Method for screening high-yield strains by utilizing biosensor to assist transposon engineering and application of method

Abstract

The invention relates to a method for screening high-yield strains by utilizing biosensor-assisted transposon engineering and application of the method. The method comprises the following steps: constructing plasmids for controlling and expressing reporter genes and resistance genes in response to a biosensor in a target host; randomly integrating the synthesis module of the target product into the genome of the target host through a transposition system; screening a bacterial colony with tolerance from the target host on a plate with corresponding resistance, and selecting a bacterial colony with high reporter gene output intensity so as to directionally screen a host strain with high yield of a target product. According to the method, a biosensor is adopted to dynamically respond to the actual yield of a target product in real time, and high-yield strains can be quickly and efficiently screened out.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a method for screening high-yield strains by utilizing biosensors to assist transposon engineering and its application. Background Art

[0002] Protocatechuic acid, also known as 3,4-dihydroxybenzoic acid, is a secondary metabolite with unique physiological activity and pharmacological properties. Protocatechuic acid has a variety of biological activities for different molecular targets: it can prevent some chronic degenerative, cardiovascular and neurodegenerative diseases; it can be used as a component of a new type of synthetic polymer and plastic; it has a complex with good electrochemical activity combined with polysaccharides; it can synthesize bioplastics; it has antibacterial, anti-inflammatory, antioxidant, anti-aging and other pharmacological properties, and can also be potentially used in drugs and functional foods. Based on the excellent properties of protocatechuic acid, its demand in the fields of medicine, feed, environmental protection, etc. is growing, and it has very obvious production significance.

[0003] Transposon mutant strain technology is one of the commonly used strain construction strategies in the field of synthetic biology and metabolic engineering. By placing the target gene and the resistance gene within the transposon recognition sequence, the strain with the target gene integration can be quickly obtained through resistance screening. The lack of high-throughput screening methods for biosynthetic protocatechuic acid strains based on related technologies is the main challenge for systematic optimization of their synthesis pathways. In addition, the residual problem of transposon resistance also brings additional metabolic burden to the strain, and in industrial applications, it also faces the potential risk of resistance genes escaping from the environment due to horizontal transfer. Summary of the invention

[0004] The present invention aims to solve one of the technical problems in the related art at least to a certain extent. To this end, one object of the present invention is to provide a method for screening high-yield strains using biosensor-assisted transposon engineering and its application. The method uses a biosensor to respond to the actual yield of the target product in real time and dynamically, and can quickly and efficiently screen out high-yield strains.

[0005] To this end, in one aspect of the present invention, the present invention provides a method for screening high-yield strains using biosensor-assisted transposon engineering, which comprises:

[0006] constructing plasmids that express reporter genes and resistance genes in response to biosensor control in the target host;

[0007] Randomly integrating the synthetic module of the target product into the genome of the target host through a transposition system;

[0008] The target host is screened for tolerant colonies on a plate with corresponding resistance, and colonies with high reporter gene output intensity are selected to selectively screen host strains with high yield of target products.

[0009] According to the present invention, a method for screening high-yield strains using biosensor-assisted transposon engineering uses a biosensor to dynamically respond to the actual yield of the target product in real time, thereby quickly and efficiently screening high-yield strains. Since the biosensor response can directly respond to the target product, the product synthesis module can be directly used in a non-resistant transposon system, so that the modified strain does not carry any resistance screening label after the biosensor plasmid is removed, thereby eliminating the metabolic burden brought by the resistance gene in downstream industrial applications.

[0010] In addition, the method for screening high-yield strains using biosensor-assisted transposon engineering according to the above embodiment of the present invention may also have the following additional technical features:

[0011] Optionally, the target product is protocatechuic acid.

[0012] Furthermore, the target host is Pseudomonas putida, and may also be other chassis cells.

[0013] Furthermore, the biosensor is a protocatechuic acid response system from Streptomyces coelicolor, in which PcaV acts as a repressor protein and releases P LV The inhibition of the promoter turns on the expression of downstream genes. Depending on the target product, the choice of the biosensor module can also be a system that responds to other target products.

[0014] Furthermore, the reporter gene is green fluorescent protein eGFP, and the resistance gene is gentamicin resistance gene Gm. The reporter gene and resistance gene can be other genes that are compatible with the target host.

[0015] Optionally, the transposition system is a non-replicating Tn5 transposition system, the resistance screening tag is removed from the transposition system, and the gene required for the synthesis of the target product is included in the transposition system. The transposition system can also be Himar1, PiggyBac, etc.

[0016] Furthermore, the target host is screened on a gentamicin resistance plate, and colonies with strong green fluorescent protein signals are selected to selectively screen host strains with high yield of the target product. The screening method may also be a high-throughput screening technology such as flow cytometry or microfluidics.

[0017] Optionally, the method comprises the following steps:

[0018] The nucleotide sequence is as shown in SEQ ID NO:1. LVThe -eGDP-Gm vector was cut with EcoRI-HindIII and ligated with the pRK415 vector cut with EcoRI-HindIII to obtain the protocatechuic acid-responsive plasmid pRK415-PcaV-P LV -eGDP-Gm;

[0019] The protocatechuic acid response plasmid pRK415-PcaV-P LV -eGDP-Gm was electrotransformed into Pseudomonas putida competent cells to obtain a protocatechuic acid responsive strain;

[0020] The pRL27-Tn5 transposition vector with a nucleotide sequence as shown in SEQ ID NO: 2 was cut by BamHI-XhoI, and the nucleotide sequence after BamHI-XhoI cutting was as shown in SEQ ID NO: 3 as the protocatechuic acid synthesis module AsbF-AroG fbr Ligation to obtain the transposition plasmid pRL27-Tn5-AsbF-AroG fbr -Tn5;

[0021] The transposition plasmid pRL27-Tn5-AsbF-AroG fbr -Tn5 was electrotransformed into the protocatechuic acid responsive strain, and screened on a gentamicin resistance plate to select colonies with strong green fluorescent protein signals, so as to selectively screen for protocatechuic acid high-yielding strains.

[0022] Thus, the high-yield strain can relieve PcaV from the promoter P through the biosensor response module when protocatechuic acid is produced. LV The inhibition of Tn5 can turn on the expression of downstream genes eGFP and Gm. Due to the randomness of the Tn5 transposition system, with the assistance of biosensors, it is possible to screen non-resistant high-yield transposition strains in a targeted manner. Compared with traditional transposition systems, due to the limitation of the types of resistance tags, the number of transpositions is limited in most cases, and a lot of screening work is required to obtain high-yield strains. Therefore, the method of using biosensors to assist transposon engineering to screen high-yield strains is more competitive in actual production applications.

[0023] Construction of the protocatechuic acid-responsive module PcaV-P in Pseudomonas putida LV -eGFP-Gm, protocatechuic acid production module AsbF-AroG fbrThe Tn5 transposition system is used to randomly insert the gene into the genome of Pseudomonas putida. Once the transposition takes effect, the protocatechuic acid produced by Pseudomonas putida will be responded to by its own protocatechuic acid sensor, and it will express eGFP and Gm resistance. Unlike the blind screening of the traditional transposition system, this method only needs to screen strains with strong fluorescent signals on gentamicin plates to selectively screen strains with high protocatechuic acid production. In addition, the indirect screening of sensors makes the transposition system unnecessary for antibiotic markers, which provides a new idea for how to efficiently screen with the traditional transposition method.

[0024] In the second aspect of the present invention, the present invention proposes a protocatechuic acid high-producing strain screened and prepared by the above method.

[0025] In the third aspect of the present invention, the present invention proposes the use of the above-mentioned protocatechuic acid high-yielding strain in the biosynthesis of protocatechuic acid.

[0026] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The principle of the method for screening high-yield strains using biosensor-assisted transposon engineering according to an embodiment of the present invention;

[0028] Figure 2 The protocatechuic acid yields of different transposase strains after screening according to the embodiments of the present invention. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is described below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not exclude the existence of other method steps before and after the combination step or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to limit the scope of the present invention. The change or adjustment of the relative relationship thereof shall also be regarded as the scope of the present invention without substantially changing the technical content.

[0030] In order to better understand the above technical scheme, the exemplary embodiments of the present invention are described in more detail below. Although exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present invention and to enable the scope of the present invention to be fully communicated to those skilled in the art.

[0031] The test materials used in the present invention are all common commercial products and can be purchased on the market; the experiments involved are all conventional experimental methods unless otherwise specified.

[0032] Sources of materials used: Pseudomonas putida S12, Escherichia coli strain TOP10 and DH5alpha-λ are commercially available, of which TOP10 strain and DH5alpha-λ are used for vector construction. pRK415 vector is commercially available. Phusion high-fidelity DNA polymerase and restriction endonuclease were purchased from Xiamen Lulong Biotechnology Development Co., Ltd. Plasmid extraction kit, DNA purification kit, gel recovery kit and genomic DNA extraction kit were purchased from Hangzhou Bio-Tech.

[0033] LB medium composition: 10 g L -1 Peptone, 5 g L -1 Yeast extract, 5g·L -1 NaCl, make up to 1L with double distilled water, and sterilize at 0.1Mpa pressure and 121℃ for 20min.

[0034] The composition of TB-Tet solid medium is: 10 g·L -1 Tryptone, 5 g L -1 Yeast extract, 10 g L -1 NaCl, 15 g L -1 Agar, 100 μg ml -1 Tet.

[0035] LB+Gm:10g·L -1 Tryptone, 5 g L -1 Yeast extract, 10 g L -1 NaCl, 15 g L -1 Agar, 34 μg mL -1 Gm.

[0036] The principle of the method for screening high protocatechuic acid producing strains using biosensor-assisted transposon engineering in this application is shown in Figure 1 :

[0037] The protocatechuic acid production module AsbF-AroG fbr After being randomly inserted into the genome, it responds to protocatechuic acid to express eGFP and Gm through the biosensor module. Based on this, only the engineered bacteria that successfully produce protocatechuic acid can survive the selection pressure of gentamicin. In addition, the production concentration of protocatechuic acid is reflected in the expression level of eGFP, making transposition screening more targeted and eliminating the need to carry labels during transposition, thus realizing the use of biosensors to assist in the screening of high-yield strains by transposon engineering without resistance labels.

[0038] In the following examples, the primers and templates used for PCR amplification are shown in Table 1.

[0039] Table 1 Primers and templates used for PCR amplification

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0047] Example 1 Construction of a biosensor response module

[0048] The biosensor is a protocatechuic acid response system from Streptomyces coelicolor (PcaV acts as a repressor protein that releases P LV The reporter gene is green fluorescent protein (eGFP), and the resistance gene is gentamicin resistance gene (Gm).

[0049] Specifically, the nucleotide sequence of PcaV-P shown in SEQ ID NO: 1 was synthesized by Nanjing GenScript Co., Ltd. LV -eGDP-Gm vector, cut by EcoRI-HindIII, and recover the target band by DNA purification kit to obtain gel recovery product; connect with pRK415 vector T4 cut by EcoRI-HindIII and transfer into Escherichia coli Top10 competent cells to extract plasmid pRK415-PcaV-P LV -eGDP-Gm, and sequencing was performed to ensure that the result was completely consistent with the designed plasmid DNA sequence, thus obtaining the biosensor response module.

[0050] Example 2 Obtaining a Tn5 transposition system without a screening tag

[0051] The transposition system is a non-replicating Tn5 transposition system, in which the transposon contains the gene required for the synthesis of the target product. The gene required for the synthesis of the target product is a protocatechuic acid synthesis module.

[0052] Specifically, the pRL27-Tn5 transposase vector synthesized by Nanjing GenScript Co., Ltd. (nucleotide sequence as shown in SEQ ID NO: 2) was used to remove the resistance screening tag, and then digested with BamHI-SalI, and then digested with BamHI-XhoI, and then with the protocatechuic acid synthesis module AsbF-AroG fbr (The nucleotide sequence synthesized by Nanjing GenScript Co., Ltd. is shown in SEQ ID NO: 3), and then transformed into Escherichia coli Top10 competent cells through T4 ligation to extract the plasmid pRL27-Tn5-AsbF-AroG fbr -Tn5, and sequencing was performed to ensure that the result was completely consistent with the designed plasmid DNA sequence, thus obtaining a non-selection tag Tn5 transposition system.

[0053] Example 3 Screening of high-yield strains using biosensors to assist transposon engineering

[0054] The biosensor response module (the protocatechuic acid response plasmid pRK415-PcaV-P obtained in Example 1) LV -eGFP-Gm) was electroporated into Pseudomonas putida S12 competent cells to obtain a protocatechuic acid responsive strain; then, the Tn5 transposition system (the transposition plasmid pRL27-Tn5-AsbF-AroG obtained in Example 2) was fbr -Tn5) was electroporated into a protocatechuic acid-responsive strain, activated at 30°C for 1 h, plated on a gentamicin-resistant plate, and cultured at 30°C overnight. The green fluorescent protein in the plate colonies was activated by blue light, and its fluorescence signal was monitored. Colonies with strong signals were selected for subsequent protocatechuic acid production detection.

[0055] The screened high-yield strain with strong green fluorescence signal was inoculated into 1 ml LB overnight culture as mother liquid. Different colonies were fermented under the same conditions (fermentation conditions: 20 μl of mother liquid was inoculated into 2 ml LB, placed at 30°C, cultured at 250 rpm for 96 hours), and the protocatechuic acid production in the fermentation broth was detected by liquid chromatography to obtain the target product high-yield strain.

[0056] The results are as follows Figure 2 As shown, with the help of biosensors, high-yield strains with strong green fluorescence signals can be screened in a targeted manner, namely Figure 2 PCA29 in the fermentation. It was found that the high-yield strain produced protocatechuic acid up to 4.62g / L.

[0057] On this basis, genome sequencing of high-yield strains can be carried out in the future to further reveal the high-yield mechanism.

[0058] In summary, according to the method for screening high-yield strains by biosensor-assisted transposon engineering in an embodiment of the present invention, the engineered bacteria can release the PcaV from the promoter P through the biosensor response module when protocatechuic acid is produced. LV The inhibition of Tn5 can turn on the expression of downstream genes eGFP and Gm. Due to the randomness of the Tn5 transposition system, with the assistance of biosensors, it is possible to screen non-resistant high-yield transposition strains in a targeted manner. Compared with traditional transposition systems, due to the limitation of the types of resistance tags, the number of transpositions is limited in most cases, and a lot of screening work is required to obtain high-yield strains. Therefore, the method of using biosensors to assist transposon engineering to screen high-yield strains is more competitive in actual production applications.

[0059] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms should not be understood as necessarily being directed to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification.

[0060] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for screening high-yield strains using biosensor-assisted transposon engineering, characterized in that: include: constructing plasmids that express reporter genes and resistance genes in response to biosensor control in the target host; Randomly integrating the synthetic module of the target product into the genome of the target host through a transposition system; The target host is screened for tolerant colonies on a plate with corresponding resistance, and colonies with high reporter gene output intensity are selected to selectively screen host strains with high yield of target products.

2. The method according to claim 1, characterized in that The target product is protocatechuic acid.

3. The method according to claim 2, characterized in that The target host is Pseudomonas putida.

4. The method according to claim 2, characterized in that The biosensor is a protocatechuic acid response system from Streptomyces coelicolor.

5. The method according to claim 3, characterized in that The reporter gene is green fluorescent protein eGFP, and the resistance gene is gentamicin resistance gene Gm.

6. The method according to claim 1, characterized in that The transposition system is a non-replicating Tn5 transposition system, and the resistance selection tag is removed from the transposition system.

7. The method according to claim 3, characterized in that The target host was screened on a gentamicin resistance plate, and colonies with strong green fluorescent protein signals were selected to selectively screen host strains with high yields of the target product.

8. The method according to claim 1, characterized in that The following steps are involved: The nucleotide sequence is as shown in SEQ ID NO:

1. LV The -eGDP-Gm vector was cut with EcoRI-HindIII and ligated with the pRK415 vector cut with EcoRI-HindIII to obtain the protocatechuic acid-responsive plasmid pRK415-PcaV-P LV -eGDP-Gm; The protocatechuic acid response plasmid pRK415-PcaV-P LV -eGDP-Gm was electrotransformed into Pseudomonas putida competent cells to obtain a protocatechuic acid responsive strain; The pRL27-Tn5 transposition vector with a nucleotide sequence as shown in SEQ ID NO: 2 was cut by BamHI-XhoI, and the nucleotide sequence after BamHI-XhoI cutting was as shown in SEQ ID NO: 3 as the protocatechuic acid synthesis module AsbF-AroG fbr Ligation to obtain the transposition plasmid pRL27-Tn5-AsbF-AroG fbr -Tn5; The transposition plasmid pRL27-Tn5-AsbF-AroG fbr -Tn5 was electrotransformed into the protocatechuic acid responsive strain, and screened on a gentamicin resistance plate to select colonies with strong green fluorescent protein signals, so as to selectively screen for protocatechuic acid high-yielding strains.

9. A protocatechuic acid high-producing strain obtained by screening according to any one of claims 1 to 8.

10. Use of the protocatechuic acid high-producing strain according to claim 9 in protocatechuic acid biosynthesis.

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