Aerobic methanophilic engineering bacterium for high yield of alpha-farnesene as well as construction method and application of aerobic methanophilic engineering bacterium

Abstract

The invention discloses a high-yield alpha-farnesene aerobic methanophilic engineering bacterium as well as a construction method and application thereof, and belongs to the technical field of metabolic engineering and synthetic biology. The method comprises the following steps: expressing an alpha-farnesene synthase gene AFS by using a strong promoter PmxaF in host bacteria (aerobic methanophilic bacteria), overexpressing 1-deoxy-xylulose-5-phosphate synthase genes dxs1 and dxs2, a farnesyl pyrophosphate synthase gene ispA and an isopentenyl pyrophosphate isomerase gene Bsidi in a synthetic route of alpha-farnesene, and overexpressing 6-phosphate glucose dehydrogenase zwf1, the aerobic methanophilic engineering bacteria for producing the alpha-farnesene are obtained. When the methanophilic engineering bacterium is used for fermentation production, the yield of alpha-farnesene is obviously improved, and the methanophilic engineering bacterium has industrial application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of metabolic engineering and synthetic biology, and specifically relates to an aerobic methanophilic engineering bacterium with high alpha-farnesene production, and a construction method and application thereof. Background Art

[0002] Terpenoids have attracted widespread attention due to their applications in medicine, energy, and agriculture. α-Farnesene is a sesquiterpenoid and an important component of plant essential oils in nature. Currently, α-farnesene plays an important role in industry, agriculture, and daily life. For example, in agriculture, α-farnesene, as a pheromone, can interfere with the normal feeding behavior and physiological processes of aphids to avoid aphids invading crops; in industrial production, α-farnesene is used in lubricants, surfactants, and cosmetics due to its excellent properties; in addition, α-farnesene has been identified as an important alternative to jet fuel due to its high carbon density and storage safety; α-farnesene can also be used as a precursor of vitamin E to promote the industrial synthesis of vitamin E. With the increasing demand for α-farnesene, the extraction of α-farnesene from plants can no longer meet the demand, which encourages researchers to develop more sustainable processes to produce α-farnesene.

[0003] Currently, α-farnesene can already be synthesized in cell factories using sugar as feedstock. However, the growing global population and limited arable land pose challenges to cell factories using sugar-based feedstocks, leading to potential competition between biomanufacturing and food supply. Therefore, there is a strong push to find alternative, more sustainable fermentation substrates.

[0004] Methane is a potent greenhouse gas and has become a promising carbon source for biomanufacturing applications. Using microorganisms to convert methane to α-farnesene can not only eliminate the need for sugar-based feedstocks for microbial manufacturing, but also help reduce carbon emissions and mitigate global climate change. Methanophiles can produce a range of chemicals, such as lactic acid and isobutyraldehyde, using methane as the sole carbon source. Although the natural methylerythritol phosphate (MEP) pathway has been elucidated in type I methanotrophs, the production of α-farnesene according to this method still has the following problems: 1) Low yield. When producing α-farnesene by this method, due to the poor thermal stability of α-farnesene synthase, it is difficult to maintain activity for a long time, and it takes a long time to catalyze the synthesis of α-farnesene, resulting in an α-farnesene yield of only 91.55 mg / L; 2) Demanding production requirements. The MEP pathway for synthesizing α-farnesene involves eight different enzymes, and each synthesized α-farnesene molecule requires 9 NADPH molecules, which poses a challenge to the supply of carbon flux and reducing equivalents. Therefore, there is an urgent need to improve the carbon yield and productivity of long-chain terpenoids in methane and develop corresponding high-yield fermentation processes. Summary of the invention

[0005] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide an aerobic methanophilic engineered bacteria with high α-farnesene production and its construction method and application, so as to solve the technical problems of low yield and strict production requirements of the current α-farnesene production methods.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention discloses an aerobic methanotrophic engineered bacterium with high α-farnesene production, wherein an α-farnesene synthesis pathway is constructed by introducing an α-farnesene synthase gene AFS into a host bacterium, and an endogenous strong promoter is used to overexpress 1-deoxy-xylulose-5-phosphate synthase gene dxs1, dxs2, farnesyl pyrophosphate synthase gene ispA, and isopentenyl pyrophosphate isomerase gene Bsidi to strengthen metabolic flow, and at the same time, a 6-phosphoglucose dehydrogenase gene zwf1 is overexpressed to strengthen reducing power, thereby obtaining an aerobic methanotrophic engineered bacterium with high α-farnesene production;

[0008] Wherein, the host bacteria is an aerobic methanophilic bacterium.

[0009] Preferably, the aerobic methanotrophic bacteria is Methylotuvimicrobiumburyatense 5GB1, Alkalicoccusglycogenes WONF2802 or Methylotuvibiumsanxanigenens NG09.

[0010] Preferably, the α-farnesene synthase gene AFS is derived from Malus domestica, with NCBI number AAS68019.1, and the DNA sequence is shown as SEQ ID NO.11.

[0011] Preferably, the 1-deoxy-xylulose-5-phosphate synthase genes dxs1, dxs2, the farnesyl pyrophosphate synthase gene ispA and the 6-phosphoglucose dehydrogenase gene zwf1 are all derived from methanophilic bacteria, and their NCBI numbers are WP_017839846.1, WP_017842755.1, WP_017842756.1 and WP_017841635.1, respectively.

[0012] Preferably, the isopentenyl pyrophosphate isomerase gene Bsidi is derived from Bacillus subtilis, with NCBI number WP_004399098.1, and its DNA sequence is shown in SEQ ID NO.3.

[0013] The second aspect of the present invention discloses a method for constructing the above-mentioned aerobic methanophilic engineered bacteria with high α-farnesene production, comprising the following steps:

[0014] 1) Transfer the DNA fragment Pc-Gm-P to the ispAL site of the host bacteria pqqA -zwf1-P tac -dxs1-P mxaF -ispA-dxs2, to obtain engineered bacteria 1;

[0015] 2) Inserting DNA fragment P into the glgA1L site of the engineered bacteria 1 obtained in step 1) 12965 -Bsidi-P tac -Bleo, obtain engineered bacteria 2;

[0016] 3) Insert P into pAWP89 plasmid mxaF The promoter is linked to the α-farnesene synthase gene AFS. mxaF -AFS expression cassette, and then transferred into engineering bacteria 2 to obtain aerobic methanophilic engineering bacteria that produces α-farnesene;

[0017] Among them, Pc-Gm-P pqqA -zwf1-P tac -dxs1-P mxaF The sequence of -ispA-dxs2 is shown in SEQ ID NO.1, P 12965 -Bsidi-P tac The sequence of -Bleo is shown in SEQ ID NO.2, P mxaF The sequence of -AFS is shown in SEQ ID NO.4.

[0018] The third aspect of the present invention discloses the use of the aerobic methanotrophic engineered bacteria with high α-farnesene production in the production of α-farnesene.

[0019] The fourth aspect of the present invention discloses a method for producing α-farnesene, wherein the seed liquid of the aerobic methanotrophic engineered bacteria with high α-farnesene production is inoculated into a fermentation medium for fermentation to obtain α-farnesene.

[0020] Preferably, during the fermentation process, 10% of the volume of the fermentation medium is added with n-dodecane, and methane is introduced simultaneously.

[0021] Preferably, the fermentation temperature is 22°C to 26°C.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The invention provides an aerobic methanophilic engineered bacterium with high α-farnesene production, which is prepared by using a strong promoter P in a host bacterium (aerobic methanophilic bacteria Methylotuvimicrobium buryatense 5GB1, Alkalicoccusglycogenes WONF2802 or Methylotuvibiumsanxanigenens NG09). mxaF The α-farnesene synthase gene AFS was expressed to achieve the bioconversion of methane to α-farnesene. The ability of the central metabolite to flow to terpene compounds was enhanced by overexpressing the 1-deoxy-xylulose-5-phosphate synthase genes dxs1 and dxs2; the precursor compound farnesyl pyrophosphate was accumulated in large quantities by overexpressing the farnesyl pyrophosphate synthase gene ispA; the isopentenyl pyrophosphate isomerase gene Bsidi was overexpressed to maintain the balance between isopentenyl pyrophosphate and dimethylpropylene pyrophosphate, thereby improving the metabolic flow of the pathway where the target product is located. By overexpressing the 6-phosphoglucose dehydrogenase gene zwf1, the MEP pathway needs to consume reducing power and strengthen the supply of NADPH, so that the material flow and reducing power are synergistically enhanced, and then an aerobic methanophilic engineered bacterium that produces α-farnesene was obtained. Compared with the original methanogen strain, the aerobic methanotrophic engineered bacteria with high α-farnesene production has significantly increased the production of α-farnesene, which has opened up new raw materials for the production of α-farnesene and provided a reference for the development of a bioconversion platform for producing α-farnesene from methane. It has important industrial application value and environmental significance, and provides a new route for the efficient synthesis of terpene compounds through a variety of metabolic optimization strategies.

[0024] The present invention provides a method for producing α-farnesene, which comprises introducing n-dodecane and methane, and fermenting and culturing aerobic methanophilic engineering bacteria with high α-farnesene production under low temperature conditions of 22° C. to 26° C. The catalytic activity can be better exerted under low temperature conditions, the biosynthesis of terpenoid compounds can be promoted, and the yield of α-farnesene can be significantly increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the method for constructing an aerobic methanophilic engineered bacterium with high α-farnesene production according to the present invention;

[0026] Figure 2 The mass spectrum of α-farnesene obtained by using the methanotrophic engineered bacteria FAR02;

[0027] Figure 3 The gas chromatogram of α-farnesene obtained by using the methanotrophic engineered bacteria FAR02;

[0028] Figure 4 This is a logarithmic growth curve of the methanophilic engineering bacteria FAR02 in Example 1 of the present invention;

[0029] Figure 5 This is a curve diagram of α-farnesene production by the methanophilic engineering bacteria FAR02 in Example 1 of the present invention;

[0030] Figure 6 This is a comparison chart of the production of α-farnesene by the methanophilic engineering bacteria FAR02 in Example 1 and the methanophilic engineering bacteria FAR01 in Comparative Example 1. DETAILED DESCRIPTION

[0031] In order to enable those skilled in the art to understand the characteristics and effects of the present invention, the following is a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used in the text are the common meanings understood by those skilled in the art for the present invention. In the event of a conflict, the definition in this specification shall prevail.

[0032] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0033] Herein, all features such as values, quantities, contents and concentrations defined in the form of numerical ranges or percentage ranges are for simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions).

[0034] In this document, unless otherwise specified, “includes,” “including,” “contains,” “has,” or similar terms cover the meanings of “consisting of” and “mainly consisting of,” for example, “A includes a” covers the meanings of “A includes a and other” and “A only includes a.”

[0035] In this document, in order to make the description concise, not all possible combinations of various technical features in various embodiments or examples are described. Therefore, as long as there is no contradiction in the combination of these technical features, the various technical features in various embodiments or examples can be combined arbitrarily, and all possible combinations should be considered to be within the scope of this specification.

[0036] The invention provides an aerobic methanophilic engineered bacterium with high α-farnesene production, which is obtained by expressing an α-farnesene synthase gene AFS in a host bacterium, overexpressing 1-deoxy-xylulose-5-phosphate synthase genes dxs1 and dxs2, a farnesyl pyrophosphate synthase gene ispA, a 6-phosphoglucose dehydrogenase gene zwf1, and an isopentenyl pyrophosphate isomerase Bsidi from Bacillus subtilis. In the genetically engineered bacterium, AFS encodes α-farnesene synthase, catalyzing the conversion of farnesyl pyrophosphate FPP into α-farnesene; dxs1 and dxs2 participate in the synthesis of 1-deoxy-xylulose-5-phosphate, and encode the first rate-limiting enzyme in the MEP pathway; ispA participates in the elongation of isopentenyl pyrophosphate (C5 unit); Bsidi controls the stability of the ratio of key intermediates IPP and DMAPP; and zwf1 provides reducing power for the MEP pathway. The above-mentioned engineered bacterium is fermented and produced, and the yield of α-farnesene is significantly increased.

[0037] The host bacteria are aerobic methanophilic bacteria Methylotuvimicrobiumburyatense 5GB1, Alkalicoccusglycogenes WONF2802 or Methylotuvibiumsanxanigenens NG09, and the genomes of the host bacteria contain ispAL and glgAL sites. The Alkalicoccusglycogenes WONF2802 and Methylotuvibiumsanxanigenens NG09 used in the present invention have been deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee, and the deposited bacteria numbers are CGMCC No. 28614 and CGMCC No. 30951, respectively.

[0038] The present invention provides a method for constructing the above-mentioned aerobic methanophilic engineering bacteria with high α-farnesene production, such as Figure 1 As shown, the following steps are included:

[0039] (1) Insert the DNA fragment Pc-Gm-P into the ispAL site of the host bacteria by electroporation pqqA -zwf1-P tac -dxs1-P mxaF -ispA-dxs2 (sequence as shown in SEQ ID NO.1), then cultured in liquid NMS medium at 22°C to 26°C, centrifuged and discarded the supernatant; the bacterial precipitate was spread on a solid NMS medium plate containing gentamicin and cultured at 22°C to 26°C for 4 to 7 days to obtain engineered bacteria 1;

[0040] The liquid NMS culture medium includes: (0.2-1) g MgSO 4 7H 2O, (0.008~0.012)gCaCl 2 6H 2 O, (0.8~1.2)g KNO 3 , (8-12) g NaCl, 20 mL phosphate buffer solution, 50 mL carbonate buffer solution, and the rest is supplemented with distilled water to 1 L; the pH value of the phosphate buffer solution is 6.8, and the phosphate buffer solution includes (2.77-5.44) g / L KH 2 PO 4 and (5.4~10.73) g / L Na 2 HPO 4 , the balance is water; the total concentration of the carbonate buffer solution is 1M, and the carbonate buffer solution is composed of (140 to 700) parts by volume of NaHCO 3 Aqueous solution and (60-300) parts by volume of Na 2 CO 3 Aqueous solution composition; solid NMS medium is obtained by adding 15g agar to the liquid NMS medium.

[0041] (2) The DNA fragment P inserted into the glgA1L site of the engineered bacteria 1 obtained in step (1) by electroporation 12965 -Bsidi-P tac -Bleo (sequence as shown in SEQ ID NO.2), then cultured in liquid NMS medium at 22°C to 26°C, centrifuged and discarded the supernatant; the bacterial precipitate was spread on a solid NMS medium plate containing bleomycin and cultured at 22°C to 26°C for 4 to 7 days to obtain engineered bacteria 2;

[0042] The Bsidi is derived from Bacillus subtilis, and its NCBI number is WP_004399098.1. Its DNA sequence is shown in SEQ ID NO.3.

[0043] (3) Insert P into pAWP89 plasmid mxaF The promoter is linked to the α-farnesene synthase gene AFS. mxaF -AFS (sequence as shown in SEQ ID NO.4) expression cassette, and then transferred into the engineered bacteria 2 obtained in step (2) by conjugation transfer to obtain an aerobic methanophilic engineered bacteria with high α-farnesene production.

[0044] The present invention provides a method for producing α-farnesene by using the aerobic methanophilic engineered bacteria with high α-farnesene production, comprising the following steps:

[0045] (1) Activating the engineered bacteria: The aerobic methanotrophic engineered bacteria constructed by the above method are inoculated onto a solid NMS medium and cultured at 22° C. to 26° C. for 4 to 5 days to obtain activated test colonies;

[0046] (2) Seed culture: The test bacterial colony activated in step (1) was inoculated into an injection bottle containing 50 mL of liquid NMS medium, and cultured at 22°C to 26°C and 200 rpm for 48 h, during which the upper air was renewed and 60 mL of methane was supplemented every 24 h to obtain a seed culture solution;

[0047] (3) The seed culture liquid obtained in step (2) was inoculated into a fermentation medium with a pH value of 9.0 and continued to ferment at 22° C. to 26° C., with the dissolved oxygen controlled above 20, while adding 10% of the volume of the fermentation medium of n-dodecane and introducing methane, and fermenting for 132 hours.

[0048] Among them, according to 1L, the fermentation medium includes: (0.2-1)g MgSO 4 7H 2 O, (0.008~0.012)gCaCl 2 6H 2 O, (7.0~9.0)g KNO 3 , (8-12) g NaCl, 20 mL phosphate buffer solution, 50 mL carbonate buffer solution, and the rest is made up to 1 L with distilled water.

[0049] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0050] The following examples use conventional instruments and equipment in the art. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The various raw materials used in the following examples, unless otherwise specified, are conventional commercial products, and their specifications are conventional specifications in the art.

[0051] Example 1

[0052] 1. Construction of Methanophilic Engineering Bacteria FAR02

[0053] 1. Using Methylotuvibiumsanxanigenens NG09 as the test bacteria, construct the methanophilic engineered bacteria IZDA with enhanced terpene synthesis ability

[0054] (1) Use 2×Phanta Max Master Mix high-fidelity DNA polymerase to generate DNA fragment P c -Gm-P pqqA -zwf1-P tac -dxs1-P mxaF -ispA-dxs2 (sequence as shown in SEQ ID NO.1) was used as a template, and PCR amplification was performed under the guidance of upstream primer ispAL-F (sequence as shown in SEQ ID NO.5) and downstream primer ispAR-R (sequence as shown in SEQ ID NO.6). The denaturation, annealing, and extension time and temperature in the amplification program were all based on the instructions of 2×Phanta Max Master Mix high-fidelity DNA polymerase. The annealing temperature was set to 57°C and the extension time was 390 seconds. P that can be electrically converted was obtained. c -Gm-P pqqA -zwf1-P tac -dxs1-P mxaF -ispA-dxs2 fragment;

[0055] Among them, dxs1, dxs2, ispA and zwf1 were all derived from the test bacteria MethylotuvibiumsanxanigenensNG09, and the corresponding NCBI numbers were WP_017839846.1, WP_017842755.1, WP_017842756.1, and WP_017841635.1, respectively;

[0056] Using 2×Phanta Max Master Mix high-fidelity DNA polymerase, the DNA fragment P 12965 -Bsidi-P tac -Bleo (sequence as shown in SEQ ID NO.2) was used as a template, and PCR amplification was performed under the guidance of upstream primer glgA1L-F (sequence as shown in SEQ ID NO.7) and downstream primer glgA1R-R (sequence as shown in SEQ ID NO.8). The denaturation, annealing, and extension time and temperature in the amplification program were all based on the instructions of 2×Phanta Max Master Mix high-fidelity DNA polymerase. The annealing temperature was set to 57°C and the extension time was 210 seconds. P 12965 -Bsidi-P tac -Bleo fragment.

[0057] (2) The test bacteria Methylotuvibiumsanxanigenens NG09 was inoculated into liquid NMS medium and cultured until the logarithmic growth phase. 6002; centrifuge the bacterial solution at 4°C and 5000×g for 10 min, discard the supernatant; resuspend the bacteria with 50mL of cold water, centrifuge at 4°C and 5000×g for 10 min, discard the supernatant; resuspend the bacteria with 1mL of distilled water to obtain competent cells;

[0058] The formula of liquid NMS medium is: MgSO 4 7H 2 O 0.2g, CaCl 2 ·2H 2 O 0.01g, KNO 3 1.0g, NaCl 8g, phosphate buffer solution 20mL, carbonate buffer solution 50mL, add distilled water to make up to 1L. Phosphate buffer solution (pH = 6.8): KH 2 PO 4 5.44 g / L and Na 2 HPO 4 5.68 g / L, the balance is water. Carbonate buffer solution (total concentration is 1 M): NaHCO 3 75.6g / L and Na 2 CO 3 10.5g / L, the balance is distilled water.

[0059] (3) Take 50 μL of the competent cells obtained in step (2) and add 500 ng of P obtained in step (1). c -Gm-P pqqA -zwf1-P tac -dxs1-P mxaF -ispA-dxs2 fragment and gently mix to insert fragment P into the ispAL site of host bacteria Methylotuvibiumsanxanigenens NG09 c -Gm-P pqqA -zwf1-P tac -dxs1-P mxaF -ispA-dxs2. Then, the mixture was transferred to a low-temperature electric shock cup with a gap of 1 mm, and electroporated using an electroporator (conditions were set to 1.5 kV, 25 μF and 200 Ω); then, 10 mL of liquid NMS medium was used for resuscitation culture at 26°C for 24 h; centrifuged at room temperature and 5000 × g for 10 min, the supernatant was discarded, and the bacterial precipitate was spread on a solid NMS medium plate containing 50 μg / mL gentamicin, and cultured at 26°C for 4 days to screen the recombinants. The correct recombinants were recorded as engineered bacteria 1.

[0060] (4) The host bacteria Methylotuvibiumsanxanigenens NG09 was replaced by the engineered bacteria 1, and the DNA fragment was replaced by the P obtained in step (1).12965 -Bsidi-P tac -Bleo fragment, replace the antibiotic in the solid NMS medium plate with 30 μg / mL bleomycin, repeat steps (2) and (3), and insert P into the glgA1L site of engineered bacteria 1. 12965 -Bsidi-P tac -Bleo fragments;

[0061] (5) After the recombinant is identified by PCR, if there is no base mutation in the sequencing, the methanophilic engineered bacterium IZDA (i.e., engineered bacterium 2) is obtained.

[0062] (6) The methanotrophic engineered bacteria IZDA obtained in step (5) was coated on a solid NMS medium containing 50 μg / mL gentamicin and 30 μg / mL bleomycin and cultured for 3 days to complete the expansion culture, and the methanotrophic engineered bacteria IZDA on the solid NMS medium was transferred to a liquid NMS medium containing 50 μg / mL gentamicin and 30 μg / mL bleomycin, wherein the liquid volume was 50 mL. The culture was carried out at 26° C. and 200 rpm until the logarithmic growth phase, and the strain was preserved.

[0063] 2. Using the methanotrophic engineering bacteria IZDA as the test bacteria, construct the methanotrophic engineering bacteria FAR02 that produces α-farnesene

[0064] (1) P mxaF -AFS (sequence as shown in SEQ ID NO.4) as a template, PCR amplification was performed under the guidance of upstream primer PmxaF-F (sequence as shown in SEQ ID NO.9) and downstream primer AFS-R (sequence as shown in SEQ ID NO.10) to obtain P mxaF -AFS destination fragment;

[0065] The sequence of the α-farnesene synthase gene AFS is shown in SEQ ID NO.11, which is derived from Malus domestica and has a NCBI number of AAS68019.1.

[0066] (2) Primers P89-F (sequence shown in SEQ ID NO.12) and P89-R (sequence shown in SEQ ID NO.13) were designed based on the pAWP89 sequence, and PCR amplification was performed. The pAWP89 linearized vector was obtained after restriction endonuclease DpnI was used for digestion at 37°C for 2 h.

[0067] (3) The P obtained in step (1) was cloned using the Novozyme One-Step Cloning Kit (Novozyme, C115-01). mxaF-AFS target fragment and the pAWP89 linearized vector obtained in step (2) were recombined. The recombination system contained an equimolar ratio of linearized vector and gene fragments, and the multi-fragment recombination conditions were 50°C for 45 min. After the recombination reaction was completed, CaCl 2 The product was transferred to E. coli S17 by transformation method. The correct band size was identified by PCR and no mutation was found by sequencing, indicating that the recombinant plasmid was successfully constructed.

[0068] (4) The E. coli S17 containing the recombinant plasmid obtained in step (3) was mixed with the methanotrophic engineered bacteria IZDA obtained after activation on the plate in equal proportions, and after culturing on a solid NMS medium containing 15% LB medium for 48 h, the mixed bacteria were spread on a solid NMS medium containing 50 mg / L of LB medium, and the recombinants were screened to obtain the methanotrophic engineered bacteria FAR02.

[0069] 2. Production of α-farnesene using the methanotrophic engineered bacteria FAR02

[0070] (1) Activating engineered bacteria: inoculate the preserved methanotrophic engineered bacteria FAR02 onto solid NMS medium and culture at 26°C for 4-5 days to obtain activated test colonies;

[0071] (2) Seed culture: The test bacterial colony activated in step (1) was inoculated into an injection bottle containing 50 mL of liquid NMS medium, and cultured at 26° C. and 200 rpm for 48 h, during which the upper air was renewed and 60 mL of methane was supplemented every 24 h to obtain a seed culture solution;

[0072] (3) The seed culture liquid obtained in step (2) was inoculated into a fermentation medium with a pH value of 9.0 and continued to ferment at 26° C., with the dissolved oxygen controlled at above 20, while adding 10% of the total volume of the fermentation medium of n-dodecane and introducing methane, and the fermentation was cultured for 132 hours.

[0073] Among them, according to 1L, the fermentation medium includes: 1g MgSO 4 7H 2 O, 0.01 g CaCl 2 6H 2 O, 8g KNO 3 , 8g NaCl, 20mL phosphate buffer solution, 50mL carbonate buffer solution, add distilled water to 1L; the phosphate buffer solution (pH = 6.8): KH 2 PO 4 5.44 g / L and Na 2 HPO 4 5.68 g / L, the balance is water. Carbonate buffer solution (total concentration is 1 M): NaHCO3 75.6g / L and Na 2 CO 3 10.5g / L, the balance is distilled water.

[0074] (4) Take 1 mL of the cultured bacterial liquid, centrifuge it at 4°C and 10,000 rpm for 5 min, take the upper organic phase, and use a gas chromatography-mass spectrometer (instrument model: Shimadzu GCMS-QP2020NX; chromatographic column: SH-Rxi-5Sil MS) to conduct qualitative analysis of α-farnesene. The carrier gas flow rate is 2 mL / min, and the oven temperature is set to 80°C for 1 min, then increased to 250°C at a rate of 10°C / min and maintained for 1 min. The MS source, MS quadrupole, connection and inlet are maintained at 230, 150, 280 and 250°C, respectively. The mass spectrum is shown in Figure 2. Figure 2 As shown, α-farnesene began to accumulate in large quantities at 36 h, and the accumulation basically stopped at 132 h. The final α-farnesene production reached 369.16 mg / L.

[0075] (5) Take 1 mL of the cultured bacterial liquid, centrifuge at 4°C and 10,000 rpm for 5 min, take the upper organic phase, and use gas chromatography (gas chromatograph: Agilent 7820A; chromatographic column: HP-5,) to quantitatively analyze α-farnesene, with a carrier gas flow rate of 2 mL / min. The oven temperature was set to 80°C for 1 min, then increased to 250°C at a rate of 10°C / min and maintained for 1 min. The gas chromatogram is shown in the figure below. Figure 3 shown.

[0076] The growth of FAR02 strain was as follows Figure 4 As shown in Figure 1, the strain grew vigorously from 0 to 48 hours, and basically stopped growing after 48 hours. The curve results of the FAR02 strain producing α-farnesene are shown in Figure 1. Figure 5 As shown, compared with NG09, FAR02 strain was able to successfully produce α-farnesene.

[0077] Comparative Example 1

[0078] 1. Construction of Methanophilic Engineering Bacteria FAR01

[0079] (1) Using AFS (sequence as shown in SEQ ID NO.11) as a template, PCR amplification was performed under the guidance of upstream primer AFS-F (sequence as shown in SEQ ID NO.14) and downstream primer AFS-R (sequence as shown in SEQ ID NO.10) to obtain the AFS target fragment.

[0080] (2) Using pAWP89 as a template, upstream primer P89-F (sequence shown in SEQ ID NO. 12) and downstream primer P tac-R (sequence as shown in SEQ ID NO.15) was used for PCR amplification, and then restriction endonuclease DpnI was used for digestion at 37°C for 2h to obtain the pAWP89 linearized vector.

[0081] (3) The AFS target fragment and the pAWP89 linearized vector obtained in step (2) were recombined using the Novagen One-Step Cloning Kit. The recombinant system contained an equimolar ratio of the AFS target fragment and the pAWP89 linearized vector, and the multi-fragment recombination conditions were 50°C for 45 min. After the recombination reaction, CaCl 2 The product was transferred to E. coli S17 by transformation method. The correct band size was identified by PCR and no mutation was found by sequencing, indicating that the recombinant plasmid was successfully constructed.

[0082] (4) The recombinant plasmid was transferred into the test bacterium Methylotuvibium sanxanigenens NG09 by biparental conjugation to obtain the methanotrophic engineered bacterium FAR01.

[0083] 2. Production of α-farnesene using the methanotrophic engineered bacteria FAR01

[0084] (1) Activating engineered bacteria: The preserved methanotrophic engineered bacteria FAR01 was inoculated onto a solid NMS medium (the formulation is the same as in Example 1), and cultured at 26° C. for 4 to 5 days to obtain activated test colonies;

[0085] (2) Take the engineered bacteria on the solid NMS medium and inoculate one loop into a 300 mL shake flask filled with liquid NMS medium (the formula is the same as in Example 1), where the liquid volume is 50 mL. Cultivate at 26°C and 200 rpm for 48 hours to obtain seed liquid. The OD of the seed liquid is 600 The culture system adopts a closed gas-liquid two-phase system, and methane is added to the culture system at the beginning and during the culture process, and the amount of methane added is 20% of the gas phase volume of the culture system.

[0086] (3) Take 5 mL of the seed solution in step (2) and inoculate it into a 300 mL shake flask containing liquid NMS medium. The liquid volume after inoculation is 50 mL, and culture is carried out at 26° C. and 200 rpm. 5 mL of n-dodecane is added 48 hours after inoculation, and culture is continued at 26° C. and 200 rpm for 72 hours.

[0087] (4) Take 1 mL of the cultured bacterial liquid, centrifuge at 4°C and 10,000 rpm for 5 min, take the upper organic phase, and use a gas chromatography-mass spectrometer (instrument model: Shimadzu GCMS-QP2020NX; chromatographic column: SH-Rxi-5Sil MS) to qualitatively analyze α-farnesene. The carrier gas flow rate is 2 mL / min, and the oven temperature is set to 80°C for 1 min, then increased to 250°C at a rate of 10°C / min and maintained for 1 min. The MS source, MS quadrupole, connection and inlet are maintained at 230, 150, 280 and 250°C, respectively.

[0088] (5) Take 1 mL of the cultured bacterial liquid, centrifuge at 4°C and 10,000 rpm for 5 min, take the upper organic phase, and use gas chromatography (gas chromatograph: Agilent 7820A; chromatographic column: HP-5) to quantitatively analyze α-farnesene. The carrier gas flow rate is 2 mL / min, and the oven temperature is set to 80°C for 1 min, then increased to 250°C at a rate of 10°C / min and maintained for 1 min. Quantitative analysis shows that the yield of α-farnesene is 6.23 mg / L.

[0089] Comparative Example 1 and Comparative Example 1, the results are as follows Figure 6 As shown, after the methanotrophic bacteria were metabolically engineered to obtain methanotrophic bacteria, they were applied to produce α-farnesene, and the α-farnesene yield increased to 59 times that of the initial strain, reaching 369.16 mg / L. This result shows that the modified methanotrophic engineered bacteria FAR02 can greatly increase the yield of α-farnesene synthesized using methane as a carbon source.

[0090] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. An aerobic methanophilic engineered bacterium with high α-farnesene production, characterized in that: The α-farnesene synthesis pathway was constructed by introducing the α-farnesene synthase gene AFS into the host bacteria, and the 1-deoxy-xylulose-5-phosphate synthase genes dxs1 and dxs2, the farnesyl pyrophosphate synthase gene ispA, and the isopentenyl pyrophosphate isomerase gene Bsidi were overexpressed using an endogenous strong promoter to strengthen the metabolic flow, and the 6-phosphoglucose dehydrogenase gene zwf1 was overexpressed to strengthen the reducing power, thereby obtaining an aerobic methanotrophic engineered bacterium with high α-farnesene production; Wherein, the host bacteria is an aerobic methanophilic bacterium.

2. The aerobic methanophilic engineered bacteria for high production of α-farnesene according to claim 1, characterized in that: The aerobic methanophilic bacteria are Methylotuvimicrobiumburyatense 5GB1, AlkalicoccusglycogenesWONF2802 or Methylotuvibiumsanxanigenens NG09.

3. The aerobic methanophilic engineered bacteria for high production of α-farnesene according to claim 1, characterized in that: The α-farnesene synthase gene AFS is derived from Malus domestica, with NCBI number AAS68019.1 and DNA sequence as shown in SEQ ID NO.

11.

4. The aerobic methanophilic engineered bacteria for high production of α-farnesene according to claim 1, characterized in that: The 1-deoxy-xylulose-5-phosphate synthase genes dxs1, dxs2, the farnesyl pyrophosphate synthase gene ispA and the 6-phosphoglucose dehydrogenase gene zwf1 are all derived from methanophilic bacteria, and their NCBI numbers are WP_017839846.1, WP_017842755.1, WP_017842756.1 and WP_017841635.1, respectively.

5. The aerobic methanophilic engineered bacteria with high α-farnesene production according to claim 1, characterized in that: The isopentenyl pyrophosphate isomerase gene Bsidi is derived from Bacillus subtilis, the NCBI number is WP_004399098.1, and its DNA sequence is shown in SEQ ID NO.

3.

6. The method for constructing an aerobic methanophilic engineered bacterium with high α-farnesene production according to any one of claims 1 to 5, characterized in that: The steps include: 1) Transfer the DNA fragment Pc-Gm-P to the ispAL site of the host bacteria pqqA -zwf1-P tac -dxs1-P mxaF -ispA-dxs2, to obtain engineered bacteria 1; 2) Inserting DNA fragment P into the glgA1L site of the engineered bacteria 1 obtained in step 1) 12965 -Bsidi-P tac -Bleo, obtain engineered bacteria 2; 3) Insert P into pAWP89 plasmid mxaF The promoter is linked to the α-farnesene synthase gene AFS. mxaF -AFS expression cassette, and then transferred into engineering bacteria 2 to obtain aerobic methanophilic engineering bacteria that produces α-farnesene; Among them, Pc-Gm-P pqqA -zwf1-P tac -dxs1-P mxaF The sequence of -ispA-dxs2 is shown in SEQ ID NO.1, P 12965 -Bsidi-P tac The sequence of -Bleo is shown in SEQ ID NO.2, P mxaF The sequence of -AFS is shown in SEQ ID NO.

4.

7. Use of the aerobic methanophilic engineered bacteria with high α-farnesene production according to any one of claims 1 to 5 in producing α-farnesene.

8. A method for producing α-farnesene, characterized in that: The seed liquid of the aerobic methanophilic engineered bacteria with high α-farnesene production according to any one of claims 1 to 5 is inoculated into a fermentation medium for fermentation to obtain α-farnesene.

9. A method for producing α-farnesene according to claim 8, characterized in that: During the fermentation process, 10% of the volume of the fermentation medium, n-dodecane, was added and methane was introduced.

10. The method for producing α-farnesene according to claim 8, characterized in that: The fermentation temperature is 22℃~26℃.

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