CN114426982B - Method for reprogramming cell metabolism and application thereof - Google Patents
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Abstract
The invention discloses a method for reprogramming cell metabolism and application thereof, which belongs to the field of genetic engineering, and utilizes sensitive nodes of a cell metabolism network to reprogram the metabolism network, and mainly comprises three parts of CRISPRi-based double-gene combination knockout, CRISPRi-based high-throughput screening and CRISPRi-based gene interaction analysis.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to a method for intelligent metabolic reprogramming of cells.
Background
Engineering biology is a interdisciplinary field of engineering and science in the 21 st century, and is of great help to explore basic laws of life activities and biotechnology breakthrough innovation. It enables us to design and build new biomolecular assemblies, networks and pathways to reprogram organisms. Its core goal is to apply engineering biotechnology to precisely control, regulate and remodel the metabolic network of microbial cell factories to achieve the synthesis of the intended product.
Control, regulation and remodeling of the cellular metabolic network, also known as metabolic reprogramming, was first widely found in the cancer field. In the field of engineering biology or synthetic biology, metabolic reprogramming is the fundamental approach to achieve engineering biological goals. Thus, accurate reprogramming of cell metabolism is critical for rapid construction of cell factories. However, it is very difficult to efficiently study the reprogramming of microbial metabolism. This is primarily because there are thousands of metabolites and genes in the cell that cross-link to form a highly complex metabolic network with chaotic character. In addition, the operation concept and the method of the metabolic network are very deficient, and an effective, simple and low-cost operation tool is lacked, and a corresponding theory is lacked to help people select an entry point for the operation of the metabolic network. Therefore, the reprogramming of cells by the conventional method can basically only use an exhaustive error-testing mode, and a great deal of time, manpower, material resources and cost are required to obtain high yield of the target product.
Different environmental signals, such as light, temperature, pH, pressure, etc., can stimulate biological cells to initiate the natural metabolic reprogramming of the cells. The natural metabolic reprogramming is the stimulation of cells in response to different physiological signals by sensing signals through signal sensors on the surface and then inducing changes in the metabolic network within the cells. Such sensors of the cell-aware environment and their associated pathways formed during evolution are critical for the survival of cells in complex and diverse environments, and it is believed that these sensors control the sensitive nodes of the cellular metabolic network. The present invention therefore proposes a method for achieving reprogramming of cellular metabolism using the combined intervention of these natural signal sensors. As shown in figure 1, the method can directly perform combined operation on sensitive nodes of a cell metabolism network, besides the sensitive nodes, the method can also perform operation on any nodes on the whole genome level, has the characteristic of high efficiency, and can be considered as intelligent metabolism reprogramming.
Currently, there are few methods of interaction research on multiple genes from the global layer, and the cost is high. It would therefore be of great interest and value to develop a method for intelligent metabolic reprogramming of organisms that is simple to implement, inexpensive and efficient to operate.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide a method for reprogramming cellular metabolism.
The basic principle of the invention is to construct a CRISPRi-based double-gene knockout library and a biosensor-based high-throughput screening method, couple a survivor-based gene interaction analysis platform, and select vibrio FA2 which rapidly grows on the next-generation industrial chassis as an expression strain.
In a first aspect, the present invention provides a method for reprogramming cellular metabolism, comprising the steps of:
s1, constructing a CRISPRi-based double sgRNA knockdown strain library;
s2, performing high-throughput screening on cells;
s3, gene interaction analysis based on survival rate.
Further, the step S1 includes the steps of:
s1.1, constructing a tool plasmid:
constructing two sgRNA expression frames on a pE1A plasmid, and inserting enzyme cutting sites of toxic proteins ccdB and Golden Gate between the two expression frames;
the tool plasmid can be used for carrying out Golden Gate connection with fragments of the library, so as to realize construction of double sgRNA plasmid libraries with different gene targets.
S1.2, constructing a recombinant fragment of a double sgRNA library:
synthesizing n pairs of primers of a target library, using the tool plasmid obtained in the step S1.1 as a template, amplifying double sgRNA library fragments by PCR, amplifying the library fragments by PCR, and recovering the double sgRNA library recombinant fragments by agarose gel electrophoresis and gel running;
enzyme digestion and enzyme ligation are carried out through a Golden Gate assembly method, and the library fragments are connected to the tool plasmid vector obtained in the S1.1 to obtain a recombination system of a double sgRNA library;
s1.3, constructing a plasmid of a double sgRNA library:
the recombinant system of the double sgRNA library is transformed into E.coli K12 and coated on a solid plate with screening resistance, so that the recombinant system approximately grows 10×n 2 A plurality of transformants;
eluting with a liquid culture medium to obtain a double sgRNA library plasmid strain, using the strain as seed liquid, and extracting plasmids after expanding culture in a screening resistance culture medium to obtain plasmids of the double sgRNA library;
s1.4, constructing a CRISPRi-based double sgRNA knockout library strain:
the double sgRNA library plasmid obtained in S1.3 is transformed into a strain containing pS8K plasmid of dCAS9 expression module, thus obtaining a CRISPRi-based double sgRNA knockdown library strain, and activity verification is carried out.
Further, the sequences of the N20 portions of the N pairs of primer sequences in S1.2 were designed according to different library-building genes.
Preferably, the strain described in S1.4 is Vibrio FA2.
Further, the step S2 includes the steps of:
and coupling a biosensor corresponding to a target product of the double sgRNA knockdown library strain with a downstream reporter gene egfp, constructing into a pS8K plasmid containing a dCAS9 expression module, converting into the strain obtained in S1.4, and performing cell sorting by a flow cell sorting technology.
Preferably, the biosensor is selected from any one of a ribosome switch, a regulatory protein or a transcriptional regulator.
A CRISPRi double gene knockout system can be used for generating a knockout library, then a biosensor is used for sensing metabolite changes in cells, further downstream fluorescent protein expression can be activated, and high-throughput screening can be performed by measuring the fluorescent intensity of different cells.
By means of the flow cell sorting technology, the cells with high fluorescence values in different cells of the knockdown library can be sorted out in a high throughput mode.
Further, the step S3 includes the steps of:
extracting cell plasmids with higher fluorescence values obtained in S2 compared with the control group, amplifying a variety of fragments through PCR (polymerase chain reaction), carrying out second generation sequencing, and carrying out gene interaction analysis based on survival rate by using an analysis program package, wherein the interaction analysis by using the analysis program package comprises the following steps:
s3.1, establishing a NiNj sequence library of a target gene;
s3.2, extracting the sequence of the double N20 region of the detected sequence by using a Python3 program;
s3.3, comparing the measured sequence with an established double N20 sequence library by using a Bowtie2 algorithm;
the results of the S3.4 comparison are specifically presented as Excel tables and heatmaps.
In a second aspect, the invention provides the use of a method of reprogramming cell metabolism in microfabrication or reprogramming microorganism metabolism.
In a third aspect, the present invention provides a knockdown library strain, characterized in that: including CRISPRi-based double sgRNA library plasmids and biosensors for high throughput screening.
Preferably, the strain is vibrio FA2.
In summary, the invention is a novel, simple and effective, low cost intelligent metabolic reprogramming platform that can be used to operate a wider network of microbial metabolism.
Drawings
FIG. 1 is a schematic diagram of metabolic reprogramming in accordance with the present invention;
FIG. 2 is a technical roadmap of the invention;
FIG. 3 is a flow chart of the analysis of gene interactions of the present invention.
Detailed Description
The following description of the preferred embodiments refers to the accompanying drawings, which are included to provide a clear and easy understanding of the technical contents. The present invention may be embodied in many different forms of embodiments and the scope of the present invention is not limited to only the embodiments described herein.
The experimental methods used in the examples are conventional methods unless otherwise specified. The materials, reagents and the like used, unless otherwise specified, are all commercially available.
The invention provides a method for reprogramming cell metabolism and application thereof.
The following will specifically describe the Vibrio FA2 as an example:
LB liquid Medium (1L): 5g of yeast powder, 10g of peptone and 10g of sodium chloride.
LB3 liquid Medium (1L): 5g of yeast powder, 10g of peptone and 30g of sodium chloride.
LB3 solid Medium (1L): 1.6-2.0% (w/v) agar powder is added into LB3 liquid culture medium. Sterilizing with steam at 121deg.C under high temperature and high pressure for 20min.
VN inorganic salt medium (VN, 1L): 5g (NH) 4 ) 2 SO 4 ，15gNaCl，1gKH 2 PO 4 ，1gK 2 HPO 4 ，0.25gMgSO 4 ，0.01gCaCl 2 ，16.4mgFeSO 4 ·7H 2 O，10mgMnSO 4 ·H 2 O，0.3mgCuSO 4 ·5H 2 O，1mgZnSO 4 ·7H 2 O，0.02mgNiCl 2 ·6H 2 O, preparing distilled water.
The culture method comprises the following steps: vibrio FA2 was inoculated into LB3 liquid medium and cultured at 37℃and 220 rpm. If the bacteria are modified, corresponding antibiotics are added. Alternatively, vibrio FA2 may be inoculated into a VN mineral salt medium. Vibrio with dCAS9 and sgRNA double plasmids, and corresponding inducer, such as 0.1mM IPTG, is added to induce fluorescent protein expression, and 10mM arabinose is added to induce dCAS9 expression. When constructing the library, the recombinant system is transformed into vibrio, spread on LB3 solid medium, added with corresponding antibiotics, and placed in a 37 ℃ incubator for culture.
Example 1 construction of double sgRNA library tool plasmid
As shown in FIG. 2, two sgRNA expression cassettes were constructed on the pE1A plasmid, one containing the J23100 constitutive promoter, N20, the gRNA scaffold and the T0 terminator.
Then inserting a toxic protein ccdB between the two expression frames, simultaneously introducing a enzyme cutting site BbSI of Golden Gate, and adding a recognition site and a protecting base of the BbSI into a tool plasmid through primers SEQ ID NO.3 and SEQ ID NO. 4.
The specific sequence is as follows:
NO.3：5-GATCGAAGACGTGTTTTAGA GCTAGAAATAGCAAGT-3；
NO.4：5-GATCGAAGACACTAGTATTATACCTAGGACTGAG-3。
by utilizing the principle of homologous recombination, two sgRNA expression frames, the ccdB and BbSI restriction sites and recognition sites of toxic proteins are simultaneously introduced on the pE1A plasmid by adopting a full-plasmid PCR method through the above primers.
Example 2 construction of recombinant fragments of double sgRNA library
As shown in FIG. 2, n pairs of primers were synthesized to construct the corresponding library, and then fragments of the double sgRNA library were amplified by PCR using a plasmid containing the two sgRNA expression cassettes of example 1 as templates.
The sequences of the N pairs of primers are identical except for the N20 part (NNN in the sequence), and the N20 part sequences are designed according to different library genes. Specific primer sequences are shown in SEQ ID NO.3 and SEQ ID NO.4:
NO.3：5-GATCGAAGACACACTAGTNNNGTTTTAGAGCTAGAAATAGCAAGT-3；NO.4：5-GATCGAAGACTAAAACNNNACTAGTATTATACCTAGGACTGAG-3。
after PCR amplification of the library fragments, agarose gel electrophoresis was performed, after running the gel, the gel with the correct band cut was recovered by gel to obtain library fragments. The library fragments were then efficiently ligated to the tool plasmid described in example 1 by the enzyme digestion method using the Golden Gate assembly method to form a recombinant system of the double sgRNA library.
Example 3 construction of a double sgRNA library plasmid
After the recombinant system of the double sgRNA library was obtained in example 2, the recombinant system was constructed into E.coli K12 by a chemical transformation method, and then the recombinant system was coated on a solid plate having screening resistance (5. Mu.g/mL tetracycline in this example) to grow approximately 10n 2 Each transformant (about 1000 transformants per plate have been verified).
Eluting with LB liquid culture medium, mixing, adding 15% glycerol, and storing in-80deg.C refrigerator. The obtained plasmid strain of the double sgRNA library is used as seed bacteria, and plasmids are extracted after the obtained strain is amplified and cultured in LB culture medium containing 5 mug/mL tetracycline, namely the plasmids of the constructed double sgRNA library.
Taking this example as an example, a double sgRNA library targeting different targets can be constructed.
Example 4 construction and testing of CRISPRi-based double sgrnas knockdown library strains
After obtaining the plasmid of the double sgRNA library in example 3, the plasmid of the constructed double sgRNA library was transformed into a strain containing pS8K plasmid containing dCAS9 expression module.
In particular, different bacteria need to first verify whether the CRISPRi-based double sgRNA knockdown system is active.
For example, the reporter genes egfp and mrfp can be integrated by homologous recombination into the chromosome of Vibrio, and then N20 corresponding to both genes can be designed, and the double sgRNA expression cassette constructed on the plasmid of pE1A as described in example 2.
Then, pS8K plasmid containing dCAS9 expression module and plasmid containing double sgRNA expression cassette constructed into pE1A were transformed into Vibrio. The engineered Vibrio was inoculated into LB3 liquid medium containing 5. Mu.g/mL tetracycline and 5. Mu.g/mL chloramphenicol, and then 0.1mM IPTG and 10mM arabinose were added to induce expression of dCS 9 and fluorescent protein. By comparing the measured fluorescence intensity with the control group, it is demonstrated that the system is active in Vibrio if the fluorescence value is significantly reduced.
Example 5 construction and testing of Glycine-based biosensors
In example 4, a CRISPRi-based double sgRNA knockdown library strain was obtained, i.e. a combined knockdown effect on the corresponding genes of vibrio was produced. To enable high throughput screening of mixed libraries, the present invention constructs biosensors to sense changes in the corresponding metabolites inside the cell, while simultaneously stimulating expression of downstream reporter genes (e.g., egfp). Similar cell signal receptors include ribosomal switches, regulatory proteins, transcriptional regulatory factors, and the like. Thus, cells with high metabolite concentrations can be screened by detecting fluorescent signals.
In particular, glycine-ribosome switch was used as a screening tool for system verification in this example. On the basis of example 4, a glycine-ribosome switch was coupled to the downstream reporter gene egfp, and then constructed into the pS8K plasmid containing the dCAS9 expression module and transformed into Vibrio FA2. The library plasmid was then transformed into engineered Vibrio FA2. This senses a change in intracellular glycine concentration through the ribosome switch, and the intracellular eGFP fluorescence value increases accordingly.
Example 6 high throughput screening based on Glycine biosensors
After the entire system was constructed in example 5, a library of combinatorial knockdown of the target gene was generated by CRISPRi, and a glycine ribosome switch was able to sense the change in the corresponding glycine concentration in the cell. The downstream egfp gene is activated when the intracellular glycine concentration increases, and the fluorescence value of different cells varies. Further, cells with higher fluorescence values than the control group were sorted out by the flow cell sorting technique, i.e., cells with high intracellular glycine concentration were selected.
Example 7: CRISPRi-based gene interaction analysis platform for coupling survival rate of multi-gene knockout library
After the target cell populations were screened out using the high throughput screening method of example 6, plasmids for these cell populations were extracted. Then, the extracted plasmid is used as a template, a variety of fragments are amplified by PCR, and then the fragments are sent to a company for second generation sequencing. After high throughput sequencing results, no suitable procedure can be performed because the present invention is an open-ended method. Thus, the present invention specifically designs an analysis package that can be performed on these high throughput sequencing data, which is then subjected to this survival-based gene interaction analysis. By the program of the invention, the genes with ideal yield or phenotype can be obtained by single knockout, the genes with ideal yield or phenotype can be obtained by combined knockout, and the genes with growth disadvantages caused by knockout can be found out.
Specifically, as shown in the flowchart 3, the analysis method is divided into three steps:
1) Establishing a NiNj sequence library of a target gene (NiNj is a double N20 sequence);
2) Extracting the sequence of the double N20 region of the measured sequence by using a Python3 program;
3) Comparing the measured sequence with an established double N20 sequence library by using a Bowtie2 algorithm;
4) The results obtained by comparison are specifically presented as Excel tables and heat maps.
Example 8 application of microorganism Metabolic reprogramming based on the platform of the present invention in microbial manufacturing
The final strains and methods described in examples 1-7 were used to study the effect on intracellular metabolite production using genes targeting two-component systems. Specifically, histidine kinase in Vibrio FA2 is selected as a target. Because histidine kinase and its downstream response regulator together form a two-component system, changes in external environment, such as temperature, pH, pressure, etc., can be sensed, and thus the intracellular metabolic network changes, resulting in metabolic reprogramming.
Furthermore, we designed primers targeting all histidine kinases of Vibrio FA2 and constructed a gene interaction analysis platform based on the CRISPRi-based multi-gene knockout library coupling survival rate by the method of examples 1-7. The combination of knockdown of the series of histidine kinases obtained by screening then slightly increased the intracellular yield. And finally, independently constructing and verifying the genotypes obtained by sequencing analysis, wherein the final result is consistent with the result of interaction analysis.
EXAMPLE 9 application of microorganism Metabolic reprogramming on the basis of the platform of the present invention in microbial manufacturing
The final strains and methods described in examples 1-7 were used to study the effect on intracellular metabolite production using genes targeting the glycine metabolic pathway. Specifically, 24 genes related to glycine metabolism in vibrio FA2 are selected as targets. Among these genes are genes upstream of glycine metabolism and downstream of glycine metabolism.
Furthermore, we designed primers targeting all glycine metabolic pathway genes of Vibrio FA2, and constructed a gene interaction analysis platform based on the coupling survival rate of the multiple gene knockout library of CRISPRi by the method of examples 1-7. Then, a series of glycine metabolic pathway gene knockdown combinations are obtained through screening, so that the intracellular glycine yield can be improved. Finally, the genotypes obtained by sequencing analysis are independently constructed and verified, and finally, the result shows that the knockdown of the gene downstream of glycine metabolism can improve the glycine yield, which is consistent with the result of interaction analysis.
Implementation 10, use of metabolic reprogramming based on the platform of the present invention in physiological research of microorganisms
The final strains and methods described in examples 1-7 were used to study the effect on Vibrio FA2 resistance using genes targeting all two-component systems of Vibrio FA2. Specifically, histidine kinase in the vibrio FA2 is selected as a target point, and the influence of the gene on the resistance of the arc FA2 to ampicillin is explored by carrying out combined knockout on genes of 35 histidine kinases in the vibrio FA2.
Furthermore, we designed primers targeting all histidine kinases of Vibrio FA2 and constructed a gene interaction analysis platform based on the CRISPRi-based multi-gene knockout library coupling survival rate by the method of examples 1-7. Then, screening to obtain a series of histidine kinase knockdown can improve the drug resistance of vibrio FA2 to ampicillin; the knockdown of a series of histidine kinases can reduce the drug resistance of vibrio FA2 to ampicillin; the combined knockdown of a series of histidine kinases can improve the resistance of vibrio FA2 to ampicillin.
Furthermore, independent knockdown and combined knockdown construction and verification are carried out on the genotypes obtained by sequencing analysis, and the final result is consistent with the result of interaction analysis.
The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention without requiring creative effort by one of ordinary skill in the art. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.
Claims (5)
1. A method of reprogramming cellular metabolism comprising the steps of:
s1, constructing a CRISPRi-based double sgRNA knockdown strain library;
s2, performing high-throughput screening on cells;
s3, gene interaction analysis based on survival rate;
the step S1 comprises the following steps:
s1.1, constructing a tool plasmid:
constructing two sgRNA expression frames on a pE1A plasmid, and inserting enzyme cutting sites of toxic proteins ccdB and Golden Gate between the two expression frames;
s1.2, constructing a recombinant fragment of a double sgRNA library:
synthesizing n pairs of primers of a target library, using the tool plasmid obtained in the step S1.1 as a template, amplifying double sgRNA library fragments by PCR, purifying, and then, carrying out enzyme digestion and enzyme ligation by a Golden Gate method, and connecting the library fragments to the tool plasmid obtained in the step S1.1 to obtain a recombination system of the double sgRNA library;
the specific primer sequences are shown in SEQ ID NO.3 and SEQ ID NO.4:
NO.3：5-GATCGAAGACACACTAGTNNNGTTTTAGAGCTAGAAATAGCAAGT-3；
NO.4：5-GATCGAAGACTAAAACNNNACTAGTATTATACCTAGGACTGAG-3；
s1.3, constructing a plasmid of a double sgRNA library:
the recombinant system of the double sgRNA library is transformed into E.coli K12, coated on a solid plate with screening resistance, eluted by adopting a liquid culture medium to obtain a plasmid strain of the double sgRNA library, the strain is used as seed liquid, and the plasmid is extracted after being amplified in the culture medium with the screening resistance to obtain the plasmid of the double sgRNA library;
s1.4, constructing a CRISPRi-based double sgRNA knockout library strain:
transforming the double sgRNA library plasmid obtained in the step S1.2 into a strain containing pS8K plasmid of dCS 9 expression module to obtain a CRISPRi-based double sgRNA knockdown library strain, and performing activity verification;
the high throughput screening in S2 comprises the steps of:
coupling a biosensor corresponding to a target product of the double sgRNA knockdown library strain with a downstream reporter gene egfp, constructing a pS8K plasmid containing a dCAS9 expression module, converting the plasmid into the strain obtained in S1.4, and performing cell sorting by a flow cell sorting technology, wherein the biosensor is selected from any one of a ribosome switch, a regulatory protein or a transcription regulatory factor;
the step S3 comprises the following steps:
extracting cell plasmids with higher fluorescence values obtained in S2 compared with the control group, amplifying a variety of fragments through PCR (polymerase chain reaction), carrying out second generation sequencing, and carrying out gene interaction analysis based on survival rate by using an analysis program package, wherein the interaction analysis by using the analysis program package comprises the following steps:
s3.1, establishing a NiNj sequence library of a target gene, wherein NiNj is a double N20 sequence;
s3.2, extracting the sequence of the double N20 region of the detected sequence by using a Python3 program;
s3.3, comparing the measured sequence with an established double N20 sequence library by using a Bowtie2 algorithm;
the results of the S3.4 comparison are specifically presented as Excel tables and heatmaps.
2. The method of reprogramming cellular metabolism of claim 1, wherein the sequences of N20 portions of the N pairs of primer sequences in S1.2 are designed according to different pooling genes.
3. The method of reprogramming cellular metabolism of claim 1, wherein the strain in S1.4 is vibrio FA2.
4. A knockdown library strain constructed by the method of any one of claims 1-3, characterized in that: including CRISPRi-based double sgRNA library plasmids and biosensors for high throughput screening.
5. The knockdown library strain of claim 4, wherein: the strain is vibrio FA2.
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