A polyhydroxyalkanoate-producing Pseudomonas putida bacterium, construction method and application thereof

Abstract

The present invention belongs to the field of genetic engineering and relates to a high-yield polyhydroxyalkanoate Pseudomonas putida, a construction method and an application thereof. The present invention is achieved by overexpressing the nucleotide sequence shown in SEQ ID NO.4 in Pseudomonas putida KT2440. intrc By mutating the gene, a strain of Pseudomonas putida KT was obtained that can ferment crab shells and significantly increase the production of polyhydroxyalkanoate (PHA). + NtrcT‑D55E Compared with Pseudomonas putida KT2440, the Pseudomonas putida KT provided by the present invention + NtrcT‑D55E The yield of PHA increased by 225.67%, and the contents of the PHA monomers C6, C8, C10, and C12 all showed significant changes. The strain provided by the present invention not only produces high levels of polyhydroxyalkanoates but also enables the resourceful processing of crab shell waste, demonstrating promising application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and in particular relates to a polyhydroxyalkanoate-high-yielding Pseudomonas putida bacterium, a construction method and an application thereof. Background Art

[0002] Polyhydroxyalkanoates (PHA) are biodegradable polymers synthesized by microorganisms. They possess excellent mechanical properties, biocompatibility, and biodegradability, and hold broad application prospects in areas such as food packaging and biomedical materials, promising environmentally friendly alternatives to petroleum-based plastics. However, the high cost of PHA synthesis substrates, approximately three times that of polypropylene of the same mass, severely limits the development and diverse applications of the PHA industry.

[0003] Shrimp and crab shell waste is a common waste generated during seafood consumption, with an annual output of more than 6 million tons. Simple low-value applications of these wastes mainly include composting and making feed. Due to the lack of high-value conversion technology, most crab shells are discarded as by-products, which not only leads to waste of resources, but also has a negative impact on the environment.

[0004] Pseudomonas ( Pseudomonas ) has become a research hotspot in the field of organic waste refining because it can use a variety of organic substances as carbon sources and energy sources and convert a variety of low-value substances as substrates to high-value conversions. Many studies have shown that Pseudomonas can be used to synthesize polyhydroxyalkanoates (PHA). Pseudomonas putida KT2440 ( Pseudomonas putida KT2440) is an important environmental microbial model strain that can degrade a variety of organic pollutants, especially in treating petroleum pollution, and has strong metabolic and environmental remediation capabilities.

[0005] Previous research by the inventor's team found that Pseudomonas putida KT2440 can use crab shells as substrates to ferment and synthesize PHA. However, due to the high nitrogen content in crab shells, PHA is usually synthesized in large quantities under conditions of C / N imbalance, such as high carbon content and low nitrogen content in cells. Therefore, the accumulation of PHA will be inhibited when using crab shells as substrates for fermentation, which limits the application of Pseudomonas putida KT2440 in the production of PHA by fermentation using crab shells as substrates. Summary of the Invention

[0006] In order to overcome the above-mentioned deficiencies of the prior art, the present invention provides a high-yield polyhydroxyalkanoate-producing Pseudomonas putida, a construction method and an application thereof. ntrcThe gene was subjected to point mutation, so that the aspartic acid at position 55 of the amino acid sequence of the NtrC protein was mutated to glutamic acid, and then overexpressed in Pseudomonas putida KT2440. ntrc By mutating the gene, a strain of Pseudomonas putida KT was obtained that can ferment crab shells and significantly increase the production of polyhydroxyalkanoate (PHA). + NtrcT-D55E Compared with Pseudomonas putida KT2440, the yield of polyhydroxyalkanoates produced by this strain was increased by 225.67%. The strain provided by the present invention can not only produce high-yield polyhydroxyalkanoates, but also realize the resource treatment of crab shell waste, and has good application prospects.

[0007] The first aspect of the present invention provides a high-yield polyhydroxyalkanoate Pseudomonas putida, wherein the Pseudomonas putida is overexpressed in Pseudomonas putida KT2440. ntrc Obtained after mutation of gene; ntrc The nucleotide sequence of the mutant gene is shown in SEQ ID NO.4.

[0008] The second aspect of the present invention provides a method for preparing the above-mentioned Pseudomonas putida, and the specific preparation steps are as follows:

[0009] Amplify the DNA template shown in SEQ ID NO. 4 by PCR using upstream primers and downstream primers to obtain an amplified product;

[0010] The amplified product was ligated with the pUCP18 vector to obtain the recombinant plasmid pPR-NtrcT-D55E;

[0011] Then, the recombinant plasmid is transformed into Pseudomonas putida KT2440 to obtain the Pseudomonas putida;

[0012] Upstream primer: 5′-tggtaaagagctcatgagccgaagtg-3′;

[0013] Downstream primer: 5′-cacctcagtggtcatcaccttcctc-3′.

[0014] Furthermore, each 25µL PCR reaction system includes: 12.5µL of 2×Mix enzyme, 1µL~2µL of upstream primer, 1µL~2µL of downstream primer, 50ng~100ng of DNA template, and sterile enzyme-free water to make up to 25µL.

[0015] Furthermore, the PCR reaction system is as follows: (1) initial denaturation; (2) denaturation; annealing: 58°C~62°C, 15s; extension; a total of 30~35 cycles; (3) terminal extension.

[0016] Furthermore, the PCR reaction system is: (1) 98°C, 2 min; (2) 98°C, 10 s; 60°C, 15 s; 15 s; 75°C; a total of 35 cycles; (3) 72°C, 5 min.

[0017] The third aspect of the present invention provides a fermentation method of the above-mentioned Pseudomonas putida, comprising the following steps:

[0018] Inoculate Pseudomonas putida into crab shell culture medium, ferment at 21°C to 33°C for 12h to 60h, and collect the fermentation liquid;

[0019] The crab shell culture medium is obtained by mixing crab shells and water, and the mixing ratio of crab shells to water is 3g~7g:100mL.

[0020] Preferably, the fermentation temperature is 30°C and the fermentation time is 42 hours.

[0021] Preferably, the mixing ratio of crab shell and water is 7g:100mL.

[0022] A fourth aspect of the present invention provides a fermentation liquid obtained by the above fermentation method.

[0023] The fifth aspect of the present invention provides a bacterial agent, which includes the above-mentioned Pseudomonas putida or fermentation broth, and industrially acceptable excipients or auxiliary agents.

[0024] Furthermore, the bacterial agent is a liquid preparation or a powder.

[0025] Furthermore, the number of viable Pseudomonas putida bacteria per milliliter in the bacterial agent is 3×10 8 CFU~3×10 10 CFU.

[0026] A sixth aspect of the present invention is a use of the aforementioned Pseudomonas putida, fermentation broth or bacterial agent in the production of polyhydroxyalkanoates.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] (1) High-yield polyhydroxyalkanoates: The present invention regulates nitrogen utilization-related genes of Pseudomonas putida KT2440 by homologous ntrc The gene was subjected to point mutation, so that the aspartic acid at position 55 of the amino acid sequence of the NtrC protein was mutated to glutamic acid, and then overexpressed in Pseudomonas putida KT2440. ntrc Mutated the gene to obtain a strain of Pseudomonas putida KT +NtrcT-D55E Pseudomonas putida KT +NtrcT-D55E It can produce polyhydroxyalkanoates, and compared with the strain Pseudomonas putida KT2440 before transformation, Pseudomonas putida KT+NtrcT-D55E The yield of polyhydroxyalkanoates was significantly increased by 225.67%, providing a more efficient strain for the industrial production of polyhydroxyalkanoates.

[0029] (2) Efficient utilization of resources: Pseudomonas putida KT provided by the present invention +NtrcT-D55E The production of polyhydroxyalkanoates by fermentation using crab shells as substrates realizes the resource utilization of crab shells, a type of aquatic waste, reduces resource waste, and at the same time reduces the production cost of polyhydroxyalkanoates and improves their market competitiveness.

[0030] (3) Promote the high-value utilization of crab shells: provide new ways for the high-value transformation of crab shells, develop high-protein utilization strains, establish a more suitable microbial chassis, provide a good platform for the production of high-value chemicals such as polyhydroxyalkanoates using crab shell fermentation, and promote the development of related applications of crab shell resource transformation and development.

[0031] (4) Meeting market demand: As a biodegradable polymer with broad application prospects, polyhydroxyalkanoates (PHA) have an increasing market demand. By increasing the production of PHA, we can meet the market demand for environmentally friendly materials and further promote industrial development. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Recombinant plasmid pPR -NtrcT-D55E Schematic diagram.

[0033] Figure 2 Pseudomonas putida strain KT +NtrcT-D55E Production of polyhydroxyalkanoates.

[0034] Figure 3 Pseudomonas putida strain KT +NtrcT-D55E Monomer yield in the production of polyhydroxyalkanoates; the detection indicators in Figures A, B, C and D are C6 monomer, C8 monomer, C10 monomer and C12 monomer of polyhydroxyalkanoates, respectively. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0036] Unless otherwise specified, the experimental methods described in the following examples are all conventional methods; the reagents and materials described are all commercially available unless otherwise specified.

[0037] Pseudomonas putida KT2440 can use crab shells as substrates to ferment and synthesize polyhydroxyalkanoates (PHA). However, due to the high nitrogen content of crab shells, PHA is usually synthesized in large quantities under the C / N imbalance conditions of high carbon content and low nitrogen content in cells. Therefore, the accumulation of PHA will be inhibited when using crab shells as substrates for fermentation, which limits the application of Pseudomonas putida KT2440 in producing PHA by fermentation using crab shells as substrates.

[0038] The present invention provides a high-yield polyhydroxyalkanoate Pseudomonas putida, a construction method and an application thereof. The present invention regulates nitrogen utilization-related genes of Pseudomonas putida KT2440 by homologous ntrc The gene was subjected to point mutation, so that the aspartic acid at position 55 of the amino acid sequence of the NtrC protein was mutated to glutamic acid, and then overexpressed in Pseudomonas putida KT2440. ntrc By mutating the gene, a strain of Pseudomonas putida KT was obtained that can ferment crab shells and significantly increase the production of polyhydroxyalkanoate (PHA). + NtrcT-D55E The present invention provides Pseudomonas putida KT + NtrcT-D55E Compared with Pseudomonas putida KT2440, its production of polyhydroxyalkanoates increased by 225.67%, which has important application value in the resource treatment of crab shell waste or the production of polyhydroxyalkanoates.

[0039] Example 1: Pseudomonas putida strain KT +NtrcT-D55E Construction

[0040] 1. Amplification of the target fragment: Synthesize the NtrcT-D55E gene fragment with the nucleotide sequence shown in SEQ ID No. 4. Design a primer combination according to primer design principles. The primer combination includes the upstream primer shown in SEQ ID NO. 1 and the downstream primer shown in SEQ ID NO. 2. Amplify the DNA template according to the following PCR reaction system and procedure to obtain the target fragment.

[0041] SEQ ID NO.1: 5'-tggtaaagagctcatgagccgaagtg-3';

[0042] SEQ ID NO. 2: 5'-cacctcagtggtcatcaccttcctc-3'.

[0043] PCR reaction system: 12 µL of 2× PrimeSTAR® Mix (purchased from Takara), 2 µL of upstream primer, 2 µL of downstream primer, 2 µL of DNA template, and sterile enzyme-free water to make up to 25 µL.

[0044] PCR reaction program: first step, 98°C, 2 min; second step, 98°C, 10 s; 60°C, 15 s; 75°C, 30 s, repeated 35 cycles; third step, 72°C, 5 min.

[0045] 2. Construction of recombinant plasmid using pUCP18 plasmid

[0046] The target fragment was connected to the pUCP18 plasmid to obtain Figure 1 The recombinant plasmid pPR-NtrcT-D55E is shown.

[0047] 3. Construction of Pseudomonas putida strain KT +NtrcT-D55E

[0048] The recombinant plasmid pPR-NtrcT-D55E was transformed into E. coli In DH5α competent cells. Because the pUCP18 plasmid contains gentamicin (Gm) resistance, monoclonal colonies were obtained by co-screening and culture using resistance medium. After DNA sequencing verification, the recombinant plasmid with the correct sequence was transformed into Pseudomonas putida KT2440 competent cells. The electroporation parameters were as follows: 1200V, 400Ω, 25μF, and the Pseudomonas putida strain KT was obtained. +NtrcT-D55E .

[0049] The amino acid sequence of NtrC after mutation is shown in SEQ ID No. 3, where * indicates a stop codon. ntrc The nucleotide sequence of the mutant gene is shown in SEQ ID No.4.

[0050] SEQ ID No.3:

[0051] MSRSETVWIVDDDRSIRWVLEKALQQEGMTTQSFDSADGVMGRLARQQPDVIISEIRMPGTSGLDLLAQIREQHPRLPVIIMTAHSDLDSAVASYQGGAFEYLPKPFDVDEAVSLVKRANQHAQEQQGLDVPQNLARTPEIIGEAPAMQEVFRAIGRLSHFNITVLINGESGTGKELVAHALHRHSPRAASPFIALNMAAIPKDLMESELFGHEKGAFTGAANLRRGRFEQADGGTLFLDEIGDMPADTQTRLLRVLADGEFYRVGGHVPVKVDVRIIAATHQNLESLVQAGKFREDLFHRLNVIRIHIPRLADRREDIPALARHFLARAAQELAVEPKILKPETEEFIRNLPWPGNVRQMENTCRWITVMASSREVLIGDLPPELLNLPHDAAPVTNWEQALRQWADQALARGQTSLLDSAVPSFERIMIETALKHTAGRRRDAALLLGWGRNTLTRKIKELGMNVAGGDDEEGDDH*。

[0052] SEQ ID No.4:

[0053]

[0054] Example 2: Pseudomonas putida strain KT +NtrcT-D55E Fermentation culture

[0055] 1. Preparation of crab shell fermentation medium

[0056] ① Preparation of crab shell fragments: Steam the swimming crab at 105℃ for 15 min, then cool to room temperature, separate the crab shells, rinse with clean water, and dry at 55℃ for 12 h. Crush the shells in a mortar to 4 mesh size and dry at 55℃ to constant weight.

[0057] ②Preparation of crab shell fermentation medium

[0058] Add crab shell fragments to a 250 mL conical flask, mix the crab shell fragments with deionized water at a solid-liquid ratio of 7 g:100 mL, cover the bottle mouth with sealing film, and autoclave at 121°C for 20 min.

[0059] 2. KT +NtrcT-D55E Fermentation culture

[0060] The Pseudomonas putida strain KT constructed in Example 1 was selected +NtrcT-D55E A single colony was inoculated into 5 mL of LB liquid medium containing Gm resistance and cultured at 30°C and 200 rpm for 24 hours to obtain a seed solution. The seed solution was then inoculated into crab shell fermentation medium at 30°C and 200 rpm for 42 hours. PHA production was measured.

[0061] Example 3: Extraction and determination of polyhydroxyalkanoate (PHA)

[0062] 1. PHA extraction method

[0063] ① Collect bacteria: Pseudomonas putida strain KT obtained in Example 1 +NtrcT-D55E After 48 h of fermentation, the bacterial solution was transferred to a centrifuge tube and centrifuged at 5500 rpm for 10 min. The supernatant was discarded, and the bacterial cells at the bottom were collected and stored in a -80°C refrigerator overnight.

[0064] ② Freeze-drying: Take out the bacteria stored at -80℃, place them in a freeze dryer and vacuum freeze-dry for 48 hours to obtain dry bacterial powder, i.e., cell dry powder.

[0065] ③ Dissolve in chloroform: Transfer the dried cell powder to an anaerobic tube and dissolve it in 7 mL of chloroform in a fume hood (add 25 mL of chloroform to 1 g of lyophilized cells). Incubate at 100°C for 4 h, cool to room temperature, and remove from the tube.

[0066] ④ Water Extraction: Add 2 mL of ddH2O to the anaerobic tube and vortex for 10 minutes. Transfer to a 15 mL centrifuge tube and centrifuge at 8500 rpm at 4°C for 10 minutes. After centrifugation, the liquid will appear in three layers: the top layer is the aqueous phase, the middle layer contains impurities such as proteins, and the bottom layer is the chloroform layer containing dissolved lipids such as PHA. Use a 5 mL syringe to aspirate the lower organic phase and filter through a 0.45 μm PTFE filter into a clean 15 mL centrifuge tube.

[0067] ⑤ Nitrogen purge: Place the obtained PHA chloroform solution in a nitrogen purge apparatus in a fume hood and purge with nitrogen until the liquid is concentrated to 1 mL. During nitrogen purge, carefully adjust the air valve to prevent splashing of the liquid in the centrifuge tube.

[0068] ⑥ Precipitate PHA: Add 10 mL of pre-chilled methanol to 1 mL of concentrated PHA chloroform solution and mix thoroughly by inversion on a rotator for 30 minutes. Centrifuge at 8500 rpm, 4°C for 10 minutes. Discard the supernatant; the crude PHA remains attached to the tube wall. Add 1 mL of chloroform to dissolve the crude PHA. Repeat steps ⑤-⑥ to further purify the PHA.

[0069] ⑦PHA acquisition: After three centrifugations, discard the supernatant from the centrifuge tube and blow nitrogen through the PHA to obtain pure PHA particles at the bottom. Blow nitrogen through the tube until all the organic reagents are volatilized to obtain the desired PHA product. Store in a -80°C refrigerator.

[0070] 2. PHA content detection: GC-MS is used to detect the content and monomer composition of PHA. The specific operation is as follows:

[0071] ① PHA esterification: Remove the extracted PHA from a -80°C refrigerator and add 4 mL of chloroform in a fume hood. Vortex for 2 minutes. Then, add 4 mL of the esterification solution and transfer the solution to an anaerobic tube. Vortex for 5 minutes to mix thoroughly. Incubate at 100°C for 4 hours. After cooling to room temperature, add 1 mL of ddH2O and vortex for 5 minutes to mix thoroughly. Transfer the solution to a 15 mL centrifuge tube. Centrifuge at 7000 rpm for 5 minutes. Remove the upper aqueous phase with a pipette and aspirate 1 mL of the lower organic phase using a 2 mL syringe. Filter the solution through a 0.22 µm nylon filter into a clean 1.5 mL chromatography vial.

[0072] ② Sample dilution: Take 100 µL of the sample solution from step ①, add 900 µL of n-hexane, repeat once more to complete a 10-fold gradient dilution to obtain the test solution, seal the container, store at -20°C, and prepare for sample loading.

[0073] GC-MS analysis: The instrument was operated according to the Agilent 7693A / 5977B gas chromatograph manual, using an Agilent microinjector with an injection volume of 1 μL and helium as the carrier gas at a flow rate of 1 mL / min. The inlet temperature was 250°C, the injection mode was splitless, and the injection time was 1 minute. The injection chamber temperature was initially set at 100°C and held for 1 minute, then increased from 100°C to 280°C at a rate of 30°C / min and held for 5 minutes. Since KT2440 is a known producer of medium-chain polyhydroxyalkanoates (mcl-PHAs), the inventors selected a mixture of C6, C8, C10, and C12 C chain length standards for the calibration curve. The detection range of the calibration curve was 5 ppm to 25 ppm.

[0074] The C6 curve is y = 103806x + 34451, R² = 0.99;

[0075] The C8 standard curve is y=117041x+36745, R²=0.99;

[0076] The C10 standard curve is y=159770x+40639, R²=0.99;

[0077] The C12 calibration curve is y=138590x+25481, R²=0.99.

[0078] The relative content of PHA was calculated and compared by peak area calculation method.

[0079] The experimental results are as follows Figure 2 As shown, the wild-type strain Pseudomonas putida KT2440 was used as the control group, and its PHA production was 20.18 mg / L. The Pseudomonas putida strain KT +NtrcT-D55E The yield of PHA production was 65.72 mg / L. Compared with the control group, the Pseudomonas putida strain KT provided by the present invention +NtrcT-D55E The yield of PHA production increased by 225.67%.

[0080] from Figure 3 As seen, Pseudomonas putida strain KT +NtrcT-D55E The content of the produced PHA monomers (C6, C8, C10, and C12) also showed significant changes compared to the control: the C6 monomer content increased from 1.15% to 2.79%; the C8 monomer content increased from 21.98% to 24.63%; the C10 monomer content increased from 40.18% to 44.75%; and the C12 monomer content decreased from 36.69% to 27.83%. This indicates that metabolic engineering not only increased PHA titer but also influenced the downstream synthesis pathway, thereby altering the relative content of the monomer components.

[0081] It should be noted that when numerical ranges are mentioned in the present invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints may be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes preferred embodiments. Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0082] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A polyhydroxyalkanoate-producing Pseudomonas putida, characterized in that: The Pseudomonas putida is overexpressed in Pseudomonas putida KT2440 ntrc Obtained after mutation of gene; ntrc The nucleotide sequence of the mutant gene is shown in SEQ ID NO.

4.

2. A method for preparing Pseudomonas putida according to claim 1, characterized in that: The specific preparation steps are as follows: Amplify the DNA template shown in SEQ ID NO. 4 by PCR using upstream primers and downstream primers to obtain an amplified product; The amplified product was connected to the pUCP18 vector to obtain the recombinant plasmid pPR -NtrcT-D55E ; The recombinant plasmid pPR -NtrcT-D55E transferring into Pseudomonas putida KT2440 to obtain the Pseudomonas putida; Upstream primer: 5′-tggtaaagagctcatgagccgaagtg-3′; Downstream primer: 5′-cacctcagtggtcatcaccttcctc-3′.

3. The preparation method according to claim 2, characterized in that Each 25µL PCR reaction system includes: 12.5µL 2× Mix enzyme, 1µL~2µL upstream primer, 1µL~2µL downstream primer, 50ng~100ng DNA template, and sterile enzyme-free water to make up to 25µL.

4. The preparation method according to claim 2, characterized in that The PCR reaction system is as follows: (1) initial denaturation; (2) denaturation; annealing: 58°C to 62°C, 15s; extension; a total of 30 to 35 cycles; (3) terminal extension.

5. A bacterial agent, characterized in that The bacterial agent consists of the Pseudomonas putida according to claim 1 and industrially acceptable excipients.

6. The microbial agent according to claim 5, characterized in that The bacterial agent is a liquid preparation or a powder.

7. The microbial agent according to claim 5, characterized in that The number of viable Pseudomonas putida bacteria per milliliter in the bacterial agent is 3×10 8 CFU~3×10 10 CFU.

8. Use of the Pseudomonas putida according to claim 1 or the bacterial agent according to claim 5 in the production of polyhydroxyalkanoates.

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