Dynamic regulation and control system based on quorum sensing and application thereof

Abstract

The invention belongs to the field of synthetic biology, and particularly relates to a dynamic regulation and control system based on quorum sensing and application thereof. The gene comprises a constitutive promoter, a signal molecule AHLs synthetase coding gene esaI, a segment of RBS sequence, a transcriptional regulatory factor synthetase coding gene esaRI70V, a quorum sensing target promoter PesaS and a threonine synthesis key gene thrB regulated by the quorum sensing target promoter PesaS. Accurate dynamic regulation and control of a threonine synthesis route are realized by adjusting the strength of the constitutive promoter in the system, addition of necessary amino acid threonine in the fermentation process of escherichia coli engineering bacteria is eliminated, an inducer does not need to be added, the regulation and control complexity and the fermentation cost in the fermentation process are effectively reduced, and the method is suitable for large-scale industrial production. The method has good production and application values.

Description

Technical Field

[0001] The present invention belongs to the field of synthetic biology, and specifically relates to a dynamic control system based on quorum sensing and its application. Background Art

[0002] L-homoserine, also known as (S)-2-amino-4-hydroxybutyric acid, is a naturally occurring, non-essential amino acid with an L-α-amino acid skeleton. Its γ-hydroxyl group exhibits diverse chemical activities and serves as a precursor to several important industrial compounds, including γ-butyrolactone, L-homoserine lactone, and 1,4-butanediol. L-homoserine can also be used to synthesize the novel pesticide glufosinate ammonium, a popular broad-spectrum, low-toxic herbicide, leading to growing demand in the downstream market for L-homoserine. Currently, L-homoserine is primarily obtained through chemical synthesis and biofermentation. Chemical synthesis of L-homoserine suffers from long reaction steps, harsh reaction conditions, and low safety. Compared to chemical synthesis, microbial fermentation for L-homoserine synthesis offers advantages such as low cost and environmental friendliness, gradually replacing chemical synthesis as the mainstream method for L-homoserine production. Escherichia coli, a model organism with a clear genetic background, rapid reproduction, and efficient genetic manipulation methods, is widely used as a base strain for metabolic engineering of high-producing L-homoserine strains.

[0003] During the transformation of an L-homoserine Escherichia coli cell factory, knocking out the synthesis pathways for its byproduct amino acids (L-lysine, L-threonine, and L-methionine) effectively increased the fermentation yield of L-homoserine. However, the resulting engineered bacteria require exogenous addition of the corresponding essential amino acids to meet the growth needs of the E. coli cells during subsequent fermentation, which increases the cost and control difficulty of the fermentation process to varying degrees. Therefore, finding an appropriate dynamic control strategy to achieve a balance between essential amino acid synthesis and L-homoserine production is particularly important for improving fermentation performance. Summary of the Invention

[0004] The present invention aims to overcome the defects of the prior art in that the biosynthesis of L-homoserine requires the addition of threonine, the high fermentation cost and the complicated fermentation operation. I70V A dynamic control system of quorum sensing and its application are used to overcome the above-mentioned defects.

[0005] To achieve the above-mentioned purpose, the present invention is implemented through the following technical solutions: In the first aspect, the present invention discloses a method based on esaI / esaR I70VThe dynamic regulatory system of quorum sensing includes a constitutive promoter, a gene encoding the signal molecule AHLs synthase esaI, an RBS sequence, and a gene encoding the transcriptional regulator synthase esaR. I70V , quorum sensing target promoter P esaS And the key gene thrB for threonine synthesis regulated by it.

[0006] Quorum sensing (QS) systems are novel dynamic regulatory systems that, through autoinduction, activate the expression of relevant genes at specific cell stages and gradually reduce it when bacterial populations reach a certain threshold. This effectively avoids bottlenecks such as growth arrest and metabolic imbalances caused by static regulation, providing a new means to increase the production of target metabolites. In particular, the Esa QS bidirectional regulatory system, derived from the maize bacterial wilt pathogen Pantoea stewartia, can simultaneously activate and deactivate gene expression, enabling precise, staged control of multiple genes and showing promising applications in the biosynthesis of high-value-added products.

[0007] The present invention constructs and optimizes an esaI / esaR system based on the esaI / esaR system of corn bacterial wilt fungus (Pantoea stewartia) to obtain a I70V The dynamic regulation system of quorum sensing was introduced into the engineered Escherichia coli bacteria with high L-homoserine production, dynamically regulating the synthesis pathway of by-product amino acids (such as L-threonine), eliminating the deficiency of the engineered bacteria in the corresponding essential amino acids, reducing the complexity and cost of fermentation operations, and improving the fermentation level of L-homoserine, laying a certain research foundation for the subsequent industrial production of L-homoserine by fermentation.

[0008] Furthermore, the regulatory system includes a regulatory portion, and a response portion placed downstream of the regulatory portion; The regulatory part is composed of a constitutive promoter and esaI gene, RBS sequence, esaR I70V Genes are connected in sequence; The response part is composed of promoter P esaS It is connected to the threonine synthesis gene thrB.

[0009] Furthermore, the vector of the regulatory system is pACYCDuet-1.

[0010] Furthermore, the nucleotide sequence of the esaI gene is shown in SEQ ID NO.1, the RBS sequence is shown in SEQ ID NO.2, and the esaR I70V The nucleotide sequence of the gene is shown in SEQ ID NO.3, and the promoter PesaS The nucleotide sequence of the thrB gene is shown in SEQ ID NO.4, and the nucleotide sequence of the thrB gene is shown in SEQ ID NO.5.

[0011] Furthermore, the nucleotide sequence of the constitutive promoter is selected from one or more of sequences SEQ ID NO. 6 to 14. Preferably, the nucleotide sequence of the constitutive promoter is selected from sequences SEQ ID NO. 6 and SEQ ID NO. 7.

[0012] In a second aspect, the present invention discloses the application of the above-mentioned dynamic control system based on quorum sensing in the fermentation production of L-homoserine.

[0013] In a third aspect, the present invention discloses a recombinant Escherichia coli comprising the above-mentioned esaI / esaR-based I70V A dynamic regulatory system for quorum sensing.

[0014] Furthermore, the recombinant Escherichia coli uses HS25 and HSY43 as host cells.

[0015] In a fourth aspect, the present invention further discloses the use of the above-mentioned recombinant Escherichia coli in the fermentation production of L-homoserine.

[0016] In a fifth aspect, the present invention also discloses a method for efficiently fermenting and producing L-homoserine, comprising the following steps: using the above-mentioned recombinant Escherichia coli as a fermentation strain, and optimizing the strength of the constitutive promoter in the control system to regulate the synthesis of by-product amino acids, and finding the quorum sensing esaI / esaR I70V The optimal promoter strength in front of the expression cassette was used to balance carbon flow distribution and produce L-homoserine by fermentation.

[0017] Therefore, the present invention has the following beneficial effects: The present invention has developed a novel esaI / esaR-based I70V The transformation strategy and optimization method of the quorum sensing dynamic regulation system for constructing high-yield L-homoserine Escherichia coli can achieve dynamic regulation of by-product essential amino acids without the need for exogenous inducers during the fermentation process, reducing carbon source loss while eliminating the deficiency of the engineered bacteria in essential amino acids, reducing the complexity of fermentation regulation and fermentation costs, enriching the toolbox for genetic manipulation of Escherichia coli engineered bacteria for efficient synthesis of L-homoserine, and laying a certain theoretical foundation for the construction of Escherichia coli L-homoserine cell factories. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The esaI / esaR-based I70V Schematic diagram of the quorum sensing dynamic regulation system.

[0019] Figure 2 Schematic diagram of the different promoter strength determination systems constructed in the present invention.

[0020] Figure 3 Schematic diagram of eGFP expression intensity using different promoter strength determination systems constructed in the present invention.

[0021] Figure 4 Optimization of esaI / esaR for different promoters constructed in the present invention I70V Schematic diagram of the quorum sensing target promoter strength measurement system under the expression cassette strategy.

[0022] Figure 5 Optimization of esaI / esaR for different promoters constructed in the present invention I70V Strength change trend of quorum sensing target promoter in HSY43 strain under the expression cassette strategy.

[0023] Figure 6 The invention is based on esaI / esaR I70V Schematic diagram of the shake flask fermentation results of HSY43 Escherichia coli engineered bacteria in the quorum sensing dynamic regulation system.

[0024] Figure 7 The invention is based on esaI / esaR I70V Schematic diagram of the fed-batch fermentation results of the HSY43-gapA-QS-thrB strain in a 5L fermenter of the quorum sensing dynamic regulation system.

[0025] Figure 8 The invention is based on esaI / esaR I70V Schematic diagram of the shake flask fermentation results of HS25 Escherichia coli engineered bacteria in the quorum sensing dynamic regulation system.

[0026] Figure 9 The invention is based on esaI / esaR I70V Schematic diagram of the fed-batch fermentation results of the HS25-J23119-QS-thrB strain in a 5L fermenter using the quorum sensing dynamic regulation system. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention described below are generally only embodiments of a portion of the present invention, rather than all embodiments. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative work should fall within the scope of protection of the present invention. The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The test materials used in the following examples, unless otherwise specified, are all conventional biochemical reagents.

[0028] The culture medium formula used in the embodiment of the present invention is as follows: (1) LB medium (g / L): peptone 10, sodium chloride 10, yeast powder 5, add 2% (w / v) agar powder to make a solid medium. L-homoserine shake flask fermentation medium (g / L): glucose monohydrate 40, (NH4)2SO4 17, yeast powder 4, MgSO4 1, KH2PO4 1, FeSO4·7H2O 0.005, MnSO4·7H2O 0.005, ZnSO4 0.005, CaCO3 25 (sterilized separately), essential amino acid: L-threonine 0.2 (whether to add or not depends on the auxotrophic type of the strain) (2) 5 L fermentation tank medium for HS25 strain (g / L): 22g of glucose monohydrate, 10g of (NH4)2SO4, 8g of yeast extract, 2g of MgSO4, 5g of KH2PO4, 10g of peptone, 5g of NaCl, 2g of betaine, 0.005g of FeSO4·7H2O, 0.005g of MnSO4·7H2O, 0.005g of ZnSO4, and 0.15% of defoaming agent. (3) Feed medium for 5 L fermentation of HS25 strain (g / L): glucose monohydrate 700, KH2PO4 16, (NH4)2SO4 13.5, betaine 1. (4) 5 L fermentation tank medium for HSY43 strain (g / L): 22g of glucose monohydrate, 8g of yeast powder, 12g of peptone, 4.02g of K3PO4, 3g of NaCl, 2.1g of citric acid monohydrate, 0.3g of ammonium ferric citrate, 2.5g of ammonium sulfate, 0.5g of magnesium sulfate heptahydrate, 1g of betaine, 0.15% of defoaming agent, and 0.7g of threonine (whether to add depends on the auxotrophic type of the strain). (5) Feed medium for 5 L fermentation tank of HSY43 strain (g / L): glucose monohydrate 660, KH2PO4 15, (NH4)2SO4 12.5, betaine 1, threonine 1 (whether to add depends on the auxotrophic type of the strain). The final concentration of the relevant antibiotics involved in the present invention: chloramphenicol (CHL): 20 μg / mL.

[0029] Example 1: Based on esaI / esaR I70V Characterization and optimization of dynamic regulatory systems for quorum sensing like Figure 1 As shown, this embodiment constructs a esaI / esaR I70V The dynamic regulatory system of quorum sensing consists of a constitutive promoter, a codon-optimized signal molecule AHLs synthase encoding gene esaI, an RBS sequence, and a codon-optimized transcriptional regulator synthase encoding gene esaR. I70V and quorum sensing target promoter P esaS To optimize and modify the strength of the quorum sensing expression cassette in the above system, a number of promoters with different strengths were screened using the Promoter Strength Calculator on the De Novo DNA Docs website (https: / / docs.denovodna.com / docs / operon-calculator), and the following promoters were constructed: Figure 2 The promoter strength measurement system shown in Figure 1 is used to measure the expression of the green fluorescent protein gene EGFP by different promoters. The green fluorescence intensity obtained is used to characterize the expression strength of the gene driven by the different promoters. The vector used is pACYCDuet-1.

[0030] The method for constructing the promoter strength determination system in this embodiment is as follows: Taking the construction of pACYC-gapA-egfp plasmid as an example, the specific construction method of the promoter strength determination system is explained. (1)P gapA Preparation of -EGFP Donor fragment The green fluorescent protein (eGFP) gene sequence used as the reporter in this invention was directly synthesized by Beijing Qingke Biotechnology Co., Ltd. Primers gapA-egfp-P1 and egfp-P2, as shown in Table 12, were designed. PCR amplification using a vector carrying the eGFP gene as a template yielded the egfp gene fragment. The PCR amplification system components and PCR amplification procedures are shown in Tables 1 and 2, respectively.

[0031] Table 1: PCR amplification system

[0032] Table 2: PCR amplification reaction program

[0033] The PCR amplified products were subjected to agarose gel electrophoresis, and the bands with the same length were purified and stored in a -20°C refrigerator for future use. gapA -P1 and P gapA -P2 primers and PCR amplification were performed to obtain P gapA The PCR amplification system composition and PCR amplification procedure are the same as those shown in Tables 2 and 3. After agarose gel electrophoresis, the bands with the same length were purified.

[0034] Furthermore, using the above two fragments as templates, gapA Fusion PCR was performed using primers P-P1 and EGFP-P2 to obtain P gapA The reaction procedures and systems for fusion PCR are shown in Tables 2 and 3.

[0035] Table 3: Fusion PCR reaction system

[0036] The products obtained after fusion PCR were subjected to agarose gel electrophoresis, and the bands with the same length were purified and the concentration was determined to obtain P gapA -egfp Donor.

[0037] (2) Preparation of linearized pACYCDuet-1 plasmid fragment Using the pACYCDuet-1 plasmid as a template, primers pACYC-line-F and pACYC-line-R (see Table 12) were designed and PCR amplified. After agarose gel electrophoresis, the amplified products were digested with DpnI enzyme for bands of the appropriate length to eliminate the original template and reduce the probability of false positives in post-transfection colonies. The digestion reaction system and procedure are shown in Tables 4 and 5.

[0038] Table 4: Digestion reaction system

[0039] Table 5: Digestion reaction procedure

[0040] Purify the digested product to remove residual protein and other impurities in the DNA fragment to obtain the linearized pACYCDuet-1 plasmid fragment. For purification steps, refer to the Clean up kit instructions.

[0041] (3) One-step cloning of the Donor fragment and the linearized pACYCDuet-1 fragment The linearized pACYCDuet-1 plasmid fragment and P gapA -egfp Donor via Clon Express TM IIOne Step Cloning Kit was used to ligate into the complete pACYC-P gapA -egfp plasmid. The PCR reaction system and procedure for one-step cloning are shown in Tables 6 and 7.

[0042] Table 6: One-step cloning system ingredients

[0043] Table 7: One-step cloning PCR program

[0044] (4) Colony PCR verification and plasmid extraction All the products after one-step cloning were added to DH5α competent cells. After transformation, they were spread on solid LB plates (containing chloramphenicol resistance) and cultured in a constant temperature incubator for 12-14 hours until single colonies grew. 8-16 evenly growing single colonies were selected and colony PCR was performed using the pACYC universal verification primers pACYC-YZ-F / pACYC-YZ-R. The colony PCR procedure and reaction system are shown in Tables 8 and 9.

[0045] Table 8: Colony PCR program

[0046] Table 9: Colony PCR system components

[0047] The products with correct PCR bands were sent for testing, and the sequences were compared. Single colonies corresponding to the samples with correct sequences were picked for test tube culture (with chloramphenicol resistance added). The culture was carried out overnight at 37°C and 180 rpm. The plasmid in the cultured bacterial solution was extracted to obtain pACYC-P gapA -egfp plasmid.

[0048] The green fluorescence determination method of the promoter strength determination system in this embodiment is as follows: The vector for promoter strength determination was transformed into Escherichia coli DH5α strain, plated onto LB plates containing chloramphenicol resistance, and transformed single colonies were obtained after overnight culture at 37°C. The transformed strain containing the promoter strength determination system was verified and obtained by colony PCR. The transformed single colony was inoculated into a test tube containing 10mL LB (containing chloramphenicol resistance) and cultivated at 37°C, 200rpm for 12h. The inoculum size was then inoculated into a 250mL shake flask containing 50mL LB medium (containing chloramphenicol resistance) and cultivated at 37°C, 180rpm for 12h. After the culture was completed, 1mL of bacterial solution was taken, centrifuged at 4000rpm for 1min to remove the supernatant, and 1mL of PBS buffer was added to resuspend the bacterial cells after washing the cells twice with pH 7.0 PBS buffer. 200 μL of the resuspended bacterial solution was measured for absorbance at an absorbance wavelength of 600nm. Take another 200 μL of the resuspended bacterial solution and use a microplate reader to measure the relative fluorescence value, i.e., the RFU value, under the conditions of excitation wavelength 488 nm and emission wavelength 520 nm. 600 If the instrument exceeds the linear range, dilute it with PBS buffer for a certain multiple and measure again. 600 The value of RFU / OD is used to measure the strength of the promoter. 600 The higher the value, the stronger the promoter strength of the bacteria.

[0049] The expression strength of different promoters was measured as follows Figure 3 As shown in the figure, the results show that the green fluorescence intensity of the promoter intensity determination system strains carrying different promoters driving the expression of green fluorescent protein is different, indicating that the different promoters in the intensity gradient are used to regulate the quorum sensing system esaI / esaR I70V Driving with an expression cassette can effectively regulate its expression intensity, help screen out the appropriate expression level to optimize the quorum sensing dynamic regulation system, and more accurately regulate the metabolism of the bacteria.

[0050] Furthermore, the construction is Figure 4 The system for measuring the dynamic regulation strength of quorum sensing under different promoter regulation strategies shown in FIG. 4 is a system for measuring the dynamic regulation strength of quorum sensing under different promoter regulation strategies. The system uses different promoters to regulate esaI / esaR I70V The quorum sensing expression box expression intensity is driven to achieve the control of intracellular signaling molecules AHLs and transcriptional regulator EsaR during bacterial growth. I70V The regulation of the content, thereby regulating the quorum sensing target promoter P esaS The expression level of eGFP green fluorescent protein gene was driven by pACYCDuet-1 vector. The green fluorescence intensity was measured to characterize the expression intensity of the target gene by the quorum sensing dynamic regulatory system under different promoter driving conditions.

[0051] The construction method of the quorum sensing dynamic regulation strength measurement system under different promoter strategies in this embodiment is as follows: Taking the construction of the pACYC-gapA-QS-egfp plasmid as an example, the specific construction method of the quorum sensing dynamic regulation strength measurement system under different promoter strategies is explained. (1)esaI-RBS-esaR I70V Preparation of Donor Fragments In the present invention, esaI / esaR I70V The expression cassette gene sequence was directly synthesized by Beijing Qingke Biotechnology Co., Ltd. The esaI-P1 / P2 and esaR-P1 / P2 primers shown in Table 12 were designed to carry the esaI and esaR I70V The gene encoding vector was used as a template for PCR amplification to obtain esaI / esaR I70V The composition of the PCR amplification system and the PCR amplification procedure are shown in Tables 1 and 2, respectively. Furthermore, the above two fragments were used as templates and esaI-P1 and esaR-P2 were used as primers for fusion PCR to obtain esaI-RBS-esaR I70V The fusion fragments are characterized by base pairing of the RBS sequences between the two fragments during primer design. The reaction procedures and systems for the fusion PCR are shown in Tables 2 and 3. (2) Quorum sensing target promoter P esaS Fragment preparation In the present invention, P esaS The gene sequence was directly synthesized by Beijing Qingke Biotechnology Co., Ltd. The design was as shown in Table 12. esaS -P1 and P esaS -P2 primer, carrying P esaS The vector of the gene sequence is used as a template for PCR amplification to obtain P esaS The composition of the PCR amplification system and the PCR amplification procedure are shown in Tables 1 and 2, respectively. (3)esaI-RBS-esaR I70V -P esaS Donor Preparation The esaI-RBS-esaR I70V Donor and P esaS Donor was used as a template and primers esaI-P1 and P esaS -P2 fusion PCR, after agarose gel electrophoresis, the product with the correct band was purified to obtain esaI-RBS-esaR I70V -P esaSDonor. The reaction procedures and systems for fusion PCR are shown in Tables 2 and 3. (4) Linearized opened pACYC-P gapA -EGFP plasmid fragment preparation Using the promoter strength determination system pACYC-gapA-egfp plasmid constructed above as a template, primers P as shown in Table 12 were designed. gapA -QS-line-F and P gapA -QS-line-R and PCR amplification were performed. After agarose gel electrophoresis, the amplified products were digested with DpnI enzyme for bands of the same length, and the digested products were purified. The digestion reaction system and procedures are shown in Tables 4 and 5. (5) Donor fragment and linearized pACYC-P gapA One-step cloning of -egfp plasmid fragments The linearized pACYC-P gapA -egfp plasmid fragment and esaI-RBS-esaR I70V -P esaS Donor via Clon Express TM II One Step Cloning Kit to form the complete pACYC-gapA-QS-egfp plasmid. The PCR reaction system and procedure for one-step cloning are shown in Tables 6 and 7.

[0052] Furthermore, all the above-mentioned one-step cloning products were added to DH5α competent cells, and after transformation, they were spread on solid LB plates (containing chloramphenicol resistance) and cultured for 12-14 h to obtain single colonies. 8-16 single colonies with uniform growth were selected, and colony PCR was performed using the universal verification primers pACYC-YZ-F / pACYC-YZ-R of the pACYCDuet-1 plasmid. The colony PCR procedure and reaction system are shown in Tables 8 and 9.

[0053] The products with correct PCR bands in the colonies were sent for testing, and the sequences were compared. The correct samples were picked and verified, and the corresponding single colonies were cultured in test tubes (chloramphenicol resistance was added) at 37°C and 180 rpm overnight. The plasmid in the cultured bacterial solution was extracted to obtain the pACYC-gapA-QS-egfp plasmid.

[0054] The green fluorescence measurement method of the quorum sensing dynamic regulation intensity measurement system under different promoter strategies is as follows:

[0055] The vector for quorum sensing dynamic regulation strength determination was transformed into the HSY43 strain, spread onto an LB plate containing chloramphenicol, and cultured overnight at 37°C to obtain a single transformed colony. The transformed strain containing the quorum sensing dynamic regulation strength determination system was verified and obtained by colony PCR. The transformed single colony was inoculated into a test tube containing 10mL LB and cultured at 37°C and 200rpm for 12h. Then, it was inoculated into 50mL LB medium at a 2% inoculum volume, and the culture conditions were 37°C and 180rpm. Every 2h, 1mL of bacterial solution was sampled and centrifuged at 4000rpm for 1min to remove the supernatant. The bacteria were washed twice with pH 7.0 PBS buffer and then resuspended in 1mL PBS buffer. 200μL of the resuspended bacterial solution was taken to measure the absorbance at an absorbance wavelength of 600nm. Take 200 μL of the resuspended bacterial solution and use a microplate reader to measure the relative fluorescence value, i.e., RFU value, under the conditions of excitation wavelength 488 nm and emission wavelength 520 nm. Calculate and mark the final RFU / OD value. 600 The values ​​were used to characterize different promoters driving esaI / esaR I70V The expression cassette is the quorum sensing target promoter P esaS driving strength.

[0056] HSY43 strains drive esaI / esaR with different promoters I70V Quorum sensing target promoter P under expression cassette strategy esaS The driving strength change diagram is as follows Figure 5 As shown in the figure, it can be seen that after replacing different promoters to drive the esaI / esaR expression cassette, the quorum sensing target promoter P esaS The expression intensity of the quorum sensing target promoter P esaS There were differences in the peak intensity, intensity reduction rate, and minimum intensity in the HSY43 strain, which indicates that the quorum sensing dynamic regulation system constructed in this example can be used to regulate the dynamic expression of key genes in engineered bacteria.

[0057] It is worth noting that different promoters drive esaI / esaR I70V Quorum sensing target promoter P under expression cassette strategy esaSThe intensity of esaI / esaR may vary in different engineered bacteria due to different growth conditions of the strains. This is because quorum sensing is a dynamic regulatory strategy closely related to the growth of the strains. The peak intensity, downward trend and low intensity value of its target promoter in different strains may be different. Therefore, the optimization method of the quorum sensing dynamic regulatory system proposed in the present invention can change the regulatory ability of the target promoter on the regulated key gene by replacing the constitutive promoter in the system, which helps to find the optimal esaI / esaR under different strains or even different target gene conditions. I70V The expression cassette strength can meet the needs of bacterial metabolism and product synthesis.

[0058] Table 10: Plasmids constructed in this example

[0059] Table 11: Strains involved in this example

[0060] Table 12: esaI / esaR used in this example to construct different promoter strength measurement systems and optimize different promoters I70V Primers used in the quorum sensing target promoter strength measurement system under the expression cassette strategy

[0061] Example 2: Based on esaI / esaR I70V Construction of quorum sensing dynamic control system Figure 1 As shown in Table 14, this embodiment constructed a batch of esaI / esaR-based I70V The gene system of the quorum sensing dynamic regulation strategy consists of a constitutive promoter, a codon-optimized signal molecule AHLs synthase encoding gene esaI, an RBS sequence, and a codon-optimized transcriptional regulator synthase encoding gene esaR I70V and quorum sensing target promoter P esaS It is composed of proteins and regulates the expression of the threonine synthesis gene thrB.

[0062] Table 13: Plasmids constructed in this example

[0063] Table 14: The methods used in this example to construct the esaI / esaR-based I70V Primer sequences for the quorum sensing dynamic regulation system

[0064] In this embodiment, based on esaI / esaR I70V The construction method of the quorum sensing dynamic regulation system is as follows: Taking the construction of the pACYC-gapA-QS-thrB plasmid as an example, the specific construction method of the promoter strength measurement system is explained. (1) Preparation of thrB Donor Fragment Using the extracted wild-type E. coli W3110 genome as a template, thrB-P1 and thrB-P2 primers (Table 14) were designed and PCR amplified to obtain thrB Donor. The PCR amplification system components and procedures are also shown in Tables 1 and 2. After agarose gel electrophoresis, the bands of the appropriate length were purified, their concentrations determined, and stored at -20°C until ready for use. (2) Preparation of linearized opened vector fragments Using the previously constructed pACYC-gapA-QS-egfp plasmid as a template, primers pACYC-thrB-line-F and pACYC-thrB-line-R (see Table 14) were designed and PCR amplification was performed. After agarose gel electrophoresis, the amplified products were digested with DpnI enzyme for bands of the appropriate length, and the digested products were purified. The digestion reaction system and procedures are shown in Tables 4 and 5. (3) One-step cloning of the donor fragment and the linearized opened vector fragment The linearized vector fragment and thrB fragment were transferred to Clon Express TM II One Step Cloning Kit was used to ligate the complete pACYC-gapA-QS-thrB plasmid. The PCR reaction system and procedure for one-step cloning are shown in Tables 6 and 7. Furthermore, all the above-mentioned one-step cloning products were added to DH5α competent cells, and after transformation, they were spread on solid LB plates (containing chloramphenicol resistance) and cultured until single colonies were obtained. Colony PCR was performed using the pACYCDuet-1 plasmid universal verification primers pACYC-YZ-F / pACYC-YZ-R. The colony PCR procedure and reaction system are shown in Tables 8 and 9. Products with correct colony PCR bands were sent for testing, sequence comparison, and samples that were verified to be correct were selected. The corresponding single colony was cultured in a test tube (with chloramphenicol resistance added) and cultured overnight at 37°C and 180 rpm. The plasmid in the culture solution was extracted to obtain the pACYC-gapA-QS-thrB plasmid.

[0065] Example 3: Containing esaI / esaR-based I70VConstruction of Escherichia coli engineered bacteria with a quorum sensing dynamic regulation system (1) Preparation of competent cells of Escherichia coli HSY43 / HS25 Take a small amount of bacterial liquid from the glycerol preservation tube, streak it on an antibiotic-free LB solid plate, and invert it in a 37°C incubator for 12-14 hours to obtain a single colony. Pick a single colony on the plate and inoculate it into a test tube containing 10mL sterile LB liquid culture medium. Incubate it at 37°C and 180rpm for 12 hours to obtain the activated seed liquid. Take 1mL of the activated seed liquid and inoculate it into a 250mL shake flask containing 50mL LB culture medium. Incubate it at 37°C and 200rpm for 2-3 hours until the bacterial liquid OD reaches 0. 600 The value is around 0.6, place the shake flask on ice to cool for 10 minutes. Transfer the cooled bacterial solution to a pre-cooled 50mL centrifuge tube, centrifuge at 4℃, 5300rpm for 5 minutes, and discard the supernatant. Add 40mL of pre-cooled sterile 0.1mol / L CaCl2, gently pipette until the bacteria are suspended, place on ice for 30 minutes, centrifuge at 4℃, 5300rpm for 5 minutes, and discard the supernatant. Add 1mL of pre-cooled 15% (V / V) glycerol mixture containing sterile 0.1mol / L CaCl2, gently pipette until the bacteria are suspended, and divide into sterile 1.5mL centrifuge tubes, 100μL per tube, and store in a -80℃ ultra-low temperature refrigerator for storage. (2) Based on esaI / esaR I70V The quorum sensing dynamic regulation system was transferred into Escherichia coli competent cells Take a competent cell and place it on ice, add 5 μL of plasmid, and let it stand for 30 minutes to allow the plasmid to adsorb on the competent cells. After standing, place it in a 42°C constant temperature water bath for heat shock for 90 seconds. After the end, quickly take it out and place it on ice for 5 minutes. After cooling, add 700 μL of sterile LB liquid culture medium and culture at 37°C and 200 rpm for 1 hour. After the culture is completed, centrifuge at 5300 rpm for 3 minutes, discard the supernatant to collect the bacteria, blow and mix the remaining bacteria, transfer and spread them on a solid LB plate, and culture in a constant temperature incubator for 14-16 hours until a single colony grows. Select 8-16 single colonies with uniform growth, and use the pACYC universal verification primers pACYC-YZ-F / pACYC-YZ-R for colony PCR. The colony PCR procedure and reaction system are shown in Tables 9 and 10. The products with the correct colony PCR bands are sent for testing, and the sequences are compared to construct the gene containing the esaI / esaR-based I70V Quorum sensing dynamic regulation system of Escherichia coli engineered bacterial cells.

[0066] Example 4: Fermentation of E. coli engineered bacteria containing the quorum sensing dynamic control system in shake flasks and 5L fermenters (1) Fermentation of HSY43 series strains in shake flasks The HSY43 strains and the HSY43 series strains constructed in Example 3 were tested in shake flasks to compare the effects of different promoters on the esaI / esaR I70V The quorum sensing expression cassette was used to examine the effect of the quorum sensing system on L-homoserine synthesis in engineered bacteria. Shake flask fermentation experiments were conducted according to the following protocol: The constructed different strains were streaked onto LB plates (if containing pACYCDuet-1 vector, 20 mg / L chloramphenicol was added to the LB plates, otherwise not added), cultured overnight in a 37°C incubator, and single colonies were picked and inoculated into 10 mL of LB medium. Seed liquid was obtained by overnight culture at 180-200 rpm and 37°C. 20 mL of HSY43 shake flask fermentation medium was added to a 500 mL shake flask, and 1 mL of seed liquid of each strain was inoculated into the shake flask medium (if containing pACYCDuet-1 vector, chloramphenicol was added at a final concentration of 20 mg / L, otherwise not added). In addition, CaCO3 in the shake flask medium formula was added together with the inoculated liquid, and the addition amount was 0.5 g / bottle. The shake flask was fermented at 180 rpm and 30°C for 48 hours. After the fermentation was completed, 1 mL of fermentation broth was taken from the shake flask, centrifuged at 12000 rpm for 5 min, and the supernatant was diluted 20 times with ultrapure water. After derivatization and filtration through a membrane (pore size 0.22 μm), the L-homoserine content in the fermentation broth was analyzed using HPLC liquid chromatography. The amount of L-homoserine produced by each strain was compared. The results are shown in Table 15. Figure 6 shown.

[0067] Table 15: Shake flask fermentation results of HSY43 series strains

[0068] Combined with Table 15 and Figure 6 The results show that the quorum sensing esaI / esaR is regulated by promoters of different strengths. I70V The expression cassette will significantly affect the intracellular signaling molecules AHLs and transcriptional regulatory factors EsaR of the engineered bacteria. I70V The expression level of some promoters is not suitable for the expression cassette, which makes the strain accumulate a considerable level of threonine outside the cell while meeting its own needs. This leads to an imbalance in the carbon and sulfur distribution between threonine and L-homoserine in some strains during shake flask fermentation, and the production of L-homoserine decreases significantly. The promoter strength used by other strains is more appropriate, and no accumulation of threonine is detected outside the cell. Judging from the shake flask fermentation results of the HSY43 series strains, the P gapAPromoter to achieve quorum sensing esaI / esaR I70V The expression cassette is the most suitable. In the absence of exogenous threonine, the L-homoserine content in the fermentation broth of HSY43-gapA-QS-thrB reached 20.66 g / L after 48 h of shake flask fermentation, which was 28.56% higher than that of the bottom plate bacteria. I70V The transformation strategy and optimization method of the quorum sensing dynamic regulation system for constructing a high-yield L-homoserine synthesis strain can effectively eliminate the essential amino acid deficiency of the high-yield L-homoserine Escherichia coli engineered bacteria, and to a certain extent can optimize the carbon flow distribution between essential amino acids and the target product L-homoserine, which is beneficial to the increase of L-homoserine production. It lays a theoretical foundation for the subsequent construction of high-yield L-homoserine Escherichia coli and enriches the genetic toolbox.

[0069] (2) Fed-batch fermentation of HSY43-gapA-QS-thrB strain in a 5 L fermenter In order to further explore the I70V The ability of the L-homoserine high-yielding strain obtained by constructing the quorum sensing dynamic regulation system to continuously produce L-homoserine was demonstrated. Based on the shake flask fermentation results of the above-mentioned HSY43 series strains, the HSY43-gapA-QS-thrB strain with the best fermentation performance was selected for batch fed-batch fermentation in a 5 L fermentor.

[0070] 10 μL of bacterial solution was transferred from the glycerol preservation tube of strain HSY43-gapA-QS-thrB to a test tube containing 10 mL of sterile LB liquid medium, and cultured at 37°C for 12 h to obtain a primary seed solution. Subsequently, the primary seed solution was aspirated at a 15% inoculum size and inoculated into two 500 mL shake flasks containing 150 mL of sterile LB liquid medium, and cultured at 37°C and 220 rpm for 12 h to obtain a secondary seed solution. Finally, all the secondary seed solution was inoculated into a fermentor containing 1.7 L of HSY43 upper tank fermentation medium (added with chloramphenicol) for batch fed fermentation. During the fermentation process, the glucose concentration was controlled at 0 g / L to 2 g / L by pH feedback batch feeding, the pH of the culture medium was maintained at around 6.8 with 17% ammonia solution, the dissolved oxygen was maintained at 20% 12 hours before fermentation (OD ≤ 25), and the stirring paddle cascade speed linkage was turned on, the fermentation temperature was 33°C, the dissolved oxygen was maintained at 15% after 12 hours of fermentation (OD > 25), the fermentation temperature was 30°C, the stirring speed was adjusted in the range of 300 rpm to 950 rpm, and samples were taken regularly to monitor the changes of various parameters during the fermentation process in real time.

[0071] The results of fed-batch fermentation of HSY43-gapA-QS-thrB strain in 5L fermenter are as follows Figure 7As shown, after 60 h of fermentation, the strain could produce 122.09 g / L of L-homoserine, with a sugar-to-acid conversion rate of 0.55 g / g glucose and a productivity of 2.03 g / L / h. No threonine accumulation was detected in the fermentation broth.

[0072] The above results show that the optimized quorum sensing dynamic control system can effectively balance the carbon flow balance between the target product L-homoserine and the essential amino acid threonine in the Escherichia coli engineered bacteria. Without the addition of any inducer, the engineered bacteria can self-regulate to continuously produce L-homoserine in large-scale fermentation, and no exogenous addition of L-threonine is required to maintain bacterial growth. Compared with the fermentation results of the HSY43 strain without the system obtained in the laboratory earlier (threonine was added), the batch fed fermentation yield and fermentation cycle of the 5L fermentor of the HSY43-gapA-QS-thrB strain without exogenous threonine addition were the same as when threonine was not added. It is foreseeable that the quorum sensing dynamic control system and optimization method designed by the present invention can also be applied to other biosynthetic pathways of Escherichia coli, and even to other microorganisms, by shortening the microbial fermentation cycle and obtaining high-value natural products without the addition of inducers.

[0073] (3) Shake flask fermentation of HS25 series strains In order to verify the esaI / esaR I70V The effectiveness of the quorum sensing dynamic regulation system in regulating the expression of key amino acids in the process of L-homoserine synthesis by different E. coli engineered bacteria was tested by introducing the quorum sensing dynamic regulation system into another high-yield L-homoserine E. coli engineered bacteria stored in the laboratory and conducting shake flask fermentation experiments. The specific process of shake flask fermentation is as described above. The experimental results are shown in Figure 2. Figure 8 , as shown in Table 16.

[0074] Table 16: Shake flask fermentation results of HS25 series strains

[0075] Combined with Table 16 and Figure 8 The results show that the esaI / esaR I70V The dynamic regulation system of quorum sensing also played a role in the high-yield L-homoserine Escherichia coli engineered strain HS25. However, the most effective P in HSY43 gapA The promoter strength in the HS25 strain did not achieve the expected effect, which may be due to the differences in the growth conditions of different engineered bacteria and the different requirements for the essential amino acid threonine. I70VThe optimization method of the quorum sensing dynamic regulation system has practical application value. When the system is applied to different L-homoserine production strains, the esaI / esaR I70V The driving strength of the promoter in front of the expression cassette is used to find the expression strength that is most suitable for the host engineering bacteria, to optimize the carbon and sulfur distribution between the target product and the essential amino acids, and to maintain cell growth and target product yield while eliminating the essential amino acid deficiency.

[0076] (4) Fed-batch fermentation of HS25-J23119-QS-thrB strain in a 5 L fermenter In order to further explore the I70V The ability of the high-homoserine-producing strain generated by the quorum sensing dynamic control system to continuously produce L-homoserine and its compatibility across different engineered strains were evaluated. Based on the shake flask fermentation results of the aforementioned HS25 series strains, the strain with the best fermentation performance, HS25-J23119-QS-thrB, was selected for fed-batch fermentation in a 5-L fermentor. The experimental protocol for 5-L fed-batch fermentation in a 5-L fermentor was as previously described, and the 5-L fermentation and feed medium formulations for the HS25 series strains were as previously described.

[0077] The results of fed-batch fermentation of HS25-J23119-QS-thrB strain in 5L fermenter are as follows Figure 9 As shown in the figure, after 96 hours of fermentation, the strain produced 101.81 g / L of L-homoserine, the sugar-acid conversion rate was 0.41 g / g glucose, and the yield was 1.06 g / L / h. No threonine accumulation was detected in the fermentation broth. This result shows that the optimized quorum sensing dynamic control system proposed in the present invention can also achieve the carbon flow balance between the target product L-homoserine and the essential amino acid threonine in different Escherichia coli engineered bacteria. We speculate that by replacing esaI / esaR I70V The strength of the promoter before the expression cassette enables the system to be adapted to different Escherichia coli engineered bacteria, and even other microbial cell factories and the synthesis of target products, and to achieve the synthesis of target products in different chassis bacteria without the need for exogenous addition of inducers, laying a solid foundation for subsequent research.

[0078] The shake flask fermentation medium, 5L fermentation tank fermentation medium, and feed medium formulations for the HSY43 / HS25 strain in this example are as described above. The derivatization reaction system and method in this example are as follows: 100 μL sample diluent + 300 μL CNBF (4-chloro-3,5-dinitrobenzotrifluoride, 0.27 g CNBF / 10 mL acetonitrile) + 500 μL borate borax buffer (0.2 mol / L boric acid + 0.05 mol / L borax, pH 9.0), 60°C, 600 rpm, protected from light for 1 hour.

[0079] The L-homoserine detection method in this embodiment is as follows: An Agilent HPLC was used for detection: an Agilent EC18 (4.6×100 mm, 4 μm) column was used, with a detection wavelength of 260 nm, an injection volume of 10 μL, a column temperature of 30°C, and a flow rate of 1 mL / min. Mobile phase A was pure acetonitrile; mobile phase B was buffer (830 mL ultrapure water + 170 mL acetonitrile + 2 mL triethylamine + 3 mL acetic acid). After passing through the membrane, the solution was degassed by ultrasonication for 30 minutes. The HPLC elution program is shown in Table 17:

[0080] Table 17: HPLC elution procedure for L-homoserine

[0081] The biomass (OD 600 ) The detection method is as follows: 1 mL of fermentation broth after shake flask fermentation was placed in a 2 mL EP tube and centrifuged at 12000 rpm at room temperature for 5 min. The supernatant was transferred to a new 1.5 mL EP tube for detection of L-homoserine production and other by-products (stored in a refrigerator at 4°C for future use). 1 mL of 2 mol / L HCl was added to the collected bacteria, the bacteria were resuspended and allowed to stand for 5 min until CaCO3 reacted completely without bubbles. The supernatant was discarded after centrifugation at 12000 rpm for 2 min, and 1 mL of pure water was added to resuspend the bacteria. The sample was diluted according to the bacterial concentration to make its OD value between 0.2 and 0.8. The OD was measured using a spectrophotometer. 600 Absorbance at.

[0082] Finally, it should be noted that the above experiments and embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A dynamic control system based on quorum sensing, characterized by: It includes a constitutive promoter, a signal molecule AHLs synthase encoding gene esaI, an RBS sequence, and a transcriptional regulator synthase encoding gene esaR I70V , quorum sensing target promoter P esaS and the threonine synthesis gene thrB regulated by it.

2. A dynamic control system based on quorum sensing according to claim 1, characterized in that: The regulatory system includes a regulatory part and a response part placed downstream of the regulatory part; The regulatory part is composed of a constitutive promoter and esaI gene, RBS sequence, esaR I70V Genes are connected in sequence; The response part is composed of promoter P esaS It is connected to the threonine synthesis gene thrB.

3. The dynamic control system based on quorum sensing according to claim 1, characterized in that: The vector of the regulatory system is pACYCDuet-1.

4. A dynamic control system based on quorum sensing according to claim 2, characterized in that: The nucleotide sequence of the esaI gene is shown in SEQ ID NO.1, the RBS sequence is shown in SEQ ID NO.2, and the esaR I70V The nucleotide sequence of the gene is shown in SEQ ID NO.3, and the promoter P esaS The nucleotide sequence of the thrB gene is shown in SEQ ID NO.4, and the nucleotide sequence of the thrB gene is shown in SEQ ID NO.

5.

5. The dynamic control system based on quorum sensing according to claim 4, characterized in that: The nucleotide sequence of the constitutive promoter is selected from one or more of sequences SEQ ID NO. 6-14.

6. Use of the quorum sensing-based dynamic control system according to any one of claims 1 to 5 in the fermentative production of L-homoserine.

7. A recombinant Escherichia coli, characterized in that: It comprises a dynamic control system based on quorum sensing as claimed in any one of claims 1 to 6.

8. The recombinant Escherichia coli according to claim 7, characterized in that: The recombinant Escherichia coli uses HS25 and HSY43 as host cells.

9. Use of the recombinant Escherichia coli according to claim 7 or 8 in the fermentation production of L-homoserine.

10. A method for producing L-homoserine by efficient fermentation, characterized in that: The steps include: Using a recombinant Escherichia coli as claimed in claim 7 or 8 as a fermentation strain, and optimizing the strength of the constitutive promoter in the control system to regulate the synthesis of by-product amino acids, and finding the quorum sensing esaI / esaR I70V The optimal promoter strength in front of the expression cassette was used to balance carbon flow distribution and produce L-homoserine by fermentation.

Images