Application of Splitting Genes in Regulating Threonine Synthesis, Cell Morphology, and Growth

Abstract

The present invention belongs to the fields of genetic engineering and microbial technology, and specifically relates to the use of split genes in regulating threonine synthesis, cell morphology, and growth. Experiments have demonstrated that regulating the expression intensity of split genes through biological elements can affect threonine synthesis, cell morphology, and growth of target microorganisms. For example, when ftsZ expression is too strong, it has little effect on microbial cell growth and threonine production, and may even be detrimental to threonine production. However, when ftsZ expression is relatively weak, it is beneficial to the accumulation of cell biomass and promotes threonine synthesis. Therefore, by regulating the expression intensity of the split genes, dynamic regulation of threonine production by bacterial strain fermentation is achieved, which is not only beneficial for actual production but also can be used as a model strain for research on the mechanism of threonine synthesis, cell morphology, and growth, thus having excellent practical application value.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and microbial technology, and particularly relates to the application of split genes in regulating threonine synthesis and cell morphology and growth. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Threonine is an essential amino acid that forms proteins in humans and animals and is widely used in a variety of fields, including biopharmaceuticals, chemical reagents, food, and feed. Major threonine producers worldwide include Ajinomoto Co., Ltd. of Japan, Degussa AG of Germany, Archer Daniels Midland Co., Ltd. of the United States, and Kyowa Hakko Kogyo Co., Ltd. of Japan. These companies account for approximately 90% of global production. However, domestic threonine production is still in its developmental stages, with production levels lagging behind those of international markets. Currently, threonine production primarily involves fermentation, protein hydrolysis, and chemical synthesis. Microbial fermentation has become the mainstream method for threonine production. Improving fermentation efficiency and reducing costs are pressing technical challenges within this method.

[0004] The cell division process is one of the key events in individual development. Through cell division, the genetic material of the parent is replicated, doubled, and evenly distributed to the daughter cells, thus ensuring the stability of the genetic material and the continuation of the species. According to previous research results, it has been found that before prokaryotes such as bacteria undergo division, certain fission proteins (such as FtsZ, FtsA, zipA, etc.) must first assemble at the division site to form a ring-shaped fission complex, thereby promoting the formation of the fission septum, and then triggering the occurrence of cell division events. Given the important role of these isolated proteins in bacterial cell separation, new antibacterial drugs are currently usually developed using these isolated proteins or their coding genes as targets. However, to date, there have been no reports on the role of these cell division genes in regulating threonine synthesis in microorganisms. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention provides the use of cell division genes in regulating threonine synthesis and cell morphology and growth. Through experimental research, the present invention has found that varying the expression intensity of cell division genes not only affects cell morphology and growth but also has varying effects on the strain's threonine production performance, thus possessing promising practical application value.

[0006] In order to achieve the above technical objectives, the technical solutions adopted by the present invention are as follows:

[0007] The first aspect of the present invention provides the use of split gene-related biological materials in regulating microbial threonine synthesis and / or microbial cell morphology and growth.

[0008] The split gene related biological materials are as follows:

[0009] a) any one or more of ftsZ, ftsB, ftsL, ftsQ, ftsA, ftsK, ftsN, and zipA;

[0010] b) a biological element that regulates the expression intensity of any one or more of ftsZ, ftsB, ftsL, ftsQ, ftsA, ftsK, ftsN and zipA.

[0011] The biological elements may include promoters of different strengths and ribosome binding sites (RBS), wherein the promoters include but are not limited to J23103, J23113, J23109, J23116, J23110, J23100 and PrhtC, and the RBSs include but are not limited to BBa_B0033 and BBa_B0034.

[0012] The microorganism is specifically a prokaryotic organism, specifically a bacterium. The bacterium is any threonine-producing bacterium. In a specific embodiment of the present invention, the bacterium is Escherichia coli and its derivatives.

[0013] The second aspect of the present invention provides a strain comprising at least the aforementioned split gene-related biological material;

[0014] The split gene related biological materials are as follows:

[0015] a1) any one or more of ftsZ, ftsB, ftsL, ftsQ, ftsA, ftsK, ftsN, and zipA;

[0016] a2) A biological element that regulates the expression intensity of any one or more of ftsZ, ftsB, ftsL, ftsQ, ftsA, ftsK, ftsN and zipA.

[0017] By regulating the expression intensity of the above-mentioned split genes, dynamic regulation of the strain's fermentation production of threonine can be achieved, which is not only beneficial to actual production, but also can be used as a model strain for related research on the threonine synthesis mechanism, cell morphology and growth mechanism.

[0018] Therefore, the third aspect of the present invention provides the use of the above strain in any one or more of the following:

[0019] b1) fermentative production of threonine;

[0020] b2) Study on the mechanism of threonine synthesis;

[0021] b3) Study on cell morphology and growth mechanism.

[0022] A fourth aspect of the present invention provides a method for dynamically regulating the fermentation production of threonine, the method comprising fermenting and culturing the above-mentioned strain, and separating and extracting threonine.

[0023] It should be noted that the threonine mentioned in the present invention is specifically L-threonine.

[0024] Beneficial technical effects of one or more of the above technical solutions:

[0025] The above technical solution provides the application of split genes in regulating threonine synthesis, cell morphology, and growth. Specifically, experiments have demonstrated that regulating the expression intensity of the split genes through biological elements can affect threonine synthesis, cell morphology, and growth of target microorganisms. For example, when ftsZ expression is too strong, it has little effect on microbial cell growth and threonine production, and may even be detrimental to threonine production. However, when ftsZ expression is weak, it promotes cell biomass accumulation and promotes threonine synthesis. Therefore, by regulating the expression intensity of the split genes, dynamic regulation of threonine fermentation production by the strain is achieved, which not only facilitates actual production but also allows the strain to be used as a model strain for research on the mechanisms of threonine synthesis, cell morphology, and growth, thus possessing excellent practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0027] Figure 1 This is the promoter strength characterization in the examples of the present invention.

[0028] Figure 2 1 shows the effect of ftsZ expression at different intensities on cell morphology in the examples of the present invention.

[0029] Figure 3 Figure 3 shows the effect of regulating ftsZ expression on threonine synthesis and cell growth in the examples of the present invention; A shows the quantitative response analysis of PrhtC by adding different concentrations of threonine to the model strain Escherichia coli MG1655 in vitro; B shows the characterization of PrhtC by shake flask fermentation in the production strain TH-103Z; C shows the effect of different intensities of ftsZ on the cell growth of the strain; D shows the effect of different intensities of ftsZ on the threonine synthesis of the strain.

[0030] Figure 4This is a graph showing the results of sampling and measuring the transcriptome in an embodiment of the present invention.

[0031] Figure 5 Graph showing transcriptome analysis results in an embodiment of the present invention.

[0032] Figure 6 This is an example of controlling the effects of other division gene expression on threonine and cell growth in the present invention.

[0033] Figure 7 This is a diagram showing the results of threonine production by fermentation in a 5-L fermenter in an example of the present invention. DETAILED DESCRIPTION

[0034] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0035] It should be noted that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0036] In a typical embodiment of the present invention, there is provided the use of split gene-related biomaterials in regulating microbial threonine synthesis and / or microbial cell morphology and growth.

[0037] The split gene related biological materials are as follows:

[0038] a) any one or more of ftsZ, ftsB, ftsL, ftsQ, ftsA, ftsK, ftsN, and zipA;

[0039] b) a biological element that regulates the expression intensity of any one or more of ftsZ, ftsB, ftsL, ftsQ, ftsA, ftsK, ftsN and zipA.

[0040] The biological element may include promoters of different strengths and ribosome binding sites (RBS), wherein the strength of the promoter (expressed as relative fluorescence intensity RFP / OD 600The present invention has found that by regulating the expression intensity of the above-mentioned split genes by the above-mentioned biological elements, the threonine synthesis of the target microorganism and its cell morphology and growth will be affected. The present invention has found that when ftsZ expression is too strong, it has little effect on the growth of microbial cells (such as the intensity of the exogenously introduced promoter is not less than 600, specifically J23116, J23110, J23100), and has little effect on threonine production, and is even not conducive to threonine production. When ftsZ expression is weak, it is beneficial to the accumulation of cell biomass and promotes threonine synthesis. The present invention has confirmed through research that Escherichia coli exogenously introduced with the promoter J23103 (promoter strength 33) exhibits the strongest threonine synthesis capacity and the highest cell biomass. Furthermore, the aforementioned fission genes of varying strengths also lead to changes in microbial cell morphology. For example, when J23103, J23113, J23109, PrhtC, and J23116 control the expression of ftsZ, cell length increases. Furthermore, when J23116 controls the expression of ftsZ, cells exhibit filamentous growth, while when J23100 controls the expression of ftsZ, cells exhibit elliptical growth. Furthermore, when PrhtC controls the expression of the aforementioned fission genes, threonine production increases to varying degrees, particularly when the ftsZ, ftsB, ftsL, ftsQ, and zipA genes are expressed, resulting in varying degrees of increase in cell biomass.

[0041] In a specific embodiment of the present invention, the biological element may be terminator-promoter-RBS, wherein the terminator may be BBa_B1006.

[0042] The microorganism is specifically a prokaryotic organism, specifically a bacterium. The bacterium is any threonine-producing bacterium. In a specific embodiment of the present invention, the bacterium is Escherichia coli and its derivatives.

[0043] In one embodiment of the present invention, the Escherichia coli derivative can be an Escherichia coli strain obtained by genetically engineering wild-type Escherichia coli, such as Escherichia coli TH-103Z, which has the ability to produce threonine and is deposited with the China Center for Type Culture Collection (address: Wuhan University, Luojia Mountain, Wuchang, Wuhan, Hubei Province) on December 5, 2022, with a biological deposit number of CCTCC NO: M 20221861.

[0044] In one embodiment of the present invention, a strain is provided, the strain comprising at least the aforementioned split gene-related biological material;

[0045] The split gene related biological materials are as follows:

[0046] a1) any one or more of ftsZ, ftsB, ftsL, ftsQ, ftsA, ftsK, ftsN, and zipA;

[0047] a2) A biological element that regulates the expression intensity of any one or more of ftsZ, ftsB, ftsL, ftsQ, ftsA, ftsK, ftsN and zipA.

[0048] The biological element may include promoters of different strengths and ribosome binding sites (RBS), wherein the strength of the promoter (expressed as relative fluorescence intensity RFP / OD 600 promoters include but are not limited to J23103, J23113, J23109, J23116, J23110, J23100 and PrhtC, and the RBSs include but are not limited to BBa_B0033 and BBa_B0034.

[0049] The present invention has discovered that different expression intensities of ftsZ have significantly different effects on threonine synthesis and cell growth. As mentioned above, when ftsZ expression is too strong, it has a smaller impact on microbial cell growth (e.g., when the strength of the exogenously introduced promoter is not less than 600, specifically J23116, J23110, or J23100), and has a smaller impact on threonine production, or even hinders threonine production. When ftsZ expression is relatively weak, it is beneficial for the accumulation of cell biomass and promotes threonine synthesis. The present invention has confirmed that Escherichia coli with the exogenously introduced promoter J23103 (promoter strength of 33) has the strongest threonine synthesis capacity and the highest cell biomass. Furthermore, these different levels of fission genes also led to changes in microbial cell morphology. For example, when J23103, J23113, J23109, PrhtC, and J23116 controlled the expression of ftsZ, cell length increased. When J23116 controlled ftsZ expression, cells even exhibited filamentous growth, while when J23100 controlled ftsZ expression, cells exhibited elliptical growth. Furthermore, when PrhtC controlled the expression of these fission genes, threonine production increased to varying degrees, particularly for the ftsZ, ftsB, ftsL, ftsQ, and zipA genes, which also resulted in varying increases in cell biomass.

[0050] In a specific embodiment of the present invention, the biological element may be terminator-promoter-RBS, wherein the terminator may be BBa_B1006.

[0051] The strain can be Escherichia coli and its derivatives. In a specific embodiment of the present invention, the strain can be obtained by introducing the above-mentioned split gene-related biological material into the starting strain, wherein the starting strain is Escherichia coli TH-103Z, which has the ability to produce threonine. The strain is deposited in the China Center for Type Culture Collection (address: Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province), the preservation date is December 5, 2022, and its biological preservation number is CCTCC NO: M 20221861.

[0052] By regulating the expression intensity of the above-mentioned split genes, dynamic regulation of the strain's fermentation production of threonine can be achieved (the strain's performance in producing threonine can be enhanced or weakened). This is not only beneficial to actual production, but also can be used as a model strain for related research on the threonine synthesis mechanism, cell morphology, and growth mechanism.

[0053] Therefore, in one embodiment of the present invention, the use of the above strain in any one or more of the following is provided:

[0054] b1) fermentative production of threonine;

[0055] b2) Study on the mechanism of threonine synthesis;

[0056] b3) Study on cell morphology and growth mechanism.

[0057] The present invention has been found through research.

[0058] In a specific embodiment of the present invention, a method for dynamically regulating the fermentation production of threonine is provided, the method comprising fermenting and culturing the above-mentioned strain, and separating and extracting threonine.

[0059] It should be noted that the threonine mentioned in the present invention is specifically L-threonine.

[0060] The present invention is further explained by the following examples, but is not intended to limit the present invention. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0061] Example

[0062] 1. Experimental Materials

[0063] 1.1 Strains, plasmids, and primers

[0064] The strains, plasmids, and primers used in this example are listed in Table 1 and Table 2.

[0065] Table 1. Strains and plasmids used in this example

[0066]

[0067]

[0068] Table 2. Primer sequences used in this example

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075] 1.2 Culture medium

[0076] The culture media used in this example are as follows: LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl. Shake flask fermentation medium: 15 g / L (NH₄)₂SO₄, 2 g / L KH₂PO₄, 1 g / L MgSO₄·7H₂O, 2 g / L yeast extract, 0.02 g / L FeSO₄, 40 g / L glucose, 20 g / L CaCO₃ for pH adjustment. Tank culture medium: 20 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, 5 / 10 / 15 g / L corn steep liquor, 2 g / L magnesium sulfate heptahydrate, 5 mg / L ferrous sulfate heptahydrate, 5 mg / L manganese sulfate tetrahydrate, 20 g / L glucose, 0.5 g / L glycine, 0.5 g / L betaine, and 200 μg / L biotin.

[0077] 1.3 Commonly used enzymes in molecular biology

[0078] The high-fidelity DNA polymerase Phanta Max Super-Fidelity DNA Polymerase used in this example for gene cloning was purchased from Novozymes Inc. The 2×Es Taq MasterMix (Dye) premixed enzyme used for colony PCR verification was purchased from Kangwei Century Co., Ltd. (Beijing).

[0079] 1.4 Commonly used test kits

[0080] Agarose gel recovery kit, plasmid mini-extraction kit, plasmid mini-extraction midi kit, and genome extraction kit were all purchased from Tiangen Biochemical Technology (Beijing); the DNA fragment rapid purification and recovery kit Cycle-Pure was purchased from OMEGA.

[0081] 2. Experimental Methods

[0082] 2.1 Construction of plasmids and engineered strains

[0083] The plasmids, strains and primers used in this example are listed in Table 1 and Table 2. To characterize promoter strength and threonine response analysis, this example used the low-copy plasmid pCL1920 as the recombinant plasmid backbone. The rhtC gene promoter was amplified from the Escherichia coli MG1655 genome. The J23103, J23113, J23109, J23116, J23110, and J23100 promoters, as well as the ribosome binding sites B0033 and B0034, were derived from iGEM synthetic biology components (http: / / parts.igem.org / Main_Page). The pCL1920 plasmid backbone was combined with the promoter, RBS, and reporter gene rfp to construct the following plasmids: PrhtC-RFP, J23103-RFP, J23113-RFP, J23109-RFP, J23116-RFP, J23110-RFP, and J23100-RFP.

[0084] PCR amplify 300-500 bp flanking the target genes ftsZ, ftsB, ftsL, ftsQ, ftsA, ftsK, ftsN, and zipA to obtain upstream and downstream homology arms. Fragments containing FRT sites and a chloramphenicol selection marker at both ends were PCR amplified from the pKD3 plasmid. The upstream and downstream homology arms were fused to the chloramphenicol selection fragment to form a genomic integration fragment. Purification was performed using gel extraction kits to obtain a water-soluble genomic integration fragment. The pTKRed plasmid carrying the homologous recombinase was transformed into the TH-103Z strain. After induction of homologous recombinase expression with ITPG at 30°C, the cells were harvested, resuspended in 10% glycerol by repeated centrifugation 3-5 times, and finally resuspended in 100 μL of 10% glycerol to obtain electrocompetent cells. After electroporation at 2.5 kV and recovery incubation at 37°C for 1-3 hours, the electroporated solution was plated onto LB plates with chloramphenicol and cultured overnight at 37°C. Single colonies were then picked for PCR verification of genomic integration. After successful integration, the thermosensitive pTKRed plasmid was removed by culturing at 42°C. Finally, the chloramphenicol selection marker with the FRT site was removed using the thermosensitive pCP20 plasmid to obtain the recombinant engineered strain.

[0085] 2.2 Scanning electron microscopy observation of cell morphology

[0086] Sample pretreatment: Take a large amount of bacterial liquid and centrifuge at 1000rpm-4000rpm, remove the supernatant, add 1× PBS with an appropriate pH of 6.8-7.4 to wash, and remove the supernatant.

[0087] Fixation: Add 2.5% glutaraldehyde prepared in 1×PBS and fix at 4°C for 3 hours, remove the supernatant, wash three times with 1×PBS, and remove the supernatant.

[0088] Dehydration: Dehydrate the sample with ethanol aqueous solution according to the concentration gradient of 30%, 50%, 70%, 80%, and 90%. After discarding the supernatant, dehydrate in 100% ethanol twice.

[0089] Drying: Use a critical point dryer to dry. Pipette the bacterial solution and drop it on the cover slip. Place the cover slip containing the sample in the critical point dryer to dry.

[0090] Gold spraying: After the sample is fully dried, adhere the cover glass to the sample stage with conductive tape and coat it with gold.

[0091] Observe under electron microscope.

[0092] 2.3 Characterization of threonine sensors and promoters

[0093] In this example, the threonine-responsive promoters PrhtC and J23103, J23113, J23109, J23116, J23110, and J23100 were characterized by analyzing the expression levels of reporter genes. Plasmids PrhtC-RFP, J23103-RFP, J23113-RFP, J23109-RFP, J23116-RFP, J23110-RFP, and J23100-RFP were transformed into Escherichia coli MG1655, cultured at 37°C, and single colonies were picked and cultured in a 12-well plate containing 2 mL of LB medium for ~12 h. The colonies were then transferred to a 24-well plate containing 1 mL of LB medium at 2% (v / v) and cultured at 37°C for 24 h. The OD values ​​of the bacteria were measured using a microplate reader. 600 The fluorescence intensity of the reporter gene RFP was measured at an excitation wavelength of 590 nm and an emission wavelength of 645 nm. At the same time, PrhtC-RFP and J23109-RFP were transferred into the production strain TH-103Z, and shake flask fermentation was performed with 20 mL of fermentation medium. The cell density was measured by UV spectrophotometer, the fluorescence intensity of the reporter gene RFP was measured by microplate reader, and the fluorescence intensity of the reporter gene RFP was measured by RFP / OD. 600 To calculate the promoter strength.

[0094] 2.4 Transcriptome Sequencing

[0095] In this example, the transcriptomes of the starting strain TH-103Z and the engineered strain J23103-ftsZ were measured. The mid-exponential growth phase of the bacteria was collected by shake flask fermentation and sequenced by Qingdao Ouyi Biotechnology Co., Ltd. with a sequencing capacity of 2G / sample.

[0096] 2.5 Threonine fermentation

[0097] Shake flask fermentation: Pick a single colony in a 12-well plate containing 2 mL of LB, culture at 37°C for 12 h, inoculate 1% (v / v) volume into a 300 mL shake flask containing 20 mL of shake flask fermentation medium, and culture at 37°C, 220 rpm for 36 h.

[0098] Fermentation in tanks: (1) Primary seed culture: Pick a single colony and place it in a 300 mL shake flask containing 50 mL LB and incubate at 37°C for ~12 h. (2) Secondary seed culture: Transfer the primary seed at 10% (v / v) volume to a 2 L shake flask containing 400 mL tank culture medium and incubate at 37°C, 220 rpm until the exponential phase. (3) Tank culture: Inoculate the secondary seed at 10% (v / v) volume into a 5 L tank, adjust the pH to 7.0 with ammonia water, control the dissolved oxygen at 20-30%, and incubate at 37°C.

[0099] 2.7 Threonine detection method

[0100] Preparation of derivatization reagents: (1) Reagent 1: Mix 1.4 mL of triethylamine with 8.6 mL of acetonitrile; (2) Reagent 2: Mix 25 μL of phenyl isothiocyanate with 2 mL of acetonitrile.

[0101] Sample Preparation: Take the fermentation broth supernatant and dilute it 20-fold with deionized water. Add 100 μL each of Reagent 1 and Reagent 2 to 200 μL of the diluted sample and mix thoroughly. Let it stand at room temperature for 1 hour. Then, add 400 μL of n-hexane, vortex and mix thoroughly, and let it stand for 10 minutes. Take 200 μL of the lower layer liquid and add 800 μL of deionized water. After mixing thoroughly, filter through a 0.22 μm organic filter membrane to obtain the test sample.

[0102] Threonine Detection: This example uses high-performance liquid chromatography to measure threonine production in the fermentation broth. Chromatographic column: Venusil AA (4.6×250 mm, 5 μm, Agela Technologies), column oven: 40°C, mobile phase A: 15.2 g of anhydrous sodium acetate dissolved in 1850 mL of double-distilled water, then added with 140 mL of acetonitrile and mixed thoroughly, mobile phase B: 80% (v / v) acetonitrile, flow rate: 1 mL, detection program: 0-2 min 0% mobile phase B, 2-14 min 7% mobile phase B, 14-29 min 30% mobile phase B, 29-32 min 50% mobile phase B, 32-33 min 100% mobile phase B, 33-39 min 100% mobile phase B, 39.1-45 min 0% mobile phase B.

[0103] 3. Experimental Results and Discussion

[0104] 3.1 Analysis of the effects of ftsZ expression at different intensities on cell morphology

[0105] We first analyzed the effects of ftsZ expression at different intensities on cell morphology. We selected six promoters with different intensities (J23103, J23113, J23109, J23116, J23110, and J23100) and two ribosome binding sites (RBS) with different intensities (B0033 and B0034) from the iGEM database. PrhtC is the promoter of the threonine transporter RhtC. The intensity characterization results are shown in Figure 2. Figure 1 As shown, the intensities of J23103, J23113, J23109, PrhtC, J23116, J23110, and J23100 increase in sequence, which are 33, 36, 55, 260, 670, 1000, and 2000, respectively.

[0106] We used the different strength promoters characterized above to control the expression of ftsZ in the genome. The original promoter of ftsZ in the genome of threonine-producing strain TH-103Z is located within its upstream gene ftsA. In order not to affect the normal expression of ftsA, the original promoter of ftsZ cannot be replaced. Therefore, we inserted a terminator-promoter-RBS sequence before the start codon of the ftsZ gene, namely BBa_B1006-promoter-B0033 / B0034. After culturing at 37°C for ~12 hours, the bacteria were harvested, fixed, dried, and sprayed with gold, and the cell morphology was observed by scanning electron microscopy. The results are shown in Figure 2. Figure 2 As shown, when J23103, J23113, J23109, PrhtC, and J23116 controlled the expression of ftsZ, the cell length increased, and even when J23116 controlled the expression of ftsZ, the cells grew in a filamentous shape, and when J23100 controlled the expression of ftsZ, the cells grew in an elliptical shape.

[0107] 3.2 Analysis of the effects of different expression intensities of ftsZ on threonine synthesis and cell growth

[0108] To analyze the effects of regulating cell division on threonine synthesis, we used two ftsZ regulation methods. The first method used threonine response activation to dynamically control ftsZ expression by autoinduction. Since the transporter RhtC specifically transports intracellular threonine to the extracellular space, we speculated that the promoter of the rhtC gene (PrhtC) responds to threonine to activate rhtC expression. To verify the response of PrhtC to threonine, we connected an RBS (B0034) and a reporter gene (RFP) after PrhtC, namely PrhtC-B0034-RFP, for characterization. The results are shown in the figure. Figure 3 As shown in A and B, different concentrations of threonine were added to the model strain Escherichia coli MG1655 in vitro for quantitative response analysis. The results showed that the response threshold of PrhtC ranged from 0 to 30 g / L threonine, with a dynamic range of 1.8 times. To verify the applicability of the PrhtC sensor in threonine-producing bacteria, we performed shake flask fermentation characterization in the producing bacteria TH-103Z ( Figure 3 B) Using J23109 constitutively expressing RFP as a control, we found that the RFP intensity controlled by the PrhtC sensor increased with fermentation time and threonine accumulation, indicating that PrhtC is a threonine-responsive sensor. We used PrhtC to control the dynamic expression of ftsZ in the genome. Figure 3 As shown in C and D, threonine production and biomass increased by 62% and 54%, respectively. The second cell division regulation method used constitutive promoters of different strengths (J23103, J23113, J23109, J23116, J23110, and J23100) to control ftsZ expression in the genome. Figure 3 As shown in Figures C and D, different expression intensities of ftsZ have different effects on threonine synthesis and cell growth. High expression of ftsZ has little effect on growth and threonine production, and may even be detrimental to threonine production. Weak expression of ftsZ promotes cell biomass accumulation and threonine synthesis.

[0109] 3.3 Transcriptome analysis of threonine-producing strains regulating ftsZ

[0110] To analyze the changes in gene transcription levels in the strains when ftsZ was regulated, we performed transcriptome sequencing on the starting strain TH-103Z and the modified strain J23103-ftsZ. During the shake flask fermentation, the bacterial culture medium at the mid-growth exponential stage was sequenced ( Figure 4 The transcriptome analysis results are shown in Figure 2. Figure 5As shown in Table 3 , a total of 141 genes in the KEGG pathway genes were significantly changed, including 125 up-regulated genes and 16 down-regulated genes. Twenty-four genes were up-regulated and one gene was down-regulated in the amino acid pathway. Aspartate kinase III (lysC) in the threonine synthesis pathway was up-regulated by 3.47 times.

[0111] Table 3. Comparison of TH-103Z and J23103-ftsZ transcriptomes

[0112]

[0113]

[0114]

[0115] 3.3 Dynamically controlling other division genes to increase threonine production

[0116] Currently, there are limited fission genes used in metabolic synthesis. We analyzed the feasibility of other fission genes in threonine synthesis. ftsZ, ftsB, ftsL, ftsQ, ftsA, ftsK, ftsN, and zipA are essential genes for cell division. The loss of these genes leads to filamentous cell growth. Therefore, we used PrhtC to control the expression of these genes. The results are shown in Figure 2. Figure 6 As shown, regulating the expression of the above genes increased threonine production to varying degrees, especially for the ftsZ, ftsB, ftsL, ftsQ, and zipA genes. Cell biomass also increased to varying degrees. This study enriched other fission genes that can be used to promote threonine synthesis. The final soluble threonine yield in the fermentation broth after fermentation in a 5-L fermenter was ~152.9 g / L. Threonine crystals precipitated at the bottom of the tank. After removal, drying, and dissolution, the crystals were found to be ~19.2 g / L. Therefore, a total of ~172.1 g / L threonine was produced during the 5-L fermenter culture ( Figure 7 ).

[0117] It should be noted that the above examples are only intended to illustrate the technical solutions of the present invention and are not intended to limit the same. Although the present invention has been described in detail with reference to the examples given, those skilled in the art may modify or replace the technical solutions of the present invention as needed without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. Application of split gene-related biomaterials in improving microbial threonine synthesis; The split gene related biological materials are as follows: Biological elements that regulate the expression intensity of ftsZ; inserting a biological element before the start codon of the ftsZ gene of the microorganism; The biological element is BBa_B1006-promoter-B0033 / B0034; The promoter is selected from J23103, J23113, J23109 and PrhtC; The microorganism is Escherichia coli TH-103Z, this strain was deposited in China Center for Type Culture Collection on December 5, 2022, and its biological deposit number is CCTCC NO: M 20221861; The split gene is ftsZ.

2. Application of split gene-related biomaterials in improving microbial threonine synthesis; The split gene related biological materials are as follows: a biological element that regulates the expression intensity of any one or more of ftsB, ftsL, ftsQ, ftsA, ftsK, ftsN, and zipA; The biological element is BBa_B1006-promoter-B0033 / B0034; The promoter is PrhtC; The microorganism is Escherichia coli TH-103Z, this strain was deposited in China Center for Type Culture Collection on December 5, 2022, and its biological deposit number is CCTCC NO: M 20221861; The split genes are ftsB, ftsL, ftsQ, ftsA, ftsK, ftsN and zipA.

3. A strain characterized by: The strain at least comprises the split gene-related biological material used in the application of claim 1 or 2; The strain is obtained by introducing the above-mentioned split gene-related biological materials into the starting strain; the starting strain is Escherichia coli TH-103Z, this strain was deposited in the China Center for Type Culture Collection, the preservation date is December 5, 2022, and its biological preservation number is CCTCC NO: M 20221861.

4. Use of the strain according to claim 3 in the fermentation production of threonine.

5. A method for improving the production of threonine by fermentation, characterized in that: The method comprises fermenting and culturing the strain according to claim 3, and separating and extracting threonine.

Images