Aerobic methanophilic engineering bacterium for producing succinic acid as well as construction method and application of aerobic methanophilic engineering bacterium

Abstract

The invention relates to an aerobic methanophilic engineering bacterium for producing succinic acid as well as a construction method and application of the aerobic methanophilic engineering bacterium, a coding gene ppc of heterologous phosphoenolpyruvate carboxylase and / or a coding gene pck of phosphoenolpyruvate carboxykinase are / is introduced into host bacteria, construction of a serine pathway is realized, and acetokinase and / or lactic dehydrogenase are / is knocked out, so that the methanophilic engineering bacterium for producing succinic acid is obtained. A synthesis route of acetic acid and / or lactic acid byproducts is blocked, and a ribulose monophosphate route and a serine circulation route are used for synergistically assimilating methane to produce succinic acid; the invention further provides a construction method of various aerobic methanophilic engineering bacteria and application of the aerobic methanophilic engineering bacteria in succinic acid production, 13C labeling experiments prove that fixed carbon dioxide enters succinic acid, and the constructed engineering bacteria remarkably improve the yield and yield of succinic acid, have good industrial application prospects and are suitable for industrial production. A foundation is laid for efficient synthesis and industrial production of succinic acid, and meanwhile, a new way is opened up for efficiently converting methane and carbon dioxide into sustainable chemical products.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and particularly relates to an aerobic methanophilic engineering bacterium producing succinic acid, and a construction method and application thereof. Background Art

[0002] Succinic acid (also known as butyric acid) is an important green chemical C4 platform compound, widely used in the production of surfactants, food additives (E363), pharmaceutical intermediates and biodegradable polyesters (such as PBS). Therefore, the U.S. Department of Energy lists it as the top of the 12 most promising bulk bio-based chemicals. At present, according to statistics, the global succinic acid market size in 2022 is US$234.8 million, and it is expected that the compound growth rate of the market size will reach 9.3% during the period 2023-2032.

[0003] The production methods of succinic acid mainly include traditional petrochemical technology routes and the increasingly emerging biomanufacturing. The former uses non-renewable strategic resource petroleum products as raw materials. Its raw material supply is unsustainable, and there is serious environmental pollution in the production process, which limits the development of the succinic acid industry. With the development of the economy, the global demand for succinic acid continues to increase. In order to achieve sustainable economic and social development, it is necessary to replace non-renewable petrochemical resources with renewable biomass resources, and replace polluting and inefficient petrochemical technology routes with clean and efficient biorefining processing methods. By using microbial fermentation to produce succinic acid, a fundamental change in resource utilization and material processing methods can be achieved, thereby promoting the large-scale production of bio-based succinic acid. At the same time, the characteristic of succinic acid fermentation process consuming carbon dioxide can help reduce carbon dioxide emissions and alleviate the greenhouse effect.

[0004] Currently, the method for synthesizing succinate by type I aerobic methanotroph (a type of bacteria that can grow with methane as the only carbon source and energy source) is mainly through the introduction of the glyoxylate cycle "Metabolic engineering of the type I methanotroph Methylomonas sp. DH-1 for production of succinate from methane". Although the succinate yield is increased to 195 mg / L, there is still a technical difficulty: formic acid will be converted into carbon dioxide during metabolism, resulting in a large amount of carbon resource excretion, thereby limiting the production capacity of succinate.

[0005] By analyzing the gene map, it was found that aerobic methanogens also have some type II aerobic methanogen-specific serine cycle pathways. This pathway can not only assimilate formaldehyde, but also has the function of capturing carbon dioxide. However, to date, there has been no report on the optimization of aerobic methanogen metabolic biochemical processes, maximization of carbon metabolic flow, and rapid guidance of target products by regulating the balance of cofactors in aerobic methanogens and constructing heterologous serine cycle pathways. Summary of the invention

[0006] In order to solve the problems existing in the above-mentioned prior art, the purpose of the present invention is to provide an aerobic methanophilic engineered bacterium that produces succinic acid, and a construction method and application thereof. By introducing exogenous genes into aerobic methanophilic bacteria to construct a serine cycle pathway, the problem of low carbon flux in the cofactor regeneration process is effectively avoided, thereby achieving the high production of succinic acid by synergistically assimilating methane through the ribulose monophosphate pathway and the serine cycle pathway, thereby enhancing the carbon dioxide fixation ability of the strain; by knocking out acetate kinase and lactate dehydrogenase, the production efficiency of methane to succinic acid is improved. The present invention optimizes the utilization rate of carbon resources and has important industrial application value and environmental significance.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] A succinic acid-producing aerobic methanotrophic engineered bacterium is provided. A heterologous phosphoenolpyruvate carboxylase encoding gene ppc and / or a phosphoenolpyruvate carboxykinase encoding gene pck are introduced into a host bacterium to achieve the construction of a serine pathway, and acetate kinase and / or lactate dehydrogenase are knocked out to block the synthesis pathway of acetate and / or lactate byproducts, thereby achieving the coordinated assimilation of methane to produce succinic acid using a ribulose monophosphate pathway and a serine cycle pathway.

[0009] The host bacteria are aerobic methanophilic bacteria Methylotuvimicrobium buryatense5GB1, Methylomicrobium alcaliphilum 20Z or Alkalicoccus glycogenes WONF2802, wherein the Alkalicoccus glycogenes WONF2802 has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, and its deposit number is CGMCC No.28614.

[0010] The phosphoenolpyruvate carboxylase is derived from aerobic methanophilic bacteria Methylosinustrichosporium OB3b; the amino acid sequence of the phosphoenolpyruvate carboxylase is shown in SEQ ID NO:1.

[0011] The phosphoenolpyruvate carboxykinase is from Escherichia coli ATCC 8739; the amino acid sequence of the phosphoenolpyruvate carboxykinase is shown in SEQ ID NO:2.

[0012] The blocking of the lactate synthesis pathway is achieved by knocking out the gene encoding lactate dehydrogenase, ldh (EQU24_19220).

[0013] The blocking of the acetate synthesis pathway is achieved by knocking out the gene encoding acetate kinase, ack (EQU24_07890).

[0014] A method for constructing an aerobic methanophilic engineered bacterium that produces succinic acid includes the following three approaches:

[0015] (1) By constructing four types of aerobic methanotrophic engineered strains SC01-SC04, the serine pathway was completed:

[0016] (1.1) The exogenous gene ppc was connected to the plasmid pAWP89 to construct the recombinant plasmid pAWP89-S1, which was then transferred into the host bacteria to obtain the aerobic methanotrophic engineered strain SC01;

[0017] (1.2) The exogenous gene pck was connected to the plasmid pAWP89 to construct the recombinant plasmid pAWP89-S2, which was then transferred into the host bacteria to obtain the aerobic methanotrophic engineered strain SC02;

[0018] (1.3) The exogenous genes ppc and pck were connected to the plasmid pAWP89, wherein ppc and pck were connected by rbs to construct the recombinant plasmid pAWP89-S3, which was then transferred into the host bacteria to obtain the aerobic methanotrophic engineered strain SC03;

[0019] (1.4) Connect the exogenous genes ppc and pck to the plasmid pAWP89, with P between ppc and pck. tac The recombinant plasmid pAWP89-S4 was constructed by ligation and then transformed into a host bacterium to obtain an aerobic methanotrophic engineered strain SC04.

[0020] (2) Blocking the pathway of byproduct acetic acid and / or lactic acid:

[0021] (2.1) The ack gene on the host bacteria was replaced with Gm to block the acetate production pathway and obtain the aerobic methanotrophic engineered strain SC05;

[0022] (2.2) The ldh gene on the host bacteria was replaced with Bleo to block the lactic acid production pathway and obtain the aerobic methanophilic engineered strain SC06;

[0023] (2.3) Block the lactic acid and acetic acid production pathways at the same time, replace the ack gene on the host bacteria with Gm, and replace the ldh gene with Bleo, to obtain the aerobic methanotrophic engineered strain SC07.

[0024] (3) Serine pathway combined with byproduct knockout pathway

[0025] (3.1) The exogenous genes ppc and pck were connected to the plasmid pAWP89, wherein ppc and pck were connected with rbs to construct the recombinant plasmid pAWP89-S3;

[0026] (3.2) Blocking the lactic acid and acetic acid production pathways at the same time, replacing the ack gene on the host bacteria with Gm, and replacing the ldh gene with Bleo, to obtain the aerobic methanotrophic engineered strain SC07;

[0027] (3.3) The recombinant plasmid pAWP89-S3 was transferred into the aerobic methanotrophic engineered strain SC07 to obtain the aerobic methanotrophic engineered strain SC08.

[0028] The use of an aerobic methanophilic engineered bacterium producing succinic acid prepared according to any of the above methods is used in fermentation to produce succinic acid.

[0029] The aerobic methanophilic engineered bacteria are used in fermentation to produce succinic acid, specifically comprising the following steps:

[0030] (1) Seed culture: After the aerobic methanotrophic engineered strain with high succinic acid production is activated on a plate, 10%-20% of the inoculum is inoculated into an injection bottle and cultured for 24-48 hours to obtain a seed solution;

[0031] (2) The specific process of fermenting succinic acid in the fermenter is as follows: the seed liquid is inoculated into the NMS2 medium in the fermenter, the inoculation amount is 10% to 15% (v / v); the ventilation volume of the ventilation gas is 1.5-2.5 vvm; the ventilation gas is CH 4 and air, CH 4 The volume ratio of the fermentation agent to air is 1:3.5-4.5, the fermentation temperature is 25-30°C, the fermentation culture time is 24-96h, and the yield of succinic acid is determined;

[0032] The NMS2 medium includes: MgSO 4 7H 2 O 0.2-1g, CaCl 2 6H 2 O0.008-0.012g, KNO 36-12 g, NaCl 8-12 g, phosphate buffer solution 40 mL, carbonate buffer solution 50 mL and trace element solution 8 mL, and the rest is made up to 1 L with distilled water;

[0033] The phosphate buffer solution, pH 6.8, is calculated based on 1L, KH 2 PO 4 2.77-5.44 g·L -1 and Na 2 HPO 4 5.4-10.73 g·L -1 , the balance is water;

[0034] The total concentration of the carbonate buffer solution is 1M, which is composed of 140-700 parts by volume of NaHCO 3 aqueous solution and 60-300 parts by volume of Na 2 CO 3 Aqueous solution composition;

[0035] The trace element solution: Na 2 -EDTA·2H 2 O 1-2g·L -1 , FeSO 4 7H 2 O 1-2g·L -1 , ZnSO 4 7H 2 O 0.5-0.8 g·L -1 , MnCl 2 ·4H 2 O 0.01-0.03 g·L -1 , H 3 BO 3 0.01-0.03g·L -1 , CoCl 2 6H 2 O 0.1-0.2 g·L -1 , CuCl 2 ·2H 2 O 0.4-0.6 g·L -1 , NiCl 2 6H 2 O 0.01-0.02 g·L -1 , Na 2 MO 4 ·2H 2 O 0.02-0.05 g·L -1 , the balance is distilled water.

[0036] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0037] 1. The present invention introduces exogenous phosphoenolpyruvate carboxylase encoding gene ppc and / or phosphoenolpyruvate carboxykinase encoding gene pck to construct a metabolic pathway for synergistically utilizing ribulose monophosphate pathway and serine cycle pathway to produce succinic acid, thereby realizing the construction of aerobic methanotrophic engineered bacteria that produce succinic acid. The strain can utilize ribulose monophosphate pathway and serine cycle pathway to synergistically assimilate methane to produce succinic acid, thereby solving the technical problem that type I aerobic methanotrophic bacteria oxidize formic acid into carbon dioxide, resulting in a large amount of carbon resource excretion, thereby resulting in a low succinic acid yield.

[0038] 2. The present invention improves the conversion efficiency from methane to succinic acid by blocking the synthesis pathway of lactic acid and / or acetic acid. Since lactic acid and acetic acid are synthesized from pyruvic acid and acetyl-CoA, respectively; excessive lactic acid and acetic acid synthesis will reduce acetyl-CoA entering the TCA cycle, thereby reducing the content of succinic acid. Therefore, by knocking out acetate kinase and lactate dehydrogenase, this problem can be effectively avoided. This innovation not only optimizes the assimilation process of methane, but also provides a feasible and environmentally friendly new method for the industrial production of succinic acid.

[0039] 3. The aerobic methanotrophic engineered bacteria producing succinic acid provided by the present invention have a succinic acid production of more than 299.36 mg / L and a succinic acid productivity of more than 199.6 mg / L / day, providing a new strategy for the utilization of methane.

[0040] 4. The aerobic methanophilic engineered bacteria producing succinic acid provided by the present invention have been 13 C labeling experiments demonstrated that the fixed carbon dioxide entered succinate, indicating that the ribulose monophosphate pathway and the serine cycle pathway were used to synergistically assimilate methane to produce high amounts of succinate, thus enhancing the carbon dioxide fixation capacity of the strain.

[0041] 5. The aerobic methanophilic engineered bacteria for producing succinic acid provided by the present invention have a succinic acid yield that is 57.3% higher than that of Methylomonas sp.DH-1, which is currently reported to produce succinic acid using methane, and a productivity that is 440% higher. In addition, compared with cyanobacteria producing succinic acid using carbon dioxide, the productivity is increased by 7% to 207%.

[0042] In summary, the aerobic methanotrophic engineered bacteria provided by the present invention significantly improve the yield of succinic acid based on the strategy of co-utilization of methane and carbon dioxide, and provide a new method for the efficient bioconversion of methane and carbon dioxide; the present invention not only has the climate benefit of slowing down the rate of short-term climate warming, the environmental benefit of coordinated control of air pollution, but also has the economic benefit of early harvesting of natural gas as an alternative fuel and industrial raw material. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1This is a construction map of the recombinant plasmid pAWP89-S1 of the present invention.

[0044] Figure 2 Schematic diagram of the ack gene knock-in complex.

[0045] Figure 3 This is a graph showing the succinic acid production of aerobic methanotrophic engineered bacteria SC01-SC08.

[0046] Figure 4 This is a graph showing the production of succinic acid by aerobic methanophilic engineered bacteria SC08 in a fermenter. DETAILED DESCRIPTION

[0047] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0048] The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified. Unless otherwise specified, the quantitative tests in the following examples are all set up with three batches of repeated experiments, and each repeated test is set up with 3 repeated treatments, and the results are averaged.

[0049] The present invention will be described in detail below in conjunction with the accompanying drawings.

[0050] Example 1

[0051] Construction of serine pathway, taking host bacteria M. buryatense 5GB1 as an example, construction of aerobic methanotrophic engineered bacteria SC01-SC04

[0052] (1) A pair of primers was designed based on the ppc sequence of the gene in the genome of Methylosinus trichosporium OB3b, and the ppc gene was obtained by PCR. The pair of primers was designed as follows:

[0053] ppc-F:5'-tattcacacaggaaacagctATGCAGGTCGAAAACGACTCC-3'

[0054] ppc-R:5'-atcacgcatcttcccgacaaTCACCCCGTATTCCGCAA-3'

[0055] (2) According to the pAWP89 sequence, a pair of primers was designed to obtain the linearized pAWP89 vector by PCR. The pair of primers was designed as follows:

[0056] pAWP89-F:5'-TTGTCGGGAAGATGCGT-3'

[0057] pAWP89-R:5'-AGCTGTTTTCCTGTGTGAATACC-3'

[0058] (3) High-fidelity polymerase 2×PhantaMax MasterMix was used to amplify the pAWP89 plasmid with primers pAWP89-F and pAWP89-R. The resulting PCR product was digested with restriction endonuclease DpnI at 37°C for 2 h and kept at 80°C for 20 min to inactivate the DpnI enzyme. The resulting digestion product was purified by gel recovery to obtain the pAWP89 linearized vector;

[0059] (4) Using high-fidelity polymerase 2×PhantaMax MasterMix, primer pair ppc-F / ppc-R and the genome of Methylosinus trichosporium OB3b as a template, the corresponding gene fragment was amplified and the fragment was purified by gel recovery;

[0060] (5) The recovered fragments were homologously recombined using the Novagen One-Step Cloning Kit. The recombination system contained an equimolar ratio of the linearized vector (the product obtained in step 3) and the gene fragment (the product obtained in step 4). The multi-fragment recombination conditions were 50°C for 15-30 minutes. After the recombination reaction, the product was transferred to E. coli DH5α by heat shock transformation. The PCR identification showed that the band size was correct, and sequencing confirmed that there was no base mutation, indicating that the plasmid pAWP89-S1 was successfully constructed (such as Figure 1 ). The same method was used to construct plasmids pAWP89-S2, pAWP89-S3 and pAWP89-S4;

[0061] (6) The recombinant plasmid pAWP89-S1 was transferred into M. buryatense 5GB1 by triparental conjugation. The specific steps are as follows: First, the recipient bacteria M. buryatense 5GB1 and the donor bacteria E. coli DH5α were revived on the corresponding selective medium.

[0062] / pAWP89-S2 and helper bacteria E.coli PRK600; secondly, use a 10μL inoculation loop to scrape 1 loop of M.buryatense 5GB1, and evenly spread it on the conjugation plate for overnight culture; then, scrape 3 / 4 loop of donor bacteria E.coli DH5α / pAWP89-S1 and 1 / 2 loop of helper bacteria E.coliPRK600 onto the conjugation plate, evenly spread it, and culture at 30℃ for 48h; after the culture, scrape the mixed bacteria and spread it on NMS solid culture medium containing 50mg / L kanamycin, and continue to culture in a 30℃ incubator; after a single colony grows out, PCR identification and sequencing of base-free mutations are performed to obtain aerobic methanotrophic engineered bacteria SC01; the same method is used to construct aerobic methanotrophic engineered bacteria SC02, SC03 and SC04.

[0063] Example 2

[0064] 13 C labeling experiment, taking the host bacteria M. buryatense 5GB1 as an example.

[0065] By adding wild-type M. buryatense 5GB1 and aerobic methanotrophic engineered bacteria SC03 13 C-labeled sodium bicarbonate in NMS medium. 13 The proportion of C-labeled organic acids determines the fixed CO 2 The data show that 13 C-labeled succinic acid (m+1) and 13 C unlabeled succinate ( 13 The ratio of C-SUC / SUC) in the aerobic methanotrophic engineered bacteria SC03 was increased by about 1.3 times compared with the wild-type M. buryatense 5GB1. These results verified that the introduction of exogenous genes into M. buryatense 5GB1 to reconstruct the serine cycle achieved CO 2 The fixed CO 2 Enter succinic acid.

[0066] Example 3

[0067] Blocking of byproducts acetic acid and / or lactic acid, taking host bacteria M. buryatense 5GB1 as an example, i.e., construction of aerobic methanotrophic engineered bacteria SC05-SC07

[0068] (1) Two pairs of primers were designed based on the gene sequence in the NCBI aerobic methanogen ASM593109v1 genome, and the upstream and downstream gene fragments of the ack gene were obtained by PCR. The two pairs of primers were designed as follows:

[0069] ackL-F:5'-AACCGAACACCGAACATCCTC-3'

[0070] ackL-R:5'-ccgaacaggcttatgtcaaATCCTTAAAATGATTTTGGTAAACGC-3'

[0071] ackR-F:5'-ccaagtaccgccacctaaAAATATTTAGACTGAACGGTTTACCG-3'

[0072] ackR-R:5'-ATGCCCAACCGAATACCGT-3'

[0073] (2) Design a pair of primers based on the gm sequence in pCM351, obtain the gm gene by PCR, and design a pair of primers as follows:

[0074] Gm-F:5'-tTTGACATAAGCCTGTTCGGTTCG-3'

[0075] Gm-R:5'-ttTTAGGTGGCGGTACTTGGGTC-3'

[0076] (3) Using high-fidelity polymerase 2×Phanta Max MasterMix, the left and right homologous arms of the ack gene and the gm gene were used as templates to obtain the 'ackgm' gene knock-in complex by overlapping PCR amplification, such as Figure 2 .

[0077] (4) After sequencing, the 'ackgm' gene knock-in complex was transformed into aerobic methanogens by electroporation. The preparation method of aerobic methanogens was as follows: the bacteria were inoculated into NMS2 medium and cultured at 30°C until the OD 600 =1.5-2.0, pour into a centrifuge tube, centrifuge at 5000rpm / min for 10min, discard the supernatant, add 50mL of sterilized low-temperature deionized water and wash three times to complete the preparation of competent cells.

[0078] (5) Take 50 μL of competent cells obtained in step 4), add 400-600 ng of knock-in complex obtained in step 3) and mix gently. Gently transfer the mixture to a 1 mm gap low-temperature electroporator and perform electroporation (conditions are 1.3-1.8 kV, 25 μF and 200 Ω). Then quickly transfer to 10 mL of NMS liquid medium containing 0.1% (w / v) methanol and resuscitate at 30°C for 12-16 h. Collect the cells and spread them on a plate containing 10 μg mL -1The cells were cultured in NMS2 solid medium containing gentamicin to screen for recombinants.

[0079] (6) After the recombinant was identified by PCR, if there was no base mutation in the sequencing, the aerobic methanotrophic engineered strain SC05 that blocked the acetate production pathway was obtained. The same gene knockout method was used to obtain the aerobic methanotrophic engineered strain SC06 that blocked the lactate production pathway, and the aerobic methanotrophic engineered strain SC07 that blocked both the acetate production pathway and the lactate production pathway.

[0080] Example 4

[0081] The serine pathway was combined with the byproduct knockout pathway to achieve the construction of the aerobic methanophilic engineered bacteria SC08 with high succinic acid production, taking the host bacteria M. buryatense 5GB1 as an example.

[0082] The recombinant plasmid pAWP89-S3 was transferred to the aerobic methanotrophic engineered strain SC07 of Example 3 by the triparental conjugation method in Example 1, thereby obtaining the aerobic methanotrophic engineered strain SC08.

[0083] Example 5

[0084] Cultivation of aerobic methanotrophic engineered bacteria SC01-SC08, taking the host bacteria M. buryatense 5GB1 as an example.

[0085] The seed solution was inoculated into a new NMS2 medium and placed in a shaker at a speed of 200 rpm and cultured at 30°C until the late growth stage. During the culture process, air was replaced every 12 hours. After the culture was completed, the succinic acid yield was determined.

[0086] The NMS2 medium includes: MgSO 4 7H 2 O 0.2-1g, CaCl 2 6H 2 O0.008-0.012g, KNO 3 0.8-1.2g, NaCl 8-12g, phosphate buffer solution 20mL, carbonate buffer solution 50mL and trace element solution 2-4mL, and the rest is made up to 1L with distilled water;

[0087] The phosphate buffer solution, pH 6.8, is calculated based on 1L, KH 2 PO 4 2.77-5.44 g·L -1 and Na 2 HPO 4 5.4-10.73 g·L -1 , the balance is water;

[0088] The total concentration of the carbonate buffer solution is 1M, which is composed of 140-700 parts by volume of NaHCO 3 aqueous solution and 60-300 parts by volume of Na 2 CO 3 Aqueous solution composition;

[0089] The trace element solution: Na 2 -EDTA·2H 2 O 1-2g·L -1 , FeSO 4 7H 2 O 1-2g·L -1 , ZnSO 4 7H 2 O 0.5-0.8 g·L -1 , MnCl 2 ·4H 2 O 0.01-0.03 g·L -1 , H 3 BO 3 0.01-0.03g·L -1 , CoCl 2 6H 2 O 0.1-0.2 g·L -1 , CuCl 2 ·2H 2 O 0.4-0.6 g·L -1 , NiCl 2 6H 2 O 0.01-0.02 g·L -1 , Na 2 MO 4 ·2H 2 O 0.02-0.05 g·L -1 , the balance is distilled water.

[0090] Specific production volume Figure 3 As shown, from Figure 3 It can be seen that the aerobic methanophilic engineered strain SC08 obtained in Example 3 has the highest succinic acid production, while the four succinic acid productions obtained in Example 1 are second.

[0091] Example 6

[0092] Aerobic methanotrophic engineered bacteria SC08 were cultured in batches in a fermenter, taking the host bacteria M. buryatense 5GB1 as an example.

[0093] Aerobic methanophilic engineered bacteria SC08 were activated on plates and seed cultured and then inoculated into the fermenter at a rate of 10%. During the fermentation process, the temperature was controlled at 30°C and the pH was controlled at 9.0±0.1. During the fermentation process, the fermenter was continuously aerated at a rate of 1 vvm; the aeration gas consisted of CH 4 and air, CH 4 The flow rate of the gas was 400sccm and the flow rate of the air was 1600sccm. During the fermentation process, the speed of the fermentation tank was adjusted according to the dissolved oxygen. Figure 4 As shown, the succinic acid production reached 299.36 mg / L in 36 h.

[0094] Example 7

[0095] Application of succinic acid secreted by aerobic methanotrophic engineered bacteria

[0096] The succinic acid produced by aerobic methanotrophic engineered bacteria can be directly used as a raw material for food additives (European food additive number is E363), pharmaceutical intermediates and biodegradable polyesters (such as polybutylene succinate (PBS)) after purification.

Claims

1. An aerobic methanophilic engineered bacterium producing succinic acid, characterized in that: The heterologous phosphoenolpyruvate carboxylase encoding gene ppc and / or the phosphoenolpyruvate carboxykinase encoding gene pck are introduced into the host bacteria to realize the construction of the serine pathway, and the acetate kinase and / or lactate dehydrogenase are knocked out to block the synthesis pathway of acetate and / or lactate by-products, thereby realizing the coordinated assimilation of methane to produce succinate using the ribulose monophosphate pathway and the serine cycle pathway.

2. The aerobic methanophilic engineered bacteria producing succinic acid according to claim 1, characterized in that: in, The host bacteria are aerobic methanophilic bacteria Methylotuvimicrobium buryatense 5GB1, Methylomicrobium alcaliphilum 20Z or Alkalicoccusglycogenes WONF2802, wherein the Alkalicoccus glycogenes WONF2802 has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, and its deposit number is CGMCC No.28614.

3. The aerobic methanophilic engineered bacteria producing succinic acid according to claim 1, characterized in that: The phosphoenolpyruvate carboxylase is from the aerobic methanogen Methylosinus trichosporium OB3b; the amino acid sequence of the phosphoenolpyruvate carboxylase is shown in SEQ ID NO:

1.

4. The aerobic methanophilic engineered bacteria producing succinic acid according to claim 1, characterized in that: The phosphoenolpyruvate carboxykinase is from Escherichia coli ATCC 8739; the amino acid sequence of the phosphoenolpyruvate carboxykinase is shown in SEQ ID NO:

2.

5. The aerobic methanophilic engineered bacteria producing succinic acid according to claim 1, characterized in that: The blocking of the lactate synthesis pathway is achieved by knocking out the gene encoding lactate dehydrogenase, ldh (EQU24_19220).

6. The aerobic methanophilic engineered bacteria producing succinic acid according to claim 1, characterized in that: The blocking of the acetate synthesis pathway is achieved by knocking out the gene encoding acetate kinase, ack (EQU24_07890).

7. A method for constructing an aerobic methanophilic engineering bacterium producing succinic acid according to claim 2, characterized in that: Including four ways to complete the serine pathway by constructing aerobic methanotrophic engineered strains SC01-SC04: (1.1) The exogenous gene ppc was connected to the plasmid pAWP89 to construct the recombinant plasmid pAWP89-S1, which was then transferred into the host bacteria to obtain the aerobic methanotrophic engineered strain SC01; (1.2) The exogenous gene pck was connected to the plasmid pAWP89 to construct the recombinant plasmid pAWP89-S2, which was then transferred into the host bacteria to obtain the aerobic methanotrophic engineered strain SC02; (1.3) The exogenous genes ppc and pck were connected to the plasmid pAWP89, wherein ppc and pck were connected by rbs to construct the recombinant plasmid pAWP89-S3, which was then transferred into the host bacteria to obtain the aerobic methanotrophic engineered strain SC03; (1.4) Connect the exogenous genes ppc and pck to the plasmid pAWP89, with P between ppc and pck. tac The recombinant plasmid pAWP89-S4 was constructed by ligation and then transformed into a host bacterium to obtain an aerobic methanotrophic engineered strain SC04.

8. A method for constructing an aerobic methanophilic engineering bacterium producing succinic acid according to claim 2, characterized in that: There are three pathways to block byproducts: (2.1) The ack gene on the host bacteria was replaced with Gm to block the acetate production pathway and obtain the aerobic methanotrophic engineered strain SC05; (2.1) The ldh gene on the host bacteria was replaced with Bleo to block the lactic acid production pathway and obtain the aerobic methanophilic engineered strain SC06; (2.3) Block the lactic acid and acetic acid production pathways at the same time, replace the ack gene on the host bacteria with Gm, and replace the ldh gene with Bleo, to obtain the aerobic methanotrophic engineered strain SC07.

9. A method for constructing an aerobic methanophilic engineering bacterium producing succinic acid according to claim 2, characterized in that: The serine pathway is combined with the byproduct knockout pathway, specifically: (3.1) The exogenous genes ppc and pck were connected to the plasmid pAWP89, wherein ppc and pck were connected with rbs to construct the recombinant plasmid pAWP89-S3; (3.2) Blocking the lactic acid and acetic acid production pathways at the same time, replacing the ack gene on the host bacteria with Gm, and replacing the ldh gene with Bleo, to obtain the aerobic methanotrophic engineered strain SC07; (3.3) The recombinant plasmid pAWP89-S3 was transferred into the aerobic methanotrophic engineered strain SC07 to obtain the aerobic methanotrophic engineered strain SC08.

10. Use of an aerobic methanophilic engineered bacterium for producing succinic acid prepared according to any of the above methods, wherein the aerobic methanophilic engineered bacterium is used in fermentation to produce succinic acid, specifically comprising the following steps: (1) Seed culture: After the aerobic methanotrophic engineered strain with high succinic acid production is activated on a plate, 10%-20% of the inoculum is inoculated into an injection bottle and cultured for 24-48 hours to obtain a seed solution; (2) The specific process of fermenting succinic acid in the fermenter is as follows: the seed liquid is inoculated into the NMS2 medium in the fermenter, the inoculation amount is 10% to 15% (v / v), the ventilation volume of the ventilation gas is 1.5-2.5vvm; the ventilation gas is composed of CH4 and air, the volume ratio of CH4 to air is 1:3.5-4.5, the fermentation temperature is 25-30°C, the fermentation culture time is 24-96h, and the yield of succinic acid is measured; The NMS2 culture medium comprises: MgSO4·7H2O 0.2-1g, CaCl2·6H2O 0.008-0.012g, KNO3 6-12g, NaCl 8-12g, phosphate buffer solution 40mL, carbonate buffer solution 50mL and trace element solution 8mL, and the rest is supplemented with distilled water to 1L; The phosphate buffer solution, pH 6.8, KH2PO4 2.77-5.44 g·L per 1L -1 and Na2HPO45.4-10.73g·L -1 , the balance is water; The total concentration of the carbonate buffer solution is 1M, and it is composed of 140-700 parts by volume of a NaHCO3 aqueous solution and 60-300 parts by volume of a Na2CO3 aqueous solution; The trace element solution: Na2-EDTA·2H2O 1-2g·L -1 , FeSO4·7H2O 1-2g·L -1 , ZnSO4·7H2O0.5-0.8g·L -1 , MnCl2·4H2O 0.01-0.03g·L -1 , H3BO30.01-0.03g·L -1 , CoCl2·6H2O 0.1-0.2g·L -1 , CuCl2·2H2O 0.4-0.6g·L -1 , NiCl2·6H2O 0.01-0.02g·L -1 , Na2MO4·2H2O0.02-0.05g·L -1 , the balance is distilled water.

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