Engineering strain for producing poly-beta-hydroxybutyric acid by using methane as well as application and method thereof

Abstract

The invention belongs to the technical field of bioengineering, and relates to an engineering strain for producing poly-beta-hydroxybutyric acid by using methane as well as application and a method of the engineering strain. The engineering strain is constructed by using methanogenic archaea M.acetivorans C2A as a starting strain, and the methanogenic archaea M.acetivorans C2A is constructed by The genome of the gene contains a methane regulation promoter as shown in SEQ ID NO.4 in a sequence table and key poly-beta-hydroxybutyric acid synthesis gene clusters phaA, phaB and phaC as shown in SEQ ID NO.1-3 in the sequence table. According to the engineering strain constructed by the invention, the conversion process from methane to poly-beta-hydroxybutyric acid is completed through self growth and metabolism of microorganisms; in addition, the yield and the yield of poly-beta-hydroxybutyric acid are improved by means of adding an extracellular electron acceptor.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and relates to an engineering strain for producing poly-β-hydroxybutyrate by utilizing methane, and a use and method thereof. Background Art

[0002] Methane, as the main component of natural gas, is both a clean energy source and the second largest greenhouse gas in the world, which has an important impact on climate change. Its release sources include natural habitats such as natural gas and oil extraction, agricultural anaerobic decomposition, landfills and wastewater treatment. In these methane-releasing environments, converting methane into high-value-added products is seen as a sustainable strategy to reduce methane emissions. The use of corn straw to prepare biochar can simultaneously address the dual challenges of agricultural waste disposal and greenhouse gas emission reduction. The biochar generated by high-temperature oxygen-limited pyrolysis of corn straw (such as 500°C) has a high specific surface area and abundant surface active sites (such as quinone groups, carbonyl groups and persistent free radicals), which can prolong its interaction time with methane oxidizing bacteria through the micropore confinement effect. Its surface active components (such as Fe3O4 nanoparticles) can also serve as electron transfer media to accelerate the coordinated metabolism of anaerobic methane-oxidizing archaea (ANME) and heterotrophic bacteria, thereby improving the efficiency of methane utilization. In addition, biochar applied to the soil can improve water retention and nutrient circulation, reduce PM2.5 emissions caused by straw burning (reduced by more than 60%), and its alkaline properties (pH 9.2±0.3) inhibit the activity of methanogens in farmland, reducing methane release from the source.

[0003] The methane conversion in the traditional chemical industry relies on expensive large-scale equipment and requires the use of a large amount of catalysts under high temperature and high pressure conditions. There are problems such as harsh reaction conditions, high energy consumption, and high equipment investment costs. In contrast, biological conversion of methane has the advantages of mild reaction conditions, directional conversion of substrates, and precise synthesis of products, and its performance can be optimized through genetic modification and metabolic regulation. Therefore, the development of cell factories based on microorganisms to convert methane into high-value-added biofuels has broad research and application prospects.

[0004] Microbial utilization of methane can be divided into two processes: aerobic and anaerobic oxidation. Aerobic methanotrophs, mainly belonging to the Proteobacteria, activate methane through methane monooxygenase and convert it into C-OH groups, which are then assimilated through the ribulose monophosphate or serine cycle. Although metabolic engineering has achieved the production of a variety of chemicals from methane, the aerobic process has low carbon and energy conversion efficiency due to oxygen loss. In contrast, anaerobic methanotrophs (ANME) belong to Euryarchaeota ANME is a phylum that uses the reverse methanogenic pathway to oxidize methane and relies on extracellular electron acceptors for energy synthesis. However, ANME grows slowly and cannot be cultured in pure culture, which limits its research and application. Phylogenetic analysis shows that the methanogenic Sarcinales is closely related to ANME, including Methanosarcina acetivorans ( M. acetivorans) as a model methanogenic archaeon, which is capable of anaerobic methane oxidation growth when FeIII is used as the electron acceptor. M. acetivorans Not only can it be cultivated in pure culture and genetically modified, but it can also achieve anaerobic methanotrophic growth through the reverse methanogenic pathway, making it possible to build cell factories for the anaerobic conversion of methane into biofuels. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides an engineered strain for producing poly-β-hydroxybutyrate using methane, and its uses and methods. The engineered strain can efficiently convert methane into poly-β-hydroxybutyrate (PHB) through methyl nutritional metabolism. The present invention also establishes a biochar-strain synergistic system, which utilizes the adsorption characteristics of biochar to improve the gas-liquid mass transfer efficiency, while its surface functional groups can promote the fixed growth of the strain. While realizing the value-added utilization of agricultural waste, this technical system can simultaneously achieve the dual goals of greenhouse gas (CH4 / CO2) emission reduction and biodegradable material production, providing an innovative solution for the circular economy model under the background of carbon neutrality.

[0006] The technical solution provided by the present invention is: an engineering strain for producing poly-β-hydroxybutyrate using methane, wherein the engineering strain is a methanogenic archaeon M. acetivorans C2A was constructed as the starting bacterium; its genome contains the key gene clusters phaA, phaB, and phaC for poly-β-hydroxybutyrate synthesis as shown in the sequence list SEQ ID NO.1-3, and a methane-regulated promoter as shown in the sequence list SEQ ID NO.4.

[0007] The present invention also provides a method for constructing the engineered strain, using a liposome-mediated method to transform a plasmid containing a methane-regulating promoter encoding gene and a poly-β-hydroxybutyrate synthesis key gene cluster phaA, phaB, and phaC into a methanogenic archaeon. M. acetivorans C2A.

[0008] The present invention also provides the use of the engineered strain, which is used to produce poly-β-hydroxybutyric acid by converting methane into poly-β-hydroxybutyric acid.

[0009] The present invention also provides a method for producing poly-β-hydroxybutyric acid using methane, comprising: (1) Inoculate the engineered strain into fresh culture medium and adjust the OD to 0. 600 nm Between 0.70-0.80; (2) Centrifuge the bacterial solution at low temperature and wash the bacteria 2-3 times with sterile basal medium; (3) Resuspend the washed cells in a selective medium containing puromycin and transfer to an anaerobic serum bottle; (4) The anaerobic serum bottle was aerated with pure methane and placed in a constant temperature shaker at 37±0.5℃ for microaerobic shaking culture.

[0010] Preferably, the anaerobic serum bottle in step (3) is added with an electron acceptor; the electron acceptor is biochar, Fenton-modified biochar, nano-zero-valent iron-modified biochar or magnetite-modified biochar.

[0011] Preferably, the working concentration of the biochar or Fenton-modified biochar is 0.2-0.25 g / L.

[0012] Preferably, the working concentration of the nano zero-valent iron modified biochar or magnetite modified biochar is 0.25-0.30 g / L.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The present invention uses model methanogenic archaea M. acetivorans Using C2A as the starting bacteria, a cell factory was constructed to convert methane into poly-β-hydroxybutyrate (PHB). This process achieves efficient conversion of methane into poly-β-hydroxybutyrate through the growth and metabolism of the microorganisms themselves. This technology can not only effectively utilize agricultural waste (such as corn stalks), but also reduce greenhouse gas leakage during the transportation, distribution and storage of methane. In addition, this invention helps to alleviate the impact of excessive use of fossil fuels on global climate change and convert methane into chemicals with more economic value; (2) The present invention uses a combination of genetic biology and environmental ecology methods to significantly improve the production and yield of poly-β-hydroxybutyrate (PHB) through genetic engineering and the addition of extracellular electron shuttles. This research result not only optimizes the production process of PHB, but also provides important theoretical guidance and practical reference for the construction and regulation of other microbial cell factories. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The methanogenic archaea in the present invention M. acetivorans C2A Effect diagram of anaerobic methane oxidation using biochar; A. Methane consumption after adding different carbon materials; B. SEM electron microscope image of bacteria and biochar; C. Changes in the electron accepting capacity (EAC) of biochar before and after cultivation; D. Fourier infrared spectrum of biochar before and after cultivation; Figure 2 The effect diagram of biochar before and after modification in the embodiment of the present invention; wherein, A. Fourier infrared spectrum of biochar before and after modification; B. methane consumption of biochar before and after modification; Figure 3 For the engineering strains in the embodiment of the present invention M. acetivorans-PHBPlasmid map used for genetic modification; A. pES plasmid backbone; B. recombinant plasmid pES-Pmc; Figure 4 For the engineering strains in the embodiment of the present invention M. acetivorans-PHB Transformant verification; A. Electrophoresis diagram of specific puromycin encoding gene fragments in the engineered strain; B. Sequencing diagram of characteristic fragments in the engineered strain; Figure 5 Schematic diagram of the metabolic principle of anaerobic oxidation of methane to produce poly-β-hydroxybutyrate (PHB) in the implementation of the present invention; wherein, A. a diagram of the plasmid transformation process; B. methane consumption after adding biochar to the engineered strain; C. a schematic diagram of the metabolism of poly-β-hydroxybutyrate (PHB); D. a schematic diagram of gas phase determination of PHB yield; Figure 6 The engineered strain constructed in the embodiment of the present invention M. acetivorans-PHB The production of poly-β-hydroxybutyrate (PHB) using methane; A. An engineered strain containing an empty plasmid pES; B. An engineered strain containing the plasmid pES-PHB. DETAILED DESCRIPTION

[0015] In order to facilitate the understanding of the present invention, the present invention is described in more detail below in conjunction with the accompanying drawings and specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0016] The present invention provides an engineered strain for converting methane into poly-β-hydroxybutyrate (PHB) using corn straw-based biochar, and its construction method and process are described in detail as follows.

[0017] Example 1: Preparation and modification of corn straw-based biochar: (1) Preparation of biochar: a. Pretreatment: Gradient dehydration technology (70℃-80℃ hot air circulation drying for 12h) is used to reduce the moisture content of the straw to below 8%; the straw is processed by an ultra-fine grinder to a particle size of ≤2mm to construct a porous structure precursor; b. Pyrolysis carbonization: Multi-stage pyrolysis is carried out using a tubular atmosphere furnace: ① Fill the pre-treated crucible (made of corundum) with 20.00±0.05g of raw material; ② Program temperature control (N2 atmosphere, 500℃ constant temperature for 6h, heating rate 10℃ / min); ③ Achieve selective pyrolysis of cellulose, with a biochar yield of 38.2±0.5%; c. Post-treatment strengthening process: washing with distilled water to pH = 7.0 ± 0.2; vacuum drying at 70 ° C for 12 hours to obtain a specific surface area ≥ 300m2 / g of functionalized biochar; stored in a sealed and light-proof place (humidity ≤ 10% RH) to ensure the stability of surface functional groups.

[0018] (2) The present invention constructs a multi-dimensional biochar modification platform to achieve directional enhancement of material functions through surface and interface engineering regulation: a. Nano-Zero-Valent Iron Loading Modification Liquid phase reduction: Under strict anaerobic conditions (N2 aeration for 1 h, dissolved oxygen ≤ 0.5 ppm), 1.5 g of biochar was dispersed in 1 L of FeSO4·7H2O solution (0.1 mol / L); 1 L of NaBH4 solution (0.108 mol / L, drop rate 5 mL / min) was added dropwise using a constant pressure dropping funnel; after centrifugation (8000 rpm, 15 min), it was ultrasonically cleaned with anhydrous ethanol for 3 times and vacuum dried at 60 °C for 12 h to obtain nZVI@BC composite material (nano zero-valent iron modified biochar), Fe 0 Loading amount: 12.3wt%.

[0019] b. Magnetic Fe3O4 composite modification Under N2 protection, 6.1g FeCl3·6H2O and 4.2g FeCl2·4H2O (molar ratio 2:1) were dissolved in 100mL ultrapure water and stirred at 90℃ to form an iron precursor solution; 1g biochar carrier was added in batches, and 20mL NH3·O2 (26%, flow rate 2mL / min) was injected by a peristaltic pump; solid-liquid separation was achieved by NdFeB permanent magnet (0.5T), and Fe3O4 / BC magnetic material (magnet-modified biochar) was obtained after vacuum drying at 60℃.

[0020] c. Construction of Fenton-like active sites 2 g of biochar was immersed in 100 mL of H2O2 solution (30%, pH = 3.0), and 27.8 g of FeSO4·7H2O (molar ratio H2O2:Fe 2+ =10:1); shake at 25 °C for 24 h (200 rpm); filter through a 0.22 μm filter membrane and vacuum dry at 60 °C to obtain Fenton-BC catalyst (Fenton modified biochar).

[0021] Example 2: Construction of corn straw biochar-methane oxidation electron symbiosis: 1. Bacteria strains and culture conditions The present invention uses commercial methanogenic archaea Methanosarcina acetivoransC2A (available from commercial sources) was cultured in 100 mL anaerobic serum bottles (sealed with butyl rubber stoppers and aluminum caps): 150 mM methanol (HPLC grade, Sigma-Aldrich) was used as the carbon source, the headspace was filled with a N2 / CO2 mixture (80:20 v / v), and the culture was incubated at 37°C. Dissolved oxygen (≤0.1 ppm) was monitored in real time by resazurin (0.0001% w / v).

[0022] 2. Addition of electron acceptors and working concentration The working concentration of biochar and Fenton-modified biochar was 0.22±0.01 g / L, respectively using surface quinone groups and ·OH-mediated Fe 2+ / Fe 3+ Cyclic enhanced electron transfer; the concentration of nano zero-valent iron and magnetite modified biochar was 0.28±0.01g / L, respectively through Fe 0 The core-shell structure and Fe3O4 lattice defects enhance the reduction potential.

[0023] Weigh 0.22 g of the prepared biochar in 100 mL of oxygen-free water, stir overnight with a magnetic stirrer, and mix evenly. Then, aerate with pure N2 for 1 hour to deoxygenate; weigh 0.22 g of the prepared Fenton reagent modified biochar, and then stir and aerate in the same way as the original biochar; weigh 0.28 g of the prepared nano zero-valent iron modified biochar and magnet modified biochar, and then stir and aerate in the same way as the original biochar; store the prepared solution in an oxygen-free environment away from light.

[0024] Under strict anaerobic conditions (dissolved oxygen ≤ 0.1 ppm), the cells washed three times with PBS buffer (pH = 7.4) were resuspended in a 50 mL serum bottle, and high-purity methane (99.999%) was introduced to a headspace volume ratio of 2:1 and a positive pressure of 0.3 MPa was maintained; functionalized electron acceptors were added using a double concentration gradient strategy.

[0025] 3. Experimental results In order to explore the electron transfer regulation mechanism of conductive carbon materials in the methane oxidation process, the present invention constructed an anaerobic microcosm experimental system: three typical carbon materials were added to a strictly anaerobic serum bottle (DO≤0.1ppm): ① multi-walled carbon nanotubes (CNT, 0.22g / L); ② graphene oxide (GO, sheet thickness 1.2nm, C / O ratio 2.3, 0.22g / L); ③ corn straw biochar (BC, pore size distribution 2-50nm, 0.22g / L), and pure methane (99.999%) was used as the substrate for 32 days of culture (37°C, 100rpm). Figure 1As shown in Figure A, compared with the carbon-free control group, all carbon material systems significantly accelerated methane oxidation (p<0.01, ANOVA), and the methane consumption rate of the biochar group increased by 50.0% and 35.5% compared with CNT and GO. Fourier infrared analysis further confirmed that biochar has a higher electron transfer efficiency, and its abundant surface carboxyl groups (-COOH) and phenolic hydroxyl groups (-OH) form an efficient electron superpath through π-π stacking with the outer membrane cytochrome c of the bacteria.

[0026] Scanning electron microscopy (SEM, accelerating voltage 15 kV, magnification 10 k×) characterization showed ( Figure 1 Figure B). After 32 days of co-cultivation, the bacteria were anchored on the surface of 2-50 nm biochar and in the mesoporous structure, confirming the multi-level pore structure of biochar (BET specific surface area 380 m 2 / g) provides an ideal topological support for electron transfer at the microorganism-material interface.

[0027] To confirm the electron acceptor function mechanism of biochar, an electrochemical workstation (CHI760E, three-electrode system, scan rate 50mV / s) was used for quantitative characterization and found that ( Figure 1 In the middle C, the electron transfer capacity (EEC) of the bacterial carbon composite system increased from the initial 0.024 mmol·e - / g dropped sharply to 0.255 mmol·e on the 32nd day - / g (a decrease of 10.6 times); simultaneous FTIR analysis (NicoletiS50, resolution 4cm -1 , scanned 32 times) displays ( Figure 1 Middle D), 1440cm -1 (CO stretching vibration) and 1620cm -1 The characteristic peak intensities of (quinone C=O vibration) decreased by 63.2% and 58.7%, respectively, confirming that the oxygen-containing functional groups on the biochar surface continuously participate in electron transfer as redox active sites. This result, for the first time, uses the electron flux-functional group decay dual evidence chain to explain that biochar drives methane oxidation through the "electron deposition-release" cycle (ΔG=-112 kJ / mol), breaking through the traditional electron acceptor theoretical framework in order to further improve the efficiency of biochar in promoting the anaerobic oxidation of methane.

[0028] The present invention further modifies the biochar, mainly in the following ways: (1) modification with nano-zero-valent iron; (2) modification with magnet; and (3) modification with Fenton reagent. The biochar before and after modification is characterized, and the 1440 cm -1 CO and 1620cm -1 The abundance of C=O oxygen-containing functional groups increased to varying degrees ( Figure 2(A) XPS data in the supplementary materials further demonstrate the changes in functional groups in different materials. Compared with unmodified biochar, the C=O abundance of biochar modified with Fenton's reagent increased by 3.93%, and the abundance of CO increased by 23%; the C=O abundance of biochar modified with magnet increased by 2.08%, and the abundance of CO increased by 4.97%; the C=O abundance of nano-zero-valent iron decreased, and the abundance of CO increased by 10%; at the same time, magnet-modified biochar and nano-zero-valent iron introduced ferrosoferric oxide and zero-valent iron.

[0029] By comparing the regulatory effects of Fenton-modified and original corn straw biochar on the anaerobic oxidation of methane, the present invention found that the modified treatment increased the specific surface area of ​​biochar by 2.8 times, increased the density of oxygen-containing functional groups on the surface, and XPS analysis results showed that the hydroxyl content increased from 8.3% to 34.7%. The experimental results show that in the anaerobic oxidation culture system, adding biochar modified by Fenton reagent can increase the methane utilization efficiency by more than 4%. Figure 2 Further analysis showed that the promotion of biochar on the anaerobic oxidation of methane was positively correlated with the content of oxygen-containing functional groups on its surface, that is, the higher the abundance of oxygen-containing functional groups, the stronger its catalytic ability for anaerobic oxidation of methane. This finding provides a theoretical basis for the application of biochar in the anaerobic oxidation of methane.

[0030] Example 3: Engineering strains M. acetivorans-PHB Build: 1. Bacteria strains and culture conditions The present invention uses commercial methanogenic archaea Methanosarcina acetivorans C2A, the culture system was established in a 100 mL anaerobic serum bottle (sealed with Butyl rubber stopper and aluminum cap). 150 mM HPLC grade methanol was used as the carbon source, the headspace was filled with N2 / CO2 mixed gas (80:20 v / v, 0.2 MPa positive pressure), and the culture was statically cultured at 37°C. The dissolved oxygen (≤0.1 ppm) was monitored in real time by a copper-based anaerobic indicator (resazurin, 0.0001% w / v).

[0031] Engineering strain screening module: Add 2 μg / mL puromycin to the basal culture medium (sterilized by 0.22 μm filter membrane).

[0032] 2. Cloning and genetic modification All plasmids were constructed using 2×MultiF Seamless Assembly Mix (ABclonal, Wuhan, China) and In-Fusion TM Each amplified fragment was cloned by cloning technology. PrimeSTAR HS DNA polymerase (Takara, Japan) was used for fragment PCR. The plasmid map used in the present invention is shown in Figure 3 .

[0033] (1) Construction of pES-Pmcr The present invention uses synthetic biology technology to construct a genetic engineering plasmid system, in which the basic vector pES plasmid is synthesized by a biological company and stored in glycerol at -80°C in the laboratory (20% v / v). By replacing the promoter element in the pES plasmid backbone, the methane-responsive promoter Pmcr (sequence see SEQ ID NO.4) from ANME archaea is directed to replace the original cdh constitutive promoter to construct the recombinant plasmid pES-Pmcr. The Pmcr promoter is synthesized by a biological company (Qingke Bio, Beijing, China) and verified by Sanger sequencing.

[0034] The present invention constructs an engineering plasmid pES-Pmcr-pha by synthetic biology technology: the pES-Pmcr plasmid is used as the backbone (containing the methane-responsive promoter Pmcr), and the vector is linearized by inverse PCR (PrimeSTAR® HS DNA polymerase, denaturation at 98°C for 10s, annealing at 55°C for 15s, extension at 72°C for 5min, 30 cycles), and the pES-Pmcr-pha plasmid ... Rhodopseudomonas palustris ) genome (phaA, phaB, phaC, sequences see SEQ ID NO.1-3); the linearized vector was ligated with phaA, phaB, and phaC fragments using the In-Fusion® HD Cloning Kit (Takara), and the constructed recombinant plasmid was verified by Sanger sequencing (coverage 100%, Q30>99.9%) and functional verification, ultimately forming a methane-regulated PHB synthesis plasmid.

[0035] (3) Conversion All plasmids were transformed into methanogenic archaea using a liposome-mediated method. M. acetivorans C2A and screened with a medium containing 2µg / mL puromycin to obtain an engineered strain.

[0036] Example 4: Engineering strains M. acetivorans-PHB Verification: The present invention utilizes PCR method and sequencing technology to verify whether the constructed engineering strain is successfully constructed.

[0037] 1. PCR verification of specific puromycin encoding gene fragments in engineering strains Extract the engineered strains selected from the screening medium M. acetivoran-PHB The specific puromycin encoding gene fragment was amplified by PCR and electrophoresed. Figure 4As shown in A, lane 1 is the wild-type control group; lane 2 is the engineered strain containing the empty plasmid pES; lane 3 is the engineered strain containing the plasmid pES-PHB; lane 4 is the DL2000 DNA Marker.

[0038] 2. Engineering strains M. acetivorans-PHB Sequencing verification of characteristic fragments in Engineering strains M. acetivorans-PHB The promoter Pmcr sequence in the , as well as phaA, phaB, and phaC sequences were sequenced (Qingke Biotechnology), and the results are as follows Figure 4 As shown in B, Pmcr successfully replaced the Pcdh promoter, and the fragments of phaA, phaB, and phaC were successfully connected.

[0039] This proves that the engineering strain of the present invention was successfully constructed.

[0040] Example 5: Engineering strains M. acetivorans-PHB Functional assay for poly-β-hydroxybutyrate (PHB) production: 1. Using the engineered strains M. acetivorans-PHB Preparation of cell factories for the production of poly-β-hydroxybutyrate (PHB): The engineered strains M. acetivorans-PHB Resuscitation to OD 600 nm About 0.70-0.80, collect 10 mL of cell suspension, centrifuge (7500 rpm, 10 min), and wash 3 times with culture medium to remove residual methanol. The washed cells were resuspended in 10 mL of culture medium containing puromycin and transferred to a 50 mL anaerobic serum bottle. Biochar was added to some experimental groups. Pure methane (99.999%) was filled into the headspace of each bottle and cultured slowly at 37°C and 100 rpm with shaking. One cycle of the metabolic process of anaerobic oxidation of methane to produce poly-β-hydroxybutyrate (PHB) is 32 days.

[0041] 2. The yield of poly-β-hydroxybutyric acid (PHB) was determined by gas phase method: a. Take 200 ml of the bacterial culture sample after 32 days of culture, centrifuge at 4700 g x 15 min, wash the precipitate three times with PBS and freeze-dry the cells; b. Add 1% (w / v) SDS solution to 2 g of lyophilized cells, stir for 10 min, centrifuge at 4700 g for 15 min, and wash the precipitate with deionized water; c. Add 30% (w / v) NaCl0 solution, stir for 3 min, centrifuge at 4700 g x 20 min to obtain a precipitate, and dry the precipitate at 60-70°C; d. Add 2 ml of chloroform and 2 ml of benzoic acid-methanol mixed solution (4 g / 100 m) to the dried sample, mix evenly, and digest at 105 °C for 6 h; e. Add 1 ml of deionized water to the dried sample, mix and shake vigorously to remove the residue in the chloroform, let stand to separate the layers, pipette the lower chloroform phase, measure the PHB content on the machine, and calculate the PHB content in the sample according to the standard curve configured with the PHB standard.

[0042] The yield of poly-β-hydroxybutyric acid was quantitatively determined using a gas chromatograph (GC-2014, Shimadzu, Japan). The chromatographic column was a capillary column (SK-FFAP, Shimadzu, Japan), the detector was a flame ionization detector (FID), and the injection volume was 5 μL. The injector and detector temperatures were maintained at 225°C and 265°C, respectively, the column temperature was maintained at 40°C for 3 min, then increased to 220°C at a rate of 20°C / min, and then maintained at 220°C for 5 min. The carrier gas flow rate (N2) was maintained at 15.4 mL / min, the hydrogen flow rate was 30 mL / min, and the air flow rate was 300 mL / min.

[0043] 3. Experimental results Pyruvate can be synthesized into poly-β-hydroxybutyrate (PHB) by decarboxylation reduction or acetyl-CoA by three-step reduction. Since acetyl-CoA is a direct intermediate in the anaerobic oxidation metabolism of methane, the present invention will Rhodopseudomonas palustris The genes encoding acetyl-CoA synthetase phaA, acetoacetyl-CoA synthetase phaB, and pha synthetase phaC were cloned into M. acetivorans The metabolic pathway from acetyl-CoA to poly-β-hydroxybutyrate (PHB) is realized in Rhodopseudomonas palustris The three enzymes encoded in the catalytic three-step reduction catalyze the production of poly-β-hydroxybutyrate (PHB) ( Figure 5 In pES plasmid, Methanosarcina ( Methanosarcina )of cdh Since methane is the substrate for methanotrophic growth, in order to make phaA, phaB and phaC regulated by methane, the present invention has M. acetivorans The origin of replication (Orip C2A) in the plasmid pES cdh The promoter was replaced with the methane-regulated promoter Pmcr present in ANME species to generate the pES-Pmcr plasmid; the phaA gene, phaB gene, and phaC gene were cloned into the plasmid pES-Pmcr to generate the plasmid pES-PHB, such as Figure 5 As shown in A.

[0044] Figure 5 Figure B shows that the engineered strain containing plasmid pES-PHB consumed more methane during methanotrophic growth and improved methane utilization efficiency.

[0045] Figure 5 The results of D gas phase assay showed that the engineered strain containing the empty plasmid pES did not produce observable poly-β-hydroxybutyrate (PHB) titer during methanotrophic growth, while the engineered strain containing the plasmid pES-PHB could be detected to produce more than 0.10 mg / L of poly-β-hydroxybutyrate (PHB) extracellularly on the 15th day of methanotrophic growth.

[0046] Example 6: Adding electron acceptors to increase the production level of poly-β-hydroxybutyrate (PHB): 1. Experimental methods (1) Material preparation and reagent preparation The preparation of the electron acceptor is the same as in Example 1.

[0047] (2) Addition of electron shuttle and working concentration The addition and working concentration of the electron acceptor are the same as those in Example 2.

[0048] (3) Determination of poly-β-hydroxybutyric acid (PHB) production Nile red fluorescence was used as a rapid quantification method for PHA.

[0049] The staining method was as follows: the cell suspension (1 ml) was centrifuged at 12000 g for 5 min and the pellet was resuspended in 1 ml of distilled water. Subsequently, 40 uL of Nile Red (80 ug / mL dissolved in 7.742 uL dimethyl sulfoxide (DMSO)) was added to the suspension to a final concentration of 3.1 ug Nile Red / mL suspension and incubated at room temperature for 30 min. The stained suspension was centrifuged at 12000 g for 5 min and the supernatant was discarded. Distilled water (1 mL) was added and the resulting pellet was vortexed vigorously. An equal amount of the suspension was dropped into a 96-well microplate. Fluorescence was then read using monocolor in an EnVision MultilabelPlate Reader (Perkin Elmer, Waltham, USA) using the Wallac EnVision Manager 1.12 software program at excitation and emission wavelengths of 535 and 605 nm, respectively.

[0050] (4) Experimental results In order to further improve the production titer and yield of poly-β-hydroxybutyrate (PHB), electron acceptors were used to promote intracellular and extracellular electron transfer. Four prepared electron acceptors, biochar, Fenton reagent modified biochar, nano zero-valent iron modified biochar, and magnet modified biochar were added to the engineered strain. M. acetivorans-PHB in the anaerobic methane oxidation culture system. Figure 6 The results of the A staining method showed that for the engineered strain containing the empty plasmid pES, a very low poly-β-hydroxybutyrate (PHB) titer was detected in the culture system with the addition of electron acceptors, which was lower than 0.12 g / L, and the titer detected continued to decrease with the increase of culture time. It may be that some nutrients were converted into PHB during the culture process with the addition of methanol, and this part of PHB was consumed during the anaerobic oxidation process due to the lack of carbon source.

[0051] For the engineered strain containing plasmid pES-PHB, significant PHB titers can be detected in the culture system with the addition of four electron acceptors. Compared with the experimental system without the addition of electron acceptors, the electron acceptors in the form of four biochars all significantly increased the PHB titers ( Figure 6 In B); the culture system in which the Fenton reagent modified biochar was added detected the highest concentration of PHB titer, reaching 0.25 g / L, which is consistent with the fact that the Fenton reagent modified biochar in Example 1 of the present invention can most efficiently improve the efficiency of the bacteria in utilizing methane for anaerobic oxidation of methane.

Claims

1. An engineered strain for producing poly-β-hydroxybutyrate using methane, characterized in that: The engineered strain is composed of methanogenic archaea M. acetivorans C2A was constructed as the starting bacterium; its genome contains the key gene clusters phaA, phaB, and phaC for poly-β-hydroxybutyrate synthesis as shown in the sequence list SEQ ID NO.1-3, and a methane-regulated promoter as shown in the sequence list SEQ ID NO.

4.

2. The method for constructing an engineered strain for producing poly-β-hydroxybutyrate using methane according to claim 1, characterized in that: The plasmid containing the methane-regulated promoter encoding gene and the key gene cluster phaA, phaB, and phaC for poly-β-hydroxybutyrate synthesis was produced into methanogenic archaea using a liposome-mediated method. M. acetivorans C2A.

3. The use of the engineered strain for producing poly-β-hydroxybutyrate using methane as claimed in claim 1, characterized in that: The engineered bacteria is used to produce poly-β-hydroxybutyric acid by converting methane into poly-β-hydroxybutyric acid.

4. A method for producing poly-β-hydroxybutyric acid using methane, characterized in that: include: (1) Inoculate the engineered strain described in claim 1 into fresh culture medium and adjust the OD to 40 in an anaerobic workstation. 600 nm Between 0.70-0.80; (2) Centrifuge the bacterial solution at low temperature and wash the bacteria 2-3 times with sterile basal medium; (3) Resuspend the washed cells in a selective medium containing puromycin and transfer to an anaerobic serum bottle; (4) The anaerobic serum bottle was aerated with pure methane and placed in a constant temperature shaker at 37±0.5℃ for microaerobic shaking culture.

5. The method according to claim 4, characterized in that: The anaerobic serum bottle in step (3) is added with an electron acceptor; the electron acceptor is biochar, Fenton-modified biochar, nano-zero-valent iron-modified biochar or magnet-modified biochar.

6. The method according to claim 5, characterized in that: The working concentration of the biochar or Fenton-modified biochar is 0.2-0.25 g / L.

7. The method according to claim 5, characterized in that: The working concentration of the nano zero-valent iron modified biochar or magnet modified biochar is 0.25-0.30 g / L.

Images