S1-F ttcgaggtcaatcacctggtctccNNKtcctggggcggtatctccacgctgctg 54
102-S1-F ttcgaggtcaatcacctggtctccgcgNNKtggggcggtatctccacgctgctg 54
103-S1-F ttcgaggtcaatcacctggtctccgcgtccNNKggcggtatctccacgctgctg 54
104-S1-F ttcgaggtcaatcacctggtctccgcgtcctggNNKggtatctccacgctgctg 54
105-S1-F ttcgaggtcaatcacctggtctccgcgtcctggggcNNKatctccacgctgctg 54
10-17-S2-R cgataccaacagact 15
18-25-S2-R atgtacccgcaggcc 15
26-33-S2-R gggatcctgcccaac 15
34-41-S2-R gaccagcatcaccgt 15
42-49-S2-R ggcggtggtcgccat 15
50-57-S2-R gcacttgcaggtccg 15
58-65-S2-R caccacgttgaaatg 15
66-73-S2-R cccggcgaagggcag 15
74-81-S2-R ctgcgggttgtgctg 15
82-89-S2-R gtcgtccttggtgat 15
90-97-S2-R cagcgccaggaggat 15
98-105-S2-R gtgattgacctcgaa 15
A(S1-R) agccttcaacccagtcagctccttc 25
B(S2-F) actgggttgaaggctctcaagggca 25
基因rhlA序列(SEQ ID No.111)：
Figure PCTCN2021133816-appb-000001
基因rhlB序列(SEQ ID No.112)：
Figure PCTCN2021133816-appb-000002
1.2自动化核酸提取
本实施例使用凯杰自动化核酸提取仪以及天根PCR产物纯化试剂盒进行上述PCR-1和PCR-2产物的纯化，验证了天根PCR产物纯化试剂盒在凯杰仪器上的适配性，节省了纯化成本，凯杰原装PCR纯化试剂盒的价格为16.7元/反应，天根PCR纯化试剂盒的价格为2.38元/反应。纯化操作仅需将PCR产物和相应试剂耗材放到仪器指定位置，仪器自动运行程序，每次可对12个PCR产物进行纯化，将纯化后的片段放在4℃冰箱保存，下一步使用前放在自动化功能岛的耗材堆栈上即可。
1.3自动化Gibson组装
本实施例利用自动化移液工作站和自动化PCR仪对上一步纯化过的核酸片段进行Gibson组装，每个发应将一个PCR-1和一个PCR-2组装成完整的质粒(即为饱和突变质粒)，按照PCR引物的设计，每8个不同的PCR-1可分别于一个PCR-2进行组装，最后共组装获得96个饱和突变质粒，该工艺流程可以同时进行96个组装反应。实验前预先配置好Gibson组装反应液，包含320μl 5X ISO缓冲液、0.64μl T5核酸外切酶(10U/ul，购自美国NEB公司)、20μl Phusion聚合酶(2U/μl，购自美国NEB公司)、160μl Taq连接酶(40U/ul，购自美国NEB公司)、双蒸水补足至1.2mL后置于-20℃保存。将Gibson组装反应所需组分放置到自动化移液工作站的试剂槽中，反应体系为：Gibson组装反应液7.5ul、纯化后的核酸片段1 1.25ul、纯化后的核酸片段2 1.25ul，总反应体系10ul。自动化移液工作站根据编辑好的自动化Gibson组装程序脚本将Gibson组装反应中的各组混合并通过自动化机械臂送至PCR仪中进行反应，50℃反应1h。
1.4自动化转化
本实施例使用自动化移液工作站将上述饱和突变质粒转化至大肠杆菌Dh5a感受态细胞中(购自康体生物)，该工艺可同时进行96个转化实验。根据编辑好的自动化质粒转化至大肠杆菌感受态细胞程序脚本将50ul感受态细胞添加到饱和突变质粒中，并在自动化移液工作站的4℃低温控制模块上孵育1h，通过工作站机械抓手转移到42℃加热控制模块上热激45s，转移至4℃低温控制模块上孵育2min，后添加500ul无抗生素的LB培养基，再转移至37℃加热模块上孵育1h，离心去除上清液300ul，剩下200ul菌液吹打重悬后均匀滴加至含有抗生素的平皿中，放在37℃微生物培养箱中倒置培养过液。
1.5饱和突变文库测序验证
本实施例通过对转化后的饱和突变文库进行测序评价构建是否成功，测序结果显示在目标编号位置存在NNK套峰即判定文库构建成功。将上一步培养过夜的平皿取出，在 平皿上滴加3mL LB培养基，使用涂布棒将平皿中所有菌落刮下，收集到新的Ep管中，从中吸取500ul菌液和500ul 40％甘油溶液混匀后置于-80℃冰箱冻存，剩余菌液送至上海生工生物有限公司测序，测序结果如图4所示。
实施例2、通量混池测序文库的自动化构建
本实施例对构建的96个鼠李糖脂酰基转移酶饱和突变文库进行了自动化高通量混池测序文库的构建，根据混池测序结果可找出饱和突变文库中单一氨基酸突变的突变株。工艺过程包括：饱和突变文库的高通量挑选、自动化突变株混合、自动化构建高通量混池测序文库。该过程在自动化功能岛及其他自动化设备上的实施路径如图5所示，此外耗材堆材存放每个实验过程中使用到的试剂耗材，自动化机械臂负责仪器与仪器、仪器与耗材堆栈之间的交互。
2.1饱和突变文库的高通量挑选
本实施例将饱和突变文库的质粒按照实施例1中的方法自动化转化至大肠杆菌BL21(DE3)表达感受态细胞中(购自康体生命)，并于37℃微生物培养箱中培养过夜。将长有菌落的平皿放置在自动挑克隆仪规定的板位上，运行挑克隆程序，将平皿中的单克隆挑取接种到装有LB液体培养的96孔深孔板中，每一个饱和突变文库挑取192个单克隆(2块96孔深孔板)。将深孔板置于高速摇床中800rpm、37℃培养24h，之后放在功能岛的耗材堆栈上，自动化机械臂将96孔深孔板转移至自动化移液工作站中，根据编辑好的自动化质粒转化至大肠杆菌感受态细胞程序脚本先将100ul菌液与100ul 40％甘油溶液混合在新的96孔酶标板中，自动化机械臂转移至封膜机上进行封膜处理，后转至-80℃冰箱冻存。再将不同编号饱和突变文库挑取出来的突变株菌液进行混合，混合规则为：不同饱和突变文库挑取出来的2块96孔深孔板的同一位置的突变株进行混合(如图6中index PCR引物组合示意中每一个位点饱和突变文库挑取出的A1混合，以此类推)，最终将所有饱和突变文库挑取的突变株混合到新的2块96孔深孔板中，并于-80℃冰箱中过夜静置。
2.2高通量混池测序文库的自动化构建
本实施例使用自动化移液工作站等自动化构建高通量混池测序文库，实现了一次测序即可查找到多个饱和突变文库中21种氨基酸(含终止密码子)突变的突变株，并可以重定位至挑取出的2块96孔板上。在一些实施案例中，本发明对96个饱和文库文库中18432个突变株进行高通量混池测序，最终挑出1667株不重复的突变株。自动化构建的过程包含2次自动化PCR(菌液PCR和index PCR)，2次自动化PCR产物纯化以及DNA 定量均一化。首先本发明对上述冻存的混合菌液取出解冻，利用自动化移液工作站进行菌液PCR扩征目的测序序列，由于目的基因全长888bp，为了满足二代测序要求，设计了3对引物分别对1-300bp、301-600bp和601-888bp区域进行PCR扩增，引物序列如表2所示。
表2
Figure PCTCN2021133816-appb-000003
菌液PCR反应体系：10ul PrimeSTAR预混高保真聚合酶、1ul正向引物(10uM)、1ul反向引物(10uM)、1ul菌液、7ul双蒸水，总反应体系20ul，自动化移液工作站将反应体系各组分混合，通过自动化机械臂送至自动化PCR仪中仪器运行菌液PCR反应程序：首先98℃预变性5min，接着运行18个循环的98℃变性10s、58℃退火10s、72℃延伸30s，然后再72℃延伸5min，最后于4℃保存。反应完成后由自动化移液工作站进行PCR产物的磁珠纯化，通过编辑好的高通量混池测序文库自动纯化程序脚本对PCR产物进行磁珠吸附、乙醇洗脱、去上清、添加超纯水洗脱等自动化纯化步骤。纯化完的PCR产物放置在自动化移液工作站的4℃降温控制模块上，进行下一步index PCR反应，index引物序列如表3所示，N501-N512为index引物1，N701-716为index引物2。
表3
Figure PCTCN2021133816-appb-000004
Figure PCTCN2021133816-appb-000005
Index引物的组合按照图6进行，A1孔的index引物分别为N501和N701。
Index PCR反应的体系为：10ul PrimeSTAR预混高保真聚合酶、2ul index引物1(10uM)、2ul index引物2(10uM)、6ul上一步PCR纯化后的产物，总反应体系20ul，自动化移液工作站混合好反应体系后，自动化机械臂转移至自动化PCR仪中运行index PCR反应程序：首先98℃预变性5min，接着运行10个循环的98℃变性10s、58℃退火10s、72℃延伸30s，然后再72℃延伸5min，最后于4℃保存，反应完成后由自动化移液工作站进行PCR产物的磁珠纯化。
2.3高通量混池测序文库均一化
本实施例使用自动化毛细管电泳仪对上述高通量混池测序文库进行毛细管电泳，检测建库的正确性，结果参见图7。
毛细管电泳图中没有条带的样品重新跑电泳验证或者重新建库，将验证正确的文库置于自动化移液工作站的4℃低温控温模块，按照编辑的自动化DNA定量程序脚本将1ul文库DNA与199ul Qubit TM dsDNA检测试剂(购自赛默飞公司)混合，通过自动化机械臂转移至酶标仪上，进行DNA含量的测定。再转移至自动化移液工作站的4℃冷凝模块，每一个样本吸取50ng DNA到新的Ep管中，最后送至中国科学院深圳先进院合成生物学所公共平台进行高通量测序，数据分析完成后可获得表4中的突变株所在挑取96孔板上的位置。有部分突变株基因型并没有找到，找出的1667株突变株覆盖了全部测序文库的86.8％。
表4：文库中突变株所在位置信息表
Figure PCTCN2021133816-appb-000006
Figure PCTCN2021133816-appb-000007
Figure PCTCN2021133816-appb-000008
Figure PCTCN2021133816-appb-000009
实施例3、蛋白质催化性质的自动化、高通量质谱表征
本实施例对编号10-29号的突变文库进行了自动化的基质辅助激光解吸电离飞行时间质谱(MALDI-ToF-MS)检测，工艺流程包括突变株自动化培养产目标代谢物、样品自动化前处理、MALDI自动化检测等。在实施例3中，对20个饱和突变文库进行了自动化质谱表征，4天即可获得所有突变株的质谱数据，其中利用MALDI-ToF-MS可在1h内可检测1536个样品，而传统LC-MS检测方法完成相应数量样本检测所需的时间超过250h。该工艺流程在自动化功能岛与其他仪器之间的实施路径如图8所示，此外耗材堆材存放每个实验过程中使用到的试剂耗材，自动化机械臂负责仪器与仪器、仪器与耗材堆栈之间的交互。
3.1突变株自动化产目标代谢物
本实施例对编号10-29饱和突变文库(共20个)中找到的突变株进行了自动化培养产目标代谢产物，将保存在-80℃中的20个饱和突变文库冻存样品取出，置于自动化功能岛的耗材堆栈上，等菌液解冻以后，立即通过自动化机械臂转移至自动化移液工作站，按照编辑好的自动化突变文库培养程序脚本从孔板的特定位置吸取20ul突变株菌液到含有LB琼脂培养基的24孔中，置于37℃微生物培养箱中培养过夜。自动化移液工作站蘸取少量菌体到含有LB培养基的24孔深孔板中，通过自动化机械臂转移至自动化振荡培养箱，37℃振荡培养3-4h后再转移到自动化移液工作站中，对菌液进行保种和转接操作。吸取100ul菌液到含有100ul 40％甘油溶液的酶标板中混合，再吸取20ul菌液到新的含有LB培养基的24孔深孔板中，每一个深孔板中添加3个野生型作为对照样本，且每个突变株的接种实验需要进行三重复。转接后转移至自动化振荡培养箱中，37℃振荡培养3h，使得菌液的OD600在0.6-0.8之间，转移至自动化移液工作站中，待菌液温度降低至30℃左右时，添加终浓度为1mM的IPTG溶液，30℃自动化振荡培养箱诱导培养24h。
3.2样品自动化前处理
本实施例使用自动化移液工作站对诱导完成后的菌液进行乙酸乙酯萃取，并将萃取后的样本自动化添加到MALDI-ToF-MS金属靶板上。将诱导后的24孔深孔板转移到自动化移液工作站上，添加等体积的乙酸乙酯溶液，在自动化振荡培养箱中振荡10min后去取出，放在自动化离心机中离心2min。自动化移液工作站预先在MALDI-ToF-MS金属靶板上滴加0.5ul溶于50％乙腈(添加0.1％TFA溶液)的α-氰基-4-羟基肉桂酸基质溶液，待基质溶液晾干后，再吸取1ul萃取后的乙酸乙酯层溶液到金属靶板上，自然晾干即可。
3.3 MALDI-ToF-MS自动化检测
本实施例使用布鲁克公司autoflex MALDI-ToF-MS进行检测，实施案例中检测的代谢产物为鼠李糖脂，有四种同系物(5a、5b、5c、5d)结构如图9所示。
将上一步待检样品金属板放置到MALDI-ToF-MS中,运行自动化采集方法，检测完成后，数据导出成表格形式，进行数据处理，将野生型产物的中四种同系物的占比都设定为1，突变株产物中的四种同系物相较于野生型产物的占比结果如表5。
表5：突变株产物中四种同系物相较于野生型的占比
编号 ％5a ％5b ％5c ％5d
野生型 1 1 1 1
010V-终止子 1.278 0.361 5.575 0.000
010V-A 0.435 1.065 1.591 0.000
010V-C 0.474 1.093 1.288 0.000
010V-D 0.151 1.151 1.461 0.000
010V-E 1.472 0.707 2.421 0.000
010V-F 0.538 0.796 3.543 0.000
010V-G 0.385 1.024 2.015 0.000
010V-H 1.088 0.432 5.376 0.000
010V-I 0.350 1.093 1.531 0.000
010V-K 0.796 0.729 3.568 0.000
010V-L 0.425 1.100 1.326 0.000
010V-M 1.442 0.638 3.032 0.000
010V-N 1.174 0.811 2.170 0.000
010V-P 0.409 1.078 1.537 0.000
010V-Q 0.340 1.058 1.830 0.000
010V-R 0.851 0.747 3.321 0.000
010V-S 0.430 1.050 1.721 0.000
010V-T 0.445 1.116 1.162 0.000
010V-V 0.900 1.041 0.871 0.000
010V-W 1.677 0.415 4.354 0.000
010V-Y 1.474 0.482 4.218 0.000
011C-终止子 1.222 0.463 5.108 0.000
011C-A 0.811 1.044 1.112 0.000
011C-C 0.884 1.050 0.862 0.000
011C-D 0.585 1.105 1.166 0.000
011C-E 0.432 1.074 1.842 0.000
011C-F 0.818 1.027 1.243 0.000
011C-G 0.725 1.097 0.876 0.000
011C-H 0.754 1.090 0.858 0.000
011C-I 0.871 1.042 0.971 0.000
011C-K 0.725 1.055 1.239 0.000
011C-L 0.858 1.032 1.095 0.000
011C-M 0.627 1.081 1.266 0.000
011C-N 0.796 1.059 1.014 0.000
011C-P 0.774 1.082 0.880 0.000
011C-Q 0.861 1.046 0.965 0.000
011C-R 0.581 1.087 1.336 0.000
011C-S 0.609 1.054 1.555 0.000
011C-T 1.075 0.747 3.013 0.000
011C-V 1.079 0.709 3.329 0.000
011C-W 0.640 1.008 1.871 0.000
011C-Y 0.948 1.034 0.844 0.000
012K-终止子 1.186 0.308 2.848 24.307
012K-A 0.409 1.065 1.433 1.845
012K-C 0.332 1.085 1.403 1.928
012K-D 0.869 1.014 1.099 1.187
012K-E 0.542 1.043 1.411 1.550
012K-F 0.330 1.066 1.545 2.013
012K-G 0.995 0.996 1.066 0.817
012K-H 0.355 1.047 1.609 2.291
012K-I 0.205 1.081 1.524 2.936
012K-K 0.000 0.000 0.000 0.000
012K-L 0.297 1.078 1.493 2.143
012K-M 0.405 1.067 1.400 2.056
012K-N 0.000 0.000 0.000 0.000
012K-P 0.290 1.070 1.593 1.956
012K-Q 0.493 1.048 1.434 1.719
012K-R 0.968 1.010 1.007 0.812
012K-S 0.516 1.081 1.221 1.099
012K-T 0.377 1.064 1.499 1.857
012K-V 0.347 1.093 1.286 2.134
012K-W 0.875 1.028 1.000 1.010
012K-Y 0.458 1.050 1.490 1.687
013G-终止子 1.318 0.324 2.977 19.829
013G-A 0.413 1.037 1.398 2.737
013G-C 0.366 1.016 1.587 3.041
013G-D 0.938 0.987 1.089 1.768
013G-E 0.568 1.020 1.317 2.489
013G-F 0.716 0.778 1.898 9.735
013G-G 1.114 0.995 0.900 0.684
013G-H 0.510 1.042 1.376 1.550
013G-I 0.434 1.031 1.379 2.942
013G-K 0.575 0.987 1.372 3.766
013G-L 0.624 0.816 1.764 9.625
013G-M 0.865 0.703 2.065 10.940
013G-N 0.809 1.022 1.133 1.014
013G-P 0.726 0.795 1.917 8.595
013G-Q 0.000 0.000 0.000 0.000
013G-R 0.441 1.067 1.270 1.694
013G-S 0.568 1.050 1.211 1.627
013G-T 0.550 0.914 1.517 6.895
013G-V 0.626 0.819 1.985 7.947
013G-W 1.367 0.474 2.308 15.899
013G-Y 0.698 0.796 1.747 9.981
014L-终止子 0.689 0.259 5.163 26.011
014L-A 0.000 0.000 0.000 0.000
014L-C 0.431 1.117 1.671 2.315
014L-D 0.481 0.980 2.105 6.745
014L-E 1.132 0.373 3.000 23.696
014L-F 0.874 1.045 1.033 0.875
014L-G 0.223 1.024 2.656 6.115
014L-H 0.677 1.082 1.286 1.387
014L-I 0.318 1.116 1.968 2.887
014L-K 0.411 0.999 2.054 7.536
014L-L 0.000 0.000 0.000 0.000
014L-M 1.073 0.967 0.824 2.627
014L-N 0.501 1.096 1.676 1.938
014L-P 0.157 1.199 1.705 3.345
014L-Q 0.618 1.093 1.382 1.425
014L-R 0.801 1.046 1.236 1.091
014L-S 0.384 1.098 1.768 3.925
014L-T 0.468 0.680 2.386 24.239
014L-V 0.372 0.861 1.970 17.976
014L-W 1.230 0.950 0.681 0.999
014L-Y 0.806 1.078 0.963 0.894
015R-终止子 1.122 0.261 3.852 22.149
015R-A 0.512 1.063 1.493 1.370
015R-C 0.573 1.055 1.408 1.446
015R-D 0.580 0.781 2.337 10.574
015R-E 0.296 1.100 1.517 2.216
015R-F 0.869 1.013 1.188 0.971
015R-G 0.278 1.038 1.814 4.026
015R-H 0.849 0.482 2.844 19.968
015R-I 0.975 0.991 1.157 0.914
015R-K 1.012 1.027 0.784 0.685
015R-L 0.827 1.003 1.282 1.553
015R-M 0.959 1.012 0.959 1.136
015R-N 0.605 1.083 1.162 0.993
015R-P 0.446 0.688 2.450 16.723
015R-Q 0.929 1.014 1.058 0.880
015R-R 0.894 1.035 0.960 0.789
015R-S 0.562 1.068 1.332 1.365
015R-T 0.276 1.183 0.973 1.210
015R-V 0.904 1.020 1.050 0.922
015R-W 0.000 0.000 0.000 0.000
015R-Y 0.513 1.075 1.336 1.622
016V-终止子 1.214 0.488 4.710 0.000
016V-A 0.554 0.929 2.697 0.000
016V-C 0.735 1.049 1.252 0.000
016V-D 1.395 0.428 4.759 0.000
016V-E 0.989 0.451 5.577 0.000
016V-F 1.150 0.392 5.658 0.000
016V-G 1.077 0.401 5.772 0.000
016V-H 1.102 0.337 6.238 0.000
016V-I 0.704 1.053 1.292 0.000
016V-K 1.151 0.502 4.752 0.000
016V-L 0.371 1.077 1.923 0.000
016V-M 0.483 1.011 2.190 0.000
016V-N 1.093 0.521 4.737 0.000
016V-P 1.203 0.369 5.723 0.000
016V-Q 1.003 0.526 4.916 0.000
016V-R 1.223 0.432 5.154 0.000
016V-S 0.735 0.743 3.788 0.000
016V-T 0.468 1.026 2.100 0.000
016V-V 0.558 1.137 0.958 0.000
016V-W 1.521 0.433 4.407 0.000
016V-Y 0.968 0.488 5.322 0.000
017H-终止子 1.278 0.437 5.746 0.000
017H-A 0.344 1.106 1.807 0.000
017H-C 0.563 0.979 2.444 0.000
017H-D 1.176 0.587 4.566 0.000
017H-E 0.626 0.879 3.250 0.000
017H-F 0.526 1.077 1.579 0.000
017H-G 0.626 0.908 2.963 0.000
017H-H 0.000 0.000 0.000 0.000
017H-I 0.401 1.076 1.942 0.000
017H-K 0.537 1.000 2.309 0.000
017H-L 0.777 0.880 2.804 0.000
017H-M 0.474 1.052 1.979 0.000
017H-N 0.362 1.120 1.627 0.000
017H-P 1.398 0.406 5.715 0.000
017H-Q 0.337 1.120 1.690 0.000
017H-R 0.347 1.129 1.575 0.000
017H-S 0.932 0.829 2.876 0.000
017H-T 0.581 0.956 2.620 0.000
017H-V 0.331 1.148 1.439 0.000
017H-W 0.556 1.079 1.482 0.000
017H-Y 0.854 1.029 1.127 0.000
018V-终止子 0.944 0.408 2.383 20.205
018V-A 0.404 1.078 1.685 2.017
018V-C 0.757 1.055 1.170 1.048
018V-D 0.811 0.354 3.045 20.447
018V-E 0.757 0.300 3.753 19.230
018V-F 0.799 0.318 4.511 12.988
018V-G 0.693 0.277 5.092 12.764
018V-H 0.676 0.221 5.215 14.786
018V-I 0.341 1.081 1.675 2.908
018V-K 0.977 0.329 3.079 18.914
018V-L 0.369 0.907 2.031 8.209
018V-M 0.279 1.054 1.660 5.177
018V-N 0.876 0.308 3.294 20.001
018V-P 0.859 0.340 3.900 14.929
018V-Q 0.875 0.255 4.349 15.769
018V-R 0.906 0.272 4.035 16.504
018V-S 0.000 0.000 0.000 0.000
018V-T 0.299 1.120 1.623 2.052
018V-V 0.903 0.770 1.598 9.200
018V-W 0.980 0.298 3.115 20.072
018V-Y 0.913 0.320 3.091 20.189
019E-终止子 1.042 0.338 3.163 15.205
019E-A 0.345 1.074 1.835 1.755
019E-C 0.534 1.075 1.456 1.372
019E-D 0.382 1.100 1.505 1.981
019E-E 0.000 0.000 0.000 0.000
019E-F 0.305 1.071 1.901 2.020
019E-G 0.280 1.014 2.134 3.364
019E-H 0.395 1.094 1.626 1.436
019E-I 0.804 1.043 1.095 1.184
019E-K 0.748 1.051 1.202 1.033
019E-L 0.448 1.035 1.677 2.828
019E-M 0.450 1.082 1.518 1.819
019E-N 0.439 1.096 1.519 1.409
019E-P 1.001 0.454 2.838 12.834
019E-Q 0.685 1.066 1.244 0.973
019E-R 0.563 1.063 1.456 1.482
019E-S 0.548 1.075 1.427 1.356
019E-T 0.722 1.076 1.079 0.974
019E-V 0.740 1.048 1.228 1.101
019E-W 0.415 1.051 1.678 2.624
019E-Y 0.392 1.105 1.517 1.621
020R-终止子 0.790 0.423 3.046 18.764
020R-A 0.230 1.135 1.852 2.407
020R-C 0.559 1.115 1.290 1.262
020R-D 0.517 0.828 2.104 10.316
020R-E 0.325 0.943 2.252 7.277
020R-F 0.266 1.103 1.954 2.659
020R-G 0.171 1.169 1.690 2.801
020R-H 0.511 1.107 1.441 1.498
020R-I 0.665 1.087 1.186 1.409
020R-K 0.633 1.092 1.287 1.115
020R-L 0.915 1.048 0.895 0.848
020R-M 0.583 1.087 1.392 1.546
020R-N 0.326 1.121 1.758 2.005
020R-P 0.520 0.764 2.613 10.118
020R-Q 0.396 1.128 1.483 2.209
020R-R 0.754 1.075 1.117 0.941
020R-S 0.293 1.136 1.596 2.876
020R-T 0.507 1.098 1.516 1.517
020R-V 0.235 1.134 1.845 2.395
020R-W 0.316 1.069 1.685 5.017
020R-Y 0.415 1.122 1.572 1.626
021V-终止子 1.070 0.257 3.810 16.443
021V-A 0.430 1.015 1.686 2.733
021V-C 0.619 1.029 1.402 1.649
021V-D 0.507 0.810 1.795 10.244
021V-E 0.337 1.000 1.890 3.261
021V-F 0.371 1.060 1.579 2.059
021V-G 0.795 0.545 3.132 10.852
021V-H 0.405 1.069 1.515 1.658
021V-I 0.852 1.007 1.182 1.282
021V-K 0.419 0.954 1.850 4.620
021V-L 0.000 0.000 0.000 0.000
021V-M 0.591 0.963 1.586 3.805
021V-N 0.357 0.984 1.821 4.159
021V-P 0.917 0.540 2.710 12.146
021V-Q 0.453 1.019 1.711 2.161
021V-R 0.916 1.003 1.123 1.097
021V-S 0.542 0.814 2.296 6.767
021V-T 0.747 1.044 1.133 1.123
021V-V 0.996 1.036 0.760 0.873
021V-W 0.456 1.058 1.331 2.647
021V-Y 0.580 1.079 1.186 1.183
022G-终止子 0.784 0.261 3.997 27.907
022G-A 0.708 1.074 1.138 1.251
022G-C 0.800 1.049 1.097 1.242
022G-D 0.613 0.704 2.385 16.082
022G-E 0.689 1.042 1.439 1.504
022G-F 0.530 1.060 1.700 1.724
022G-G 0.354 1.077 1.951 2.366
022G-H 0.781 1.031 1.300 1.303
022G-I 0.645 1.055 1.339 2.192
022G-K 0.496 1.074 1.661 1.789
022G-L 0.901 0.305 3.835 24.246
022G-M 0.749 1.032 1.363 1.466
022G-N 0.667 1.049 1.442 1.453
022G-P 0.264 0.990 2.471 5.629
022G-Q 0.733 0.498 4.394 12.720
022G-R 0.433 1.069 1.890 1.747
022G-S 0.559 0.862 2.249 8.964
022G-T 0.679 1.069 1.271 1.200
022G-V 0.867 1.036 1.047 1.076
022G-W 0.472 1.072 1.707 2.013
022G-Y 0.546 1.081 1.510 1.417
023Q-终止子 1.262 0.322 3.105 17.229
023Q-A 0.381 1.053 1.626 1.700
023Q-C 0.734 0.991 1.365 2.201
023Q-D 0.666 1.024 1.257 2.015
023Q-E 0.593 1.041 1.398 1.295
023Q-F 0.357 1.016 1.701 3.225
023Q-G 0.365 1.030 1.722 2.394
023Q-H 0.000 0.000 0.000 0.000
023Q-I 0.368 1.040 1.674 2.185
023Q-K 0.620 1.064 1.186 1.110
023Q-L 0.561 0.995 1.591 2.627
023Q-M 0.599 1.023 1.396 2.024
023Q-N 0.000 0.000 0.000 0.000
023Q-P 0.867 1.016 1.110 1.097
023Q-Q 0.000 0.000 0.000 0.000
023Q-R 0.603 1.032 1.467 1.184
023Q-S 0.717 1.031 1.266 1.145
023Q-T 0.578 1.037 1.456 1.308
023Q-V 0.361 1.079 1.464 1.636
023Q-W 0.794 0.936 1.500 3.329
023Q-Y 0.307 1.052 1.652 2.411
024D-终止子 1.130 0.306 3.162 16.355
024D-A 0.550 0.981 1.473 3.339
024D-C 0.360 1.059 1.432 2.129
024D-D 0.000 0.000 0.000 0.000
024D-E 0.599 0.843 1.987 5.722
024D-F 0.409 0.985 1.764 2.861
024D-G 0.958 0.996 1.050 1.297
024D-H 0.295 1.045 1.633 2.180
024D-I 0.688 0.978 1.401 2.585
024D-K 0.443 1.003 1.528 3.139
024D-L 0.000 0.000 0.000 0.000
024D-M 0.986 0.729 1.924 7.149
024D-N 1.234 0.396 2.778 13.804
024D-P 0.647 0.825 2.002 5.926
024D-Q 0.000 0.000 0.000 0.000
024D-R 0.541 0.960 1.587 3.660
024D-S 0.624 0.821 2.074 5.884
024D-T 0.522 0.953 1.648 3.783
024D-V 0.455 0.978 1.673 3.230
024D-W 0.755 0.778 1.853 7.686
024D-Y 0.375 1.051 1.435 2.324
025P-终止子 1.284 0.291 3.080 17.381
025P-A 1.132 0.980 0.928 1.082
025P-C 1.327 0.982 0.656 0.824
025P-D 1.346 0.956 0.777 1.059
025P-E 0.783 1.022 1.108 1.407
025P-F 1.084 0.990 0.939 1.021
025P-G 0.823 1.008 1.183 1.195
025P-H 1.229 0.482 2.713 11.583
025P-I 0.996 0.975 1.077 1.693
025P-K 1.151 0.983 0.907 0.911
025P-L 1.025 0.992 0.971 1.296
025P-M 1.018 0.995 0.916 1.593
025P-N 1.221 0.479 2.624 12.344
025P-P 0.444 0.993 1.407 4.067
025P-Q 1.241 0.480 2.568 12.459
025P-R 0.984 1.003 1.016 0.891
025P-S 1.161 0.973 0.903 1.275
025P-T 1.110 0.980 0.979 0.992
025P-V 0.968 1.003 0.992 1.235
025P-W 1.097 0.989 0.895 1.244
025P-Y 0.942 0.991 1.115 1.222
026G-终止子 0.000 0.000 0.000 0.000
026G-A 0.906 0.879 2.229 0.000
026G-C 0.000 0.000 0.000 0.000
026G-D 0.000 0.000 0.000 0.000
026G-E 1.076 0.858 1.963 0.000
026G-F 0.000 0.000 0.000 0.000
026G-G 0.000 0.000 0.000 0.000
026G-H 0.000 0.000 0.000 0.000
026G-I 0.000 0.000 0.000 0.000
026G-K 0.000 0.000 0.000 0.000
026G-L 0.546 1.100 1.361 0.000
026G-M 0.000 0.000 0.000 0.000
026G-N 0.709 1.060 1.265 0.000
026G-P 0.000 0.000 0.000 0.000
026G-Q 0.000 0.000 0.000 0.000
026G-R 0.445 1.109 1.551 0.000
026G-S 0.000 0.000 0.000 0.000
026G-T 0.000 0.000 0.000 0.000
026G-V 0.000 0.000 0.000 0.000
026G-W 0.812 0.793 3.179 0.000
026G-Y 0.000 0.000 0.000 0.000
027R-终止子 1.034 0.469 2.556 13.261
027R-A 0.319 1.039 1.574 1.818
027R-C 0.315 1.025 1.632 2.086
027R-D 1.229 0.489 2.787 9.640
027R-E 0.452 0.867 2.127 4.560
027R-F 0.288 1.053 1.533 1.745
027R-G 0.468 0.981 1.521 3.200
027R-H 0.245 1.064 1.496 1.856
027R-I 0.443 0.929 1.762 4.148
027R-K 0.861 1.035 0.984 0.847
027R-L 0.286 1.045 1.509 2.205
027R-M 0.274 1.057 1.522 1.751
027R-N 0.288 1.075 1.397 1.624
027R-P 0.232 1.040 1.567 2.538
027R-Q 0.372 0.931 1.998 3.371
027R-R 0.810 1.032 1.062 0.915
027R-S 0.320 1.021 1.533 2.754
027R-T 0.347 0.991 1.643 3.133
027R-V 0.321 1.028 1.593 2.127
027R-W 0.281 1.033 1.528 2.627
027R-Y 0.285 1.041 1.564 2.100
028S-终止子 1.095 0.276 3.999 15.716
028S-A 1.082 1.003 0.833 0.811
028S-C 0.465 1.062 1.479 1.806
028S-D 0.586 1.045 1.445 1.393
028S-E 0.959 0.295 3.142 20.789
028S-F 0.342 1.046 1.799 2.221
028S-G 0.873 1.020 1.115 0.933
028S-H 0.862 0.564 2.740 12.366
028S-I 0.474 0.854 2.328 6.307
028S-K 0.823 1.010 1.244 1.269
028S-L 0.448 1.068 1.498 1.666
028S-M 0.493 1.052 1.502 1.800
028S-N 1.087 0.426 3.077 14.094
028S-P 1.012 1.010 0.919 0.870
028S-Q 1.230 0.344 3.157 15.589
028S-R 1.145 0.984 0.872 0.724
028S-S 0.974 1.019 0.919 0.879
028S-T 0.550 1.064 1.369 1.340
028S-V 0.463 0.908 2.207 4.743
028S-W 0.409 0.936 2.020 5.087
028S-Y 0.372 1.068 1.614 1.878
029T-终止子 1.760 0.419 4.382 0.000
029T-A 0.732 0.867 2.446 0.000
029T-C 0.454 1.007 1.775 0.000
029T-D 1.513 0.377 5.088 0.000
029T-E 1.248 0.629 3.520 0.000
029T-F 1.152 0.471 4.899 0.000
029T-G 1.319 0.594 3.689 0.000
029T-H 0.765 0.840 2.607 0.000
029T-I 0.625 0.923 2.170 0.000
029T-K 1.091 0.769 2.665 0.000
029T-L 1.110 0.654 3.535 0.000
029T-M 0.494 0.976 1.961 0.000
029T-N 0.482 1.023 1.610 0.000
029T-P 1.469 0.434 4.706 0.000
029T-Q 0.636 0.880 2.493 0.000
029T-R 0.757 0.876 2.338 0.000
029T-S 0.435 0.994 1.908 0.000
029T-T 1.183 0.981 0.866 0.000
029T-V 0.487 0.986 1.890 0.000
029T-W 0.960 0.800 2.622 0.000
029T-Y 0.996 0.770 2.801 0.000
本发明的上述对鼠李糖脂酰基转移酶的实验，可在6周内完成系统性单点突变文库构建、NGS测序分析和突变分选、细胞发酵和产物质谱分析，获得系统性突变序列－催化性能对应关系，并且筛选得到具有期望催化性能的突变株。
此外，本发明的上述各实施例可使用一种自动化功能岛，其结构可参见图10所示，岛内仪器设备有自动化机械臂、自动化移液工作站、自动化PCR仪、自动化离心机、耗材堆栈、封膜机、撕膜机、酶标仪以及自动化振荡培养箱，其中自动化移液工作站操作系统Evoware由生产商提供，功能岛的操作系统Momentum由赛默飞提供。自动化移液工作站配备了灵活八通道移液吸头、高通量96通道移液吸头、多功能抓手机械臂、低温控制模块、加热控制模块、振荡模块、孔板板位、吸头板位和耗材载架若干。该功能岛与其他自动化设备，如：自动化挑克隆仪、自动化核酸提取仪等，为本发明涉及的蛋白质饱和突变文库高通量、自动化构建流程；饱和突变文库的自动化分选流程；高通量混池测序文库构建流程；以及蛋白质催化性质自动化质谱表征等流程提供了有力支持，将相关工艺流程编辑成自动化程序脚本来实现相应的自动化实验流程。

Claims (10)

  1. 一种蛋白质自动化工程改造优化方法，该方法包括自动化构建蛋白质饱和突变文库的过程；其中，自动化构建蛋白质饱和突变文库的过程包括：
    在突变位点设计简并引物引入氨基酸突变，进行自动化PCR、自动化核酸提取、自动化组装、自动化转化，转化后获得饱和突变文库。
  2. 根据权利要求1所述的方法，其中，采用Gibson assembly方法或基因合成引入蛋白质突变。
  3. 根据权利要求1所述的方法，其中所述自动化PCR按照以下操作进行：
    通过引入简并密码子NNK产生突变，每一个基因编号位点饱和突变文库由以重组质粒为模板扩增出来的2个片段进行组装获得；其中两片段组装时，将组装试剂放在自动化移液工作站的试剂槽内，工作站按照编辑好的自动化PCR扩增反应程序脚本将反应体系中的各组分混合到PCR反应板中，通过自动化移液臂转移至自动化PCR仪中反应；反应结束后再由自动化机械臂运回自动化移液工作站，向每一个反应中添加DpnI酶，再于自动化PCR仪中进行反应，以消化模板质粒；反应结束后放置在耗材堆栈上，分别对片段1和片段2进行凝胶电泳。
  4. 根据权利要求1所述的方法，其中所述自动化组装按照以下操作进行：
    利用自动化移液工作站和自动化PCR仪对纯化过的核酸片段进行Gibson组装；将Gibson组装反应所需组分放置到自动化移液工作站的试剂槽中，自动化移液工作站根据编辑好的自动化Gibson组装程序脚本将Gibson组装反应中的各组混合并通过自动化机械臂送至PCR仪中进行反应。
  5. 根据权利要求1所述的方法，其中所述自动化转化按照以下操作进行：
    使用自动化移液工作站将饱和突变质粒转化至感受态细胞中，根据编辑好的自动化质粒转化至感受态细胞程序脚本将感受态细胞添加到饱和突变质粒中，并在自动化移液工作站的低温控制模块上孵育，通过工作站机械抓手转移到加热控制模块上热激，之后转移至低温控制模块上孵育，后添加无抗生素的培养基，再转移至加热模块上孵育，离心去除上清液，剩下菌液吹打重悬后均匀滴加至含有抗生素的平皿中，放在微生物培养箱中倒置培养过液。
  6. 根据权利要求1所述的方法，该方法还包括：
    以饱和突变文库为基础，进行自动化高通量混池测序文库的构建，根据混池测序结果找出饱和突变文库中单一氨基酸突变的突变株。
  7. 根据权利要求6所述的方法，其中，自动化高通量混池测序文库的构建的过程包括：
    饱和突变文库的高通量挑选：将长有菌落的平皿放置在自动挑克隆仪规定的板位上，运行挑克隆程序，将平皿中的单克隆挑取接种到装有LB液体培养的深孔板中；将深孔板置于摇床中培养，之后放在功能岛的耗材堆栈上，自动化机械臂将深孔板转移至自动化移液工作站中，根据编辑好的自动化质粒转化至感受态细胞程序脚本先将菌液与甘油溶液混合在新的酶标板中，自动化机械臂转移至封膜机上进行封膜处理；
    自动化突变株混合：将不同编号饱和突变文库挑取出来的突变株菌液进行混合，混合规则为：不同饱和突变文库挑取出来的2块深孔板的同一位置的突变株进行混合，最终将所有饱和突变文库挑取的突变株混合到新的2块深孔板中；并于-80℃冰箱中过夜静置；
    自动化构建高通量混池测序文库：进行2次自动化PCR：菌液PCR和index PCR，对2次PCR产物进行自动化磁珠纯化，进行DNA定量均一化。
  8. 根据权利要求1所述的方法，该方法还包括：
    对饱和突变文库进行了自动化质谱表征；
    优选地，使用MALDI离子源和TOF检测器质谱、ESI离子源质谱仪、QQQ检测器中的一种或多种对蛋白质催化性质进行表征。
  9. 根据权利要求8所述的方法，其中，基于全细胞产物、纯化蛋白或细胞裂解物对蛋白质催化性能进行分析。
  10. 根据权利要求1-9中任一项所述的方法，其是对鼠李糖脂酰基转移酶进行自动化工程改造。
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