CN116162584A - Application of split gene in regulating threonine synthesis and cell morphology and growth - Google Patents
Application of split gene in regulating threonine synthesis and cell morphology and growth Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K14/245—Escherichia (G)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
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Abstract
The invention belongs to the technical fields of genetic engineering and microorganisms, and particularly relates to application of a split gene in regulating threonine synthesis and cell morphology and growth. Experiments prove that the threonine synthesis of target microorganisms and the cell morphology and growth of the target microorganisms can be influenced by regulating the expression intensity of the split genes through biological elements. Such as less impact on microbial cell growth and less impact on threonine production, even adverse threonine production, when ftsZ expression is too strong, and accumulation of cellular biomass and promotion of threonine synthesis, when ftsZ expression is weak. Therefore, the dynamic regulation and control of threonine produced by fermenting the strain are realized by regulating and controlling the expression intensity of the split genes, so that the dynamic regulation and control of threonine produced by fermenting the strain is not only beneficial to actual production, but also can be used as a model strain for carrying out related researches on threonine synthesis mechanism, cell morphology and growth mechanism, and has good practical application value.
Description
Technical Field
The invention belongs to the technical fields of genetic engineering and microorganisms, and particularly relates to application of a split gene in regulating threonine synthesis and cell morphology and growth.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Threonine is an essential amino acid type constituting human and animal body proteins, and is widely used in various fields such as biological medicine, chemical reagents, foods and feeds at present. Currently, major threonine production enterprises in the world comprise Japanese-style plain companies, german Desoxhauster companies, american ADM companies, japanese-style co-ordinated fermentation industry companies and the like, the yield of the large companies accounts for about 90% of the global share, and domestic threonine production is in a development stage, and the production level is still a certain gap from abroad. At present, the production method of threonine mainly comprises three methods of a fermentation method, a protein hydrolysis method and a chemical synthesis method, wherein a microbial fermentation method is a main stream method for producing threonine, and the technical problems of improving fermentation efficiency and reducing fermentation cost in the microbial fermentation method are to be solved urgently.
The process of cell division is one of the important events in the development of individuals, by which the genetic material of the parent is duplicated and equally distributed into daughter cells, thus ensuring genetic material stabilization and continuation of species. According to the previous research results, it has been found that prokaryotes such as bacteria are first assembled to form a cyclic division complex at the division site by some division proteins (such as FtsZ, ftsA, zipA, etc.) before they undergo division, and thus promote the formation of division membrane, thereby triggering the occurrence of cell division event. Based on the important role of the above isolated proteins in bacterial cell isolation, it is common to develop novel antibacterial drugs by using the above isolated proteins or their coding groups as targets. However, there has been no report so far on the regulation of the role of the above cell division genes in threonine synthesis in microorganisms.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides the application of the split gene in regulating threonine synthesis and cell morphology and growth. According to experimental research, the cell division genes expressed in different intensities have influence on cell morphology and growth, and simultaneously have different influence on threonine production performance of the strain, so that the cell division genes have good practical application value.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides the use of a split gene related biomaterial for regulating threonine synthesis and/or microbial cell morphology and growth in a microorganism.
The split gene related biological material is as follows:
a) ftsZ, ftsB, ftsL, ftsQ, ftsA, ftsK, ftsN and zipA;
b) A biological element that modulates the expression intensity of any one or more of ftsZ, ftsB, ftsL, ftsQ, ftsA, ftsK, ftsN and zipA.
Wherein the biological element may include promoters of different strengths including but not limited to J23103, J23113, J23109, J23116, J23110, J23100 and PrhtC, and Ribosome Binding Sites (RBSs) including but not limited to BBa_B0033 and BBa_B0034.
The microorganism is a prokaryote, a bacterium, a threonine producing bacterium, and a bacterium such as Escherichia coli or a derivative thereof.
In a second aspect of the present invention, there is provided a strain comprising at least the split gene-related biomaterial described above;
the split gene related biological material is as follows:
a1 ftsZ, ftsB, ftsL, ftsQ, ftsA, ftsK, ftsN and zipA;
a2 A biological element that modulates the expression intensity of any one or more of ftsZ, ftsB, ftsL, ftsQ, ftsA, ftsK, ftsN and zipA.
The dynamic regulation and control of threonine produced by fermenting the strain are realized by regulating and controlling the expression intensity of the split genes, so that the dynamic regulation and control of threonine produced by fermenting the strain is not only beneficial to actual production, but also can be used as a model strain for carrying out related researches on threonine synthesis mechanism, cell morphology and growth mechanism.
Accordingly, in a third aspect of the invention there is provided the use of a strain as described above in any one or more of the following:
b1 Fermentation to produce threonine;
b2 Threonine synthesis mechanism research;
b3 Cell morphology and growth mechanism.
In a fourth aspect of the present invention, there is provided a method for producing threonine by dynamic regulation fermentation, which comprises subjecting the above strain to fermentation culture, and separating and extracting to obtain threonine.
It is to be noted that threonine mentioned in the present invention is specifically L-threonine.
The beneficial technical effects of one or more of the technical schemes are as follows:
the technical scheme provides application of the split gene in regulating threonine synthesis and cell morphology and growth, and particularly experiments prove that the expression intensity of the split gene is regulated and controlled by a biological element, so that the threonine synthesis of target microorganisms and the cell morphology and growth of the target microorganisms are influenced. Such as less impact on microbial cell growth and less impact on threonine production, even adverse threonine production, when ftsZ expression is too strong, and accumulation of cellular biomass and promotion of threonine synthesis, when ftsZ expression is weak. Therefore, the dynamic regulation and control of threonine produced by fermenting the strain are realized by regulating and controlling the expression intensity of the split genes, so that the dynamic regulation and control of threonine produced by fermenting the strain is not only beneficial to actual production, but also can be used as a model strain for carrying out related researches on threonine synthesis mechanism, cell morphology and growth mechanism, and has good practical application value.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a representation of promoter strength in an example of the invention.
FIG. 2 shows the effect of ftsZ on cell morphology at different expression levels in the examples of the present invention.
FIG. 3 is a graph showing the effect of modulation of ftsZ expression on threonine synthesis and cell growth in examples of the present invention; a is that threonine with different concentrations is added into a model strain escherichia coli MG1655 in vitro to carry out metering response analysis on PrhtC; b is shake flask fermentation characterization PrhtC in the production bacteria TH-103Z; c is the influence of ftsZ of different intensities on the growth of cells of the strain; d is the effect of different intensities of ftsZ on threonine synthesis by the strain.
FIG. 4 is a graph showing the results of transcriptome sampling in an embodiment of the invention.
FIG. 5 is a graph showing the results of the analysis of the transcription set in the embodiment of the invention.
FIG. 6 is a graph showing the effect of controlling the expression of other split genes on threonine and cell growth in an example of the present invention.
FIG. 7 is a graph showing the results of fermentation in a 5-L fermenter to produce threonine in the examples of the present invention.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof. It is to be understood that the scope of the invention is not limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention.
In an exemplary embodiment of the present invention, there is provided the use of a split gene related biomaterial for regulating threonine synthesis and/or microbial cell morphology and growth in a microorganism.
The split gene related biological material is as follows:
a) ftsZ, ftsB, ftsL, ftsQ, ftsA, ftsK, ftsN and zipA;
b) A biological element that modulates the expression intensity of any one or more of ftsZ, ftsB, ftsL, ftsQ, ftsA, ftsK, ftsN and zipA.
Wherein the biological element may comprise promoters of different intensities and Ribosome Binding Sites (RBS), the intensities of the promoters (in terms of relative fluorescence intensity RFP/OD 600 Calculated as) is 20-2000, and further 30-300, at this time, the method is favorable for the production and synthesis of threonine by escherichia coli, and the cell biomass is higher; such promoters include, but are not limited to, J23103, J23113, J23109, J23116, J23110, J23100, and PrhtC, and such RBSs include, but are not limited to, BBa_B0033 and BBa_B0034. According to the invention, the expression intensity of the split genes is regulated and controlled through the biological elements, so that threonine synthesis of target microorganisms and cell morphology and growth of the target microorganisms are influenced. The research shows that when ftsZ expression is too strong, the effect on the growth of microbial cells is small (for example, the strength of an exogenously introduced promoter is not less than 600, and can be J23116, J23110 and J23100 in particular), and the effect on threonine production is small, and even the threonine production is not favored. Facilitates accumulation of cellular biomass and promotes threonine synthesis when ftsZ expression is weak. According to the invention, the research proves that the escherichia coli with the exogenous introduced promoter J23103 (with the promoter strength of 33) has the strongest threonine synthesis capacity and the highest cell biomass. At the same time, the division genes with different intensities can also lead to microbial cell shapeChanges in state, such as J23103, J23113, J23109, prhtC, J23116 control the increase in cell length when ftsZ is expressed, even when J23116 controls the filiform growth of cells when ftsZ is expressed, and J23100 controls the elliptic growth of cells when ftsZ is expressed. Meanwhile, threonine yield is improved to different degrees when PrhtC controls the expression of the split genes, especially ftsZ, ftsB, ftsL, ftsQ, zipA genes, and cell biomass is also improved to different degrees.
In a specific embodiment of the invention, the biological element may be a terminator-promoter-RBS, wherein the terminator may be bba_b1006.
The microorganism is a prokaryote, a bacterium, a threonine producing bacterium, and a bacterium such as Escherichia coli or a derivative thereof.
In one embodiment of the present invention, the escherichia coli derivative may be an escherichia coli strain obtained by genetically engineering a wild-type escherichia coli. Escherichia coli TH-103Z, which has threonine-producing properties, is deposited with the China center for type culture Collection (address: university of Wuhan, hubei province) with a date of deposition of 2022, 12 months, 05 days, and a biological deposit number of CCTCC NO: m20221861.
In one embodiment of the present invention, there is provided a strain comprising at least the split gene-related biomaterial described above;
the split gene related biological material is as follows:
a1 ftsZ, ftsB, ftsL, ftsQ, ftsA, ftsK, ftsN and zipA;
a2 A biological element that modulates the expression intensity of any one or more of ftsZ, ftsB, ftsL, ftsQ, ftsA, ftsK, ftsN and zipA.
Wherein the biological element may comprise promoters of different intensities and Ribosome Binding Sites (RBS), the intensities of the promoters (in terms of relative fluorescence intensity RFP/OD 600 Calculated as such) is 20 to 2000, further 30 to 300; promoter packageIncluding but not limited to J23103, J23113, J23109, J23116, J23110, J23100 and PrhtC, including but not limited to BBa_B0033 and BBa_B0034.
According to the research, the ftsZ with different expression intensities has significantly different effects on threonine synthesis and cell growth, and as described above, the ftsZ has less effect on microbial cell growth when the ftsZ is expressed too strongly (such as the strength of an exogenously introduced promoter is not less than 600, and can be J23116, J23110 and J23100 in particular), and has less effect on threonine production, and even is unfavorable for threonine production. Facilitates accumulation of cellular biomass and promotes threonine synthesis when ftsZ expression is weak. According to the invention, the research proves that the escherichia coli with the exogenous introduced promoter J23103 (with the promoter strength of 33) has the strongest threonine synthesis capacity and the highest cell biomass. Meanwhile, the division genes with different intensities can also cause the morphological change of microbial cells, such as the increase of cell length when J23103, J23113, J23109, prhtC and J23116 control the expression of ftsZ, even the filiform growth of cells when J23116 control the expression of ftsZ, and the elliptic growth of cells when J23100 control the expression of ftsZ. Meanwhile, threonine yield is improved to different degrees when PrhtC controls the expression of the split genes, especially ftsZ, ftsB, ftsL, ftsQ, zipA genes, and cell biomass is also improved to different degrees.
In a specific embodiment of the invention, the biological element may be a terminator-promoter-RBS, wherein the terminator may be bba_b1006.
The strain can be escherichia coli and derivative bacteria thereof, and in a specific embodiment of the invention, the strain can be obtained by introducing the split gene related biological material from a starting strain, wherein the starting strain is Escherichia coli TH-103Z and has threonine producing performance, and the strain is preserved in China center for type culture collection (address: wuchang Lopa nationality in Wuhan, hubei province), with a preservation date of 2022, 12 months and 05 days, and the biological preservation number is CCTCC NO: m20221861.
By regulating the expression intensity of the split genes, dynamic regulation of threonine production by fermentation of the strain (the threonine production performance of the strain can be enhanced or weakened) is realized, the actual production is facilitated, and meanwhile, the strain can be used as a model strain for carrying out related researches on threonine synthesis mechanism, cell morphology and growth mechanism.
Thus, in one embodiment of the invention there is provided the use of the strain described above in any one or more of the following:
b1 Fermentation to produce threonine;
b2 Threonine synthesis mechanism research;
b3 Cell morphology and growth mechanism.
The invention has been found by research,
in one embodiment of the invention, a method for producing threonine by dynamic regulation fermentation is provided, and the method comprises the steps of fermenting and culturing the strain, and separating and extracting to obtain threonine.
It is to be noted that threonine mentioned in the present invention is specifically L-threonine.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
Examples
1. Experimental materials
1.1 strains, plasmids and primers
The strains, plasmids and primers used in this example are shown in tables 1 and 2.
TABLE 1 strains and plasmids used in this example
TABLE 2 primer sequences used in this example
1.2 Medium
The media used in this example, LB medium, were as follows: 10g/L peptone, 5g/L yeast extract, 10g/L NaCl. Shake flask fermentation medium: 15g/L (NH) 4 ) 2 SO 4 ,2g/L KH 2 PO 4 ,1g/L MgSO 4 ·7H 2 O,2g/L Yeast extract, 0.02g/L FeSO 4 40g/L glucose, 20g/L CaCO 3 The pH was adjusted. Feeding a culture medium: 20g/L ammonium sulfate, 2g/L potassium dihydrogen phosphate, 5/10/15g/L corn steep liquor, 2g/L magnesium sulfate heptahydrate, 5mg/L ferrous sulfate heptahydrate, 5mg/L manganese sulfate tetrahydrate, 20g/L glucose, 0.5g/L glycine, 0.5g/L betaine, 200. Mu.g/L biotin.
1.3 common enzymes for molecular biology
The high fidelity DNA polymerase for gene cloning of this example was purchased from Norwegian corporation as the enzyme Phanta Max Super-Fidelity DNA Polymerase. 2 XEs Taq Master mix (Dye) premix enzyme for colony PCR validation was purchased from Kangji (Beijing) Inc.
1.4 common kit
Agarose gel recovery kit, plasmid small extract medium amount kit and genome extraction kit are purchased from Tiangen biochemical technology (Beijing); the DNA fragment rapid purification recovery kit Cycle-Pure is purchased from OMEGA company;
2. experimental method
2.1 construction of plasmids and engineering strains
The plasmids, strains and primers used in this example are listed in tables one and two. To characterize the promoter strength and threonine response analysis, this example uses a low copy plasmid pCL1920 as the backbone of the recombinant plasmid, the rhtC gene promoter, J23103, J23113, J23109, J23116, J23110, J23100 promoters, and ribosome binding sites B0033 and B0034 were derived from the iGEM synthetic biological element (http:// parts. IGEM. Org/main_Page), and the pCL1920 plasmid backbone was constructed with the promoter, RBS, reporter gene rfp by the Gibson assembly method as follows: prhtC-RFP, J23103-RFP, J23113-RFP, J23109-RFP, J23116-RFP, J23110-RFP, J23100-RFP.
The 300-500bp fragments on both sides of target gene ftsZ, ftsB, ftsL, ftsQ, ftsA, ftsK, ftsN, zipA are amplified by PCR to obtain upstream and downstream homology arms, the fragments with FRT sites and chloramphenicol screening markers on both ends are obtained from pKD3 plasmid by PCR amplification, the upstream and downstream homology arms and the chloramphenicol screening fragments are fused into genome integration fragments, and the genome integration fragments are recovered and purified by kit gel to obtain the water-soluble genome integration fragments. Transferring pTKRED plasmid with homologous recombination enzyme into strain TH-103Z, collecting bacteria after ITPG induction homologous recombination enzyme expression at 30deg.C, repeatedly centrifuging and re-suspending 3-5 times with 10% glycerol, and finally re-suspending with 100 μL 10% glycerol to obtain electrotransformation competent cells. After 2.5kV voltage transformation and recovery culture at 37 ℃ for 1-3 hours, the electrotransformation bacteria liquid is coated on a chloramphenicol LB plate for overnight culture at 37 ℃. Then, single colony is selected for PCR verification of genome integration, and temperature sensitive pTKRED plasmid is removed by culturing at 42 ℃ after successful integration. And finally, removing chloramphenicol screening markers with FRT sites by using a temperature-sensitive pCP20 plasmid to obtain the recombinant engineering strain.
2.2 scanning Electron microscope for observing cell morphology
Sample pretreatment: centrifuging a large amount of bacterial liquid at 1000rpm-4000rpm, removing supernatant, adding 1 XPBS with proper pH of 6.8-7.4, cleaning, and removing supernatant.
Fixing: 2.5% glutaraldehyde in 1 XPBS was added and fixed at 4℃for 3h, the supernatant removed, and the supernatant removed by washing three times with 1 XPBS.
Dehydrating: the samples were dehydrated with aqueous ethanol at 30%, 50%, 70%, 80%, 90% concentration gradients, and the supernatants were discarded and dehydrated 2 times in 100% ethanol.
And (3) drying: and drying by a critical point dryer, sucking fungus liquid drop on a cover glass, and placing the cover glass carrying the sample into the critical point dryer for drying.
And (5) metal spraying: after the sample was sufficiently dried, the coverslip was attached to a sample stage to which a conductive tape was attached and gold plated.
And (5) observing under an electron microscope.
2.3 characterization of threonine sensor and promoter
This example characterizes threonine-responsive promoters PrhtC and J23103, J23113, J23109, J23116, J23110, J23100 by analysis of the expression levels of the reporter gene. Transferring plasmids PrhtC-RFP, J23103-RFP, J23113-RFP, J23109-RFP, J23116-RFP, J23110-RFP and J23100-RFP into escherichia coli MG1655, culturing at 37 ℃ and picking single colony, culturing in a 12-well plate with 2mL LB culture medium for 12h, transferring 2% (v/v) of the single colony into a 24-well plate with 1mL LB culture medium for 24h at 37 ℃, and measuring the OD of the bacteria by using an enzyme-labeled instrument 600 And excitation 590nm and emission 645 nm. Meanwhile, prhtC-RFP and J23109-RFP are transferred into a production strain TH-103Z, shake flask fermentation characterization is carried out by using 20mL fermentation medium, cell density is measured by an ultraviolet spectrophotometer, fluorescence intensity of a gene RFP is measured by an enzyme-labeled instrument, and RFP/OD is adopted 600 To calculate the strength of the promoter.
2.4 transcriptome sequencing
In this example, transcriptomes of the strain TH-103Z and the engineering strain J23103-ftsZ were measured, and medium-term bacterial cells of the growth index were collected by shake flask fermentation, and were sequenced by Qingdao European Biotechnology Co., ltd, and the sequencing amount was 2G/sample.
2.5 threonine fermentation
Shaking and fermenting: single colonies were picked up and cultured in 12-well plates containing 2mL of LB at 37℃for 12h, inoculated in 300mL shake flasks containing 20mL of shake flask fermentation medium at 1% (v/v) volume, and cultured at 220rpm for 36h at 37 ℃.
And (3) fermentation in a tank: (1) first-stage seed culture: single colonies were picked and cultured in 300 mL-volume shake flasks containing 50mL of LB at 37℃for-12 h. (2) secondary seeds: primary seeds were transferred in 10% (v/v) volumes to 2L shake flasks containing 400mL of the upper tank medium and incubated at 37℃and 220rpm to exponential phase. (3) loading in a tank: inoculating the second seed into 5L tank according to 10% (v/v) volume, adjusting pH to 7.0 with ammonia water, controlling dissolved oxygen at 20-30%, and culturing at 37deg.C.
2.7 threonine detection method
Preparing a derivatization reagent: (1) reagent one: mixing 1.4mL of triethylamine with 8.6mL of acetonitrile uniformly; (2) reagent II: mu.L of phenyl isothiocyanate was taken and mixed with 2mL of acetonitrile.
Sample treatment: taking the supernatant of the fermentation liquor, diluting the supernatant by 20 times with deionized water, taking 100 mu L of each of the first reagent and the second reagent, adding the first reagent and the second reagent into 200 mu L of diluted samples, uniformly mixing, and standing at room temperature for 1h. Then 400 mu L of normal hexane is added, vortex shaking and mixing are carried out, standing is carried out for 10min, 200 mu L of lower liquid is taken, 800 mu L of deionized water is added, and after mixing, a detection sample is obtained by filtering with a 0.22 mu m organic filter membrane.
Threonine detection: in this example, high performance liquid chromatography was used to measure threonine production in fermentation broths. Chromatographic column: venusilAA (4.6X105 mm,5 μm, agela technologies), column incubator: 40 ℃, mobile phase a:15.2g anhydrous sodium acetate was dissolved in 1850mL double distilled water, 140mL acetonitrile was added and mixed well, mobile phase B:80% (v/v) acetonitrile, flow rate: 1mL, detection procedure: 0-2min 0% mobile phase B, 2-14min 7% mobile phase B, 14-29min 30% mobile phase B, 29-32min 50% mobile phase B, 32-33min 100% mobile phase B, 33-39min 100% mobile phase B, 39.1-45min 0% mobile phase B.
3. Experimental results and discussion
3.1 analysis of the influence of ftsZ of different expression intensities on cell morphology
We first analyzed the effect of ftsZ on cell morphology at different expression intensities, selecting six promoters of different intensities from the iGEM database, J23103, J23113, J23109, J23116, J23110, J23100, and two Ribosome Binding Sites (RBS) of different intensities of B0033, B0034. PrhtC is a promoter of threonine transporter RhtC, and the intensity characterization results are shown in FIG. 1, wherein the intensities of J23103, J23113, J23109, prhtC, J23116, J23110 and J23100 are sequentially enhanced, and are respectively 33, 36, 55, 260, 670, 1000 and 2000.
We used the promoters of different intensities characterized above to control the expression of ftsZ in the genome, and the ftsZ original promoter in the threonine-producing strain TH-103Z genome was located inside its upstream gene ftsA, so that the ftsZ original promoter could not be replaced in order not to affect the normal expression of ftsA, and we inserted a terminator-promoter-RBS sequence, namely BBa_B1006-promoter-B0033/B0034, in front of the ftsZ gene start codon. After culturing for 12h at 37 ℃, collecting bacteria, fixing, drying, spraying gold, observing cell morphology through a scanning electron microscope, and the results are shown in figure 2, wherein when J23103, J23113, J23109, prhtC and J23116 control expression of ftsZ, the cell length is increased, even when J23116 controls filiform growth of cells when ftsZ is expressed, and when J23100 controls elliptical growth of cells when ftsZ is expressed.
3.2 analysis of the influence of ftsZ of different expression intensities on threonine Synthesis and cell growth
To analyze the effect of regulating cell division on threonine synthesis, we studied using two ftsZ regulation modes. The first method adopts threonine response activation mode to perform self-induced dynamic control on ftsZ expression, because the transport protein RhtC specifically transports intracellular threonine to outside cells, we speculate that the promoter (PrhtC) of the rhtC gene responds to threonine activation rhtC expression, in order to verify the response condition of PrhtC to threonine, we connect a RBS (B0034) and a reporter gene (RFP), namely PrhtC-B0034-RFP, after PrhtC to perform characterization, and as shown in figure 3A, B, threonine with different concentrations is added in vitro in a model strain escherichia coli MG1655 for metering response analysis, and the result shows that the response threshold range of PrhtC is 0-30g/L threonine, and the dynamic range is 1.8 times. To verify whether PrhtC sensor is suitable for threonine-producing bacteria, we performed shake flask fermentation characterization in producing bacteria TH-103Z (FIG. 3B), and as a result found that PrhtC sensor-controlled RFP intensity was enhanced with increasing fermentation time and threonine accumulation, with J23109 constitutively expressed RFP as a control, and therefore PrhtC was a threonine-responsive activated sensor. We used PrhtC to control ftsZ dynamic expression in the genome, resulting in an increase in threonine production and biomass of-62% and 54%, respectively, as shown in FIG. 3, C, D. The second mode of regulation of cell division uses constitutive promoters of different intensities (J23103, J23113, J23109, J23116, J23110, J23100) to control ftsZ expression in the genome, and as a result, as shown in FIG. 3C, D, ftsZ of different expression intensities have different effects on threonine synthesis and cell growth, less on growth when ftsZ expression is too strong, and less on threonine production, even less on threonine production. Facilitates accumulation of cellular biomass and promotes threonine synthesis when ftsZ expression is weak.
3.3 transcriptome analysis of threonine-producing Strain that regulates ftsZ
To analyze the transcriptional level changes of genes in strains when ftsZ was regulated, we performed transcriptome sequencing of the starting strain TH-103Z and the engineered strain J23103-ftsZ, and sequenced the medium-term bacterial fluid during shake flask fermentation (FIG. 4). As a result of the transcriptome analysis, as shown in FIG. 5 and Table III, there were 141 genes in the KEGG pathway gene, including 125 up-regulated genes, 16 down-regulated genes, 24 up-regulated genes in the amino acid pathway, 1 down-regulated genes, and up-regulated aspartokinase III (lysC) in the threonine synthesis pathway 3.47-fold.
Table 3, TH-103Z and J23103-ftsZ transcriptome comparisons
3.3 dynamic control of other Split genes to increase threonine production
The current limited number of split genes used in metabolic synthesis, ftsZ, ftsB, ftsL, ftsQ, ftsA, ftsK, ftsN, zipA, which is a gene essential for cell division, was analyzed to determine the feasibility of other split genes in threonine synthesis, and the deletion of these genes resulted in filamentous cell growth, so we used PrhtC to control the expression of these genes, and as shown in FIG. 6, the threonine production was improved to various extents, especially ftsZ, ftsB, ftsL, ftsQ, zipA, when the expression of these genes was regulated, and the cell biomass was also increased to various extents. Finally, the yield of the soluble threonine in the fermentation broth after fermentation in the 5-L fermentation tank is 152.9g/L, threonine crystals are precipitated at the bottom of the tank, and the crystals are taken out, dried, dissolved and detected to be 19.2g/L, so that 172.1g/L threonine is produced in the 5-L fermentation tank during culture (FIG. 7).
It should be noted that the above examples are only for illustrating the technical solution of the present invention and are not limiting thereof. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can make modifications and equivalents to the technical solutions of the present invention as required, without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. Application of split gene related biological material in regulating and controlling threonine synthesis and/or microbial cell morphology and growth;
the split gene related biological material is as follows:
a) ftsZ, ftsB, ftsL, ftsQ, ftsA, ftsK, ftsN and zipA;
b) A biological element that modulates the expression intensity of any one or more of ftsZ, ftsB, ftsL, ftsQ, ftsA, ftsK, ftsN and zipA.
2. The use according to claim 1, wherein the biological element comprises promoters of different intensities and ribosome binding sites RBS; the strength of the promoter is 20-2000, and further 30-300.
3. The use of claim 2, wherein the promoter comprises J23103, J23113, J23109, J23116, J23110, J23100 and PrhtC, and the RBS comprises bba_b0033 and bba_b0034.
4. The use of claim 3, wherein the biological element is a terminator-promoter-RBS, and wherein the terminator is bba_b1006.
5. Use according to claim 1, wherein the microorganism is a prokaryote, in particular a bacterium, which is any threonine-producing bacterium, further wherein the bacterium is escherichia coli and derivatives thereof;
the escherichia coli is Escherichia coli TH-103Z, the strain is preserved in China center for type culture collection, the preservation date is 2022 and 12 months 05 days, and the biological preservation number is CCTCC NO: m20221861.
6. A strain comprising at least a split gene-related biological material;
the split gene related biological material is as follows:
a1 ftsZ, ftsB, ftsL, ftsQ, ftsA, ftsK, ftsN and zipA;
a2 A biological element that modulates the expression intensity of any one or more of ftsZ, ftsB, ftsL, ftsQ, ftsA, ftsK, ftsN and zipA.
7. The strain of claim 6, wherein the biological element comprises promoters of different strengths, including J23103, J23113, J23109, J23116, J23110, J23100 and PrhtC, and a ribosome binding site RBS, including bba_b0033 and bba_b0034;
the biological element is a terminator-promoter-RBS, wherein the terminator is bba_b1006;
the strain is escherichia coli and derivative bacteria thereof;
the strain is obtained by introducing the split gene related biological material into a starting strain, wherein the starting strain is Escherichia coli TH-103Z, the strain is preserved in China center for type culture collection (China center for type culture collection), the preservation date is 2022 and 12 months 05 days, and the biological preservation number is CCTCC NO: m20221861.
8. Use of a strain according to claim 6 or 7 in any one or more of the following:
b1 Fermentation to produce threonine;
b2 Threonine synthesis mechanism research;
b3 Cell morphology and growth mechanism.
9. A method for producing threonine by dynamic regulation fermentation, which comprises fermenting and culturing the strain of claim 6 or 7, and separating and extracting to obtain threonine.
10. The method of claim 9, wherein the threonine is L-threonine.
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| ELHADI, D: "CRISPRi engineering E. coli for morphology diversification", METABOLIC ENGINEERING, vol. 38, 30 November 2016 (2016-11-30), pages 358 - 369 * |
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