Recombinant streptomyces mobaraensis as well as construction method and application thereof

Abstract

The invention discloses recombinant streptomyces mobaraensis as well as a construction method and application thereof, and belongs to the technical field of bioengineering. According to the invention, smY2019 is used as an original strain, and a CRISPR / cas9 technology is utilized to construct five chassis cells of a GIs gene cluster; on the basis of smY2019-deltaG5, two kinds of chassis cells in which endogenous CRISPR / Cas system genes are knocked out are constructed. And on the basis of the smY2019-deltaG5-C2, further constructing a chassis cell smY2019-deltaG5-C3 of which a non-homologous end connection (NHEJ) repair pathway gene is knocked out. The TGase secretory enzyme activity of the chassis cell smY2019-deltaG5-C3 is improved by 37.3% compared with that of the original strain smY2019, and meanwhile, the transformation efficiency, the growth speed, the protein synthesis capability and the energy metabolism level of the chassis cell are obviously improved compared with that of the original strain smY2019.

Description

Technical Field

[0001] The present invention relates to a recombinant Streptomyces mobaraensis and a construction method and application thereof, belonging to the technical field of bioengineering. Background Art

[0002] Chassis cells, also known as gene chassis cells, are a type of microbial cells that have undergone genome reduction or genome synthesis. Through genome editing, chassis cells eliminate the unfavorable characteristics of protein expression and rewire the metabolic network, so they often exhibit better cell adaptability, recombinant protein production, and stability of exogenous expression pathways than wild-type strains. In recent years, microbial chassis cells constructed based on metabolic engineering technology and synthetic biology methods have been widely used in the production of various natural high-value-added chemicals, high-quality clean energy, and biological materials.

[0003] Streptomyces is the largest genus in Actinomycetes. Due to its possession of multiple secondary metabolic pathways and efficient protein secretion mechanisms, it is often used as a producer of biomedical and fermentation industrial enzyme proteins and is widely applied in fields such as food and medicine. Currently, multiple Streptomyces strains, such as Streptomyces lividans, Streptomyces coelicolor A3(2), Streptomyces avermitilis, Streptomyces violaceoruber, etc., have been transformed into microbial cell factories for the production of target products such as industrial enzymes, antibiotics, functional sugars, health products, and drug precursors, showing strong industrial production application capabilities.

[0004] Streptomyces mobaraensis is a spore-producing thermophilic actinomycete that can be used to produce various antibiotics and industrial enzyme proteins. Streptomyces mobaraensis (S. mobaraensis) has characteristics such as high safety, strong protein secretion ability, and mature fermentation process, and has been certified as a GRAS (generally recognized as safe) safe strain by the US Food and Drug Administration (FDA). Due to its advantages such as clear physiological and biochemical characteristics, mature genetic manipulation methods, and convenient culture and fermentation, it has been widely used in the industrial fermentation production of transglutaminase (TGase). Currently, there have been studies on the analysis of the genome and transcriptome data of S. mobaraensis, which provides strong support for further exploring its genomic information, reveals its broad potential as a chassis cell, and is expected to transform it into a general expression system more suitable for protein synthesis, natural product production, and exogenous BGC expression.

[0005] At present, there are still some obvious defects in the S. mobaraensis protein expression system, such as low or unstable heterologous protein expression yields, long bacterial growth cycles, complex and difficult-to-control morphological differentiation characteristics, low genetic transformation efficiency, few and inefficient gene editing tools, etc. These factors limit its application in large-scale industrial production. In addition, compared with some mature chassis cell strains, such as Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae, the development of S. mobaraensis chassis cells is still relatively lagging behind.

[0006] Therefore, this patent takes the mutagenized strain smY2019 of S. mobaraensis DSM40587 as the research object, and uses the CRISPR / Cas9 gene editing system to streamline its genome, thereby constructing an S. mobaraensis chassis cell with redundant genes knocked out, aiming to optimize the industrial fermentation production of TGase, which has important scientific significance and industrial application value. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a recombinant Streptomyces mobaraensis that is safe, has a simpler operation process, and can efficiently secrete and express transglutaminase in view of the deficiencies of the prior art.

[0008] The present invention also provides a method for constructing the above recombinant Streptomyces mobaraensis.

[0009] The present invention provides the application of the above recombinant Streptomyces mobaraensis in the fermentation of transglutaminase and its application as a chassis strain in the construction of a genetically engineered bacterium for fermenting transglutaminase.

[0010] The technical solution adopted by the present invention is as follows:

[0011] A recombinant Streptomyces mobaraensis provided by the present invention is a chassis strain, which uses Streptomyces mobaraensis smY2019 as the starting strain and is obtained by knocking out five largest genomic islands (GIs), all endogenous CRISPR / Cas systems and genes related to the NHEJ repair pathway. Among them, the Streptomyces mobaraensis smY2019 is a mutagenized strain of Streptomyces mobaraensis S. mobaraensis DSM40587, and its GenBank accession number is CP083590. It is a mutagenized strain that can efficiently secrete transglutaminase screened in the early stage of the laboratory. The genomic island (GIs) genes are GI-I, GI-II, GI-III, GI-IV and GI-V, and the specific nucleotide sequences and their positions in the S. mobaraensis genome are as described in Tables 3-1 to 3-6. The endogenous CRISPR / Cas system genes are CRISPR1 and CRISPR2, and the specific nucleotide sequences and their positions in the S. mobaraensis genome are as described in Table 4. The NHEJ repair pathway genes are K7I03_296010, K7I03_29605 and K7I03_29600, and the specific nucleotide sequences and their positions in the S. mobaraensis genome are as described in Table 5.

[0012] The present invention provides a chassis cell of Streptomyces mobaraensis, which is obtained by using Streptomyces mobaraensis smY2019 as the starting strain and knocking out one or more of the genomic island GIs genes, endogenous CRISPR / Cas system genes, and NHEJ repair pathway genes on its genome.

[0013] In one embodiment of the present invention, the NCBI numbers of the genes of the genomic islands GIs are K7I03_28245, K7I03_28250, K7I03_28255, K7I03_28260, K7I03_28265, K7I03_28270, K7I03_28275, K7I03_28280, K7I03_28285, K7I03_28290, K7I03_28295, K7I03_28300, K7I03_28305, K7I03_28310, K7I03_28315, K7I03_28320, K7I03_28325, K7I03_28330, K7I03_28335, K7I03_28340, K7I03_28345, K7I03_28350, K7I03_28355, K7I03_28360, K7I03_28365, K7I03_28370, K7I03_28375, K7I03_28380, K7I03_28385, K7I03_28390, K7I03_28395, K7I03_28400, K7I03_28405, K7I03_28410, K7I03_28415, K7I03_28420, K7I03_28425, K7I03_28430, K7I03_28435, K7I03_28835, K7I03_28840, K7I03_28845, K7I03_28850, K7I03_28855, K7I03_28860, K7I03_28865, K7I03_28870, K7I03_28875, K7I03_28880, K7I03_28885, K7I03_28890, K7I03_28895, K7I03_28900, K7I03_28905, K7I03_28910, K7I03_28915, K7I03_28920, K7I03_28925, K7I03_28930, K7I03_28935, K7I03_28940, K7I03_28945, K7I03_28950, K7I03_28955, K7I03_28960, K7I03_28965, K7I03_28970, K7I03_28975, K7I03_28980, K7I03_28985, K7I03_28990, K7I03_28995, K7I03_29000, K7I03_29005, K7I03_18425, K7I03_18430, K7I03_18435, K7I03_18440, K7I03_18445, K7I03_18450,K7I03_18455, K7I03_18460, K7I03_18465, K7I03_18470, K7I03_18475, K7I03_18480, K7I03_18485, K7I03_18490, K7I03_18495, K7I03_18500, K7I03_18505, K7I03_18510, K7I03_18515, K7I03_18520, K7I03_18525, K7I03_18530, K7I03_18535, K7I03_18540, K7I03_18545, K7I03_18550, K7I03_18555, K7I03_18560, K7I03_18565, K7I03_18570, K7I03_18575, K7I03_18580, K7I03_18585, K7I03_18590, K7I03_18595, K7I03_18600, K7I03_18605, K7I03_18610, K7I03_03350, K7I03_03355, K7I03_03360, K7I03_03365, K7I03_03370, K7I03_03375, K7I03_03380, K7I03_03385, K7I03_03390, K7I03_03395, K7I03_03400, K7I03_03405, K7I03_03410, K7I03_03415, K7I03_03420, K7I03_03425, K7I03_03430, K7I03_03435, K7I03_03440, K7I03_03445, K7I03_03450, K7I03_03455, K7I03_03460, K7I03_03465, K7I03_01620, K7I03_01625, K7I03_01630, K7I03_01635, K7I03_01640, K7I03_01645, K7I03_01650, K7I03_01655, K7I03_01660, K7I03_01665, K7I03_01670, K7I03_01675, K7I03_01680, K7I03_01685, K7I03_01690, K7I03_01695, K7I03_01700, K7I03_01705, K7I03_01710, K7I03_01715, K7I03_01720, K7I03_01725K7I03_01730; the endogenous CRISPR / Cas system genes are located at positions 1358697 to 1368646 and positions 1437389 to 1442520 on the genome of Streptomyces mobaraensis smY2019; the NCBI numbers of the NHEJ repair pathway genes are K7I03_29600, K7I03_29605, and K7I03_29610.,

[0014] In one embodiment of the present invention, the chassis cell is obtained by knocking out the genomic island GIs genes on its genome using Streptomyces mobaraensis smY2019 as the starting strain.

[0015] In one embodiment of the present invention, the chassis cell is obtained by knocking out the genomic island GIs genes on its genome and positions 1358697 to 1368646 on its genome using Streptomyces mobaraensis smY2019 as the starting strain.

[0016] In one embodiment of the present invention, the chassis cell is obtained by knocking out the genomic island GIs genes on its genome and the endogenous CRISPR / Cas system genes on its genome using Streptomyces mobaraensis smY2019 as the starting strain.

[0017] In one embodiment of the present invention, the chassis cell is obtained by knocking out the genomic island GIs genes, the endogenous CRISPR / Cas system genes on its genome, and the NHEJ repair pathway genes on its genome using Streptomyces mobaraensis smY2019 as the starting strain.

[0018] The present invention also provides a method for constructing the above recombinant Streptomyces mobaraensis, comprising the following steps:

[0019] (1) The starting strain used in the present invention is smY2019, which is preserved in our laboratory.

[0020] (2) In the present invention, first, through analysis using the prediction software IslandViewer4 and CRISPRCasFinder, it is found that there are 18 genomic islands (GIs) and 2 endogenous CRISPR / Cas systems in the genome of S. mobaraensis DSM40587. Then, through homologous sequence alignment, it is found that there are 3 NHEJ pathway-related genes in the genome of S. mobaraensis DSM40587.

[0021] (3) Based on the CRISPR / Cas9 editing system constructed in previous experiments, 5 of the largest GIs, all endogenous CRISPR / Cas systems, and the related genes of the NHEJ repair pathway (a total of 167.6 kb) in smY2019 are knocked out.

[0022] (4) For a new round of genome editing based on the CRISPR / Cas9 system, quickly eliminate the resistant Cas9 expression plasmid in the strain. Pick the successfully edited colonies and streak them on the antibiotic-free ISP4 medium, and grow them in an incubator at 37 °C for 3 days to eliminate the plasmid.

[0023] (5) Finally, the chassis strain smY2019-ΔG5-C3 was obtained, with its genome reduced by 2.2%. Subsequently, parameters such as its protein synthesis, growth rate, transformation efficiency, ATP energy, and reducing power were evaluated.

[0024] The present invention also provides a method for improving the growth rate and / or transformation efficiency of Streptomyces mobaraensis smY2019, and the method is to knock out one or more of the genes of the above genomic island GIs, the endogenous CRISPR / Cas system genes, and the NHEJ repair pathway genes.

[0025] In one embodiment of the present invention, the method is: knocking out the genes of the genomic island GIs on its genome.

[0026] In one embodiment of the present invention, the method is: knocking out the genes of the genomic island GIs on its genome, and the positions from the 1,358,697th to the 1,368,646th on its genome.

[0027] In one embodiment of the present invention, the method is: knocking out the genes of the genomic island GIs on its genome, and the endogenous CRISPR / Cas system genes on its genome.

[0028] In one embodiment of the present invention, the method is: knocking out the genes of the genomic island GIs on its genome, the endogenous CRISPR / Cas system genes on its genome, and its NHEJ repair pathway genes.

[0029] The present invention also provides a method for preparing TGase enzyme, and the method is to prepare it by fermentation using the above chassis cells.

[0030] The present invention also provides a method for increasing the enzyme activity of the TGase enzyme fermented by Streptomyces mobaraensis smY2019, and the method is to knock out one or more of the genes of the above genomic island GIs, the endogenous CRISPR / Cas system genes, and the NHEJ repair pathway genes.

[0031] In one embodiment of the present invention, the method is: knocking out the genes of the genomic island GIs on its genome;

[0032] In one embodiment of the present invention, the method is: knocking out the genes of the genomic island GIs on its genome, and the positions from the 1,358,697th to the 1,368,646th on its genome.

[0033] In one embodiment of the present invention, the method is: knocking out the genes of genomic islands GIs on its genome and the genes of the endogenous CRISPR / Cas system on its genome.

[0034] In one embodiment of the present invention, the method is: knocking out the genes of genomic islands GIs on its genome, the genes of the endogenous CRISPR / Cas system on its genome and the genes of its NHEJ repair pathway.

[0035] The present invention also provides a method for expressing a target protein. The method is to use the above chassis cell as a host cell to express the target protein.

[0036] In one embodiment of the present invention, the target protein includes but is not limited to: TGase enzyme.

[0037] The present invention also provides the above application of the chassis cell or the above method in the preparation of TGase enzyme or a product containing TGase enzyme or in the preparation of a target protein.

[0038] In one embodiment of the present invention, the target protein includes but is not limited to: TGase enzyme.

[0039] Beneficial effects

[0040] (1) The present invention uses the CRISPR / cas9 antibiotic-free gene knockout technology to sequentially knock out 5 largest GIs, 2 endogenous CRISPR / Cas systems and 3 related genes of the NHEJ repair pathway in the smY2019 genome, and obtains the chassis strain smY2019-ΔG5-C3 with reduced genome. Compared with smY2019, its TGase enzyme activity is increased by 37.3%, and the TGase enzyme activity is much higher than that of the original strain under the same fermentation conditions.

[0041] (2) Compared with smY2019, the chassis strain smY2019-ΔG5-C3 with reduced genome has the ATP content and NADPH / NADP+ increased by 1.3 and 0.53 times respectively; and the sporulation time on the solid plate is shortened from 6 d to 4 d, and the transformation efficiency of the Cas9 expression plasmid is increased by 4 times. As a chassis cell, all properties are superior to the starting strain, which has important scientific significance and application value for the later optimization and production of this strain. Description of the drawings

[0042] Figure 1 It is the distribution of GIs in the genome of S. mobaraensis DSM40587.

[0043] Figure 2Effect of knocking out GIs on the activity of TGase enzyme. (a) Activity of TGase enzyme in the fermentation supernatant of the knockout strain; (b) SDS-PAGE protein electrophoresis analysis of the fermentation supernatant of the knockout strain.

[0044] Figure 3 Effect of knocking out GIs on the growth and metabolism of the strain. (a) Growth status of the knockout strain; (b) Intracellular energy of the knockout strain; (c) NADPH / NADP+ level of the knockout strain.

[0045] Figure 4 Effect of knocking out the CRISPR / Cas system and NHEJ-related genes on the activity of TGase enzyme. (a) Activity of TGase enzyme in the fermentation supernatant of the knockout strain; (b) SDS-PAGE protein electrophoresis analysis of the fermentation supernatant of the knockout strain

[0046] Figure 5 Effect of knocking out the CRISPR / Cas system and NHEJ-related genes on the growth and metabolism of the strain. (a) Growth status of the knockout strain; (b) Intracellular energy of the knockout strain; (c) NADPH / NADP+ level of the knockout strain.

[0047] Figure 6 Comparison of the growth rate and transformation efficiency of smY2019 and smY2019-ΔG5-C3. (a) Comparison of growth morphology; (b) Comparison of transformation efficiency. Detailed implementation method

[0048] The starting strain involved in the following examples is smY2019, which was deposited at the China Center for Type Culture Collection on September 17, 2020, with the deposit number CCTCC NO: M 2020507, and is recorded in the Chinese invention patent text with the publication number CN112126613A; the cloning host used is E. coli JM109 purchased from Baori Biotechnology Co., Ltd.; the ATP content detection kit is purchased from Sangon Biotech (Shanghai) Co., Ltd.; the NADPH / NADP + detection kit is purchased from Beyotime Biotechnology Co., Ltd.; the One Step Cloning Kit (ClonExpress TM II) is purchased from Nanjing Novozymes Biotech Co., Ltd.; the rest of the plasmids and strains are constructed in this experiment.

[0049] In the following examples, the experimental methods are all conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0050] The media involved in the following examples are as follows:

[0051] GYM Sporulation Medium (g·L -1 ): Glucose 10, yeast extract 4, malt powder 3.

[0052] Seed Medium (g·L -1 ): Glycerol 20, yeast extract 5, tryptone 20, K2HPO4 4, MgSO4 2, pH 7.2.

[0053] Fermentation Medium (g·L -1 ): Glycerol 20, yeast extract 5, tryptone 20, soy flour 20, KH2PO4 2, K2HPO4 4, MgSO4 2, CaCO3 2, pH 7.2 - 7.4.

[0054] ISP4 Medium (g·L -1 ): Soluble starch 10, K2HPO4 1, MgSO4·7H2O 1, NaCl 1, (NH4)2SO4 2, CaCO3 2, FeSO4·7H2O 0.001, MnCl2·4H2O 0.001, ZnSO4·7H2O 0.001.

[0055] The specific construction method of the knockout strains involved in the following examples is achieved through the following steps:

[0056] (1) Screen the protospacer (20bp + NGG, a total of 23bp sequence) of the above - related genes from the target sequence, and form a double - strand through PCR single - strand annealing (system: 5uL FOR + 5uL REV + 90uL H2O; program: heat at 95℃ / 5min, cool to 4℃ at 0.1℃ / sec).

[0057] (2) Use the restriction endonuclease Bbs I to digest the plasmid pCRISPomyces - 2 (purchased from Miaoling Plasmid Platform), then ligate the purified and recovered plasmid with the sgRNA in (1) using Solution I to obtain a recombinant plasmid and cut it with the restriction endonuclease Xba I.

[0058] (3) Using the S. mobaraensis genome as a template, amplify 1kb of the upstream and downstream homologous arms of 5 GIs (GI - I to GI - V), 2 endogenous Cas protein gene clusters (CRISPR1 and CRISPR2), and 3 NHEJ - related genes respectively. Then ligate the purified and recovered plasmid in (2) with the upstream and downstream homologous arm fragments using the Novoprotein One - Step Cloning Kit for multi - fragment ligation to obtain a recombinant plasmid.

[0059] (4) Transform all the above - mentioned recombinant plasmids into E. coli JM109, and perform PCR and sequencing verification.

[0060] (5) Take 70 μL of S. mobaraensis protoplast solution and mix it with 5 - 10 μg of the integrative expression plasmid constructed in (4), transfer it into a 0.1 cm electroporation cuvette, and perform electroporation once under the conditions of 2500 V and 25 Ω; immediately add 1 mL of pre-cooled 1 M sorbitol after electroporation, and finally transfer it to a sterile 1.5 mL EP tube, and culture it in a shaker at 30 °C and 220 rpm for 2 h. Spread the culture solution onto a GYM plate containing 50 μg·mL -1 apramycin, and screen positive transformants by PCR verification and gene sequencing analysis after culturing for 5 - 6 d.

[0061] The detection methods involved in the following examples are as follows:

[0062] Electroporation of S. mobaraensis protoplasts

[0063] Take 70 μL of S. mobaraensis protoplast solution and mix it with 5 - 10 μg of the integrative expression plasmid, transfer it into a 0.1 cm electroporation cuvette (Bio-Rad, USA), and perform electroporation once under the conditions of 2500 V and 25 Ω; immediately add 1 mL of pre-cooled 1 M sorbitol after electroporation, and finally transfer it to a sterile 1.5 mL EP tube, and culture it in a shaker at 30 °C and 220 rpm for 2 h. Spread the culture solution onto a GYM plate containing apramycin, and screen positive transformants by PCR verification and gene sequencing analysis after culturing for 5 - 6 d.

[0064] Determination method of TGase enzyme activity:

[0065] Every 12 h, take 1 mL of the fermentation broth, centrifuge it at 10000 rpm for 10 min in a 4 °C centrifuge, take the supernatant and keep it on ice for use. Add 60 μL of the TGase sample solution to 150 μL of the enzyme activity test substrate and mix well. After reacting at 37 °C in a metal bath for 10 min, immediately add 60 μL of the TGase termination solution. Centrifuge the reaction solution at 10000 rpm for 1 min, take 200 μL of the supernatant and measure the absorbance at a wavelength of 525 nm. The blank control is to first add 60 μL of the enzyme activity test termination solution to 60 μL of the enzyme sample solution, react at 37 °C for 10 min, and then add 150 μL of the enzyme activity test substrate.

[0066] Determination method of mycelial growth amount:

[0067] Every 12 h, take 1 mL of the fermentation samples at different time points and transfer them to a pre-weighed empty 1.5 mL EP tube (M1). Centrifuge the bacterial liquid at 10000 rpm for 10 min to leave the bacterial cells. Open the lid of the EP tube and place it in a 65 °C oven to dry until constant weight, then take it out and weigh (M2). Calculate the weight of the bacterial cells at different time points (M2 - M1), and draw a curve of the dry cell weight.

[0068] Adenosine triphosphate (ATP) assay method:

[0069] Every 12 h, 1 mL of the fermentation samples at different time points was taken and centrifuged at 10,000 rpm for 10 min to leave the bacterial cells. The cells were washed 3 times with PBS buffer and then ultrasonically disrupted for 1 min. Subsequently, they were centrifuged at 10,000 rpm at 4 °C for 10 min, and the supernatant was transferred to another EP tube. 500 μL of chloroform was added, and the mixture was shaken well and then centrifuged at 10,000 rpm at 4 °C for 3 min. The supernatant was taken and placed on ice for measurement. The intracellular ATP assay was performed according to the instructions of the ATP detection kit.

[0070] NADPH / NADP + Assay method: Every 12 h, 1 mL of the fermentation samples at different time points was taken and centrifuged at 10,000 rpm for 10 min to leave the bacterial cells. The cells were washed 3 times with PBS buffer and then an appropriate amount of pre-cooled NADP+ / NADPH extraction solution was added. The cells were gently pipetted to promote cell lysis. Subsequently, they were centrifuged at 10,000 rpm at 4 °C for 10 min, and the supernatant was transferred to ice for measurement. The NADPH / NADP+ assay was performed according to the instructions of the coenzyme II NADP(H) content kit.

[0071] The sgRNAs used for knocking out the related genes involved in the following examples are shown in Table 1:

[0072] Table 1: sgRNAs used for knocking out the related genes

[0073]

[0074]

[0075] The primers used for amplifying the repair template sequences involved in the following examples are shown in Tables 2-1 to 2-2:

[0076] Table 2-1: Primers used for amplifying the repair template sequences

[0077]

[0078] Table 2-2: Primers used for amplifying the repair template sequences

[0079]

[0080]

[0081] The genes of the genomic islands GIs knocked out involved in the following examples are shown in Tables 3-1 to 3-6:

[0082] Table 3-1: Genes of the GIs knocked out

[0083]

[0084]

[0085] Table 3-2: Deleted GIs Genes

[0086]

[0087]

[0088] Table 3-3: Deleted GIs Genes

[0089]

[0090]

[0091] Table 3-4: Deleted GIs Genes

[0092]

[0093]

[0094] Table 3-5: Deleted GIs Genes

[0095]

[0096]

[0097] Table 3-6: Deleted GIs Genes

[0098]

[0099] The deleted endogenous CRISPR / Cas system genes involved in the following examples are shown in Table 4 as follows:

[0100] Table 4: Deleted Endogenous CRISPR / Cas System Genes

[0101]

[0102]

[0103] The deleted genes related to the NHEJ repair pathway involved in the following examples are shown in Table 5 as follows:

[0104] Table 5: Deleted Genes Related to the NHEJ Repair Pathway

[0105]

[0106] Example 1: Deletion of GIs

[0107] (1) Prediction of GIs:

[0108] The software IslandViewer4 was used to predict GIs in the genome of S. mobaraensis DSM40587. From the results, the number, range, starting position of GIs in the genome, and the functions of the genes they contain were obtained. As shown in the appendix Figure 1 As shown, different colors represent the prediction results of different analysis methods. Islandpick did not predict any GIs, while SIGI-HMM and IslandPath-DIMOB predicted 7 and 11 GIs respectively. Therefore, the genome of S. mobaraensis DSM40587 (NCBI accession number: CP083590) may contain 18 GIs with a total length of 267.1 kb.

[0109] (2) Construction and cultivation of knockout strains:

[0110] Based on the CRISPR / Cas9 gene editing system, the 5 largest GIs in the smY2019 genome (GI-I, GI-II, GI-III, GI-IV, and GI-V, as shown in Tables 3-1 to 3-6) were knocked out in sequence. The method for constructing knockout strains is as shown above. The sgRNA sequences required for knockout are shown in Table 1, and the primer sequences for amplifying the repair template sequences required for knockout are shown in Table 2.

[0111] After correct knockout verification, the recombinant strains were obtained respectively:

[0112] smY2019-ΔG1 (knocking out the GI-I gene on the smY2019 genome, the gene in Table 3-1), smY2019-ΔG2 (knocking out the GI-I and GI-II genes on the smY2019 genome, the genes in Tables 3-1 to 3-2), smY2019-ΔG3 (knocking out the GI-I, GI-II, and GI-III genes on the smY2019 genome, the genes in Tables 3-1 to 3-3), smY2019-ΔG4 (knocking out the GI-I, GI-II, GI-III, and GI-IV genes on the smY2019 genome, the genes in Tables 3-1 to 3-4), and smY2019-ΔG5 (knocking out the GI-I, GI-II, GI-III, GI-IV, and GI-V genes on the smY2019 genome, the genes in Tables 3-1 to 3-6).

[0113] After streaking the above-mentioned recombinant bacterial transformants obtained on the GYM plate, they were cultured in a constant temperature incubator at 30 °C for 4-5 d. Subsequently, the mycelium on the GYM solid plate was appropriately scraped with a sterile spreading rod, and the spore suspension was adjusted to a final concentration of 10 6 cells·mL -1Inoculate into the seed medium and culture in a shaker at 30 °C and 220 rpm for 24 - 36 h. Then, transfer to a 250 mL Erlenmeyer flask containing 30 mL of fermentation medium at an inoculum size of 8% (v·v -1 ) and continue to culture in a shaker at 30 °C and 220 rpm for 84 h; Detect the enzyme activity of TGase (glutamine transaminase, EC 2.3.2.13) in the fermentation broth respectively. The NCBI number of TGase is (GenBank: UBI36310.1).

[0114] The results are shown in Table 6 and Figures 2 - 3 as follows:

[0115] Table 6: TGase enzyme activities of recombinant strains obtained by knocking out different genes at different fermentation times

[0116]

[0117] The results show that:

[0118] 1) Effects of GIs knockout on protein synthesis:

[0119] Studies have shown that after deleting parts of the strain genome, the protein synthesis ability is enhanced. Since TGase is the most important extracellular protein of smY2019, its protein synthesis ability of GIs knockout strains was analyzed using TGase as the reporter protein. According to the fermentation results in (2), both smY2019 and GIs knockout strains reached the highest TGase enzyme activity at 60 h of fermentation, and at this time, the TGase enzyme activity increased with the increase in the number of GIs knocked out; among them, the TGase enzyme activity of smY2019 - ΔG5 reached 31.3 U·mL -1 , which was 30.6% higher than that of smY2019 (see a) in the appendix Figure 2 ).

[0120] SDS - PAGE protein electrophoresis analysis was performed on the fermentation supernatants of the knockout strains respectively. SDS - PAGE analysis showed that the TGase protein band (38 kDa) gradually became thicker with the increase in the number of GIs knocked out in the strain (see b) in the appendix Figure 2 ). The results indicate that the knockout of GIs helps to improve the protein secretion and expression of smY2019.

[0121] 2) Effects of GIs knockout on growth and metabolism:

[0122] Generally, it is considered that deleting a large number of redundant genes will help reduce the cell metabolic burden and increase the energy available for the synthesis of target products in the cell. According to the fermentation results in (2), there was almost no difference in the growth among the GIs knockout strains in the first 48 h, and after 48 h, the mycelial growth amounts were all higher than that of smY2019 to varying degrees (see the appendix Figure 3in a). In addition, it was found that with the gradual knockout of GIs, the intracellular ATP content and NADPH / NADP of the strain + were also significantly increasing gradually, and both reached the maximum at 36 h; among them, the ATP content and NADPH / NADP of smY2019-ΔG5 + were 1.1 times and 0.47 times higher than those of smY2019, respectively (see Figure 3 b and c in the appendix). The results showed that the increase in intracellular energy and reducing power levels might promote protein synthesis in the GI-knockout strains.

[0123] Example 2: Knockout of the endogenous CRISPR / Cas system and NHEJ-related genes

[0124] (1) Prediction of the CRISPR / Cas system and the NHEJ repair pathway:

[0125] The CRISPRCasFinder software was used to predict and analyze the endogenous CRISPR / Cas system in the genome of S. mobaraensis DSM40587, and two complete type I-E CRISPR / Cas systems were identified. Their Cas protein gene clusters were 9.92 kb and 5.11 kb, respectively, and they were named CRISPR1 and CRISPR2 (see Table 4). In addition, according to the homologous sequence alignment analysis, it was found that the NHEJ repair pathway consisted of a total of three proteins (one Ku protein and two DNA ligases (LigD)) (see Table 5), with a total of 3 kb.

[0126] (2) Construction and cultivation of the knockout strains:

[0127] Based on the above-constructed CRISPR / Cas9 gene editing system, two Cas protein gene clusters and the genes related to the NHEJ repair pathway were successively knocked out on the basis of smY2019-ΔG5. The construction methods of the relevant knockout strains were as shown above. The sgRNA sequences required for knockout are shown in Table 1, and the primer sequences for amplifying the repair template sequences required for knockout are shown in Table 2.

[0128] After verifying the correct knockout, the recombinant strains were obtained respectively:

[0129] smY2019-ΔG5-C1 (CRISPR1 on the genome of smY2019-ΔG5 was knocked out, as shown in Table 4), smY2019-ΔG5-C2 (CRISPR1 and CRISPR2 on the genome of smY2019-ΔG5 were knocked out, as shown in Table 4), and smY2019-ΔG5-C3 (CRISPR1, CRISPR2, and three genes encoding the NHEJ repair pathway on the genome of smY2019-ΔG5 were knocked out, as shown in Tables 4 - 5). A total of 167.6 kb of genomic fragments were deleted in smY2019-ΔG5-C3, reducing the genomic size of the strain by 2.2%.

[0130] After streaking the above recombinant bacterial transformants obtained on the GYM plate on the plate, they were cultured in a constant temperature incubator at 30 °C for 4 - 5 d. Subsequently, the mycelium on the GYM solid plate was appropriately scraped with a sterile spreading rod, and the spore suspension was inoculated into the seed medium at a final concentration of 10 6 cells·mL -1 . After culturing in a shaker at 30 °C and 220 rpm for 24 - 36 h, it was transferred to a 250 mL Erlenmeyer flask containing 30 mL of fermentation medium at an inoculation amount of 8% (v·v -1 ), and continued to be cultured in a shaker at 30 °C and 220 rpm for 84 h; the TGase enzyme activity in the fermentation broth was detected respectively.

[0131] The results are shown in Table 7 and Figures 4 - 5 as follows:

[0132] Table 7: TGase enzyme activity of recombinant strains obtained by knocking out different genes at different fermentation times

[0133]

[0134]

[0135] The results showed that:

[0136] 1) Effects of knocking out the CRISPR / Cas system and the NHEJ repair pathway on protein synthesis:

[0137] According to the analysis and comparison of the fermentation results in (2), when the CRISPR / Cas system and the NHEJ repair pathway were further knocked out, the change in TGase enzyme activity was small. Among them, after knocking out CRISPR1, the TGase enzyme activity in smY2019-ΔG5-C1 reached the highest of 32.9 U·mL -1 at 60 h, which was increased by 5.1% and 37.3% compared with smY2019-ΔG5 and smY2019 respectively. However, when the relevant genes of CRISPR2 and the NHEJ repair pathway were continuously knocked out, due to the relatively small deleted fragments, the TGase enzyme activity no longer increased further (see attachedFigure 4 in a). SDS-PAGE analysis also found that as the number of genes knocked out in the strain increased, the thickness of the TGase protein band (38 kDa) remained almost unchanged (see Appendix Figure 4 in b).

[0138] 2) Effects of knocking out the CRISPR / Cas system and the NHEJ repair pathway on growth and metabolism:

[0139] According to the fermentation result analysis in (2), it was found that the mycelial growth, ATP content, and NADPH / NADP of smY2019-ΔG5-C1, smY2019-ΔG5-C2, and smY2019-ΔG5-C3 + did not change much compared with smY2019-ΔG5 (see Appendix Figure 5 in a).

[0140] However, compared with smY2019, the ATP content and NADPH / NADP of smY2019-ΔG5-C3 + increased by 1.3 times and 0.53 times respectively (see Appendix Figure 5 in b and c), and the results showed that the deletion of redundant genes saved the energy consumption of the cells.

[0141] Example 3: Analysis of the growth rate and transformation efficiency of the chassis strain

[0142] Research has shown that the streamlined chassis cells not only grow faster but also have a more efficient transformation efficiency. The growth rate and transformation efficiency of the chassis strain smY2019-ΔG5-C3 obtained by modifying smY2019 were analyzed.

[0143] (1) Growth rate analysis:

[0144] smY2019 and smY2019-ΔG5-C3 were simultaneously inoculated onto the GYM sporulation medium and cultured in a constant temperature incubator at 30 °C. It was found that compared with smY2019, smY2019-ΔG5-C3 grew faster, the aerial mycelium was more developed and lush, and the color was darker (see Appendix Figure 6 in a). And the sporulation time of the solid plate of smY2019-ΔG5-C3 was shortened from 6 d to 4 d.

[0145] (2) Transformation efficiency analysis:

[0146] The plasmid pCRISPomyces-2 (purchased from the Miaoling plasmid platform) was simultaneously transformed into smY2019 and smY2019-ΔG5-C3. It was found that under the same transformation conditions (protoplast transformation), the number of transformants of the latter increased significantly, by more than 4 times (see Appendix Figure 6 in b).

[0147] It is speculated that the loss of large DNA fragments improves the performance of the strain and eliminates the interference of the endogenous CRISPR / Cas system, resulting in a significant increase in the transformation efficiency of the strain. On the other hand, the elimination of the interference of endogenous NHEJ repair can promote the editing efficiency of the repair template, is expected to improve the gene editing efficiency in the strain, and thus better perform genetic operations in the strain.

[0148] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A Streptomyces mobaraensis chassis cell, characterized in that, The chassis cell is obtained by knocking out one or more of the genomic island GIs genes, endogenous CRISPR / Cas system genes, and NHEJ repair pathway genes on the genome of Streptomyces mobaraensis smY2019 as the starting strain.

2. The chassis cell according to claim 1, characterized in that, The NCBI numbers of the genes in the genomic island GIs are K7I03_28245, K7I03_28250, K7I03_28255, K7I03_28260, K7I03_28265, K7I03_28270, K7I03_28275, K7I03_28280, K7I03_28285, K7I03_28290, K7I03_28295, K7I03_28300, K7I03_28305, K7I03_28310, K7I03_28315, K7I03_28320, K7I03_28325, K7I03_28330, K7I03_28335, K7I03_28340, K7I03_28345, K7I03_28350, K7I03_28355, K7I03_28360, K7I03_28365, K7I03_28370, K7I03_28375, K7I03_28380, K7I03_28385, K7I03_28390, K7I03_28395, K7I03_28400, K7I03_28405, K7I03_28410, K7I03_28415, K7I03_28420, K7I03_28425, K7I03_28430, K7I03_28435, K7I03_28835, K7I03_28840, K7I03_28845, K7I03_28850, K7I03_28855, K7I03_28860, K7I03_28865, K7I03_28870, K7I03_28875, K7I03_28880, K7I03_28885, K7I03_28890, K7I03_28895, K7I03_28900, K7I03_28905, K7I03_28910, K7I03_28915, K7I03_28920, K7I03_28925, K7I03_28930, K7I03_28935, K7I03_28940, K7I03_28945, K7I03_28950, K7I03_28955, K7I03_28960, K7I03_28965, K7I03_28970, K7I03_28975, K7I03_28980, K7I03_28985, K7I03_28990, K7I03_28995, K7I03_29000, K7I03_29005, K7I03_18425, K7I03_18430, K7I03_18435, K7I03_18440, K7I03_18445, K7I03_18450, K7I03_18455,K7I03_18460, K7I03_18465, K7I03_18470, K7I03_18475, K7I03_18480, K7I03_18485, K7I03_18490, K7I03_18495, K7I03_18500, K7I03_18505, K7I03_18510, K7I03_18515, K7I03_18520, K7I03_18525, K7I03_18530, K7I03_18535, K7I03_18540, K7I03_18545, K7I03_18550, K7I03_18555, K7I03_18560, K7I03_18565, K7I03_18570, K7I03_18575, K7I03_18580, K7I03_18585, K7I03_18590, K7I03_18595, K7I03_18600, K7I03_18605, K7I03_18610, K7I03_03350, K7I03_03355, K7I03_03360, K7I03_03365, K7I03_03370, K7I03_03375, K7I03_03380, K7I03_03385, K7I03_03390, K7I03_03395, K7I03_03400, K7I03_03405, K7I03_03410, K7I03_03415, K7I03_03420, K7I03_03425, K7I03_03430, K7I03_03435, K7I03_03440, K7I03_03445, K7I03_03450, K7I03_03455, K7I03_03460, K7I03_03465, K7I03_01620, K7I03_01625, K7I03_01630, K7I03_01635, K7I03_01640, K7I03_01645, K7I03_01650, K7I03_01655, K7I03_01660, K7I03_01665, K7I03_01670, K7I03_01675, K7I03_01680, K7I03_01685, K7I03_01690, K7I03_01695, K7I03_01700, K7I03_01705, K7I03_01710, K7I03_01715, K7I03_01720, K7I03_01725K7I03_01730; The endogenous CRISPR / Cas system genes are CRISPR1 gene and CRISPR2 gene. The CRISPR1 gene is located at positions 1358697 to 1368646 on the genome of Streptomyces mobaraensis smY2019, and the CRISPR2 gene is located at positions 1437389 to 1442520 on the genome of Streptomyces mobaraensis smY2019; The NCBI numbers of the NHEJ repair pathway genes are K7I03_29600, K7I03_29605, and K7I03_29610 respectively.

3. The chassis cell according to claim 1 or 2, characterized in that, The chassis cell is obtained by knocking out the genomic island GIs genes on the genome of Streptomyces mobaraensis smY2019 as the starting strain; or the chassis cell is obtained by knocking out the genomic island GIs genes on the genome of Streptomyces mobaraensis smY2019 and the CRISPR1 gene on its genome; or the chassis cell is obtained by knocking out the genomic island GIs genes on the genome of Streptomyces mobaraensis smY2019 and the endogenous CRISPR / Cas system genes on its genome; or the chassis cell is obtained by knocking out the genomic island GIs genes, endogenous CRISPR / Cas system genes on its genome, and NHEJ repair pathway genes on the genome of Streptomyces mobaraensis smY2019 as the starting strain.

4. A method for improving the growth rate and / or transformation efficiency of Streptomyces mobaraensis smY2019, characterized in that, The method is to knock out one or more of the genomic island GIs genes, endogenous CRISPR / Cas system genes, and NHEJ repair pathway genes recited in claim 2.

5. The method according to claim 4, characterized in that, The method is: knocking out the genomic island GIs genes on its genome; or knocking out the genomic island GIs genes on its genome and the CRISPR1 gene on its genome; or knocking out the genomic island GIs genes on its genome and the endogenous CRISPR / Cas system genes on its genome; or knocking out the genomic island GIs genes, endogenous CRISPR / Cas system genes on its genome, and NHEJ repair pathway genes on its genome.

6. A method for preparing TGase enzyme, characterized in that, The method is prepared by fermentation using the chassis cell recited in any one of claims 1 to 3.

7. A method for improving the enzyme activity of TGase enzyme fermented by Streptomyces mobaraensis smY2019, characterized in that, The method is to knock out one or more of the genomic island GIs genes, endogenous CRISPR / Cas system genes, and NHEJ repair pathway genes recited in claim 2.

8. The method according to claim 7, wherein The method is: knocking out the genomic island GIs genes on its genome; or knocking out the genomic island GIs genes on its genome and the CRISPR1 gene on its genome; or knocking out the genomic island GIs genes on its genome and the endogenous CRISPR / Cas system genes on its genome; or knocking out the genomic island GIs genes, endogenous CRISPR / Cas system genes on its genome, and NHEJ repair pathway genes on its genome.

9. A method for expressing a target protein, characterized in that, The method is to express the target protein using the chassis cell recited in any one of claims 1 to 3 as the host cell.

10. Use of the chassis cell recited in any one of claims 1 to 3, or the method recited in any one of claims 4 to 9 in the preparation of TGase enzyme or a product containing TGase enzyme or in the preparation of a target protein.

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