Pseudomonas putida for high-yield polyhydroxyalkanoate as well as construction method and application of pseudomonas putida

Abstract

The invention belongs to the field of genetic engineering, and relates to pseudomonas putida for high-yield polyhydroxyalkanoate as well as a construction method and application of the pseudomonas putida. According to the invention, a strain of pseudomonas putida KT + NtrcT-D55E capable of taking crab shells as a fermentation substrate and remarkably improving the yield of polyhydroxyalkanoate (PHA) is obtained by overexpressing an ntrc mutant gene of which the nucleotide sequence is shown as SEQ ID NO.4 in pseudomonas putida KT2440. Compared with the Pseudomonas putida KT2440, the yield of the PHA produced by the Pseudomonas putida KT + NtrcT-D55E provided by the invention is increased by 225.67%, and the contents of C6, C8, C10 and C12 of PHA monomers are all remarkably changed. The strain provided by the invention not only can produce polyhydroxyalkanoate at high yield, but also realizes resourceful treatment of crab shell wastes, and has a good application prospect.

Description

Technical Field

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

[0002] Polyhydroxyalkanoates (PHA) are biodegradable polymers synthesized by microorganisms. They have excellent mechanical properties, biocompatibility and biodegradability. They have broad application prospects in food packaging, biomedical materials and other fields, and are expected to become environmentally friendly materials to replace petroleum-based plastics. However, the cost of PHA synthesis substrates is relatively high, about three times that of polypropylene of the same quality, which seriously limits the development and diversified application of the PHA industry.

[0003] Shrimp and crab shell waste is a common waste generated in the process of seafood consumption, with an annual output of more than 6 million tons. The 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 for high-value conversion. 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 the treatment of petroleum pollution, and has strong metabolic and environmental remediation capabilities.

[0005] The team of the present invention conducted preliminary research and found that Pseudomonas putida KT2440 can use crab shells as substrates to ferment and synthesize PHA. However, due to the high nitrogen content of crab shells, PHA is usually synthesized in large quantities when cells face C / N imbalance conditions such as high carbon content and low nitrogen content. Therefore, the accumulation of PHA will be inhibited under the premise of fermentation using crab shells as substrates, limiting the application of Pseudomonas putida KT2440 in producing PHA by fermentation using crab shells as substrates. Summary of the invention

[0006] In order to overcome the 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 Mutation of genes to obtain a strain of Pseudomonas putida KT that can use crab shell as a fermentation substrate and significantly increase the production of polyhydroxyalkanoate (PHA) + NtrcT-D55E Compared with Pseudomonas putida KT2440, the yield of polyhydroxyalkanoates produced by this strain is 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 Pseudomonas putida that produces high levels of polyhydroxyalkanoates. The Pseudomonas putida is a polyhydroxyalkanoate that 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: Amplify the DNA template shown in SEQ ID NO.4 by PCR using the upstream primer and the downstream primer to obtain an amplified product; The amplified product was connected with the pUCP18 vector to obtain the recombinant plasmid pPR-NtrcT-D55E; Then, the recombinant plasmid is transferred into Pseudomonas putida KT2440 to obtain the Pseudomonas putida; Upstream primer: 5′-tggtaaagagctcatgagccgaagtg-3′; Downstream primer: 5′-cacctcagtggtcatcaccttcctc-3′.

[0009] Furthermore, 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.

[0010] Furthermore, the PCR reaction system is: (1) initial denaturation; (2) denaturation; annealing: 58°C~62°C, 15s; extension; a total of 30~35 cycles; (3) terminal extension.

[0011] 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.

[0012] The third aspect of the present invention provides a fermentation method of Pseudomonas putida described above, comprising the following steps: Inoculate Pseudomonas putida into crab shell culture medium, ferment at 21°C to 33°C for 12h to 60h, and collect the fermentation liquid; 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.

[0013] Preferably, the fermentation temperature is 30°C and the fermentation time is 42 hours.

[0014] Preferably, the mixing ratio of crab shell and water is 7g:100mL.

[0015] A fourth aspect of the present invention provides a fermentation broth obtained by the above fermentation method.

[0016] The fifth aspect of the present invention provides a bacterial agent, which includes the Pseudomonas putida or fermentation broth described above, and industrially acceptable auxiliary materials or additives.

[0017] Furthermore, the bacterial agent is a liquid preparation or a powder.

[0018] Furthermore, the number of viable Pseudomonas putida contained in each milliliter of the bacterial agent is 3×10 8 CFU~3×10 10 CFU.

[0019] A sixth aspect of the present invention is the use of the above-mentioned Pseudomonas putida, fermentation broth or bacterial agent in the production of polyhydroxyalkanoates.

[0020] Compared with the prior art, the beneficial effects of the present invention are: (1) High-yield polyhydroxyalkanoates: The present invention regulates nitrogen utilization-related genes of Pseudomonas putida KT2440 - 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 gene, obtained 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.

[0021] (2) Efficient utilization of resources: Pseudomonas putida KT provided by the present invention +NtrcT-D55EThe 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.

[0022] (3) Promote the high-value utilization of crab shells: Provide new pathways 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 applications related to the transformation and development of crab shell resources.

[0023] (4) Meeting market demand: As a biodegradable polymer with broad application prospects, polyhydroxyalkanoates have an increasing market demand. By increasing the production of polyhydroxyalkanoates, we can meet the market demand for environmentally friendly materials and further promote industrial development. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Recombinant plasmid pPR -NtrcT-D55E Schematic diagram.

[0025] Figure 2 Pseudomonas putida strain KT +NtrcT-D55E Production of polyhydroxyalkanoates.

[0026] Figure 3 Pseudomonas putida strain KT +NtrcT-D55E Monomer yield for producing polyhydroxyalkanoates; detection indicators in Figure A, Figure B, Figure C and Figure D are C6 monomer, C8 monomer, C10 monomer and C12 monomer of polyhydroxyalkanoates, respectively. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution 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 used to limit the present invention.

[0028] 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.

[0029] Pseudomonas putida KT2440 can use crab shells as substrates to ferment and synthesize polyhydroxyalkanoates (PHA), but because crab shells have a high nitrogen content, PHA is usually synthesized in large quantities when cells face C / N imbalance conditions such as high carbon content and low nitrogen content. Therefore, the accumulation of PHA will be inhibited when using crab shells as substrates for fermentation, limiting the application of Pseudomonas putida KT2440 in producing PHA using crab shells as substrates for fermentation.

[0030] The present invention provides a high-yield polyhydroxyalkanoate-producing Pseudomonas putida, a construction method and an application thereof. The present invention regulates nitrogen utilization-related genes of Pseudomonas putida KT2440 - 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 Mutation of genes to obtain a strain of Pseudomonas putida KT that can use crab shell as a fermentation substrate and significantly increase the production of polyhydroxyalkanoate (PHA) + NtrcT-D55E The Pseudomonas putida KT provided by the present invention + 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.

[0031] Example 1: Pseudomonas putida strain KT +NtrcT-D55E Construction 1. Amplify the target fragment: synthesize the NtrcT-D55E gene fragment of the nucleotide sequence shown in SEQ ID No.4, design a primer combination according to the primer design principle, the primer combination includes the upstream primer shown in SEQ ID NO.1 and the downstream primer shown in SEQ ID NO.2, and amplify the DNA template according to the following PCR reaction system and procedure to obtain the target fragment.

[0032] SEQ ID NO.1: 5'-tggtaaagagctcatgagccgaagtg-3'; SEQ ID NO. 2: 5'-cacctcagtggtcatcaccttcctc-3'.

[0033] PCR reaction system: 2× PrimeSTAR® Mix (purchased from Takara) 12µL, upstream primer 2µL, downstream primer 2µL, DNA template 2µL, and sterile enzyme-free water to make up to 25µL.

[0034] PCR reaction program: first step, 98°C, 2 min; second step, 98°C, 10 s; 60°C, 15 s; 75°C, 30 s, repeated for 35 cycles; third step, 72°C, 5 min.

[0035] 2. Construction of recombinant plasmid using pUCP18 plasmid The target fragment was connected to the pUCP18 plasmid to obtain Figure 1 The recombinant plasmid shown is pPR-NtrcT-D55E.

[0036] 3. Construction of Pseudomonas putida strain KT +NtrcT-D55E The recombinant plasmid pPR-NtrcT-D55E was transferred into 2 Transformation E. coli DH5α competent cells. Because the pUCP18 plasmid contains gentamicin (Gm) resistance, the monoclonal colony was obtained by co-screening and culture using a resistance medium. The recombinant plasmid with the correct sequence was verified by DNA sequencing and transferred 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 .

[0037] 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.

[0038] SEQ ID No.3: MSRSETVWIVDDDRSIRWVLEKALQQEGMTTQSFDSADGVMGRLARQQPDVIISEIRMPGTSGLDLLAQIREQHPRLPVIIMTAHSDLDSAVASYQGGAFEYLPKPFDVDEAVSLVKRAN QHAQEQQGLDVPQNLARTPEIIGEAPAMQEVFRAIGRLSHFNITVLINGESGTGKELVAHALHRHSPRAASPFIALNMAAIPKDLMESELFGHEKGAFTGAANLRRGRFEQADGGTLFLD EIGDMPADTQTRLLRVLADGEFYRVGGHVPVKVDVRIIAATHQNLESLVQAGKFREDLFHRLNVIRIHIPRLADRREDIPALARHFLARAAQELAVEPKILKPETEEFIRNLPWPGNVRQ MENTCRWITVMASSREVLIGDLPPELLNLPHDAAPVTNWEQALRQWADQALARGQTSLLDSAVPSFERIMIETALKHTAGRRRDAALLLGWGRNTLTRKIKELGMNVAGGDDEEGDDH*.

[0039] SEQ ID No.4:

[0040] Example 2: Pseudomonas putida strain KT +NtrcT-D55E Fermentation culture 1. Preparation of crab shell fermentation medium ①Prepare crab shell fragments: steam the swimming crab at 105℃ for 15 min, then cool to room temperature, separate the crab shell, wash with clean water, and dry at 55℃ for 12 h. Crush it into 4 mesh in a mortar, and dry at 55℃ to constant weight.

[0041] ②Preparation of crab shell fermentation medium Add crab shell fragments into 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 sterilize under high pressure at 121°C for 20 min.

[0042] 2. KT +NtrcT-D55E Fermentation culture The Pseudomonas putida strain KT constructed in Example 1 was selected. +NtrcT-D55E The monoclonal colony was inoculated into 5 mL of LB liquid medium containing Gm resistance and cultured at 30°C and 200 rpm for 24 h to obtain seed liquid. The seed liquid was inoculated into crab shell fermentation medium for fermentation at 30°C and 200 rpm. The PHA yield was detected after 42 h of fermentation.

[0043] Example 3: Extraction and determination of polyhydroxyalkanoates (PHA) 1. PHA extraction method ① Collecting 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.

[0044] 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.

[0045] ③Chloroform dissolution: Transfer the cell powder to an anaerobic tube and add 7 mL of chloroform to dissolve it in a fume hood (the ratio of chloroform addition is 25 mL chloroform: 1 g frozen stem cells). Incubate at 100°C for 4 h, cool to room temperature and remove.

[0046] ④ Add water to extract: Add 2 mL ddH 2O, vortex for 10 min. Transfer to a 15 mL centrifuge tube and centrifuge at 8500 rpm at 4°C for 10 min. After centrifugation, the liquid presents three layers, the top layer is the water phase, the middle layer is the impurity layer such as protein, and the bottom layer is the chloroform layer that dissolves lipids such as PHA. Use a 5 mL syringe to draw the lower organic phase and filter it through a 0.45 μm PTFE filter membrane into a clean 15 mL centrifuge tube.

[0047] ⑤ Nitrogen blowing: Place the obtained PHA chloroform solution in a nitrogen blowing apparatus in a fume hood and blow nitrogen to concentrate the liquid to 1 mL. During nitrogen blowing, carefully adjust the gas valve to prevent splashing of the liquid in the centrifuge tube.

[0048] ⑥Precipitation of PHA: Add 10 mL of methanol (pre-cooled) to the concentrated 1 mL of PHA chloroform solution, and mix by inversion on a rotator for 30 min. Centrifuge at 8500 rpm, 4°C for 10 min, and discard the supernatant. The crude PHA attached to the wall of the centrifuge tube is added with 1 mL of chloroform to dissolve the crude PHA, and repeat steps ⑤ to ⑥ to further purify PHA.

[0049] ⑦ Obtaining PHA: After three centrifugations, the PHA in the centrifuge tube with the supernatant discarded was nitrogen blown to obtain pure PHA particles at the bottom. The nitrogen was blown until all the organic reagents were volatilized to obtain the final desired PHA product, which was stored in a -80°C refrigerator.

[0050] 2. PHA content detection: GC-MS is used to detect the content and monomer composition of PHA. The specific operation is as follows: ①PHA esterification: Take out the extracted PHA from the -80℃ refrigerator, add 4 mL of chloroform in the fume hood, vortex for 2 min, then add 4 mL of esterification solution, transfer the liquid to the anaerobic tube, vortex for 5 min to mix it. Incubate at 100℃ for 4 h, cool to room temperature, and add 1 mL of ddH 2 O, vortex for 5 min to mix thoroughly, and transfer to a 15 mL centrifuge tube. After centrifugation at 7000 rpm for 5 min, remove the upper aqueous phase with a pipette, use a 2 mL syringe to draw 1 mL of the lower organic phase, and filter through a 0.22 µm nylon filter into a clean 1.5 mL chromatographic injection vial.

[0051] ② Sample dilution: Take 100 µL of sample solution in step ①, add 900 µL of n-hexane, repeat once, complete 10-fold gradient dilution, obtain the test solution, seal, store at -20℃, and prepare for loading.

[0052] ③GC-MS detection and analysis: According to the manual of the Agilent 7693A / 5977B gas chromatograph, an Agilent micro-injector was used, the injection volume was 1 μL, the carrier gas was helium, and the flow rate was 1 mL / min. The injection port temperature was 250°C, the injection mode was non-split, the injection time was 1 min, the injection box starting temperature was 100°C, maintained for 1 min, and then increased from 100°C to 280°C at a rate of 30°C / min, and maintained for 5 min. Since KT2440 is a known medium-chain polyhydroxyalkanoate (mcl-PHA) producing bacterium, the inventors selected C6, C8, C10, and C12 standard products with C chain lengths and mixed them to make a standard curve, and the detection range of the standard curve was 5ppm~25ppm.

[0053] The C6 curve is y = 103806x+34451, R² = 0.99; The C8 standard curve is y=117041x+36745, R²=0.99; The C10 standard curve is y=159770x+40639, R²=0.99; The C12 calibration curve is y=138590x+25481, R²=0.99.

[0054] The relative content of PHA was calculated and compared by the peak area calculation method.

[0055] 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 KT2440 provided by the present invention +NtrcT-D55E The yield of PHA produced 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%.

[0056] from Figure 3 As seen, Pseudomonas putida strain KT +NtrcT-D55E The content of PHA monomers (C6, C8, C10, C12) produced also changed significantly compared with the control group: the content of monomer C6 increased from 1.15% to 2.79%; the content of monomer C8 increased from 21.98% to 24.63%; the content of monomer C10 increased from 40.18% to 44.75%; and the content of monomer C12 decreased from 36.69% to 27.83%. This shows that metabolic engineering not only increases the titer of PHA, but also affects the downstream synthesis pathway, thereby changing the relative content of monomer components.

[0057] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can 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 a preferred embodiment. Although the 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 know the basic creative concept. Therefore, the attached 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.

[0058] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A Pseudomonas putida that produces high amounts of polyhydroxyalkanoate, 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 the upstream primer and the downstream primer to obtain an amplified product; The amplified product was connected to the pUCP18 vector to obtain the recombinant plasmid pPR -NtrcT-D55E ; PR -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-62°C, 15s; extension; a total of 30-35 cycles; (3) terminal extension.

5. A fermentation method of Pseudomonas putida according to claim 1, characterized in that: The following steps are involved: Inoculate Pseudomonas putida into crab shell culture medium, ferment at 21°C to 33°C for 12h to 60h, and collect the fermentation liquid; 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.

6. A fermentation broth obtained according to the fermentation method of Pseudomonas putida according to claim 5.

7. A bacterial agent, characterized in that: The bacterial agent comprises the Pseudomonas putida according to claim 1 or the fermentation broth according to claim 6, and industrially acceptable auxiliary materials or additives.

8. The bacterial agent according to claim 7, characterized in that The bacterial agent is a liquid preparation or a powder.

9. The bacterial agent according to claim 7, characterized in that The number of live Pseudomonas putida contained in each milliliter of the bacterial agent is 3×10 8 CFU~3×10 10 CFU.

10. Use of the Pseudomonas putida according to claim 1, the fermentation broth according to claim 6 or the bacterial agent according to claim 7 in producing polyhydroxyalkanoates.

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