CN113265412A - Multi-enzyme coupling positioning system and construction method and application thereof - Google Patents

Multi-enzyme coupling positioning system and construction method and application thereof Download PDF

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CN113265412A
CN113265412A CN202110550828.4A CN202110550828A CN113265412A CN 113265412 A CN113265412 A CN 113265412A CN 202110550828 A CN202110550828 A CN 202110550828A CN 113265412 A CN113265412 A CN 113265412A
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dcas
linker
plasmid
enzyme
target
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申晓林
童贻刚
陈昕
周朝
袁其朋
王佳
孙新晓
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Beijing University of Chemical Technology
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Beijing University of Chemical Technology
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Abstract

The invention provides a multienzyme coupling positioning system, which comprises dCas protein-linker-target enzyme fusion plasmid, guide RNA plasmid and DNA scaffold plasmid; the dCas protein-linker-target enzyme fusion plasmid is anchored to the DNA scaffold plasmid under the direction of the guide RNA plasmid. The target enzyme in the dCas protein-linker-target enzyme fusion plasmid is linked to dCas protein by a linker. The target enzyme is an enzyme required in the biosynthetic pathway of the target product. The DNA scaffold plasmid comprises a plurality of dCas binding sites. The invention also provides a construction method and application of the multi-enzyme coupling positioning system. The invention can realize the space positioning and sequential catalysis of a plurality of enzymes by adopting the multi-enzyme coupling positioning system, thereby improving the yield of target products.

Description

Multi-enzyme coupling positioning system and construction method and application thereof
Technical Field
The invention relates to the technical field of bioengineering, in particular to a multi-enzyme coupling positioning system and a construction method and application thereof.
Background
In the process of producing a target product by using microbial cells, a plurality of enzyme sequential catalytic reactions are required to produce a final product. However, poor substrate specificity of the enzyme, problems such as side reactions due to the similarity of intracellular metabolites to enzyme substrates, substrate or intermediate product escape, and disturbance of cellular metabolic flux due to the introduction of exogenous enzymes all reduce the catalytic efficiency of the enzyme and the production efficiency of the target product. Therefore, the spatial layout of intracellular microenvironment is artificially carried out by enzymes according to the catalytic sequence or catalytic capacity, the dissipation of substrates is reduced, the nonspecific catalysis of the enzymes and the non-substrates is prevented, the mass transfer efficiency of the substrates is improved, the catalytic efficiency of the enzymes is further improved, and the generation of side reactions in metabolic pathways is reduced.
The CRISPR technology is a gene editing technology which is rapidly developed in recent years, has the advantages of small operation difficulty, low editing cost, high targeting efficiency, traceless editing and the like, is widely applied to the fields of construction and synthetic biology of microbial cell factories, and develops and designs a large number of new gene editing elements, tools and gene lines. However, no studies and techniques have been reported to apply this technique to localized catalysis of enzymes to improve the catalytic efficiency of cell factories.
Disclosure of Invention
In view of the above, the present invention provides a multi-enzyme coupled localization system, and a construction method and applications thereof, so as to solve the above problems.
To this end, the present invention provides a multiple-enzyme coupled localization system comprising: a dCas protein-linker-target enzyme fusion plasmid, a guide RNA plasmid, and a DNA scaffold plasmid, and the dCas protein-linker-target enzyme fusion plasmid is anchored on the DNA scaffold plasmid under the guidance of the guide RNA plasmid; wherein the dCas protein-linker-target enzyme fusion plasmid comprises a plurality of dCas proteins, a plurality of linkers and a plurality of target enzymes, and the target enzymes are linked to the dCas proteins by the linkers; a plurality of the target enzymes are enzymes required in a biosynthetic pathway of a target product; the DNA scaffold plasmid is a DNA fragment comprising a plurality of dCas binding sites, the dCas binding sites being arranged in a sequence such that each dCas binding site corresponds to one dCas protein under the direction of the guide RNA plasmid such that a plurality of dCas proteins are anchored to the DNA scaffold plasmid.
Herein, a "dCas protein" is a functional protein, which is a Cas protein that is unable to digest a DNA scaffold but is still able to bind to a target DNA sequence specified by a guide RNA. Preferably, the distance between a plurality of the dCas binding sites in the DNA scaffold can be adjusted by inserting bases of different lengths, by adjusting the distance between the dCas binding sites to adjust the distance between a plurality of the target enzymes. Preferably, a plurality of said target enzymes are key enzymes in the biosynthetic pathway of the target product, i.e. enzymes in the biosynthetic metabolic pathway of the target product which determine the speed and direction of the reaction.
"linker" refers to a linker peptide capable of linking the corresponding dCas protein to the corresponding target enzyme. The plurality of linkers in the dCas protein-linker-target enzyme fusion plasmid can be the same peptide or a plurality of peptides with different lengths and different flexibilities. Preferably, the length and flexibility of the linker is matched to the target enzyme to which it is linked.
Based on the multi-enzyme coupling positioning system, the multi-enzyme coupling positioning system also comprises a chassis cell, and the dCas protein-linker-target enzyme fusion plasmid, the guide RNA plasmid and the DNA scaffold plasmid are jointly introduced into the chassis cell.
Based on the above, the underpan cells are bacteria, yeast or fungi, and the bacteria or fungi are wild type or modified. Preferably, the Chassis cells are Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae or Aspergillus niger.
Based on the above, the dCas protein is selected from any two or three of dCas9 protein, dCas12 protein and dCas13 protein.
Based on the above, the target product includes but is not limited to ferulic acid, acetaminophen, arbutin or dencichine and other substances capable of biosynthesis. Meanwhile, the target enzymes are preferably key enzymes in biosynthetic pathways for synthesizing ferulic acid, acetaminophen, arbutin or dencichine.
The invention also provides a construction method of the multi-enzyme coupling positioning system, which comprises the following steps:
constructing a recombinant plasmid to provide a plurality of target enzymes, a plurality of linkers and a plurality of dCas proteins, respectively carrying out PCR amplification treatment on the plurality of target enzymes and the plurality of dCas proteins, and connecting the dCas proteins with the target enzymes through the linkers to form a plurality of dCas protein-linker-target enzyme genes; connecting a plurality of dCas protein-linker-target enzyme genes to the same expression plasmid to form a dCas protein-linker-target enzyme recombinant plasmid; performing PCR amplification treatment on a DNA scaffold and guide RNA respectively to prepare a DNA scaffold recombinant plasmid and a guide RNA recombinant plasmid, wherein the DNA scaffold is a DNA fragment containing a plurality of dCas binding sites, bases with different lengths can be inserted among the dCas binding sites according to requirements to adjust the distance among the target enzymes, the dCas binding sites are arranged in sequence, and each dCas binding site corresponds to one dCas protein so that the dCas proteins are anchored on the DNA scaffold plasmid;
and (3) constructing an engineering bacterium, and simultaneously transforming the dCas protein-linker-target enzyme recombinant plasmid, the DNA scaffold recombinant plasmid and the guide RNA recombinant plasmid into a chassis cell to construct a multi-enzyme coupling positioning system.
Wherein, preferably, the expression plasmid adopted by the dCas protein-linker-target enzyme recombinant plasmid is pZE; the expression plasmid adopted by the DNA bracket recombinant plasmid and the guide RNA recombinant plasmid is pXS, so the DNA bracket recombinant plasmid can be represented as pXS-DNA, and the guide RNA recombinant plasmid can be represented as pXS-gRNA.
Based on the above, in the step of constructing a recombinant plasmid, the method for constructing the dCas protein-linker-target enzyme recombinant plasmid comprises: firstly, the target enzyme is connected with the dCas protein through the linker in an enzyme digestion connection mode to form the dCas protein-linker-target enzyme gene; and connecting a plurality of dCas protein-linker-target enzyme genes to the same expression plasmid in a plug-and-play connection manner to form the dCas protein-linker-target enzyme recombinant plasmid. Wherein the dCas protein-linker-target enzyme gene codes to form a fusion protein. The difference in length and rigidity of the linker used in the dCas protein-linker-target enzyme recombinant plasmid allows the activity of a plurality of the target enzymes to be unaffected.
Therefore, in the construction process of the above-mentioned multienzyme coupling positioning system, introducing related dCas protein-linker-target enzyme plasmid, DNA scaffold plasmid and guide RNA plasmid into original or modified bacteria, fungi and yeast, wherein, a method combining simple enzyme digestion connection mode and plug-and-play mode is adopted to connect a plurality of target enzymes with different dCas proteins respectively to form dCas protein-linker-target enzyme fusion protein; then the dCas protein in the dCas protein-linker-target enzyme plasmid is matched and combined with dCas binding sites in the DNA scaffold plasmid through the guide RNA plasmid, so that a plurality of target enzymes are arranged, spatially positioned and catalyzed on the DNA scaffold plasmid according to a preset design.
Based on the above, a plurality of the target enzymes are key enzymes in the pathway for synthesizing p-acetaminophenol, including p-pabABC (Chinese character 'aminobenzoate synthetase', 4ABH (Chinese character 'p-aminobenzoate hydroxylase') and nhoA (Chinese character 'p-aminophenol acetylase'), and the dCas protein-linker-target enzyme recombinant plasmid is pZE-dCas9-linker-pabABC-dCas12-linker-4 ABH-nhoA.
Based on the above, a plurality of the target enzymes are enzymes in the biosynthetic ferulic acid pathway, including TyrB (hereinafter, referred to as "tyrosine aminotransferase"), TAL (hereinafter, referred to as "tyrosine ammonia lyase"), HpaBC (hereinafter, referred to as "4-hydroxypropionate 3-hydroxylase"), and COMT (hereinafter, referred to as "caffeine 3-O-methyltransferase"), and the dCas protein-linker-target enzyme recombinant plasmid is pZE-dCas9-linker-TyrB-dCas 12-linker-TAL-HpaBC-COMT.
The invention also provides an application of the multi-enzyme coupling positioning system, which comprises the following steps: inoculating the multienzyme coupling positioning system into a culture medium according to the inoculation amount of 1-5% of the volume ratio, adding an inducer, and performing fermentation treatment at 28-40 ℃ to produce a target product; the carbon source in the medium is glucose, glycerol or a combination of the two.
Wherein the culture medium comprises: 1-5 g ∙ L-1 MOPS，5～40 g∙L-15-40 g of ∙ L of glucose-11-5 g of ∙ L of glycerol-15-8 g of ∙ L of yeast powder-1 NaHPO4，0.3～2 g∙L-1 NaCl，3 g∙L-1 KH2PO4，1～5 g∙L-1NH4Cl，240～250 mg∙L-1 MgSO4，14～15.5 mg∙L-1CaCl2. Preferably, the ratio of glucose to glycerol is (1-3): 1, more preferably, the ratio of glucose to glycerol is 2: 1.
Therefore, the multi-enzyme coupling positioning system provided by the invention comprises a dCas protein-linker-target enzyme fusion plasmid, a guide RNA plasmid and a DNA scaffold plasmid, wherein the dCas protein-linker-target enzyme fusion plasmid comprises a plurality of dCas proteins, and the dCas proteins are connected with a target enzyme through a linker; the DNA scaffold comprises a plurality of dCas binding sites which are arranged in sequence, each dCas binding site corresponds to one dCas protein in the dCas protein-linker-target enzyme fusion plasmid under the guidance of the guide RNA plasmid, so that the dCas proteins are anchored on the DNA scaffold plasmid, a plurality of target enzymes are arranged in a specific sequence, and meanwhile, bases with different lengths can be inserted between the adjacent dCas binding sites in the DNA scaffold plasmid according to requirements for adjusting the distance between the adjacent target enzymes, so that the multi-enzyme coupling positioning system provided by the invention can realize spatial positioning of the target enzymes and realize sequential catalysis to realize the spatial layout of microenvironment in the underpan cells, reduce the escape of substrates, prevent the non-specific catalysis of the target enzymes and non-substrates, improve the mass transfer efficiency of the substrates, further improve the catalytic efficiency of the target enzymes and reduce the generation of side reactions in metabolic pathways, thereby improving the yield of the target product.
Furthermore, simple carbon sources such as glucose and the like are utilized to produce target products of ferulic acid and acetaminophen by combining a metabolic engineering technology, the yield of the ferulic acid and the yield of the acetaminophen can respectively reach 63 mg/L and 20 mg/L, and after the corresponding target enzymes are subjected to coupling positioning optimization by adopting the multi-enzyme coupling positioning system, the metabolic engineering bacteria utilizing the simple carbon sources can reach the final yield of 601 mg/L and 220 mg/L. The experimental results prove that the multi-enzyme coupling positioning system provided by the invention can effectively improve the yield of the target product.
Drawings
FIG. 1A is a schematic diagram showing a disordered state of an enzyme in a basal disc cell in a normal case; and B is a schematic diagram of the construction of a multi-enzyme coupling positioning system provided by the embodiment of the invention.
FIG. 2 is a graph showing the results of fermentation of the engineered bacteria BW1 and BW2 of the present invention in the production of acetaminophen.
FIG. 3 is a HPLC result chart of 48h sample produced acetaminophen by engineering bacteria BW1 provided in example 1 of the present invention.
FIG. 4 is a HPLC result chart of 48h sample produced acetaminophen by engineering bacteria BW2 provided in example 1 of the present invention.
FIG. 5 is a graph showing the results of fermentation of ferulic acid produced by the engineering bacterium Q1 provided in example 2 of the present invention.
FIG. 6 is a graph showing the results of fermentation of ferulic acid produced by the engineered bacterium Q2 provided in example 2 of the present invention.
FIG. 7 is a HPLC result chart of 48h sample of ferulic acid produced by the engineered bacterium Q1 provided in example 2 of the present invention.
FIG. 8 is a graph showing the HPLC results obtained by diluting the 48h sample of ferulic acid produced by the engineered bacterium Q2 provided in example 2 of the present invention by 3 times.
In the sequence table:
SEQ ID NO.1 is the nucleotide sequence of the linker used in the examples of the present invention.
Detailed Description
The technical solution of the present invention is further described in detail by the following embodiments. In the present invention, there is no special requirement for the type of expression plasmid, and it is considered that the construction method for expressing the target gene in escherichia coli can adopt various methods commonly used in the art, for example, the target gene is connected to a vector after enzyme digestion treatment, and details are not repeated.
Referring to fig. 1, the embodiment of the present invention provides a multi-enzyme coupled localization system, which includes a bottom plate cell, a dCas protein-linker-target enzyme fusion plasmid, a guide RNA plasmid, and a DNA scaffold plasmid, and the dCas protein-linker-target enzyme fusion plasmid is anchored on the DNA scaffold plasmid under the guidance of the guide RNA plasmid.
In this example, the DNA scaffold was artificially designed to provide a plurality of dCas binding sites, and the distance between dCas binding sites was adjusted by inserting bases of different lengths according to the distance between target enzymes, mainly because the distance between dCas binding sites affects the distance between target enzymes, and the distance between target enzymes affects the yield of target products.
The dCas protein-linker-target enzyme fusion plasmid comprises a plurality of different dCas protein-linker-target enzyme genes, and the plurality of different dCas protein-linker-target enzyme genes are linked together by ligation to the same expression plasmid. Each dCas protein-linker-target enzyme gene is mainly composed of dCas protein, linker and target enzyme, the target enzyme is connected with the corresponding dCas protein through the corresponding linker, and the length and flexibility of the linker are matched with the corresponding connected target enzyme. Each dCas protein-linker-target enzyme gene is mainly formed by connecting the target enzyme with the dCas protein through a linker by means of simple enzyme digestion, and the dCas protein-linker-target enzyme fusion plasmid is mainly formed by connecting a plurality of dCas protein-linker-target enzyme genes together in a plug-and-play manner. The linker was optimized to have a cleavage site, so that the dCas protein-linker-target enzyme fusion plasmid was assembled with a plurality of target enzymes. Wherein the dCas protein is selected from any two or three of dCas9, dCas12 and dCas 13. In this example, the plurality of linkers in the dCas protein-linker-target enzyme are the same peptide, and have the same length and flexibility. In other embodiments, the plurality of linkers in the dCas protein-linker-target enzyme have different lengths and flexibilities, and the dCas protein, the linker and the target enzyme correspond one to one.
The guide RNA plasmid comprises a plurality of different gRNA sites, the front section and the rear section of the DNA scaffold plasmid are respectively provided with an enzyme cutting site, so that the design of different gRNAs is convenient, dCas protein in the dCas protein-linker-target enzyme fusion plasmid can be specifically identified and guided to be combined with dCas binding sites in the DNA scaffold plasmid, and the dCas protein-linker-target enzyme fusion plasmid is anchored on the DNA scaffold plasmid, so that target enzymes of the dCas protein-linker-target enzyme fusion plasmid are arranged in different spaces and distances, and the catalysis is convenient.
Therefore, the above-mentioned multienzyme coupling localization system provided by the embodiment of the present invention utilizes the CRISPR system composed of guide RNA plasmid and functional module dCas protein, and performs spatial localization of the target enzyme by virtue of its recognition and targeting characteristics, so as to realize artificial layout of multienzyme in microenvironment for synthesizing the target product, and further realize efficient catalysis of the enzyme of the target product in the biosynthetic pathway according to a specific sequence.
In the following examples, E.coli strains BW25113 and trans5 α, both commonly used, were commercially available, with trans5 α for vector construction and BW25113 as the fermentation strain.
EXAMPLE 1 production of Paracetamol
Recombinant plasmid: pZE-pabABC-4ABH-nhoA and pZE-dCas9-linker-pabABC-dCas12-linker-4ABH-nhoA, pXS-DNA, pXS-sgRNA
The method for constructing the recombinant plasmid provided in this example specifically includes the following steps. Screening for genes encoding p-aminobenzoate synthase (pabABC), p-aminobenzoic acid hydroxylase (4 ABH), p-aminophenol acetylase (nhoA), dCas9, dCas12, derived from bacteria, fungi or protein engineering. After obtaining a target fragment by PCR amplification, carrying out enzyme digestion on the target fragment and the vector by using a proper enzyme, recovering the enzyme-digested fragment, and then inserting the recovered fragment into an expression plasmid pZE12-luc or pXS to respectively obtain recombinant plasmids: pZE-pabABC-4ABH-nhoA, pZE-dCas9-linker-pabABC-dCas12-linker-4ABH-nhoA, pXS-DNA, pXS-sgRNA.
Wherein, the recombinant plasmid pZE-pabABC-4ABH-nhoA is mainly obtained by connecting genes pabABC, 4ABH and nhoA to an Escherichia coli expression vector pZE 12-luc. The recombinant plasmid pZE-dCas9-linker-pabABC-dCas12-linker-4ABH-nhoA is mainly obtained by connecting genes dCas9-linker-pabABC, dCas12-linker-4ABH and nhoA to an escherichia coli expression vector pZE12-luc, wherein the genes dCas9-linker-pabABC are obtained by connecting dCas9 and pabABC through the linker shown in SEQ ID NO.1, and the genes dCas12-linker-4ABH are obtained by connecting dCas12 and 4ABH through the linker shown in SEQ ID NO. 1. The recombinant plasmid pXS-DNA scaffold was mainly obtained by ligating a DNA scaffold plasmid designed based on the sites recognized by dCas9 and dCsa12 to E.coli expression vector pXS. The recombinant plasmid pXS-sgRNA is mainly obtained by connecting the sgRNA gene to an Escherichia coli expression vector pXS.
Engineering bacteria for producing acetaminophen: recombinant Escherichia coli BW1 and BW2
The engineering bacteria for producing acetaminophen provided in this example has no special requirements for the type of host strain used for constructing expression plasmids, and the embodiment of the present invention adopts BW25113 strain as the initial host for constructing plasmids.
Firstly, selecting fresh BW25113 colony to inoculate into 4 mL LB culture medium, culturing for 8-12 h at 37 ℃, then inoculating 1 mL into 100 mL LB culture medium, culturing to OD at 37 ℃600When the length reaches 0.6 deg.C, centrifuging at 6000 rpm for 10 min at 4 deg.CAnd (3) washing the thallus with 10 mL of 10% precooled glycerol, centrifuging at 6000 rpm for 10 min, repeating the glycerol washing step again, pouring out the residual glycerol as much as possible after centrifuging, and finally adding a proper amount of 10% glycerol to resuspend cells to prepare competent cells. Respectively adding 90 mu L of competence into 2 mu L of recombinant plasmids pZE-pabABC-4ABH-nhoA and pZE-dCas9-linker-pabABC-dCas12-linker-4ABH-nhoA, pXS-DNA, pXS-CRISPR and pXS-Cas-target, placing for two minutes on ice, adding 600 mu L of LB culture medium after electrotransformation, washing out the cells after electrotransformation, reviving for 1 h at 37 ℃, coating the cells on an ampicillin resistant plate, culturing overnight in a constant temperature incubator at 37 ℃, and after bacteria grow on the plate, selecting the bacteria to culture in 4 mL of LB culture medium containing ampicillin resistance at 37 ℃ for 8-10 h to obtain the engineering bacteria for producing acetaminophen: the Escherichia coli strain containing the recombinant plasmid pZE-pabABC-4ABH-nhoA is expressed by recombinant Escherichia coli BW 1; coli strain containing recombinant plasmid pZE-dCas9-linker-pabABC-dCas12-linker-4ABH-nhoA, pXS-DNA, pXS-sgRNA, expressed as recombinant E.coli BW 2.
Comparison of the applications of recombinant Escherichia coli BW1 and BW2
Fresh recombinant Escherichia coli BW1 and BW2 engineering single colonies are selected on a plate and inoculated into 4 mL LB test tubes containing corresponding antibiotics, after the culture is carried out for 8h at 37 ℃, the test tubes are transferred into 50 mL culture media containing the corresponding antibiotics to carry out fermentation culture, the inoculation amount is 2 percent of volume ratio, the fermentation temperature is 30 ℃ or 37 ℃, and the rotation speed is 200 rpm. Wherein the culture medium: 2 g ∙ L-1MOPS，20 g∙L-1Glucose, 10 g ∙ L-1Glycerol, 5 g ∙ L-1Yeast powder, 6 g ∙ L-1 NaHPO4，0.5 g∙L-1 NaCl，3 g∙L-1 KH2PO4，2 g∙L-1 NH4Cl，246.5 mg∙L-1 MgSO4，14.7 mg∙L-1 CaCl2And adding corresponding antibiotics according to actual conditions.
The inducer IPTG with the final concentration of 0.5 mM is added at the beginning of fermentation, and part of fermentation liquor is taken out every 12h for measuring the growth condition of the thalli and the yield of the target product acetaminophen, and the result is shown in figure 2. As can be seen from fig. 2: the yield of the acetaminophen synthesized by fermentation culture of the recombinant escherichia coli BW1 can reach 20 m/L, and the yield of the acetaminophen synthesized by fermentation culture of the recombinant escherichia coli BW2 can reach 220 m/L. Thus, the yield of acetaminophen synthesized using recombinant E.coli BW2 was much higher than that synthesized using recombinant E.coli BW 1.
The fermentation liquids obtained by fermentation culture of recombinant escherichia coli BW1 and BW2 were tested by HPLC analysis method, and the test results are shown in fig. 3 and fig. 4, respectively. Wherein, the detection conditions are as follows:
a chromatographic column: separating the column: diamonsil C18, ID 5 μm, 250X 4.6 mm;
mobile phase: a is methanol, B is 1 per mill trifluoroacetic acid aqueous solution, the column temperature is 40 ℃, the flow rate is 1 mL/min, and the detection wavelength is 280 nm. The gradient elution procedure is shown in table 1 below:
TABLE 1 Acetaminophen gradient elution procedure
Figure DEST_PATH_IMAGE001
As can be seen from fig. 3 and 4, the peak-off times of acetaminophen in fig. 3 and 4 are substantially consistent, from which it can be seen that: the multi-enzyme coupling positioning system provided by the embodiment can be used for synthesizing acetaminophen, and the yield of acetaminophen can be improved.
EXAMPLE 2 production of Ferulic acid
Recombinant plasmid: pZE-TyrB-TAL-HpaBC-COMT, pZE-dCas9-linker-TyrB-dCas12-linker-TAL-HpaBC-COMT, pXS-DNA, pXS-sgRNA
The method for constructing the recombinant plasmid provided in this example specifically includes the following steps. Screening coding pairs which are derived from bacteria, fungi or protein engineering, carrying out PCR amplification on TyrB, TAL, HpaBC, COMT, dCas9, dCas12, DNA and sgRNA to obtain a target fragment, then carrying out enzyme digestion on the target fragment and a vector by using a proper enzyme, recovering the fragment after enzyme digestion, and then inserting the fragment into an expression plasmid pZE12-luc or pXS to respectively obtain a recombinant plasmid: pZE-TyrB-TAL-HpaBC-COMT, pZE-dCas9-linker-TyrB-dCas12-linker-TAL-HpaBC-COMT, pXS-DNA, pXS-sgRNA.
Wherein the recombinant plasmid pZE-TyrB-TAL-HpaBC-COMT is mainly obtained by connecting genes TyrB, TAL, HpaBC and COMT to an escherichia coli expression vector pZE 12-luc. The recombinant plasmid pZE-dCas9-linker-TyrB-dCas12-linker-TAL-HpaBC-COMT is mainly obtained by connecting genes dCas9-linker-TyrB, dCas12-linker-TAL, HpaBC and COMT to an escherichia coli expression vector pZE12-luc, wherein the genes dCas9-linker-TyrB are obtained by connecting dCas9 and TyrB to a linker shown in SEQ ID NO.1, and the genes dCas12-linker-TAL are obtained by connecting dCas12 to TAL to a linker shown in SEQ ID NO. 1. The recombinant plasmid pXS-DNA scaffold was mainly obtained by ligating a DNA scaffold designed based on the sites recognized by dCas9 and dCsa12 to E.coli expression vector pXS. The recombinant plasmid pXS-sgRNA is mainly obtained by connecting the sgRNA gene to an Escherichia coli expression vector pXS.
Engineering bacteria for producing ferulic acid: recombinant Escherichia coli Q1 and Q2
The engineering bacteria for producing ferulic acid provided by the embodiment has no special requirements on the types of host strains for constructing expression plasmids, and the embodiment of the invention adopts BW25113 strain as the initial host for constructing plasmids.
Firstly, selecting fresh BW25113 colony to inoculate into 4 mL LB culture medium, culturing for 8-12 h at 37 ℃, then inoculating 1 mL into 100 mL LB culture medium, culturing to OD at 37 ℃600When the growth reaches 0.6 ℃, centrifuging at 6000 rpm for 10 min at the temperature of 4 ℃ to collect thalli, washing with 10 mL of 10% precooled glycerol, centrifuging at 6000 rpm for 10 min, repeating the glycerol washing step again, pouring the residual glycerol to the greatest extent after centrifuging, and finally adding a proper amount of 10% glycerol to resuspend cells to prepare competent cells. Respectively adding 90 mu L of competence into 2 mu L of recombinant plasmids pZE-TyrB-TAL-HpaBC-COMT and pZE-dCas9-linker-TyrB-dCas12-linker-TAL-HpaBC-COMT, pXS-DNA and pXS-sgRNA, placing the mixture on ice for two minutes, adding 600 mu L of LB culture medium after electrotransformation, washing the cells after electrotransformation, reviving the cells for 1 h at 37 ℃, coating the mixture on an ampicillin resistance plate, culturing the mixture in a constant temperature incubator at 37 ℃ overnight, and selecting the bacteria to culture the bacteria in 4 mL of LB culture medium containing ampicillin resistance at 37 ℃ for 8-10h, obtaining engineering bacteria for producing ferulic acid: the Escherichia coli strain containing the recombinant plasmid pZE-TyrB-TAL-HpaBC-COMT is expressed by recombinant Escherichia coli Q1; coli strain containing recombinant plasmid pZE-dCas9-linker-TyrB-dCas12-linker-TAL-HpaBC-COMT, pXS-DNA, pXS-sgRNA, expressed as recombinant E.coli Q2.
Comparison of applications of recombinant Escherichia coli Q1 and Q2
Fresh recombinant Escherichia coli Q1 and Q2 engineering single colonies are selected on a plate and inoculated into 4 mL LB test tubes containing corresponding antibiotics, after the culture is carried out for 8h at 37 ℃, the culture is transferred into a shake flask containing 50 mL culture medium containing the corresponding antibiotics for fermentation culture, the inoculation amount is 2 percent of volume ratio, the fermentation temperature is 30 ℃ or 37 ℃, and the rotation speed is 200 rpm. Wherein the culture medium: 2 g ∙ L ‾1MOPS，20 g∙L‾1Glucose, 10 g ∙ L ‾1Glycerol, 5 g ∙ L ‾1Yeast powder, 6 g ∙ L ‾1 NaHPO4，0.5 g∙L‾1NaCl，3 g∙L‾1 KH2PO4，2 g∙L‾1 NH4Cl，246.5 mg∙L‾1 MgSO4，14.7 mg∙L‾1 CaCl2And adding corresponding antibiotics according to actual conditions.
The final concentration of 0.5 mM inducer IPTG was added at the beginning of the fermentation, and part of the fermentation broth was taken out every 12h for measuring the growth of the cells and the yield of ferulic acid by the target product, the results are shown in FIGS. 5 and 6. Wherein, the figure 5 shows that the yield of the ferulic acid synthesized by the fermentation culture of the recombinant Escherichia coli Q1 can reach 63 m/L; FIG. 6 shows that the yield of ferulic acid synthesized by fermentation culture of recombinant Escherichia coli Q2 can reach 601 m/L. By comparing fig. 5 and fig. 6, it is found that: the yield of ferulic acid synthesized by recombinant Escherichia coli Q2 is much higher than that of ferulic acid synthesized by recombinant Escherichia coli Q1.
The fermentation liquids obtained by fermentation culture of recombinant Escherichia coli Q1 and Q2 were tested by HPLC analysis, and the test results are shown in FIG. 7 and FIG. 8, respectively. Wherein, the target product detects ferulic acid under the following conditions:
a chromatographic column: separating the column: diamonsil C18, ID 5 μm, 250X 4.6 mm;
mobile phase: a is methanol, B is 1 per mill formic acid aqueous solution, the column temperature is 40 ℃, the flow rate is 1 mL/min, and the detection wavelength is 280 nm. The gradient elution procedure is shown in table 2 below:
TABLE 2 Ferulic acid gradient elution schedule
Figure 543423DEST_PATH_IMAGE002
As can be seen from fig. 7 and 8, the ferulic acid in fig. 7 and 8 has substantially the same peak-off time, from which it can be seen that: the multi-enzyme coupling positioning system provided by the embodiment can be used for synthesizing ferulic acid and improving the yield of the ferulic acid.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention and not to limit it; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art will understand that: modifications to the specific embodiments of the invention or equivalent substitutions for parts of the technical features may be made; without departing from the spirit of the present invention, it is intended to cover all aspects of the invention as defined by the appended claims.
SEQUENCE LISTING
<110> Beijing university of chemical industry
<120> multi-enzyme coupling positioning system and construction method and application thereof
<130> 2021
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 45
<212> DNA
<213> Artificial sequence
<400> 1
GGAGG TGGCG GGTCA GGGGG CGGTG GATCC GGCGG AGGGG GTTCA

Claims (10)

1. A multi-enzyme coupled localization system, which is characterized in that: comprises a dCas protein-linker-target enzyme fusion plasmid, a guide RNA plasmid and a DNA scaffold plasmid, and the dCas protein-linker-target enzyme fusion plasmid is anchored on the DNA scaffold plasmid under the guide of the guide RNA plasmid; wherein the dCas protein-linker-target enzyme fusion plasmid comprises a plurality of dCas proteins, a plurality of linkers and a plurality of target enzymes, and the target enzymes are linked to the functional proteins via the linkers; a plurality of the target enzymes are enzymes required in a biosynthetic pathway of a target product; the DNA scaffold plasmid is a DNA fragment comprising a plurality of dCas binding sites, the dCas binding sites being arranged in a sequence such that each dCas binding site corresponds to one dCas protein under the direction of the guide RNA plasmid such that a plurality of dCas proteins are anchored to the DNA scaffold plasmid.
2. The multiple-enzyme-coupled localization system according to claim 1, wherein: the distance between a plurality of the dCas binding sites in the DNA scaffold is adjusted by inserting bases of different lengths.
3. The multi-enzyme conjugate according to claim 1 or 2, characterized in that: chassis cells are also included, and the dCas protein-linker-target enzyme fusion plasmid, the guide RNA plasmid, and the DNA scaffold plasmid are co-introduced into the chassis cells.
4. The multiple-enzyme-coupled localization system according to claim 3, wherein: the dCas protein is selected from any two or three of dCas9 protein, dCas12 protein and dCas13 protein.
5. The multiple-enzyme-coupled localization system according to claim 4, wherein: the target product is ferulic acid, acetaminophen, arbutin or dencichine.
6. A construction method of a multi-enzyme coupling positioning system comprises the following steps:
constructing a recombinant plasmid to provide a plurality of target enzymes, a plurality of linkers and a plurality of dCas proteins, respectively carrying out PCR amplification treatment on the plurality of target enzymes and the plurality of dCas proteins, and connecting the dCas proteins with the target enzymes through the linkers to form a plurality of dCas protein-linker-target enzyme genes; connecting a plurality of dCas protein-linker-target enzyme genes to the same expression plasmid to form a dCas protein-linker-target enzyme recombinant plasmid; performing PCR amplification treatment on a DNA scaffold and guide RNA respectively to prepare a DNA scaffold recombinant plasmid and a guide RNA recombinant plasmid, wherein the DNA scaffold is a DNA fragment containing a plurality of dCas binding sites, bases with different lengths can be inserted among the dCas binding sites according to requirements to adjust the distance among the target enzymes, the dCas binding sites are arranged in sequence, and each dCas binding site corresponds to one dCas protein so that the dCas proteins are anchored on the DNA scaffold plasmid;
and (3) constructing an engineering bacterium, and simultaneously transforming the dCas protein-linker-target enzyme recombinant plasmid, the DNA scaffold recombinant plasmid and the guide RNA recombinant plasmid into a chassis cell to construct a multi-enzyme coupling positioning system.
7. The method for constructing a multi-enzyme coupled localization system according to claim 6, wherein: the method for constructing the dCas protein-linker-target enzyme recombinant plasmid comprises the steps of firstly connecting the target enzyme with dCas protein through the linker in an enzyme digestion connection mode to form the dCas protein-linker-target enzyme gene; and connecting a plurality of dCas protein-linker-target enzyme genes to the same expression plasmid in a plug-and-play connection manner to form the dCas protein-linker-target enzyme recombinant plasmid.
8. The method for constructing a multi-enzyme coupled localization system according to claim 7, wherein: a plurality of the target enzymes are key enzymes in the biosynthetic paracetamol pathway, including pabABC, 4ABH and nhoA, and the dCas protein-linker-target enzyme recombinant plasmid is pZE-dCas9-linker-pabABC-dCas12-linker-4 ABH-nhoA.
9. The method for constructing a multi-enzyme coupled localization system according to claim 7, wherein: a plurality of target enzymes are key enzymes in the biosynthetic ferulic acid pathway, including TyrB, TAL, HpaBC and COMT, and the dCas protein-linker-target enzyme recombinant plasmid is pZE-dCas9-linker-TyrB-dCas 12-linker-TAL-HpaBC-COMT.
10. Use of a multi-enzyme coupled localization system according to any of claims 1 to 5, comprising: inoculating the multienzyme coupling positioning system into a culture medium according to the inoculation amount of 1-5% of the volume ratio, adding an inducer, and performing fermentation treatment at 28-40 ℃ to produce a target product; the carbon source in the medium is glucose, glycerol or a combination of the two.
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