Promoter responding to pentanediamine, pentanediamine biosensor and application of pentanediamine biosensor

Abstract

The invention belongs to the technical field of biology, and particularly relates to a promoter responding to pentanediamine, a pentanediamine biosensor and application of the pentanediamine biosensor. The original PpyrF promoter can respond to the concentration of pentamethylene diamine and regulate and control the expression of report protein GFP, the promoter PpyrF-D40 or PpyrF-F5 is obtained by truncation or mutation optimization of the promoter PpyrF, and the dynamic range and sensitivity of the pentamethylene diamine biosensor are improved. The pentane diamine biosensor further constructed on the basis of the promoter PpyrF-D40 or PpyrF-F5 has no responsiveness to a pentane diamine synthesis precursor lysine, so that the biosensor has good specificity when being applied to screening of pentane diamine production strains. The fluorescence intensity value of the pentane diamine biosensor obtained through screening shows an excellent linear response relationship with the concentration of pentane diamine, and the pentane diamine biosensor can be used for efficiently breeding a pentane diamine high-yield strain by being combined with a high-throughput screening system and the like, and has the advantages of high detection efficiency, high sensitivity and the like.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a promoter that responds to pentanediamine, a pentanediamine biosensor, and their applications. Background Technology

[0002] Pentylenediamine, as a promising bio-based monomer, shows broad application prospects in the field of polymer synthesis. It can be used to synthesize novel high-performance polyamides (such as PA54 and PA56) and polyurethanes (such as PDI). Compared with traditional petroleum-based polymers (such as PA66), materials synthesized using bio-based pentylenediamine as a monomer typically exhibit superior mechanical properties, heat resistance, and additional advantages such as stronger hygroscopicity, biodegradability, and flame retardancy, meeting the needs of green and sustainable development.

[0003] Currently, microbial fermentation is the main biomanufacturing route for producing pentanediamine. However, the microbial biosynthesis of pentanediamine involves complex system coordination, and its challenges extend beyond the rational modification of a few metabolic network genes. On the one hand, multiple metabolic genes interact through intricate regulatory networks, often requiring multi-module engineering strategies to efficiently optimize metabolic pathways. On the other hand, some seemingly unrelated metabolic pathways or cellular processes may unexpectedly affect product synthesis due to distal regulatory effects. Therefore, modern metabolic engineering strategies are evolving from traditional "local optimization" to "system reconstruction," using high-throughput genetic tools to systematically generate diverse pathways and genomic variations to develop high-performance strains. However, how to quickly and accurately screen out performance-enhancing mutants has become a bottleneck restricting its successful application. Developing matching high-throughput screening technologies is key to achieving rapid identification of industrial microbial strains.

[0004] Gene-encoded biosensors, due to their high sensitivity and automation potential, have become powerful tools for high-throughput screening and real-time monitoring, greatly accelerating the "design-build-test" cycle of strain development and optimization. Currently, many biosensors are based on transcription factors, which regulate the expression of reporter genes (such as green fluorescent protein GFP) in response to specific molecule concentrations, thereby enabling the detection of target metabolites. However, reports on responsive biosensors for diamine compounds (especially pentanediamine), particularly suitable for common industrial hosts such as *E. coli*, remain very limited.

[0005] Therefore, developing a pentanediamine biosensor with high sensitivity and specificity is crucial for establishing a high-throughput screening platform for high-performance pentanediamine-producing strains and promoting a complete closed loop of metabolic engineering from system reconstruction to efficient screening, and has significant industrial application value. Summary of the Invention

[0006] The technical problem to be solved by the first aspect of the present invention is to provide a promoter that responds to pentanediamine, in order to address the shortcomings of the prior art.

[0007] The technical problem to be solved by the second aspect of the present invention is to provide the application of the promoter in the construction of a pentanediamine biosensor.

[0008] The technical problem to be solved by the third aspect of the present invention is to provide a pentanediamine biosensor.

[0009] The technical problem to be solved by the fourth aspect of the present invention is to provide a recombinant strain.

[0010] The technical problem to be solved by the fifth aspect of the present invention is to provide the application of the pentanediamine biosensor in detecting pentanediamine content.

[0011] The technical problem to be solved by the sixth aspect of the present invention is to provide the application of the pentanediamine biosensor in screening high-yield pentanediamine strains.

[0012] The technical problem to be solved by the seventh aspect of the present invention is to provide a method for screening high-yield pentanediamine strains.

[0013] The design concept of this invention is as follows: A putrescine-specific biosensor based on the transcription factor PuuR in *E. coli* exhibits significant selectivity for putrescine and extremely low cross-reactivity with structurally similar diamines (including 1,3-diaminopropane, 1,5-pentanediamine, and 1,6-diaminohexane). Given the potential of pentanediamine to revolutionize the polyamide and polyurethane industries, it has become a key target for bio-based polymer research and industrial applications. Therefore, developing a pentanediamine-responsive promoter and a pentanediamine biosensor for screening high-yield pentanediamine-producing strains is particularly important.

[0014] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0015] In a first aspect, the present invention provides a promoter responsive to pentanediamine, said promoter being P pyrF -D40 or P pyrF -F5.

[0016] Wherein, the promoter P pyrF -D40 is an option for the original promoter P. pyrF It was obtained by truncating the -10 region downstream by 40 bp.

[0017] Specifically, the original promoter P pyrF It is the 150 bp base sequence preceding the E. coli pyrF gene, and its nucleotide sequence is shown in SEQ ID NO.1.

[0018] Wherein, the promoter P pyrF -F5 is a further method using degenerate primers (as shown in SEQ ID NO. 15~16) to perform circular PCR on the promoter P. pyrF The -D40 sequence was obtained by randomly mutagenizing the interval sequences in the -35 and -10 regions and then screening them.

[0019] Wherein, the promoter P pyrF -D40, whose nucleotide sequence is shown in SEQ ID NO.12; the promoter P pyrF -F5, whose nucleotide sequence is shown in SEQ ID NO.17.

[0020] Secondly, the present invention provides the application of the promoter described above in the construction of a pentanediamine biosensor.

[0021] Thirdly, the present invention provides a pentanediamine biosensor, wherein the pentanediamine biosensor contains a promoter P pyrF -D40 or P pyrF -F5.

[0022] The pentanediamine biosensor is a promoter P pyrF -D40 or P pyrF -F5 and the recombinant vector of the green fluorescent protein reporter gene gfp.

[0023] Specifically, the promoter can regulate the expression of the green fluorescent protein reporter gene gfp in response to the concentration of pentanediamine.

[0024] The green fluorescent protein reporter gene gfp has the nucleotide sequence shown in SEQ ID NO.30.

[0025] The pentanediamine biosensor uses a pET28a plasmid containing the knockout of the lacO and lacI genes and integrating the multiple cloning site MCS, terminator rrnB T1, and terminator rrnB T2 as a vector.

[0026] In some embodiments of the present invention, the pET28a plasmid that knocks out the lacO and lacI genes and integrates the multiple cloning site MCS, terminator rrnB T1, and terminator rrnB T2 is the recombinant plasmid pcadR-P. pyrF -GFP, the recombinant plasmid pcadR-P pyrF -GFP has been disclosed in Chinese patent CN120384090A.

[0027] In some embodiments of the present invention, the promoter-based P pyrFThe truncated pentanediamine biosensor constructed using the -D40 plasmid is described below: pyrF -GFP was used as a template to obtain the promoter P by PCR amplification. pyrF The truncated gene fragment P downstream of the -10 region pyrF -D40, further digestion of the linearized recombinant plasmid pcadR-P with Bgl II and Xba I enzymes. pyrF -GFP and truncated gene fragment P pyrF -D40 is linked via homologous recombination to obtain recombinant plasmid P. pyrF -D40-GFP, which is based on promoter P pyrF A truncated pentanediamine biosensor constructed using -D40.

[0028] In some embodiments of the present invention, the promoter-based P pyrF The mutant pentanediamine biosensor constructed using -F5 is described below: It is constructed using recombinant plasmid P... pyrF Using -D40-GFP as a template, the promoter P was targeted by circular PCR using degenerate primers. pyrF Random mutations were performed on the spacer sequences between the -35 and -10 regions of -D40 to obtain a pentanediamine biosensor mutant library. This library was then initially screened by inoculating it into 5–10 mL LB medium containing 20 g / L pentanediamine, and the fluorescence intensity (GFP / OD) was compared. 600 Value. Single-point inoculation with high fluorescence intensity was performed in 5–10 mL LB medium containing different concentrations of pentanediamine for secondary screening. The optimal mutant pentanediamine biosensor P was obtained by comparing the response sensitivity of different mutant pentanediamine biosensors to pentanediamine. pyrF -F5-GFP.

[0029] In some embodiments of the present invention, the promoter-based P pyrF A mutant pentanediamine biosensor constructed using -F5, with the original promoter P... pyrF Compared to the significantly enhanced response sensitivity of pentanediamine, it exhibits a superior linear response relationship at concentrations of 0–40 g / L pentanediamine.

[0030] In some embodiments of the present invention, the promoter P pyrF P pyrF -D40 or P pyrF The pentanediamine biosensor constructed from -F5 was inoculated into 5-10 mL LB medium containing 0-20 g / L lysine. The resulting pentanediamine biosensor did not respond to the pentanediamine precursor lysine, thus eliminating the interference of lysine on the biosensor.

[0031] Fourthly, the present invention provides a recombinant strain containing the aforementioned pentanediamine biosensor, used to evaluate the response of the biosensor to exogenous addition of pentanediamine or pentanediamine in the pentanediamine-producing recombinant strain.

[0032] The recombinant strain uses Escherichia coli as its host.

[0033] In some embodiments of the present invention, the Escherichia coli is Escherichia coli DH5α or Escherichia coli KA30.

[0034] The Escherichia coli KA30 has been disclosed in Chinese patents CN113817762B and CN118516387A.

[0035] Fifthly, the present invention provides the application of the aforementioned pentanediamine biosensor in detecting pentanediamine content.

[0036] In a sixth aspect, the present invention provides the application of the aforementioned pentanediamine biosensor in screening high-yield pentanediamine strains.

[0037] In a seventh aspect, the present invention provides a method for screening high-yield pentamethylenediamine strains, wherein the strain to be screened containing the pentamethylenediamine biosensor described in claim 3 or 4 is fermented and cultured, and the fluorescence intensity GFP / OD is calculated. 600 The content of pentanediamine in the fermentation broth was measured to screen for high-yield pentanediamine strains.

[0038] In some embodiments of the present invention, *E. coli* KA30, a lysine-producing bacterium, was used as a chassis to introduce a biosensor and the recombinant plasmid PCDF duet-trc-cadA to obtain a pentanediamine recombinant strain. The above-mentioned pentanediamine recombinant strain was randomly mutagenized using ambient pressure room temperature plasma (ARTP) mutagenesis to obtain a library of random mutant strains. After fermentation culture, the mutagenized strains were sorted using flow cytometry, and single cells showing a significant increase in fluorescence signal were selected as candidate mutagenized strains with enhanced pentanediamine production capacity. Subsequently, the candidate mutagenized strains were inoculated into fresh fermentation medium and subjected to shake-flask fermentation for secondary screening. The fluorescence intensity GFP / OD ratio was calculated by measuring the intensity and biomass of green fluorescent protein in the fermentation broth. 600 Simultaneously, the concentration of the final product, pentanediamine, was measured by high-performance liquid chromatography (HPLC) of the fermentation broth, and mutagenic strains with enhanced pentanediamine production capacity were screened. The above practices demonstrate that, based on the coupling of a pentanediamine biosensor and a high-throughput screening system, high-yield pentanediamine-producing strains can be screened efficiently and rapidly.

[0039] Beneficial effects:

[0040] (1) This invention is based on transcriptome analysis to mine response promoter P pyrFBy truncating and mutating its sequence, a promoter P that responds to pentanediamine was obtained. pyrF -D40 or P pyrF -F5, further based on P pyrF -D40 or P pyrF -F5 constructs a novel, highly efficient pentanediamine biosensor.

[0041] (2) This invention is based on promoter P pyrF -D40 or P pyrF A pentanediamine biosensor constructed using the -F5 promoter enables sensitive detection of the target substance pentanediamine. The promoter P... pyrF P pyrF -D40 or P pyrF The pentanediamine biosensor constructed from -F5 showed no response to lysine, a precursor for pentanediamine synthesis, indicating that it has good specificity when used for screening pentanediamine-producing strains.

[0042] (3) This invention is based on P pyrF The -F5-based pentamethylenediamine biosensor utilizes the mutated endogenous pentamethylenediamine response promoter P in E. coli. pyrF Using F5 and green fluorescent protein (GFP) as basic biological elements, this biosensor links pentanediamine concentration to fluorescence intensity, making pentanediamine concentration visible and enabling real-time, sensitive monitoring of intracellular pentanediamine concentration in bacterial strains. This biosensor can be combined with flow cytometry to establish a high-throughput screening system, facilitating rapid and efficient screening of high-yield pentanediamine single cells and providing technical support for the industrial production of pentanediamine. Attached Figure Description

[0043] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0044] Figure 1 pcadR-P, a pentanediamine biosensor pyrF A schematic diagram of the construction of -GFP.

[0045] Figure 2 For containing P pyrF The response intensity of the truncated pentanediamine biosensor to different concentrations of pentanediamine.

[0046] Figure 3 For containing P pyrF The response strength of the -D40 mutant pentanediamine biosensor in 20 g / L pentanediamine medium.

[0047] Figure 4 For containing P pyrFThe response strength of the -D40 mutant pentanediamine biosensor to different concentrations of pentanediamine.

[0048] Figure 5 To contain P respectively pyrF P pyrF -D40、P pyrF The response strength of the -F5 pentanediamine biosensor to different concentrations of lysine.

[0049] Figure 6 For containing P pyrF The -F5 pentanediamine biosensor was used to measure the fit between pentanediamine concentration and fluorescence intensity during ARTP mutagenesis library screening. Detailed Implementation

[0050] Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods in the art; unless otherwise specified, the reagents and materials are commercially available.

[0051] In the following embodiments, the recombinant plasmid pcadR-P pyrF -GFP has been disclosed in Chinese patent CN120384090A.

[0052] In the following embodiments, the Escherichia coli KA30 has been disclosed in Chinese patents CN113817762B and CN118516387A.

[0053] In the following examples, the PCDF duet, pTrc99a plasmid, and Escherichia coli MG1655 were purchased from Wuhan Miaoling Biotechnology Co., Ltd.

[0054] Example 1: Truncated optimization and screening of pentamethylenediamine biosensors

[0055] 1. Construction of a truncated promoter and truncated pentanediamine biosensor

[0056] With recombinant plasmid pcadR-P pyrF -GFP serves as a template for the promoter P pyrFThe downstream region of the -10 region (as shown in SEQ ID NO.1) was truncated in a gradient manner, with truncations of 10, 20, 30, 40, 50, and 60 bases (bp) designed respectively. The specific process is as follows: using a common upstream primer (as shown in SEQ ID NO.2) and downstream primers targeting different truncated lengths (as shown in SEQ ID NO.3~8), the corresponding PCR products were obtained by PCR amplification. The reaction conditions were: 95 ℃ for 3 min, 95 ℃ for 15 s, 55 ℃ for 15 s, 72 ℃ for 10 s, for a total of 30 cycles; 72 ℃ for 5 min. The obtained PCR products were subjected to 2% agarose gel electrophoresis and then purified using a purification and recovery kit (purchased from Tiangen Biotech (Beijing) Co., Ltd.) to obtain the promoter P. pyrF The truncated gene fragment P downstream of the -10 region pyrF -D10、P pyrF -D20、P pyrF -D30、P pyrF -D40、P pyrF -D50, P pyrF -D60 (hereinafter abbreviated as P) pyrF The nucleotide sequences of the truncated gene fragments (-D10 / D20 / D30 / D40 / D50 / D60) are shown in SEQ ID NO. 9~14. Sequence information is shown in Table 1.

[0057] Table 1 Sequence Information of Example 1

[0058]

[0059] The previously constructed recombinant plasmid pcadR-P pyrF -GFP was linearized by digestion with Bgl II and Xba I from Takara. The digestion reaction mixture was: 5 μL 10× buffer, 1 μL Bgl II, 1 μL Xba I, and plasmid vector pcadR-P. pyrF -GFP 25 μL, ddH2O 18 μL. The enzyme digestion system was reacted at 37 ℃ for 1 h. Then the linearized plasmid was reacted with P... pyrF The D10 / D20 / D30 / D40 / D50 / D60 gene fragments were ligated via homologous recombination. The reaction system consisted of: 2 μL of 5×CE II buffer, 1 μL of Exnase (Novizan), and P... pyrF 5 μL of the -D10 / D20 / D30 / D40 / D50 / D60 gene fragment and 2 μL of linearized plasmid were ligated at 37 °C for 30 min. The ligation product was transformed into E. coli Trans1-T1, and positive strains Trans1-T1-P were screened by PCR. pyrFThe recombinant plasmid was constructed using the -D10 / D20 / D30 / D40 / D50 / D60-GFP method and DNA sequencing was performed to verify the correct construction. The construction of the biosensor plasmid is as follows: Figure 1 As shown.

[0060] The positive strain Trans1-T1-P pyrF -D10 / D20 / D30 / D40 / D50 / D60-GFP was inoculated into 5 mL LB liquid medium containing 50 mg / L kanamycin and cultured overnight at 37 ℃ with shaking at 200 rpm. After 12 h, recombinant plasmid P was extracted according to the instructions of the Tiangen plasmid extraction kit. pyrF -D10 / D20 / D30 / D40 / D50 / D60-GFP, thus obtaining 6 truncated pentanediamine biosensors P pyrF -D10 / D20 / D30 / D40 / D50 / D60-GFP.

[0061] 2. Performance testing and screening of truncated pentanediamine biosensors

[0062] The obtained 6 types of truncated pentanediamine biosensors P pyrF -D10 / D20 / D30 / D40 / D50 / D60-GFP and pristine pentanediamine biosensor pcadR-P pyrF -GFP was introduced into *E. coli* DH5α to obtain recombinant strains. The recombinant strains were inoculated into 5–10 mL of LB medium containing 50 mg / L kanamycin at concentrations of 0 g / L, 10 g / L, 20 g / L, 30 g / L, and 40 g / L pentanediamine, respectively. After overnight incubation at 35–38°C, fluorescence was detected and OD was measured. 600 To detect fluorescence, take 1-2 mL of bacterial culture, centrifuge at 12000 rpm for 1-2 min at 4℃, discard the supernatant, and resuspend in 1-2 mL of PBS buffer; then add 200 μL of the resuspended solution to a black 96-well plate. Use a Thermo fluorescence analyzer with the excitation wavelength set to 485 nm and the emission wavelength to 526 nm to measure the GFP fluorescence value and calculate the fluorescence intensity GFP / OD ratio. 600 value.

[0063] like Figure 2 As shown, by comparing the response sensitivity of different truncated pentanediamine biosensors to different pentanediamine concentrations, it was found that promoter truncation can effectively change the response characteristics of the biosensor. Among them, a 40 bp truncated pentanediamine biosensor P was obtained. pyrF -D40-GFP showed the best performance compared to the original pentamethylenediamine biosensor pcadR-P. pyrFCompared to -GFP, not only is the maximum fluorescence intensity significantly improved, but the apparent response threshold is also increased, approximately reaching that of the original pentanediamine biosensor pcadR-P. pyrF -2 times that of GFP. Therefore, continue with P pyrF -D40-GFP is used for subsequent optimization.

[0064] Example 2: Mutation Optimization and Screening of Pentylene Diamine Biosensor

[0065] 1. Construction of a pentanediamine biosensor mutant library

[0066] The recombinant plasmid P obtained in Example 1 was screened. pyrF Using -D40-GFP as a template, the promoter P was activated by circular PCR using degenerate primers (as shown in SEQ ID NO. 15~16). pyrF Random mutations were performed on the spacer sequences of -D40 in the -35 and -10 regions. The reaction conditions were: 95 ℃ for 3 min, 95 ℃ for 15 s, 55 ℃ for 15 s, 72 ℃ for 90 s, for a total of 30 cycles; followed by 72 ℃ for 5 min. The obtained PCR products were subjected to 1% agarose gel electrophoresis, and the mutated P was recovered using a purification and recovery kit (purchased from Tiangen Biotech (Beijing) Co., Ltd.). pyrF The target fragment was -D40-GFP. Digestion was performed using the restriction endonuclease Dpn I. The digestion reaction mixture consisted of 1 μL of 10×buffer, 1 μL of Dpn I, and 8 μL of the target fragment. The digestion was carried out at 37 °C for 2 h. The digestion product containing the random mutation was then transformed into *E. coli* DH5α to construct a pentanediamine biosensor mutant library.

[0067] 2. Screening and identification of promoter mutants and mutant pentanediamine biosensors

[0068] The above-mentioned pentanediamine biosensor mutant library was seeded into 5–10 mL LB medium containing 20 g / L pentanediamine and cultured. The fluorescence intensity GFP / OD was compared. 600 The values ​​are initially filtered. For example... Figure 3 As shown, a batch of potentially beneficial mutants with fluorescence intensity higher than the initial strain were obtained.

[0069] Single-point inoculation with high fluorescence intensity was performed in 5–10 mL of LB medium containing 50 mg / L kanamycin at concentrations of 0 g / L, 10 g / L, 20 g / L, 30 g / L, and 40 g / L pentanediamine, respectively, for secondary screening. The secondary screening results are as follows: Figure 4 As shown, there are 7 mutants (P pyrFThe fluorescence intensity of (-E1 / F5 / H12 / C8 / D2 / A3 / G4) was significantly improved, with the background leakage fluorescence intensity increasing from 320 to a maximum of 1,880. pyrF -E1 mutant fluorescence intensity GFP / OD 600 The value varies non-linearly within the range of 1788 to 3767 (2.11 times), P pyrF -F5 mutant fluorescence intensity GFP / OD 600 The value changes linearly within the range of 1771 to 4885 (2.76 times), P pyrF The fluorescence intensity of the -H12 mutant is GFP / OD. 600 The value changes linearly within the range of 1880 to 4260 (2.27 times), P pyrF -C8 mutant fluorescence intensity GFP / OD 600 The value changes linearly within the range of 1631 to 3674 (2.25 times), P pyrF -D2 mutant fluorescence intensity GFP / OD 600 The value changes linearly within the range of 1776 to 4052 (2.28 times), P pyrF -A3 mutant fluorescence intensity GFP / OD 600 The value changes linearly within the range of 1372 to 3726 (2.72 times), P pyrF -G4 mutant fluorescence intensity GFP / OD 600 The value changes linearly within the range of 1516 to 3499 (2.31 times), P pyrF -D40 truncated fluorescence intensity GFP / OD 600 The value changes linearly within the range of 861 to 1870 (2.17 times), P pyrF fluorescence intensity of the original promoter GFP / OD 600 The value changes linearly within the range of 320 to 830 (2.59 times). Wherein, P... pyrF The -F5 mutant performed the best, compared to the original sensor P before optimization. pyrF Compared to the control, its background leakage fluorescence intensity increased by 5.5 times, while maintaining a wider dynamic range (1.87 vs 1.76). Furthermore, P... pyrF The -F5 mutant also showed significantly higher response sensitivity than the wild type. Sequencing results showed that P pyrF The GC content in the spacer region of the -F5 mutant increased from 40% to 80%. Therefore, the optimal mutant pentanediamine biosensor P obtained through screening was selected. pyrF -F5-GFP can be used for subsequent high-throughput screening. The nucleotide sequences of the seven beneficial mutants are shown in SEQ ID NO.17~23.

[0070] The sequence information involved in Example 2 is shown in Table 2.

[0071] Table 2 Sequence information involved in Example 2

[0072]

[0073] Example 3: Specificity test of the pentamethylenediamine biosensor

[0074] The promoter P obtained above pyrF P pyrF -D40 or P pyrF The pentanediamine biosensor constructed using F5 was introduced into *E. coli* DH5α to obtain recombinant strains. These recombinant strains were inoculated into 5–10 mL of LB medium containing 50 mg / L kanamycin and lysine at concentrations of 0 g / L, 10 g / L, and 20 g / L, respectively. After overnight incubation at 35–38°C, fluorescence detection and OD were performed. 600 Value. For example... Figure 5 As shown, the obtained pentanediamine biosensor did not respond to lysine, a precursor for pentanediamine synthesis, thus eliminating the interference of lysine on the biosensor. This indicates that the biosensor has good specificity when used for screening pentanediamine-producing strains.

[0075] Example 4: Application of pentamethylenediamine biosensor in screening high-yield pentamethylenediamine strains

[0076] 1. Construct the recombinant plasmid PCDF duet (trc)

[0077] Using PCDF duet as a template, the deleted PCDF duet expression cassette region was obtained by PCR amplification using the upstream and downstream primers shown in SEQ ID NO.24~25.

[0078] Using pTrc99a plasmid as a template, the Trc-MCS-rrnBT1-rrnBT2 fragment was obtained by PCR amplification using the upstream and downstream primers shown in SEQ ID NO.26~27.

[0079] The PCDF duet (△T7) fragment and the Trc-MCS-rrnBT1-rrnBT2 fragment were ligated via homologous recombination. The reaction mixture consisted of 2 μL of 5×CE II buffer, 1 μL of Exnase (Novizan), 5 μL of the Trc-MCS-rrnBT1-rrnBT2 fragment, and 2 μL of the PCDF duet (△T7) fragment. Ligation was carried out at 37 ℃ for 30 min. The ligation product was transformed into *E. coli* Trans1-T1. Positive strains, Trans1-T1-PCDF duet (trc), were screened by PCR and their DNA was sequenced to verify the correct construction of the recombinant plasmid.

[0080] The positive strain Trans1-T1-PCDF duet (trc) was inoculated into 5 mL LB liquid medium containing 80 mg / L streptomycin and cultured overnight at 37 ℃ and 200 rpm with shaking. After 12 h, the recombinant plasmid PCDF duet (trc) was extracted according to the instructions of the Tiangen plasmid extraction kit.

[0081] 2. Construct the recombinant plasmid PCDF duet-trc-cadA containing lysine decarboxylase.

[0082] Using *E. coli* MG1655 as a template, the cadA gene fragment was amplified by PCR using the upstream and downstream primers shown in SEQ ID NO. 28-29. The previously constructed recombinant plasmid PCDF duet (trc) was linearized by digestion with Nco I and Kpn I from Takara. The digestion reaction mixture consisted of: 5 μL 10× buffer, 1 μL Nco I, 1 μL Kpn I, 25 μL PCDF duet (trc) plasmid vector, and 18 μL ddH2O. The digestion was carried out at 37 °C for 1 h. Then, the linearized plasmid and the cadA gene fragment were ligated by homologous recombination. The reaction mixture consisted of: 2 μL 5× CE II buffer, 1 μL Exnase (Novizan), 5 μL cadA gene fragment, and 2 μL linearized plasmid. The ligation was carried out at 37 °C for 30 min. The ligation product was then transformed into *E. coli* Trans1-T1. Positive strain Trans1-T1-PCDF duet-trc-cadA was screened by PCR and its DNA was sequenced to verify that the recombinant plasmid PCDF duet-trc-cadA was constructed correctly.

[0083] The sequence information involved in Example 4 is shown in Table 3.

[0084] Table 3 Sequence information involved in Example 4

[0085]

[0086] 3. Application of pentamethylenediamine biosensors in screening high-yield pentamethylenediamine strains

[0087] The pentanediamine biosensor P obtained in Example 2 pyrF -F5-GFP and the recombinant plasmid PCDFduet-trc-cadA obtained in Example 3 were co-transformed into lysine-producing *Escherichia coli* KA30 to obtain a pentanediamine recombinant strain. The pentanediamine recombinant strain was cultured in LB medium to the logarithmic growth phase, and the bacterial suspension was diluted with 0.85% sterile physiological saline to adjust the OD of the suspension.600 Maintaining the pH between 0.6 and 0.8, the diluted bacterial solution was evenly spread onto sterile iron slides in a laminar flow hood, and mutagenesis was performed using an ARTP mutagenesis breeding system. The parameters were set as follows: power 100 W, gas flow rate 10 SLSM, and mutagenesis time 40 s. After mutagenesis, the iron slides were placed in sterile centrifuge tubes containing physiological saline, eluted by shaking, and then incubated upside down for 24–36 h. Single-site fermentation validation was performed on selected mutagenized samples. The specific steps were as follows: a single site was inoculated into seed culture medium for expansion culture, and cultured at 35–40℃ for 12–16 h until OD reached. 600 =1~2, pH=4~6, to obtain seed culture; wherein, the seed culture medium contains 40 mg / L streptomycin sulfate and 25 mg / L kanamycin resistance, and its formula is: ammonium sulfate 10 g / L, monosodium glutamate 5 g / L, peptone 5 g / L, yeast extract 10 g / L, dipotassium hydrogen phosphate 0.5 g / L, sodium pyruvate 0.55 g / L, sucrose 5 g / L, magnesium sulfate heptahydrate 1 g / L, manganese sulfate monohydrate 0.077 g / L, ferrous sulfate heptahydrate 0.032 g / L, methionine 0.3 g / L, threonine 0.3 g / L.

[0088] The obtained seed culture was inoculated into the fermentation medium at an inoculation rate of 1-5% v / v, and the initial OD was controlled. 600 The concentration of the active ingredient was 0.1–0.2 mg / L, the culture conditions were 35–40 °C, pH = 6–8, and the total fermentation time was 24–36 h. The fermentation medium contained 40 mg / L streptomycin sulfate and 25 mg / L kanamycin resistance, and its formula was: ammonium sulfate 10 g / L, peptone 5 g / L, yeast extract 2 g / L, potassium chloride 0.5 g / L, magnesium sulfate heptahydrate 1.6 g / L, MopasNa 100 mM, CaCO3 10 g / L, biotin 30 μg / L, vitamin B1 0.06 g / L, glucose 20 g / L, methionine 0.3 g / L, threonine 0.3 g / L, zinc sulfate 0.086 g / L, copper sulfate 0.077 g / L, manganese sulfate 0.032 g / L, nicotinamide 0.01 g / L, and ferrous sulfate heptahydrate 0.032 g / L.

[0089] Take 1-2 mL of fermentation broth and measure the final growth OD. 600 To measure fluorescence intensity, take 1-2 mL of bacterial culture, centrifuge at 12000 rpm for 1-2 min at 4℃, discard the supernatant, and resuspend in 1-2 mL of PBS buffer; then add 200 μL of the resuspended solution to a black 96-well plate. Use a Thermo fluorescence analyzer with excitation wavelength set to 485 nm and emission wavelength to 526 nm to measure GFP fluorescence value and calculate fluorescence intensity GFP / OD.600 value.

[0090] Centrifuge 2 mL of fermentation broth at 12000 rpm for 2 min, and collect the supernatant for pentanediamine detection. The detection method for pentanediamine is as follows: The concentration of 1,5-pentanediamine was determined using an Agilent 1290 high-performance liquid chromatography system and an Agilent YMC Carotenoid C30 column (4.6 × 250 mm). The mobile phase composition was 0.5% trifluoroacetic acid and 5% acetonitrile; the flow rate was 0.8 mL / min. -1 The column oven temperature was 35℃; the injection volume was 10 μl; and a differential detector was used. Figure 6 As shown, the fluorescence intensity GFP / OD 600 A strong linear correlation (R0) was observed between the value and the concentration of pentamethylenediamine. 2 =0.91), indicating the reliability of the biosensor, which can be applied to high-throughput screening.

[0091] This invention provides a promoter responsive to pentanediamine, a pentanediamine biosensor, and their applications. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A promoter responsive to pentanediamine, characterized in that, The nucleotide sequence of the promoter is shown in SEQ ID NO.12 or SEQ ID NO.

17.

2. The application of the promoter according to claim 1 in the construction of a pentanediamine biosensor.

3. A pentanediamine biosensor, characterized in that, It contains the promoter as described in claim 1.

4. The pentanediamine biosensor according to claim 3, characterized in that, The pentanediamine biosensor is a recombinant vector carrying the promoter of claim 1 and the green fluorescent protein reporter gene gfp, wherein the promoter regulates the expression of the green fluorescent protein reporter gene gfp.

5. The pentanediamine biosensor according to claim 3, characterized in that, The pentanediamine biosensor uses a pET28a plasmid containing the knockout of the lacO and lacI genes and integrating the multiple cloning site MCS, terminator rrnB T1, and terminator rrnB T2 as a vector.

6. A recombinant bacterial strain, characterized in that, The recombinant strain contains the pentanediamine biosensor as described in claim 3 or 4.

7. The recombinant strain according to claim 6, characterized in that, The recombinant strain uses Escherichia coli as the host, and the Escherichia coli is either Escherichia coli DH5α or Escherichia coli KA30.

8. The application of the pentanediamine biosensor according to any one of claims 3 to 5 in the detection of pentanediamine content.

9. The application of the pentanediamine biosensor according to any one of claims 3 to 5 in screening high-yield pentanediamine strains.

10. A method for screening high-yield pentamethylenediamine strains, characterized in that, The selected strain containing the pentanediamine biosensor as described in claim 3 or 4 was fermented, and the fluorescence intensity GFP / OD was calculated. 600 The content of pentanediamine in the fermentation broth was measured to screen for high-yield pentanediamine strains.

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