WO2020048125A1 - Construction and application of efficient streptomyces genome simplification system - Google Patents

Abstract

Provided is construction and application of an efficient streptomyces genome simplification system. The streptomyces genome simplification system is constructed by the step as follows: on the basis of whole-genome comparative analysis for a large-segment redundant gene region, a homologous recombination system and an efficient site-specific recombination system (Cre/loxP system) are combined for target large-segment deletion. The homologous recombination system adopts a homologous recombination strategy based on a suicide vector, targeting performance and efficiency of knockout are improved, and the efficiency reaches up to 100% when the homologous recombination system is applied to one-time deletion of about 700 Kb redundant gene region. The theoretical basis is laid for construction of chassis cells of streptomyces, efficient construction of synthetic biological chassis cells is implemented, and support is provided for pharmaceutical industrialization and new drug research and development.

Description

Construction and Application of an Efficient Streptomyces Genome Simplification System Technical field

The invention belongs to the field of microbial genetic engineering, and relates to an efficient construction and application of a streptomyces genome reduction system, in particular to a large fragment redundant gene knockout system based on a combination of a homologous recombination system and a site-specific recombination system.

Background technique

Streptomyces is a Gram-positive bacterium. For decades, Streptomyces has been the main source of research and development of new antibiotics. Many secondary metabolites derived from Streptomyces have been used as drug lead compounds, which have been widely used in clinical practice. Therapeutic drugs such as immunosuppressants, anticancer drugs, antibiotics, antifungal drugs, antiviral drugs and antihypertensive drugs, etc. Streptomyces as an industrial production of many important antibiotics, has the ability to synthesize a variety of complex natural products. The size of the Streptomyces genome is generally between 6-10Mb. In addition to the huge genome containing essential genes related to normal growth and development, It is various types of secondary metabolic biosynthetic gene cluster genes. The secondary metabolic biosynthetic gene clusters are generally evolved by microorganisms under the pressure of resisting the external environment or obtained through horizontal gene transfer. Therefore, it is generally considered that the genes related to the secondary metabolic biosynthetic gene clusters are non-functional under laboratory culture conditions. Essential genes. The Streptomyces genome contains a large number of endogenous secondary metabolic biosynthetic gene clusters. On the one hand, the expression of endogenous gene clusters will compete with exogenous synthetic pathways for precursors and energy, and on the other hand, a large number of by-products will be generated, affecting the target metabolites. The yield and downstream separation and purification can achieve the high quality and high yield of the target product by eliminating the production of by-products.

With the rapid development of bioinformatics analysis technology and synthetic biology enabling technology, deep analysis of the Streptomyces genome can find that there are a large number of redundant genes in the genome, and many redundant genes are concentratedly distributed in the subtelomere area to form large The redundant gene region of the fragment, the expression of redundant genes will consume a large amount of energy of cells such as DNA replication, transcription, and translation, which will seriously reduce the effective utilization of energy, and will also generate a large number of by-products such as non-target secondary Metabolites, amino acids, sugars, etc. affect the yield of target metabolites and downstream separation and purification projects. Therefore, the large-scale deletion of redundant genes through efficient gene editing technology can not only streamline the genome of Streptomyces, but also reduce the internal energy consumption, making the metabolic profile of the strain more simple and clear. Genome-reduced streptomyces can also be used as chassis cells to achieve efficient biosynthesis of exogenous natural products or drugs.

Summary of the Invention

The purpose of the present invention is to provide a method for constructing a highly efficient streptomyces genome reduction system. Based on comparative genomics analysis, a large segment of redundant gene regions is analyzed, and a homologous recombination system and an efficient site-specific recombination system are used in combination to realize a large segment gene. Method for efficient editing of regions, thereby streamlining the Streptomyces genome. This is a new technology for efficiently constructing Streptomyces chassis cells based on bioinformatics analysis data.

The construction method of the present invention is implemented by the following steps:

(1) The sequenced whole genome sequence of Streptomyces and Streptomyces coelicolor A3 (2) (Deposit No. CGMCC 4.7168), the whole genome sequence (sequence number GenBank: NC_003888.3) and Streptomyces avermitilis Streptomyces avermitmitilis MA-4680 (deposit number CGMCC 4.3588) and the entire genome sequence (sequence number GenBank: NC_003155.5) were submitted to the whole genome alignment analysis software Mauve for alignment analysis;

(2) Analysis of the entire genome comparison data obtained in (1), according to the gene regions that are conserved in all compared genomes are essential gene regions, and the gene regions that exist only in one or two genomes are non-essential genes Region theory, divide the streptomyces genome into essential gene regions and non-essential gene regions;

(3) Select a non-essential gene region divided in (2), a region in which biosynthetic gene clusters are relatively concentrated, and be a candidate large fragment redundant gene region;

(4) Construction of knockout plasmid and suicide plasmid. Using the pHAH plasmid as a template, design specific primers to amplify the lox66 fragment (ATAACTTCGTATAGCATACATTATACGAAcggta), and introduce XbaI and EcoRV digestion sites at both ends. After digestion and purification, T4 DNA ligase was used to ligate XbaI and EcoRV double-digested pKC1139 plasmid at 16 ° C to obtain a Streptomyces knockout plasmid pKClox66. The pSET152 vector was digested with HindIII, and a larger DNA fragment was recovered. T4 DNA ligase was added and ligated at 16 ° C. The pSET153 plasmid was obtained after concatenation. Then the pSET153 plasmid was digested with SacI and the larger DNA fragment was recovered. pIJ779 was used as a template, specific primers were designed to amplify the spectacular gene aadA fragment, and SacI digestion sites were introduced at both ends. After digestion and purification, T4DNA ligase was used to ligate SacI single digestion at 16 ° C and alkaline phosphatase was used. Dephosphorylated pSET153 plasmid to obtain pSET154 plasmid. Using pHAH as a template, design specific primers to amplify the lox71 (taccgTTCGTATA GCTACAT TATACGAAGTTAT) fragment, and introduce the restriction sites of HindIII and XbaI at both ends. After digestion and purification, use T4DNA ligase to connect HindIII and XbaI at 16 ° C The pSET154 plasmid was digested with enzyme to obtain the streptomyces suicide plasmid pSETlox71. Using pALCre as a template, specific primers were designed to amplify the Cre gene fragment, and NdeI and HindIII restriction sites were introduced at both ends. After digestion and purification, T4DNA ligase was used to connect NdeI and HindIII double-digested pIJ8668 at 16 ° C. -nit-pIJ101ori-MCS plasmid to obtain the streptomyces expression plasmid pNitCre. Design the homology arm according to the sequence of the large fragment redundant gene region described in step (3), design two homology arms at the 5'-end, and introduce the restriction sites HindIII / XbaI and EcoRV / EcoRI, 3'- A homology arm is designed at the end, and the restriction site EcoRV / EcoRI is introduced. The length is generally 1000bp-1500bp. The homology arm is amplified by PCR and then digested with HindIII / XbaI and EcoRV / EcoRI. The 5'-end The homologous arm fragments were sequentially ligated into the Streptomyces knockout plasmid pKClox66 to constitute a knockout plasmid; a 3'-end homologous arm was amplified by PCR and digested with EcoRV / EcoRI and ligated to the Streptomyces suicide plasmid pSETlox71 to constitute suicide. Plasmid

(5) The knockout plasmid obtained in step (4) is introduced into E. coli competent cells ET12567 / pUZ8002 or S17-1 or ET12567 / pUB307 by transformation;

(6) introducing the knockout plasmid in step (5) into Streptomyces by E. coli-Streptomyces conjugation transduction;

(7) The single exchange of the plasmid is knocked out, and the transformant in step (6) is subjected to apramycin resistance screening at a temperature greater than 34 ° C (generally 39 ° C). Strains that are resistant to serotonin and grown at 39 ° C are mutants that undergo single exchange;

(8) Knock out the double exchange of the plasmid, pick the single exchange mutant strain obtained in step (7) into the non-resistant TSB liquid medium and culture at 39 ° C for 36 hours, and then transfer to the non-resistant TSB liquid medium and culture at 39 ° C. After 24h, transfer to non-resistant TSB liquid medium and incubate at 30 ° C for 24h. Take 100μL of bacterial solution and draw a monoclonal on the non-resistant plate.

(9) Perform photocopy screening on the monoclonals grown in step (8). The same monoclonal is simultaneously marked on the corresponding positions of two plates, one is an antibiotic-free plate, and the other is apramycin-containing. The resistant plates were selected to be non-growth on the resistant plates, and the normal clones grown on the non-resistant plates were placed in TSB liquid medium. After culturing for 36-48 hours, the appropriate amount of bacterial solution was used to extract genomic DNA for PCR verification. Screening for positive monoclonals;

(10) The positive monoclonal line obtained in step (9) is streaked on the solid spore-producing medium of Streptomyces. After culturing for an appropriate time, the monoclonal line produces spores. Collect the spores and add sterilized glycerol to a final concentration of 20%, -80 ° C. Save for future use

(11) The suicide plasmid obtained in step (4) is introduced into E. coli competent cells ET12567 / pUZ8002 or S17-1 or ET12567 / pUB307 by transformation;

(12) introducing the suicide plasmid in step (11) into the Streptomyces mutant spores obtained in step (10) by E. coli-streptomyces conjugation transduction;

(13) Single exchange of suicide plasmids. The transformants in step (12) are screened for spectinomycin resistance at a temperature of 30 ° C. Strains capable of growing normally on spectinomycin-resistant plates are monomorphic. The exchanged mutants were transferred to TSB liquid medium. After 36-48 h of culture, appropriate amount of bacterial solution was used to extract genomic DNA for PCR verification, and positive clones were selected;

(14) The positive monoclonal line obtained in step (13) was streaked on a solid spore-producing medium of Streptomyces, and cultured for an appropriate time, the monoclonal line produced spores. Collect the spores and add sterilized glycerol to a final concentration of 20%, -80 ° C. Save for future use

(15) Introduction of a Streptomyces expression plasmid. The Streptomyces expression plasmid pNitCre is transformed into E. coli competent cells ET12567 / pUZ8002 or S17-1 or ET12567 / pUB307 by transformation, and the Streptomyces are transformed by E. coli-Streptomyces conjugation. The expression plasmid pNitCre was introduced into the Streptomyces spores obtained in step (14), and apramycin resistance screening was performed. Monoclonal clones that could grow normally on apramycin resistant plates were selected and transferred to TSB liquid culture. Medium, after 36-48h of culture, take an appropriate amount of bacterial solution to extract genomic DNA for PCR verification, and select positive monoclonals;

(16) The positive monoclonal clone obtained in step (15) is streaked on the solid spore-producing medium of Streptomyces. After culturing for an appropriate time, the monoclonal clone produces spores. Collect the spores and add sterilized glycerol to a final concentration of 20%, -80 ° C. Save for future use

(17) Cre enzyme expression. Take 100 μL of the Streptomyces spores obtained in step (16) and insert them into TSB liquid medium. After shaking culture for 24 hours, add the inducer ε-caprolactam (final concentration: 1%), and continue to culture for 10 hours. Take 100 μL of the bacteria solution and dilute it 1000 times, then spread it on a solid plate, and invert and culture for several days until the monoclonals grow;

(18) Select the clones obtained in step (17) for photocopy screening. The same clone is simultaneously placed on the corresponding positions of two plates, one plate is an antibiotic-free plate, and the other is resistant to spectinomycin. flat. Pick the non-growth on the resistant plate, and the normal growth on the non-resistance plate to TSB liquid medium. After 36-48h of culture, extract the appropriate amount of bacterial solution to extract the genomic DNA for PCR verification and select the positive monoclonal. ;

(19) The positive monoclonal clone obtained in step (18) is streaked on a solid spore-producing medium of Streptomyces, and cultured for an appropriate time, the monoclonal clone produces spores. Collect the spores and add sterilized glycerol to a final concentration of 20%, -80 ° C Save for future use

(20) Loss of Streptomyces expression plasmid, take 100 μL of frozen spores from step (19), dilute 1000 times and coat non-resistance plate. After culturing for an appropriate time, clones will grow. For resistant plates, the obtained monoclonals were subjected to photocopy screening. The same monoclonal was simultaneously placed on the corresponding positions of two plates. One plate was an antibiotic-free plate and the other was an apela-resistant plate. Non-growth on the plate. Monoclonals that normally grow on non-resistant plates are mutants with loss of expression plasmid;

(21) The positive monoclonal obtained in step (20) is the final genome-reduced Streptomyces chassis cells, and it is streaked on the Streptomyces solid spore production medium, and cultured for an appropriate time, the monoclonals produce spores, collect the spores and add Sterilize glycerol to a final concentration of 20% and store at -80 ° C;

(22) The Streptomyces chassis cells obtained in step (21) are inoculated with a fermentation medium such as TSB medium and YEME medium, and the following assessments are performed: growth cycle assessment, metabolic profile assessment, exogenous protein expression ability assessment, exogenous secondary Evaluation of metabolite production capacity.

Another object of the present invention is to provide the application of the method in constructing a high-strength version of a Streptomyces chassis cell with a reduced genome. The invention analyzes and locates a large fragment redundant gene region with low conservation as a candidate knockout region based on the comparison results of the whole genomes of multiple streptomyces. According to the knockout region, the homologous arm was connected to the Streptomyces knockout plasmid and the Streptomyces suicide plasmid. The knockout plasmid and suicide plasmid were constructed. Efficient deletion of the redundant gene regions of the fragments to construct a high-strength version of Streptomyces chassis cells. Through the determination of biomass, analysis of metabolic profiles, analysis of foreign protein expression, and analysis of foreign secondary metabolic pathways, it was determined that the genome-reduced Streptomyces high-version chassis cells have better performance and are convenient for efficient biosynthesis and industrialization of microbial drugs produce.

The method of the present invention has been successfully used to construct a genetically engineered strain of Streptomyces catarrhalis, which is classified and named: Streptomyces chattanoogensis L321 (Streptomyces chattanoogensis L321). The Common Microbiological Center (CGMCC) is deposited under the accession number: CGMCC No. 16339, the deposit date is August 27, 2018, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The genome of this strain has been genetically modified to reduce the genome of its original starting strain by about 8.1%, and has shown a series of excellent properties such as a stronger ability to express foreign proteins, a simpler metabolic profile, and a stronger foreign source Secondary metabolite production capacity.

The application is based on the comparison of multiple whole genomes of Streptomyces, analyzing and locating redundant large gene regions with low conservation as candidate knockout regions.

The application is to design a homologous arm connected to a Streptomyces knockout plasmid and a Streptomyces suicide plasmid according to the knockout area, construct a knockout plasmid and a suicide plasmid, introduce lox66 and lox71 sites to both ends of the knockout area, and induce Expression Cre mediates the efficient deletion of large redundant gene regions, thereby constructing a high-strength version of Streptomyces chassis cells.

The application is to determine that the genome-reduced streptomyces high-version chassis cells have better performance through biomass determination, metabolic profiling analysis, exogenous protein expression analysis, and exogenous secondary metabolic pathway expression analysis, which is convenient for microbial drugs. Efficient biosynthesis and industrial production.

The method of the present invention is to locate a redundant gene region of a large segment in a genome according to the theory that an essential gene is highly conserved in evolution and a non-essential gene is low in conservation. In the method of the present invention, a universal knockout vector and a suicide vector are modified by constructing a homologous recombination system and a site-specific recombination system with universal applicability for targeted integration of loxP mutation lox66 and lox71 on the streptomyces genome And large fragments are missing. The method of the present invention uses a homologous recombination system to integrate the recognition site of a site-specific recombinase to both sides of a redundant gene region of a large fragment, and uses an efficient site-specific recombination system to target the deletion of a redundant gene of a large fragment. Region to construct a genome-reduced Streptomyces chassis cell.

The invention has obvious advantages:

1. The present invention can accurately locate a large genomic redundant genome region in a Streptomyces genome using a universal whole-genome alignment technology.

2. The present invention constructs a universal Streptomyces knockout plasmid, a suicide plasmid, and a site-specific recombinase expression plasmid, which can be used for the knockout of a single gene or a gene cluster, and can also be used for the seamlessness of large redundant gene regions. Is missing.

3. The present invention is widely used in constructing highly efficient Streptomyces chassis cells, and all the genome-sequenced Streptomyces can use the method of the present invention to perform efficient editing of large fragment redundant gene regions.

The invention provides a method for constructing a highly efficient streptomyces genome reduction system. The method of the invention has the advantages of simple operation, high efficiency, strong targeting, high accuracy, and universal applicability. The invention lays a theoretical foundation for the construction of streptomyces chassis cells, facilitates the efficient construction of synthetic biology chassis cells, and provides strong support for the industrialization of drugs and the development of new drugs.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the comparison of the entire genome of Streptomyces chantanica L10 with the whole genome of Streptomyces azureus and Streptomyces avermitilis. There are two non-essential gene regions with low conservation in the genome of Streptomyces chantanica L10 The sequence located in the 7994797bp-8731201bp interval was selected as the candidate large redundant gene region.

Figure 2 is a map of Streptomyces chantanica L10 knockout plasmid pKClox66LF, suicide plasmid pSETlox71LR, and expression plasmid pNitCre.

Fig. 3 shows a strategy for targeted knockout of candidate large redundant gene regions in the L10 genome of Streptomyces catarrhica by a homologous recombination system combined with a site-specific recombination system.

Figure 4 shows the analysis of knockout efficiency of large candidate redundant gene regions in the L10 genome of S. chattanooga and PCR verification.

FIG. 5 is a growth curve of Streptomyces mutans L321 and wild-type strain L10.

FIG. 6 shows the expression of eGFP protein in the mutant strain L321 of Chattanooga and the wild-type strain L10, respectively. The differences in protein expression intensity at different periods were detected by Western blot.

FIG. 7 shows the difference in metabolic profiles between the mutant strain L321 of Chattanooga and the wild type strain L10 in different fermentation media.

FIG. 8 shows the difference in the ability to express heterogeneous secondary metabolic biosynthetic gene clusters between the mutant strain L321 of Chattanooga and the wild type strain L10, respectively.

detailed description

The detailed description is described below with reference to the accompanying drawings and specific embodiments.

Example 1

The present invention will be described in detail by using the method to streamline the genome of Streptomyces catarrhalis L10.

The specific implementation steps are as follows:

(1) Submit the sequenced whole genome sequence of Streptomyces chantanica L10 and the whole genome sequence of Streptomyces coelicolor and Streptomyces avermitilis published in NCBI to the whole genome comparison analysis software Mauve for comparison The analysis is specifically: the sequence of the entire genome of Streptomyces catarrhalis L10 and Streptomyces coelicolor A3 (2) (deposit number CGMCC 4.7168), which is well-known in NCBI, and the entire genome sequence (sequence number GenBank: NC_003888. 3) Together with Streptomyces avermitilis MA-4680 (deposit number CGMCC 4.3588), the entire genome sequence (sequence number GenBank: NC_003155.5) is submitted to the whole genome alignment analysis software Mauve for alignment analysis;

(2) Analysis of the entire genome comparison data obtained in (1), according to the gene regions that are conserved in all compared genomes are essential gene regions, and the gene regions that exist only in one or two genomes are non-essential genes Region theory, divide the L10 genome of S. chatanooga into essential gene regions and non-essential gene regions;

(3) Select a non-essential gene region in the genome of Streptomyces chantanica L10 divided in (2), and a region with a relatively concentrated biosynthetic gene cluster. The sequence located in the range of 7994797bp-8731201bp is a candidate for large segment redundancy. Gene region (see Figure 1). The Streptomyces chantanica L10 is classified and named as: Streptomyces chattanoogensis L10, deposited in the General Microbiology Center of the China Microbial Strain Collection Management Committee, deposit number: CGMCC2644, deposit date: August 27, 2008, depository address : Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101;

(4) The lox66 site was knocked into the genome of Streptomyces chantanica L10, and the Streptomyces knockout plasmid pKClox66 was used as the starting plasmid. The two homology arms at the 5 'end of the large redundant gene region were connected to transform E.coli TG1. Competent cells, construct a knockout plasmid pKClox66LF (see Figures 2 and 3);

(5) Escherichia coli-streptomyces conjugation transfer, the knockout plasmid in (4) was extracted from E. coli TG1, chemically transformed E. coli ET12567 / pUZ8002, and coated with apramycin (50 μg / mL), kanamycin (50 μg / mL), chloramphenicol (25 μg / mL) on an LB solid plate, and cultured at 37 ° C. for 16 hours;

(6) Pick the monoclonal on the plate and transfer it to a test tube containing 5 mL of LB liquid medium. Add apramycin (50 μg / mL), kanamycin (50 μg / mL), and chlorine to the medium. Mycin (25 μg / mL), 37 ° C, 220 rpm shaking culture for 16 h;

(7) Pipette 300 μL of liquid culture and transfer to a triangular flask containing 30 mL of LB liquid medium. Add apramycin (50 μg / mL), kanamycin (50 μg / mL), and chloramphenicol to the medium. (25 μg / mL), 37 ° C, 220 rpm shaking culture for 3-4 hours, until OD600nm = 0.6-0.8;

(8) Transfer the bacterial solution to a 50 mL centrifuge tube, collect the bacterial cells by centrifugation at room temperature and 6000 rpm for 5 min, and discard the supernatant;

(9) Add 10mL LB liquid medium to resuspend the bacteria, collect the bacteria by centrifugation at room temperature and 6000rpm for 5min, and discard the supernatant;

(10) Repeat step (9) twice, and finally add 1 mL of LB liquid medium to resuspend the bacterial cells for later use;

(11) Pipette 500 μL of Streptomyces chantanica L10 spores into a 1.5 mL EP tube, add 500 μL of LB liquid culture medium, mix well, collect the spores by centrifugation at room temperature and 6000 rpm for 5 min, and discard the supernatant;

(12) Add 1 mL of LB liquid medium, resuspend the spores, collect the spores by centrifugation at room temperature and 6000 rpm for 5 min, and discard the supernatant;

(13) Repeat step (12) twice, and finally add 1mL 2XYT liquid medium to resuspend the spores, put them in a 45 ℃ water bath, heat-shock for 10min, cool to room temperature and set aside;

(14) Pipette 500 μL of E. coli from step (10) and add to 500 μL of spores from step (13), mix, centrifuge at room temperature and 6000 rpm for 5 min, discard 500 μL of the supernatant, and resuspend the remaining 500 μL of liquid bacteria and spores. Spread on MS solid plate, invert and incubate at 30 ° C for 16-18h, then apply apramycin (50 μg / mL) and nalidixic acid (25 μg / mL), continue to culture for 4-5 days, and grow a monoclonal ;

(15) Transfer the monoclonal to YMG solid medium, the medium contains apramycin (50 μg / mL), and place it in a 37 ° C incubator for 3 days;

(16) Transfer to YMG solid medium (without antibiotics) and place in a 37 ° C incubator for 3 days;

(17) Repeat step (16) twice to perform photocopy screening on the monoclonals. The monoclonals are streaked on the corresponding positions of the two plates: one without antibiotics and the other with apramycin (50 μg / mL) resistant plate, cultured at 30 ° C for 5 days;

(18) Pick out the non-growth on the resistant plate, and transfer the monoclonal clones that are normally growing on the non-resistant plate to TSB liquid medium. After shaking culture at 30 ° C and 220 rpm for 36 hours, take 500 μL of bacterial solution to extract genomic DNA for PCR verification After the PCR product was purified, it was sequenced and verified. The positive clones knocked in at lox66 were selected, streaked in YMG medium, cultured for about 10 days, spores were collected, glycerol was added at a final concentration of 20%, and stored at -80 ℃. spare;

(19) The lox71 site was inserted into the genome of Streptomyces catarrhalis mutant, and the Streptomyces suicide plasmid pSETlox71 was used as the starting plasmid. A homology arm at the 3 'end of the large redundant gene region was connected to transform E.coliTG1 competent state. Cells and construct suicide plasmid pSETlox71LR (see Figures 2 and 3);

(20) Perform the conjugation and transfer between E. coli and Streptomyces, the steps are the same as (5)-(14), in which apramycin is replaced by spectinomycin at a concentration of 100 μg / mL, and Streptomyces spores are step (18) After the spores were frozen and the monoclonals appeared on the MS plate, the monoclonals were streaked on YMG solid medium, and the medium contained spectinomycin (100 μg / mL), and cultured in a 30 ° C incubator for 3 days;

(21) Monoclone was transferred to TSB liquid culture medium, containing spectinomycin (100 μg / mL) in the medium. After shaking culture at 30 ° C and 220 rpm for 36 h, genomic DNA was extracted from 500 μL of bacterial solution for PCR verification and the PCR product was purified. After sequencing verification, positive clones inserted at lox71 site were selected, streaked on YMG medium, cultured for about 10 days, spores were collected, glycerol with a final concentration of 20% was added, and stored at -80 ° C until use;

(22) Introduction and induced expression of Cre enzyme expression plasmid. The Streptomyces expression plasmid pNitCre (see Figure 2) is used to transfer and transfer between E. coli and Streptomyces. The steps are the same as (5)-(14). Streptomyces spores are the steps. (21) Frozen spores. After the monoclonals grow on the MS plate, pick the monoclonal lines on the YMG solid medium. The culture medium contains apramycin (50 μg / mL) and place them in a 30 ° C incubator. After about 10 days of medium culture, spores were collected, glycerol was added to a final concentration of 20%, and stored at -80 ° C until use;

(23) Induced expression of Cre enzyme, take 100 μL of Streptomyces catarrhalis spores obtained in step (22) and insert it into TSB liquid culture medium. After shaking culture for 24 hours, add the inducer ε-caprolactam (final concentration is 1%) After continuing to culture for 10 hours, 100 μL of the bacterial solution was diluted 1000 times, and then coated on a YMG solid plate, and cultured for several days upside down, until the monoclonals grew;

(24) Select the clones obtained in step (23) for photocopy screening. The same clone is simultaneously marked on the corresponding positions of two plates, one plate is an antibiotic-free plate and the other is a plate containing spectinomycin (100 μg / mL) resistant plates. Pick non-growth on the resistant plate, normal growth on the non-resistant plate to TSB liquid culture medium, culture 36-48h, then extract the appropriate amount of bacterial solution to extract genomic DNA for PCR verification and sequencing verification, screen out Positive monoclonal clones with large redundant gene regions deleted from the genome and calculated knockout efficiency (see Figure 4);

(25) The positive monoclonal lines obtained in step (24) were streaked on YMG solid medium, cultured for about 10 days until the monoclonals produced spores, collected the spores and added sterilized glycerol to a final concentration of 20%, and stored at -80 ° C. spare;

(26) Loss of Streptomyces expression plasmid, take 100 μL of spores frozen in step (25), dilute 1000 times and coat with non-resistant YMG plate, and grow the colony after appropriate time. Non-resistance plates. The obtained monoclonals were screened by photocopying. The same clone was simultaneously placed on the corresponding positions of two YMG plates. One plate was an antibiotic-free plate and the other was an apramycin-resistant plate. . Non-growth on resistant plates, and monoclonals that normally grow on non-resistant plates are mutants with loss of expression plasmids;

(27) The positive monoclonal obtained in step (26) is the final genome-reduced Streptomyces catarrhalis L321, which has been deposited in the Common Microbiology Center (CGMCC) of the China Microbial Species Collection and Management Committee (CGMCC). Streptomyces chattanoogensis L321, deposit number: CGMCC No. 16339, deposit date: August 27, 2018, deposit address: No. 3, Beichen West Road, Chaoyang District, Beijing. Streak on YMG solid medium, culture for about 10 days to produce spores from the monoclonal, collect spores and add sterilized glycerol to a final concentration of 20%, and store at -80 ° C.

Example 2 Comparison of Growth Curves of Streptomyces Chattanooga Mutant L321 and Wild Type Strain L10

(1) Streptomyces chantanica L321 and wild-type strain L10 spores were respectively inoculated into seed medium, and cultured at 30 ° C and 220 rpm for 24 hours;

(2) Transfer 1mL of seed culture solution to YEME medium, culture at 30 ° C and 220rpm, collect 1mL of bacterial solution at different time points (with an interval of 12 hours) to determine the dry weight, and draw the growth curve (see Figure 5). The results show There was no significant change in the growth cycle of the constructed mutant L321.

Example 3 Comparison of the intensity of expression of eGFP protein in mutant strain L321 of Chattanooga and wild type strain L10

(1) Introduction of the eGFP expression plasmid pIJ8668-ermEp * -egfp into Streptomyces chantanica L321 and wild-type strain L10 by conjugation transduction, respectively;

(2) Mutant strains were inoculated with TSB medium and YEME medium, and 1 mL of bacterial solution was collected at different time points. The bacterial cells were collected by centrifugation at 12,000 rpm, ultrasonically broken, and the supernatant was taken for quantification of Bradford protein after centrifugation. PAGE electrophoresis

(3) The expression of eGFP protein was detected by Western blot, and the results showed that the mutant L321 had stronger ability to express foreign proteins (see FIG. 6).

Example 4 Comparison of the metabolic profiles of Streptomyces mutans L321 and wild-type strain L10 in different fermentation media

(1) Streptomyces chantanica L321 and wild-type strain L10 were inoculated with different fermentation mediums (YEME, YSG, and YMG). After cultured for 120 hours, 500 μL of bacterial solution was collected and 500 μL of methanol was added. Centrifuge at 12000 rpm for 10 min. The supernatant was filtered with 0.45 μm. Membrane filtration

(2) The filtered supernatant was analyzed by HPLC to analyze the differences in metabolic spectra. HPLC conditions: Column: C18 (Aglient, Eclipse Plus XDB, 5um, 4.6mm * 250mm); Detection wavelength: Full wavelength scanning; Flow rate: 1.00mL / min; injection volume: 25ul; mobile phase A is water, containing 0.1% formic acid, mobile phase B is 100% methanol; HPLC sampling procedure: 0-20min, 5% -95% B phase; 20-25min, 5 % B phase; The conventional substance analysis and detection wavelength was selected for analysis. The results showed that the mutant strain L321 had a significantly reduced endogenous by-products, had a simpler metabolic background, and was suitable as a chassis cell (see FIG. 7).

Example 5 Comparison of the ability to express heterologous secondary metabolite biosynthetic gene clusters in mutants of Streptomyces catarrhalis L321 and wild type strain L10

(1) The actinomycete expression plasmid pSET152-act was introduced into Streptomyces chantanica L321 and wild-type strain L10 by conjugation and transduction respectively. The mutant strain was inoculated with YEME medium for fermentation, and 1 mL of bacterial solution was collected at different time points.

(2) The production of actinomycetin was measured by a spectrophotometer, and the results showed that the mutant L321 had stronger secondary metabolite synthesis ability (see FIG. 8).

Claims (7)

1. Construction of an efficient streptomyces genome reduction system, which is characterized by analyzing and locating large fragments of redundant gene regions in the Streptomyces genome through whole-genome comparison technology, and constructing a Streptomyces knockout plasmid pKClox6 and a Streptomyces suicide plasmid pSETlox71 And streptomyces expression plasmid pNitCre, in which the knockout plasmid pKClox66 carries lox66 site, streptomyces temperature-sensitive replicon pSG5, Apra resistance; suicide plasmid pSETlox71 carries lox71 site, no streptomyces replicon, Spect resistance; expression The plasmid pNitCre carries a Streptomyces codon-optimized Cre recombinase, Streptomyces free-form replicon pIJ101, and Apra resistance.
2. The construction of an efficient streptomyces genome reduction system according to claim 1, characterized in that it is achieved by the following steps:

   (1) The determination of redundant gene regions of large fragments is realized by the following steps:

   1) Submit the sequenced whole genome sequence of Streptomyces with the whole genome sequence of Streptomyces coelicolor and Streptomyces avermitilis published in NCBI to the whole genome comparison analysis software Mauve for comparison analysis;

   2) Analyze the results of whole-genome comparisons. According to the theory that all conserved gene regions in the compared genomes are essential gene regions, and gene regions that exist only in one or two genomes are non-essential gene regions, The genome is divided into essential gene regions and non-essential gene regions;

   3) Select a non-essential gene region with a relatively concentrated biosynthetic gene cluster as a candidate large fragment redundant gene region;

   (2) The construction of the Streptomyces knockout plasmid pKClox66 was achieved by the following steps:

   1) Using the pHAH plasmid as a template, design specific primers to amplify the lox66 fragment, and introduce XbaI and EcoRV digestion sites at both ends of it. After digestion, purify and reserve for use; pKC1139 plasmid is double digested with XbaI and EcoRV and recovered;

   2) The digested lox66 fragment in step 1) is ligated into the digested pKC1139 vector to obtain a Streptomyces knockout plasmid pKClox66;

   (3) The construction of streptomyces suicide plasmid pSETlox71 is realized by the following steps:

   1) pSET152 vector was digested with HindIII, and larger DNA fragments were recovered;

   2) The DNA fragment recovered in step 1) is added to T4DNA ligase to obtain pSET153 plasmid after self-cyclization;

   3) The pSET153 plasmid obtained in step 2) was digested with SacI, and a larger DNA fragment was recovered for future use;

   4) Using pIJ779 as a template, design specific primers to amplify the magnificent gene fragment, and introduce SacI restriction sites at both ends of the gene. After purification by enzyme digestion, use it for future use;

   5) The purified DNA fragment in step 4) is ligated into the DNA fragment recovered in step 3) to obtain pSET154 plasmid;

   6) Using pHAH as a template, design specific primers to amplify the lox71 fragment, and introduce HindIII and XbaI digestion sites at both ends. After digestion and purification, use the pSET154 plasmid after double digestion with HindIII and XbaI.

   7) The digested lox71 fragment in step 6) is ligated into the digested pSET154 vector to obtain the streptomyces suicide plasmid pSETlox71;

   (4) The construction of the streptomyces expression plasmid pNitCre is achieved by the following steps:

   1) Using pALCre as a template, design specific primers to amplify the Cre gene fragment, and introduce NdeI and HindIII digestion sites at both ends of the Cre gene.

   2) pIJ8668-nit-pIJ101ori-MCS plasmid was double-digested with NdeI and HindIII to recover larger DNA fragments for future use;

   3) The DNA fragment recovered in step 1) is ligated with the DNA fragment recovered in step 2) to obtain a streptomyces expression plasmid pNitCre.
3. The application of an efficient streptomyces genome reduction system according to claim 2, characterized in that it is implemented by the following steps:

   (1) The sequenced whole genome sequence of Streptomyces and Streptomyces coelicolor A3 (2), known as NCBI, deposit number CGMCC 4.7168, the sequence number of the whole genome sequence GenBank: NC_003888.3, and Streptomyces avermitilis Streptomyces avermitilis MA-4680, its deposit number CGMCC 4.3588, and the sequence number of the whole genome sequence GenBank: NC_003155.5, were submitted to the whole genome comparison analysis software Mauve for alignment analysis;

   (2) Analysis of the entire genome comparison data obtained in (1), according to the gene regions that are conserved in all compared genomes are essential gene regions, and the gene regions that exist only in one or two genomes are non-essential genes Region theory, divide the streptomyces genome into essential gene regions and non-essential gene regions;

   (3) Select a non-essential gene region divided in (2), a region in which biosynthetic gene clusters are relatively concentrated, and be a candidate large fragment redundant gene region;

   (4) Construction of knockout plasmids and suicide plasmids. Design homology arms based on the sequence of the large redundant gene region described in claim 1 or 2. Design two homology arms at the 5'-end and 3'-end. Design a homologous arm with a length of 1000bp-1500bp. The homologous arm is amplified by PCR and digested. The two homologous arm fragments at the 5'-end are sequentially connected to the Streptomyces knockout plasmid pKClox66 to form a knockout. Plasmid; a homology arm at the 3 'end is connected to the Streptomyces suicide plasmid pSETlox71 to constitute a suicide plasmid;

   (5) The knockout plasmid obtained in step (4) is introduced into E. coli competent cells ET12567 / pUZ8002 or S17-1 or ET12567 / pUB307 by transformation;

   (6) introducing the knockout plasmid in step (4) into Streptomyces by E. coli-Streptomyces conjugation transduction;

   (7) The single exchange of the plasmid is knocked out, and the transformant in step (6) is subjected to apramycin resistance screening at a temperature greater than 34 ° C (generally 39 ° C). Strains that are resistant to serotonin and grown at 39 ° C are mutants that undergo single exchange;

   (8) Knock out the double exchange of the plasmid, pick the single exchange mutant strain obtained in step (7) into the non-resistant TSB liquid medium and culture at 39 ° C for 36 hours, and then transfer to the non-resistant TSB liquid medium and culture at 39 ° C. After 24h, transfer to non-resistant TSB liquid medium and incubate at 30 ° C for 24h. Take 100μL of bacterial solution and draw a monoclonal on the non-resistant plate.

   (9) Perform photocopy screening on the monoclonals grown in step (8). The same monoclonal is simultaneously marked on the corresponding positions of two plates, one is an antibiotic-free plate, and the other is apramycin-containing. The resistant plates were selected to be non-growth on the resistant plates, and the normal clones grown on the non-resistant plates were placed in TSB liquid medium. After culturing for 36-48 hours, the appropriate amount of bacterial solution was used to extract genomic DNA for PCR verification. Screening for positive monoclonals;

   (10) The positive monoclonal line obtained in step (9) is streaked on the solid spore-producing medium of Streptomyces. After culturing for an appropriate time, the monoclonal line produces spores. Collect the spores and add sterilized glycerol to a final concentration of 20%, -80 ° C Save for future use

   (11) The suicide plasmid obtained in step (4) is introduced into E. coli competent cells ET12567 / pUZ8002 or S17-1 or ET12567 / pUB307 by transformation;

   (12) introducing the suicide plasmid in step (7) into the spores of the Streptomyces mutant strain obtained in step (10) by E. coli-streptomyces conjugation transduction;

   (13) Single exchange of suicide plasmids. The transformants in step (12) are screened for spectinomycin resistance at a temperature of 30 ° C. Strains capable of growing normally on spectinomycin-resistant plates are monomorphic. The exchanged mutants were transferred to TSB liquid medium. After 36-48 h of culture, appropriate amount of bacterial solution was used to extract genomic DNA for PCR verification, and positive clones were selected;

   (14) The positive monoclonal line obtained in step (13) was streaked on a solid spore-producing medium of Streptomyces, and cultured for an appropriate time, the monoclonal line produced spores. Collect the spores and add sterilized glycerol to a final concentration of 20%, -80 ° C. Save for future use

   (15) Introduction of a Streptomyces expression plasmid. The Streptomyces expression plasmid pNitCre is transformed into E. coli competent cells ET12567 / pUZ8002 or S17-1 or ET12567 / pUB307 by transformation, and the Streptomyces are transformed by E. coli-Streptomyces conjugation. The expression plasmid pNitCre was introduced into the spores of the Streptomyces mutant strain obtained in step (14), and was screened for apramycin resistance. Monoclonals that were able to grow normally on apramycin-resistant plates were selected and transferred to TSB. In the liquid culture medium, after 36-48 hours of culture, an appropriate amount of bacterial solution was used to extract genomic DNA for PCR verification, and positive clones were selected;

   (16) The positive monoclonal clone obtained in step (15) is streaked on the solid spore-producing medium of Streptomyces. After culturing for an appropriate time, the monoclonal clone produces spores. Collect the spores and add sterilized glycerol to a final concentration of 20%, -80 ° C. Save for future use

   (17) Induced expression of Cre enzyme, take 100 μL of the spores of the Streptomyces mutant strain obtained in step (16) into TSB liquid medium, and shake culture for 24 hours, then add the inducer ε-caprolactam to a final concentration of 1%, and continue to culture After 10 hours, 100 μL of the bacterial solution was diluted 1000 times, and then coated on a solid plate and cultured upside down for several days until the monoclonals grew.

   (18) Select the clones obtained in step (17) for photocopy screening. The same clone is simultaneously placed on the corresponding positions of two plates, one plate is an antibiotic-free plate, and the other is resistant to spectinomycin. flat. Pick the non-growth on the resistant plate, and the normal growth on the non-resistance plate to TSB liquid medium. After 36-48h of culture, extract the appropriate amount of bacterial solution to extract the genomic DNA for PCR verification and select the positive monoclonal. ;

   (19) The positive monoclonal clone obtained in step (18) is streaked on a solid spore-producing medium of Streptomyces, and cultured for an appropriate time, the monoclonal clone produces spores. Collect the spores and add sterilized glycerol to a final concentration of 20%, -80 ° C Save for future use

   (20) Loss of Streptomyces expression plasmid, take 100 μL of frozen spores from step (19), dilute 1000 times and coat non-resistance plate. After culturing for an appropriate time, clones will grow. For the resistant plates, the obtained monoclonals were subjected to photocopy screening. The same monoclonal was simultaneously placed on the corresponding positions of two plates, one was an antibiotic-free plate, and the other was an apolla-resistant plate. Non-growth on resistant plates, and monoclonals that normally grow on non-resistant plates are mutants with loss of expression plasmids;

   (21) The positive monoclonal obtained in step (20) is the final genome-reduced Streptomyces mutant strain, streaked on the solid spore-producing medium of Streptomyces, and cultured for an appropriate time. Bacterial glycerol to a final concentration of 20%, stored at -80 ° C, ready for use;

   (22) The mutant strain obtained in step (21) is evaluated such as growth cycle assessment, metabolic profile assessment, exogenous protein expression ability assessment, and exogenous secondary metabolite production ability assessment.
4. Use of the method according to claim 1 or 2 or 3 in constructing a high version of a Streptomyces chassis cell.
5. The application according to claim 4, characterized in that, based on the comparison results of the whole genomes of a plurality of Streptomyces, a large conserved redundant gene region with low conservation is analyzed and located as a candidate knockout region.
6. The application according to claim 4, characterized in that a homologous arm is connected to a Streptomyces knockout plasmid and a Streptomyces suicide plasmid according to the knockout region, a knockout plasmid and a suicide plasmid are constructed, and lox66 and lox71 sites are introduced into The two ends of the knockout region were induced to express Cre to mediate the efficient deletion of large redundant gene regions, thereby constructing a high-strength version of Streptomyces chassis cells.
7. The application according to claim 4, characterized in that it is determined that the genome-reduced Streptomyces high-version chassis cells have better quality through biomass determination, metabolic profiling analysis, foreign protein expression analysis, and foreign secondary metabolic pathway expression analysis. Performance, convenient for efficient biosynthesis and industrial production of microbial drugs.

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