Method for high-throughput screening of key genes in Escherichia coli 1, 5-pentamethylene diamine biosynthetic pathway

Abstract

The invention discloses a method for high-throughput screening of key genes in a 1, 5-pentamethylene diamine biosynthetic pathway of escherichia coli, and the high-throughput screening is carried out by using a 1, 5-pentamethylene diamine biosensor. The 1, 5-pentamethylene diamine biosensor is a recombinant plasmid obtained by integrating a lysine decarboxylase coding gene cadA and a promoter trc thereof, and a green fluorescent protein (GFP) coding gene and a promoter pyrF thereof into a plasmid skeleton. A fluorescent quantitative flow cytometer is used for screening single-cell thalli with high yield of 1, 5-pentanediamine according to the fluorescence intensity of GFP, key genes can be rapidly screened out from a large number of genes to be screened in a high-throughput mode, and the screening workload and time are greatly saved. According to the application of the key gene in the biosynthetic pathway of the escherichia coli 1, 5-pentanediamine screened by the method disclosed by the invention, the yield of the 1, 5-pentanediamine produced by fermenting the escherichia coli KA30 can be greatly improved, the substrate conversion rate in the fermentation process is improved, the production cost of the 1, 5-pentanediamine is reduced, and the economic benefit is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial genetic engineering, and particularly relates to a method for high-throughput screening of key genes in the 1,5-pentanediamine biosynthesis pathway of Escherichia coli. Background Art

[0002] 1,5-Pentanediamine (1,5-diaminopentane) is an important chemical raw material, especially playing an important role in the synthesis of high molecular compounds such as polyamides and polyureas. It is a colorless liquid with an ammonia smell, soluble in water and many organic solvents, and is a basic compound that can react with acids to form salts.

[0003] In recent years, the technology for biosynthesis of 1,5-pentanediamine has become increasingly mature and is also the mainstream method for the synthesis of 1,5-pentanediamine. Among them, the biological method includes whole-cell catalysis and fermentation. The whole-cell catalysis method uses L-lysine as a raw material and obtains 1,5-pentanediamine through the catalysis of lysine decarboxylase, and the process is relatively cumbersome. The fermentation method has advantages such as mild conditions and low production costs, but this method also has its deficiencies, such as poor tolerance of microorganisms to 1,5-pentanediamine and low substrate conversion rate. Therefore, screening for key genes in the process of microbial fermentation of 1,5-pentanediamine is of great significance for realizing the efficient biosynthesis of 1,5-pentanediamine by microorganisms. Summary of the Invention

[0004] The object of the present invention is to provide a method for high-throughput screening of key genes in the 1,5-pentanediamine biosynthesis pathway of Escherichia coli for the problems of poor tolerance of microorganisms to 1,5-pentanediamine and low production efficiency of 1,5-pentanediamine existing in the process of existing microbial fermentation production of 1,5-pentanediamine.

[0005] In order to achieve the above object, the technical solution provided by the present invention is as follows:

[0006] A 1,5-pentanediamine biosensor, comprising a lysine decarboxylase encoding gene cadA and its promoter trc, a green fluorescent protein GFP encoding gene and its promoter pyrF, and a plasmid backbone.

[0007] Among them, the nucleotide sequence of the lysine decarboxylase encoding gene cadA is as shown in SEQ ID NO.1; the nucleotide sequence of the promoter trc is as shown in SEQ ID NO.2; the nucleotide sequence of the green fluorescent protein GFP encoding gene is as shown in SEQ ID NO.3; the nucleotide sequence of the promoter pyrF is as shown in SEQ ID NO.4.

[0008] Among them, the plasmid backbone is plasmid pET28a.

[0009] Among them, the genes lacO and lacI are deleted from the plasmid pET28a; the genes lacO and lacI are the operator sequence lacO and the regulatory gene lacI encoding the repressor protein in the lactose operon, respectively.

[0010] Among them, the multiple cloning site MCS, the terminator rrnB T1, and the terminator rrnB T2 are integrated into the plasmid pET28a; the multiple cloning site MCS, the terminator rrnB T1, and the terminator rrnB T2 are all from the plasmid pTrc99a.

[0011] The present invention also provides a method for constructing the 1,5-pentanediamine biosensor described above, comprising the following steps:

[0012] (1) Delete the lacO and lacI genes from the plasmid pET28a to obtain the plasmid pET28a(ΔlacIO);

[0013] (2) Using the plasmid pET28a(ΔlacIO) as a template, perform PCR amplification with primers 28a-F and 28a-R to obtain a plasmid fragment; wherein, the nucleotide sequences of the primers 28a-F and 28a-R are shown in SEQ ID NO.12 and SEQ ID NO.13, respectively;

[0014] (3) Connect the multiple cloning site MCS, the terminator rrnB T1, and the terminator rrnB T2 to the plasmid fragment obtained in step (2) to obtain the recombinant plasmid pcadR;

[0015] (4) Integrate the green fluorescent protein GFP coding gene and its promoter pyrF, and the lysine decarboxylase coding gene cadA and its promoter trc into the recombinant plasmid pcadR obtained in step (3), and the 1,5-pentanediamine biosensor is obtained.

[0016] The present invention also provides a recombinant strain containing the 1,5-pentanediamine biosensor described above.

[0017] Among them, the starting strain of the recombinant strain is the high-lysine-producing Escherichia coli KA30, which is a strain classified as Escherichia coli, strain number: NT1003△Met△Thr, and was deposited at the China Center for Type Culture Collection (CCTCC) on May 30, 2013, with the deposit number: CCTCC NO:M2013239. The detailed information of this strain has been publicly disclosed in the patent CN 103361289 B.

[0018] The present invention also provides the application of the described 1,5-pentanediamine biosensor in screening key genes in the 1,5-pentanediamine biosynthesis pathway of Escherichia coli; wherein, the Escherichia coli is Escherichia coli KA30.

[0019] The present invention also provides a method for screening key genes in the 1,5-pentanediamine biosynthesis pathway of Escherichia coli using the described 1,5-pentanediamine biosensor, comprising the following steps:

[0020] (1) Introduce the 1,5-pentanediamine biosensor into Escherichia coli KA30 to obtain recombinant Escherichia coli KA30-GFP-cadA;

[0021] (2) Construct a single-gene recombinant expression vector for the gene to be screened to obtain a single-gene overexpression library, and introduce it into the recombinant Escherichia coli KA30-GFP-cadA obtained in step (1) to obtain library recombinant strains; wherein, the construction of the recombinant expression vector should be based on the standard that the single gene can be overexpressed in Escherichia coli KA30; the single-gene recombinant expression vectors in the single-gene overexpression library can be introduced into the recombinant Escherichia coli KA30-GFP-cadA alone, partially mixed, or all mixed;

[0022] (3) Ferment and culture the library recombinant strains obtained in step (2), and screen out single-cell bacteria with enhanced GFP fluorescence intensity and / or enhanced ratio of GFP fluorescence intensity to bacterial OD 600 (GFP / OD 600 ) compared with the negative control;

[0023] (4) Ferment and culture the single-cell bacteria obtained in step (3), screen out single-cell bacteria with increased 1,5-pentanediamine production compared with the negative control, extract their plasmids, and sequence them to screen out the key genes.

[0024] In step (2), when the GFP fluorescence intensity is used as the screening criterion, the screening method is: after the fermentation culture is completed, take an appropriate amount of the fermentation broth, centrifuge and wash it several times with sterile PBS buffer (wash away the fermentation broth to prevent contamination of the subsequent fluorescence quantitative flow cytometry instrument), control the OD of the bacteria with sterile PBS buffer 600 to be 0.2-0.4, and transfer an appropriate amount of the bacterial solution to a sterile flow tube, and perform fluorescence quantitative flow cytometry sorting to screen out single-cell bacteria with significantly enhanced GFP fluorescence intensity compared with the negative control; when the ratio of GFP fluorescence intensity to bacterial OD 600 (GFP / OD 600 ) is used as the screening criterion, the screening method is: after the fermentation culture is completed, take an appropriate amount of the fermentation broth sample, measure the OD of the bacteria in the fermentation broth 600, then centrifuge the fermentation broth at 12,000 - 13,000 rpm for 1 - 3 min at 0 - 4°C, discard the supernatant, resuspend the cells with a sterile PBS buffer solution of the same volume as the fermentation broth sample. Take an appropriate amount of the resuspended cell solution and place it in a black 96-well plate. Use a fluorescence analyzer to measure the GFP fluorescence intensity at an excitation wavelength of 485 nm and an emission wavelength of 526 nm, and calculate the ratio of the GFP fluorescence intensity to OD 600 (GFP / OD 600 ). Screen out single-cell bacteria with a significantly increased GFP / OD 600 ratio compared to the negative control.

[0025] Preferably, when the number of genes to be screened is too large, in step (3), a rough screening can be first performed based on the GFP fluorescence intensity, and then a rescreening can be carried out based on the ratio of the GFP fluorescence intensity to OD 600 (GFP / OD 600 ).

[0026] Preferably, when the number of single-cell bacteria obtained in step (3) is so large that it takes a lot of time, labor, and economic costs to measure the 1,5-pentanediamine concentration in step (4), the single-cell bacteria obtained in step (3) can be fermented and cultured, and then the screening method of step (3) can be repeated to screen out single-cell bacteria that can stably express the gene to be screened, and then the screening method of step (4) can be carried out.

[0027] In steps (3) and (4), the fermentation is microplate fermentation, shake tube fermentation, or shake flask fermentation.

[0028] Among them, the components of the fermentation medium used in the fermentation culture process are: ammonium sulfate 8 - 10 g / L, peptone 5 - 6 g / L, yeast extract powder 2 - 5 g / L, potassium chloride 0.5 - 0.75 g / L, magnesium sulfate heptahydrate 1.6 - 2 g / L, MoPasNa (3-morpholinopropanesulfonic acid) 21.143 - 23.124 g / L, glucose 20 g / L, methionine 0.15 - 0.3 g / L, threonine 0.15 - 0.3 g / L, zinc sulfate heptahydrate 0.086 - 1 g / L, copper sulfate anhydrous 0.077 - 0.85 g / L, manganese sulfate monohydrate 0.032 - 0.043 g / L, vitamin B1 0.06 - 0.01 g / L, nicotinamide 0.01 - 0.02 g / L, biotin 30 - 35 μg / L, ferrous sulfate heptahydrate 0.032 - 0.043 g / L.

[0029] Among them, the fed-batch method is adopted for feeding during the fermentation culture process, and feeding is carried out twice. Each time, 500-600 g / L of glucose aqueous solution and 400-500 g / L of ammonium sulfate aqueous solution are fed, so that after feeding, the final concentrations of glucose and ammonium sulfate in the fermentation medium are 20-25 g / L and 10-15 g / L respectively.

[0030] The biosensor of the present invention simultaneously contains the lysine decarboxylase encoding gene cadA and the green fluorescent protein GFP encoding gene and its promoter pyrF. The lysine decarboxylase encoding gene cadA expresses lysine decarboxylase, which catalyzes the decarboxylation reaction of lysine produced by Escherichia coli KA30 to generate 1,5-pentanediamine. At the same time, according to the mechanism that the promoter pyrF of the green fluorescent protein GFP can make an up-regulated response to 1,5-pentanediamine, the green fluorescent protein GFP encoding gene is used as a reporter gene, and the expression level of 1,5-pentanediamine can be quantitatively analyzed by detecting the fluorescence intensity of the green fluorescent protein GFP encoded by it. Compared with the traditional high-performance liquid chromatograph for measuring the concentration of the target metabolite, this method greatly shortens the detection time and cost, and when this technology is combined with a flow cytometer, it can improve the screening efficiency and quickly screen out the key genes in the 1,5-pentanediamine biosynthesis pathway of Escherichia coli KA30 from a large number of genes to be screened.

[0031] Beneficial effects:

[0032] (1) In the 1,5-pentanediamine biosensor of the present invention, the promoter pyrF of the green fluorescent protein GFP can make an up-regulated response to 1,5-pentanediamine. Using the green fluorescent protein GFP encoding gene as a reporter gene, the expression level of 1,5-pentanediamine can be quantitatively analyzed by detecting the fluorescence intensity of the green fluorescent protein GFP encoded by it. Compared with directly quantitatively detecting 1,5-pentanediamine, using a fluorescence quantitative flow cytometer to screen single-cell bacteria with high-yield 1,5-pentanediamine according to the GFP fluorescence intensity can quickly screen out the key genes in the 1,5-pentanediamine biosynthesis pathway of Escherichia coli from a large number of genes to be screened, greatly saving the screening workload and time.

[0033] (2) The key genes in the 1,5-pentanediamine biosynthesis pathway of Escherichia coli can be screened out by the 1,5-pentanediamine biosensor constructed by the present invention. The application of these key genes can greatly increase the yield of 1,5-pentanediamine fermented by Escherichia coli KA30, improve the substrate conversion rate during the fermentation process, reduce the cost of producing 1,5-pentanediamine, and improve economic benefits. Description of the drawings

[0034] The following further specifically describes the present invention in conjunction with the drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0035] Figure 1 Schematic diagram of the construction process of the biosensor pcadR-pyrF-GFP-trc-cadA of the present invention.

[0036] Figure 2 For GFP / OD in Example 2 600 Scatter plot of the correlation between the yield of 1,5-pentanediamine.

[0037] Figure 3 Dual-parameter scatter plot of EITC-A (GFP fluorescence intensity) and SSC-A (side scatter signal) for high-throughput screening of some overexpressed single genes in the gene library to be screened by the biosensor of the present invention in Example 3.

[0038] Figure 4 For the GFP fluorescence intensity and GFP / OD of 22 single-cell bacterial cells obtained after multi-batch fermentation and screening of single-cell bacterial cells in 96-well deep plates in Example 4 600 Statistical chart; where the abscissa is the number of single-cell bacterial cells.

[0039] Figure 5 Schematic diagram of the 7 target genes finally screened in Example 4 that can significantly improve the yield of 1,5-pentanediamine, their improvement multiples and action mechanisms. Detailed implementation manners

[0040] The following further illustrates the present invention according to the following examples. It should be understood that the following implementation manners are only used to illustrate the present invention and do not limit the present invention.

[0041] For specific technologies or conditions not specified in the examples, they shall be in accordance with the technologies or conditions described in the literature in this field or in accordance with the product specifications. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through regular channels.

[0042] In the following examples, the components of the LB (Luria-Bertani) liquid medium are 10 g / L of peptone, 5 g / L of yeast extract, 5 g / L of sodium chloride, and 0.55 g / L of sodium pyruvate; the components of the LB solid medium are 10 g / L of peptone, 5 g / L of yeast extract, 5 g / L of sodium chloride, 0.55 g / L of sodium pyruvate, and 25 g / L of agar powder; the components of the seed medium are 10 g / L of ammonium sulfate, 5 g / L of sodium glutamate, 5 g / L of peptone, 10 g / L of yeast extract, 4 g / L of dipotassium hydrogen phosphate trihydrate, 0.55 g / L of sodium pyruvate, 5 g / L of sucrose, 1 g / L of magnesium sulfate heptahydrate, 0.077 g / L of manganese sulfate monohydrate, 0.3 g / L of methionine, 0.3 g / L of threonine, and 0.032 g / L of ferrous sulfate heptahydrate.

[0043] The single-gene overexpression library ASKA Library (ASKA library) used in the following examples was purchased from NBRP E. coli Strain and is a complete set of independent gene clone libraries constructed based on the E. coli genome ORF.

[0044] The detection method for the content of 1,5-pentanediamine in the fermentation broth is as follows: For the determination of 1,5-pentanediamine concentration, an Agilent 1290 high-performance liquid chromatography system and an Agilent YMC Carotenoid C30 chromatographic column (4.6×250 mm, 5 μm) are used; the detector is a differential refractive index detector; the mobile phase is an aqueous solution of 0.5% v / v trifluoroacetic acid and an aqueous solution of 5% v / v acetonitrile with a volume ratio of 1:10, the flow rate is 0.8 mL / min; the column oven temperature is 35 °C; the injection volume is 10 μL.

[0045] Example 1 Construction of a 1,5-pentanediamine biosensor

[0046] (1) Construction of the recombinant plasmid pcadR-pyrF-GFP.

[0047] Using the pET28a plasmid as a template, and cadR-F and cadR-R as the upstream and downstream primer pairs, the pET28a plasmid was subjected to PCR amplification to delete the operator sequence lacO of the lactose operon and the regulatory gene lacI encoding the repressor protein on the pET28a plasmid, obtaining the plasmid pET28a(ΔlacIO).

[0048] Using the plasmid pET28a(ΔlacIO) as a template, and 28a-F and 28a-R as the upstream and downstream primers for PCR amplification to obtain the target fragment; at the same time, using the pTrc99a plasmid as a template, and 99a-F and 99a-R as the upstream and downstream primers for PCR amplification to obtain the target fragment containing the multiple cloning site MCS and the terminators rrnB T1 and rrnB T2. After double digestion of the above two target fragments with the restriction enzymes NcoI and XhoI, they were ligated using T4 DNA ligase to obtain the target recombinant plasmid pcadR.

[0049] The plasmid pcadR was digested with the restriction endonucleases KpnI and NcoI to obtain a linearized vector. Meanwhile, using the plasmid ptrc99a-GFPuv stored in the laboratory as a template, and gfp-F and gfp-R as the upstream and downstream primers, the GFP coding gene was amplified by PCR to obtain the GFP coding gene, which was recombined with the linearized plasmid pcadR by homologous recombination to obtain the recombinant plasmid pcadR-GFP; the construction method of the plasmid ptrc99a-GFPuv was as follows: the gene GFPuv (nucleotide sequence as shown in SEQ ID NO.14) was synthesized by Suzhou Genewiz Biotechnology Co., Ltd. and cloned on the plasmid pUC57 to obtain the recombinant plasmid pUC57-GFPuv; using the recombinant plasmid pUC57-GFPuv as a template, the GFPuv′ fragment was amplified using the primers GFPuv-99a-F / GFPuv-99a-R, and it was recombined with the linearized plasmid pTrc99a to obtain the plasmid ptrc99a-GFPuv.

[0050] Using the genome of Escherichia coli NT1003 as a template, the promoter pyrF fragment was obtained by PCR amplification using the primers pyrF-F / pyrF-R. The recombinant plasmid pcadR-GFP was digested with the restriction endonucleases XbaI and BglII to obtain a linearized vector. The above promoter pyrF fragment and the linearized pcadR-GFP plasmid were ligated by homologous recombination to obtain the recombinant plasmid pcadR-pyrF-GFP.

[0051] (2) Construction of the recombinant plasmid pCDFDuet-trc-cadA-ΔlacO

[0052] Using the recombinant plasmid pCDFDuet-cadA-△lacO constructed in Example 1 of Patent CN 118516387 A as a template, inverse PCR was performed using the primers trc-F / trc-R to replace the original T7 promoter in the recombinant plasmid pCDFDuet-cadA-△lacO with the trc promoter to obtain the recombinant plasmid pCDFDuet-trc-cadA-ΔlacO.

[0053] (3) Construction of the 1,5-pentanediamine biosensor pcadR-pyrF-GFP-trc-cadA

[0054] Using the recombinant plasmid pCDFDuet-trc-cadA-ΔlacO as a template, PCR amplification was performed with primers cadA-GFP-F (carrying a SalI restriction site) and cadA-GFP-R (carrying a HindIII restriction site). The amplification program was pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 20 s, for a total of 30 cycles; final extension at 72°C for 5 min. The obtained PCR product was subjected to 1% agarose gel electrophoresis, and the corresponding trc-cadA gene fragment was recovered using a purification and recovery kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.).

[0055] The recombinant plasmid pcadR-pyrF-GFP was double digested with restriction enzymes SalI and HindIII (restriction enzymes SalI and HindIII were purchased from Shanghai Haojia Technology Development Co., Ltd.). The enzyme digestion reaction system was 1 μL of 10× buffer, 1 μL of SalI, 1 μL of HindIII, and 7 μL of the recombinant plasmid pcadR-pyrF-GFP. After the enzyme digestion system was reacted at 37°C for 1 h, a linearized plasmid was obtained. The linearized plasmid pcadR-pyrF-GFP was ligated with the trc-cadA gene fragment by homologous recombination, and the trc-cadA gene fragment was incorporated into the MCS region of the plasmid pcadR-pyrF-GFP. The ligation reaction system (the homologous recombination enzyme Exnase II was purchased from Nanjing Novozymes Biotech Co., Ltd.) was 1 μL of Exnase II, 2 μL of 5x CE II Buffer, 5 μL of the trc-cadA gene fragment, and 2 μL of the linearized plasmid pcadR-pyrF-GFP, and the reaction was carried out at 37°C for 30 min. The homologous recombination product was transformed into Escherichia coli DH5α, and the transformed bacterial solution was spread on an LB plate containing 25 mg / L kanamycin and cultured at 37°C for 12 - 16 h. After the transformants grew out, single colonies were selected for colony PCR to screen positive transformants, and DNA sequencing verification was performed to successfully construct a recombinant plasmid, namely the biosensor pcadR-pyrF-GFP-trc-cadA. The schematic diagram of the construction process of the biosensor pcadR-pyrF-GFP-trc-cadA of the present invention is as Figure 1 shown.

[0056] The primer sequences used in the construction process of the 1,5-pentanediamine biosensor pcadR-pyrF-GFP-trc-cadA are shown in Table 1.

[0057] Table 1 Primer sequences used in the construction process of pcadR-pyrF-GFP-trc-cadA

[0058]

[0059]

[0060] Example 2 Verifying the Response Ability of 1,5-Pentanediamine Biosensor

[0061] (1) Construction of Recombinant Escherichia coli KA30-GFP-cadA Containing Biosensor

[0062] Inoculate the positive Escherichia coli DH5α strain containing the recombinant plasmid pcadR-pyrF-GFP-trc-cadA into 5 mL of LB liquid medium (containing 25 mg / L kanamycin), and culture it overnight at 37 °C with shaking at 220 rpm. Extract the recombinant plasmid pcadR-pyrF-GFP-trc-cadA according to the operation instructions of the Tiangen plasmid extraction kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., product number: DP103-03).

[0063] Mix 5 μL of the recombinant plasmid pcadR-pyrF-GFP-trc-cadA with 30 μL of Escherichia coli KA30 competent cells, incubate on ice for 25 min, then heat shock at 42 °C for 45 s, and continue to incubate on ice for 2 min. In a laminar flow hood, add 1 mL of LB liquid medium to it, and culture it on a shaker at 37 °C for 1 h. After the culture is completed, centrifuge at 4000 rpm for 4 min. In the laminar flow hood, remove 800 μL of the supernatant, resuspend the remaining cells and supernatant by pipetting, and spread them on an LB solid plate containing 25 mg / L kanamycin. After spreading, incubate it in an inverted position in a 37 °C incubator for about 20 h to obtain recombinant Escherichia coli KA30-GFP-cadA containing the biosensor.

[0064] (2) Preparation of Competent Cells of Recombinant Escherichia coli KA30-GFP-cadA

[0065] Pick a single colony of recombinant Escherichia coli KA30-GFP-cadA on the solid plate in step (1) into a 50 mL centrifuge tube containing 5 mL of LB liquid medium (containing 25 mg / L kanamycin), culture it overnight at 37 °C, and then inoculate it into 50 mL of LB liquid medium (containing 25 mg / L kanamycin) at an inoculation amount of 1% v / v. Culture it at 37 °C until OD 600When it is about 0.6, transfer the culture medium into a 50 mL centrifuge tube, pre-cool it on ice for 15 min, then centrifuge at 4000 rpm for 10 min at 4 °C. Pour off the supernatant and collect the cells. Add 10 mL of sterilized and pre-cooled 0.1 M CaCl₂ to the collected cells, resuspend them, let the cells stand for 10 - 15 min, centrifuge at 4000 rpm for 10 min at 4 °C, collect the cells, and repeat this step once to obtain the competent cells of Escherichia coli KA30-GFP-cadA. Add 1 mL of sterilized and pre-cooled 0.1 M CaCl₂ + 10% v / v glycerol to the competent cells, resuspend the cells, aliquot the cell suspension into sterilized centrifuge tubes, and place them in an -80 °C refrigerator for subsequent use.

[0066] (3) Preliminary experiment to verify the response ability of the 1,5-pentanediamine biosensor

[0067] The single-gene overexpression library ASKA Library is stored in a 96-well plate in an -80 °C refrigerator in the form of frozen bacteria. There are a total of 56 96-well plates, approximately 4122 single-gene overexpression plasmids, and each single-gene overexpression plasmid is chloramphenicol-resistant. After the library is thawed and refrozen, each 96-well plate is taken as a group. Pipette 10 μL of the culture medium from each well of each 96-well plate into a 50 mL centrifuge tube containing 5 mL of LB liquid medium (containing 25 mg / L chloramphenicol) for mixed culture. After overnight culture at 37 °C and 220 rpm, extract the mixed plasmids according to the operation instructions of the Tiangen plasmid extraction kit. A total of 56 tubes of mixed plasmids are obtained.

[0068] Randomly select two examples of the mixed plasmids, take 2 μL each and mix them with 30 μL of Escherichia coli KA30-GFP-cadA competent cells respectively for chemical transformation. The transformation method is the same as that in step (1) of this example. The LB solid plate used contains 12.5 mg / L kanamycin and 12.5 mg / L chloramphenicol.

[0069] Randomly select several single colonies on two kinds of LB solid plates and inoculate them into 5 mL of seed medium (containing 12.5 mg / L chloramphenicol and 12.5 mg / L kanamycin) respectively. Culture overnight at 37 °C and 220 rpm for 12 h; then transfer them to 100 mL of fermentation medium (containing 12.5 mg / L chloramphenicol and 12.5 mg / L kanamycin) at an inoculation amount of 5% v / v, and culture at 37 °C and 220 rpm until the OD of the bacteria in the fermentation medium 600 When it is about 0.6, add IPTG inducer to the fermentation medium to make its initial concentration in the fermentation medium 1 mM, and continue to culture at 37 °C and 220 rpm for about 24 h. After fermentation, take 1 mL of the fermentation broth sample and measure the OD of the bacteria in the fermentation broth 600Values. Then, the fermentation broth was centrifuged at 12,000 rpm for 1 min at 4°C, and the supernatant was discarded. The cells were resuspended in 1 mL of sterile PBS buffer. 200 μL of the resuspended cell solution was taken and placed in a black 96-well plate. Using a Thermo fluorescence analyzer, the excitation wavelength was set at 485 nm, and the emission wavelength was set at 526 nm to measure the GFP fluorescence intensity, and the ratio of GFP fluorescence intensity to OD 600 was calculated (GFP / OD 600 ); the concentration of 1,5-pentanediamine in the fermentation broth was detected using a high-performance liquid chromatograph. The correlation between GFP / OD 600 and the yield of 1,5-pentanediamine was analyzed. The results are shown in Figure 2 . The results showed that there was a linear positive correlation between GFP / OD 600 and the concentration of 1,5-pentanediamine, that is, GFP / OD 600 increased with the increase in the concentration of 1,5-pentanediamine. It was shown that the response mechanism construction of the 1,5-pentanediamine biosensor was completed.

[0070] Example 3 High-throughput screening using a fluorescence quantitative flow cytometer

[0071] 3 μL of plasmid was taken from each of the 56 tubes of mixed plasmids and 1 tube of control empty plasmid (control group, the control empty plasmid was the empty plasmid of the ASKA library without any overexpressed genes) described in step (3) of Example 2, and was mixed with 30 μL of Escherichia coli KA30-GFP-cadA competent cells respectively, and chemical transformation was carried out (the operation steps were the same as those in step (1) of Example 2). The transformed bacterial solution was spread on an LB solid plate containing 12.5 mg / L chloramphenicol and 12.5 mg / L kanamycin (a total of 57 solid plates), and cultured overnight at 37°C.

[0072] A large number of single colonies were enriched on 56 LB solid plates. The spreader that had been burned by an alcohol lamp was used to scrape down all the single colonies as much as possible, and they were inoculated into 5 mL of seed medium (containing 12.5 mg / L chloramphenicol and 12.5 mg / L kanamycin) respectively, and cultured overnight at 37°C and 220 rpm for 12 h to obtain seed solutions; then, they were transferred to 100 mL of fermentation medium (containing 12.5 mg / L chloramphenicol and 12.5 mg / L kanamycin) at an inoculation amount of 5% v / v, and cultured at 37°C and 220 rpm until the OD 600 of the cells in the fermentation medium was approximately 0.6. IPTG inducer was added to the fermentation medium to make its initial concentration in the fermentation medium 1 mM, and continued to culture at 37°C and 220 rpm for about 50 h. Glucose and ammonium sulfate were fed in batches twice during this period, 500 g / L glucose aqueous solution and 400 g / L ammonium sulfate aqueous solution each time, so that after feeding, the final concentrations of glucose and ammonium sulfate in the fermentation medium were 20 g / L and 10 g / L respectively.

[0073] After fermentation, 1 mL of the fermentation broth was taken and centrifuged and washed twice with sterile PBS (the purpose was to wash away the fermentation broth to prevent it from contaminating the subsequent fluorescence quantitative flow cytometry instrument). The bacterial cells were diluted with sterile PBS buffer to an OD 600 of 0.2 - 0.4, and transferred to a sterile flow tube. Single-cell bacterial cells with GFP fluorescence intensity much higher than that of the control group were screened out using a fluorescence quantitative flow cytometer.

[0074] Part of the screening results are as Figure 3 shown. In the two-parameter scatter plot of EITC-A (GFP fluorescence intensity) and SSC-A (side scatter signal), the cells in region P4 showed significant high GFP expression characteristics. Compared with the P3 control region (fluorescence intensity concentrated in the range of 10 2 ~10 3 ), the GFP fluorescence signal in region P4 spanned the range of 10 3 ~10 5 , indicating that this population was the target GFP-positive cells. By setting the sorting gate (Gate), cells with EIIC-A > 10 3 were defined as the high GFP-expression population and sorted and collected, successfully achieving the enrichment of the target cells. The single-cell bacterial cells screened out were collected using a sterile 96-well plate, and each single-cell bacterial cell was placed in a separate well. To ensure the viability of the cells, 200 μL of LB liquid medium (containing 12.5 mg / L chloramphenicol and 12.5 mg / L kanamycin) was added to each well of the 96-well plate, which served as a buffer and also provided certain nutrients to the cells. A total of 14 96-well plates were collected, containing 1344 single-cell bacterial cells in total. To ensure the accuracy of the experiment, well A1 of each 96-well plate was used as the control group for subsequent repeated screening.

[0075] Single-cell bacterial cells with GFP fluorescence intensity much higher than that of the control group were screened out (P4 in Figures a and b). A total of about 1344 single-cell bacterial cells were screened out for subsequent screening, and these bacterial cells contained overexpressed single genes that were duplicates.

[0076] Example 4 Screening, verification and rational analysis of key genes in the 1,5-pentanediamine biosynthesis pathway

[0077] Approximately 1344 single-cell bacterial cells with GFP fluorescence intensity far higher than that of the control group and single-cell bacterial cells of the control group obtained by screening in Example 3 were respectively inoculated into 96-well shallow plates to culture seed liquid, with each single-cell bacterial cell placed separately into one well (each well containing 200 μL of seed medium), and then the seed liquid was respectively transferred to 96-well deep plates (each well containing 1.5 mL of fermentation medium) at an inoculation amount of 5% v / v for three batches of fermentation culture and screening to obtain genes to be screened that can promote GFP expression and can stably express themselves; in the first and second batches, the GFP fluorescence intensity was used as the screening criterion, and in the third batch, GFP / OD 600 was used as the screening criterion. Finally, 22 single-cell bacterial cells were obtained, and their GFP fluorescence intensity and GFP / OD 600 are as Figure 4 shown. The methods for culturing the seed liquid, fermentation culture, and the screening method using the GFP fluorescence intensity as the criterion are all the same as those in Example 3; the screening method using GFP / OD 600 as the criterion is as follows: referring to the GFP / OD 600 measurement method in step (3) of Example 2, single-cell bacterial cells with a GFP / OD 600 value more than 5 times that of the control group were screened out.

[0078] The seed liquids of the above 22 single-cell bacterial cells and the control group bacterial cells were inoculated into 30 mL of fermentation medium for shake-flask fermentation culture. The methods for culturing the seed liquid and shake-flask fermentation culture are the same as those in Example 3. After fermentation, high-performance liquid chromatography was used to detect the 1,5-pentanediamine content in each fermentation broth, and single-cell bacterial cells with a 1,5-pentanediamine yield higher than 1.2 times that of the control group were screened out. Finally, 7 single-cell bacterial cells were collected in this experiment, plasmids were extracted, and after sequencing, 7 target genes that can significantly up-regulate the 1,5-pentanediamine yield were obtained. The genes contained in these 7 single-cell bacterial cells and the 1,5-pentanediamine yield obtained from their fermentation are shown in Table 1.

[0079] Table 2 Seven single-cell bacterial cells finally screened, the target genes they contain, and the 1,5-pentanediamine yield

[0080]

[0081] The results are as Figure 5 shown. These 7 significantly up-regulated overexpression genes are respectively KdsA, rihA, nadA, hypB, gltA, alaC, and dcuC, and their nucleotide sequences are clearly shown in SEQ ID NO.5 - 11. They mainly play roles in aspects such as redox, transcriptional regulation, biofilm formation, and energy metabolism. Among them, the overexpression of the rihA gene has the most obvious effect on the increase in the 1,5-pentanediamine yield, and the yield is increased to 1.43 times compared with the control.

[0082] The present invention provides a method for high-throughput screening of key genes in the 1,5-pentanediamine biosynthesis pathway of Escherichia coli. There are many methods and approaches to specifically implement this technical solution. The above description is only a preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be implemented using existing technologies.

Claims

1. A 1,5-pentanediamine biosensor, characterized in that, The described 1,5-pentanediamine biosensor is a recombinant plasmid obtained by integrating the lysine decarboxylase-encoding gene cadA and its promoter trc, the green fluorescent protein GFP-encoding gene and its promoter pyrF into a plasmid backbone.

2. The 1,5-pentanediamine biosensor according to claim 1, characterized in that, The nucleotide sequence of the lysine decarboxylase-encoding gene cadA is as shown in SEQ ID NO.1; the nucleotide sequence of the promoter trc is as shown in SEQ ID NO.2; the nucleotide sequence of the green fluorescent protein GFP-encoding gene is as shown in SEQ ID NO.3; the nucleotide sequence of the promoter pyrF is as shown in SEQ ID NO.

4.

3. The 1,5-pentanediamine biosensor according to claim 1, characterized in that, The plasmid backbone is plasmid pET28a.

4. The 1,5-pentanediamine biosensor according to claim 3, characterized in that, Genes lacO and lacI are deleted from the plasmid pET28a.

5. The 1,5-pentanediamine biosensor according to claim 3, wherein A multiple cloning site MCS, a terminator rrnB T1 and a terminator rrnB T2 are integrated into the plasmid pET28a.

6. The method for constructing a 1,5-pentanediamine biosensor according to any one of claims 1 to 5, characterized in that, It includes the following steps: (1) Genes lacO and lacI in the plasmid pET28a are deleted to obtain plasmid pET28a(ΔlacIO); (2) Using plasmid pET28a(ΔlacIO) as a template, PCR amplification is performed with primers 28a-F and 28a-R to obtain a plasmid fragment; wherein, the nucleotide sequences of the primers 28a-F and 28a-R are as shown in SEQ ID NO.12 and SEQ ID NO.13 respectively; (3) The multiple cloning site MCS, the terminator rrnB T1 and the terminator rrnB T2 are ligated to the plasmid fragment obtained in step (2) to obtain a recombinant plasmid pcadR; (4) The green fluorescent protein GFP-encoding gene and its promoter pyrF, and the lysine decarboxylase-encoding gene cadA and its promoter trc are integrated into the recombinant plasmid pcadR obtained in step (3), and thus the 1,5-pentanediamine biosensor is obtained.

7. A recombinant strain containing the 1,5-pentanediamine biosensor described in any one of claims 1 to 5.

8. The recombinant strain according to claim 7, characterized in that, The starting strain of the recombinant strain is Escherichia coli KA30.

9. Use of the 1,5-pentanediamine biosensor described in any one of claims 1 to 5 in screening for key genes in the 1,5-pentanediamine biosynthesis pathway of Escherichia coli.

10. The application according to claim 9, characterized in that, The Escherichia coli is Escherichia coli KA30.

11. A method for screening key genes in the 1,5-pentanediamine biosynthesis pathway of Escherichia coli using the 1,5-pentanediamine biosensor according to any one of claims 1 to 5, characterized in that, It includes the following steps: (1) The 1,5-pentanediamine biosensor is introduced into Escherichia coli KA30 to obtain recombinant Escherichia coli KA30-GFP-cadA; (2) A single-gene recombinant expression vector of the gene to be screened is constructed to obtain a single-gene overexpression library, which is introduced into the recombinant Escherichia coli KA30-GFP-cadA obtained in step (1) to obtain library recombinant strains; (3) Ferment and culture the library recombinant strains obtained in step (2), and screen out single-cell bacterial cells with increased GFP fluorescence intensity and / or increased ratio of GFP fluorescence intensity to bacterial OD 600 compared with the negative control; (4) The single-cell bacteria obtained in step (3) are fermented and cultured, and single-cell bacteria with an increased 1,5-pentanediamine yield compared to the negative control are screened out, and their plasmids are extracted and sequenced, and thus the key genes can be screened out.

12. Use of genes KdsA, rihA, nadA, hypB, gltA, alaC and dcuC screened by the method according to claim 11 in the biosynthesis of 1,5-pentanediamine.

13. The application according to claim 12, wherein The nucleotide sequences of the genes KdsA, rihA, nadA, hypB, gltA, alaC and dcuC are respectively as shown in SEQ ID NO.5 to 11.

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