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

Abstract

The invention relates to an aerobic methanophilic engineering bacterium for producing taxadiene as well as a construction method and application of the aerobic methanophilic engineering bacterium. A strong promoter P16200 is used for expressing a taxadiene synthase gene TXS in host bacteria; 1-deoxy-xylulose-5-phosphate synthase genes dxs1 and dxs2, geranyl pyrophosphate synthase gene ispA and isopentenyl pyrophosphate isomerase gene Bsidi from bacillus subtilis in a taxadiene synthesis route are overexpressed, the metabolic flux of the route in which a target product is located is improved, meanwhile, 6-glucose phosphate dehydrogenase zwf1 is overexpressed, supply of NADPH (nicotinamide adenine dinucleotide phosphate) is enhanced, and the yield of the target product is increased. Therefore, the material flow and the reducing power are synergistically enhanced; bioconversion from methane to taxadiene is achieved for the first time, the yield of the taxadiene of the constructed engineering methanophilic bacteria is obviously increased, the engineering methanophilic bacteria have industrial application prospects, and a new way is provided for efficient synthesis of terpenoids.

Description

Technical Field

[0001] The invention belongs to the technical field of metabolic engineering and synthetic biology, and specifically relates to an aerobic methanophilic engineered bacterium producing taxadiene, and a construction method and application thereof. Background Art

[0002] Terpenoids have attracted attention in clinical practice due to their pharmacological activity in treating a variety of diseases. Paclitaxel is a well-known terpenoid chemotherapy drug extracted from the bark of the yew tree. However, the plant extraction process is inefficient and can also lead to the destruction of vegetation. By 2025, the global market size of paclitaxel is expected to expand at an annual growth rate of 8.2%. There is an urgent need to find more sustainable methods to obtain this high-value compound. The metabolic engineering strategy that has emerged in recent years has enabled microorganisms to have the potential to synthesize paclitaxel, which may increase the supply of paclitaxel. At present, microbial cell factories have achieved the synthesis of a variety of paclitaxel precursor compounds. Among them, taxadiene is a key intermediate compound for the production of paclitaxel. The biosynthesis of paclitaxel must be processed and modified on the skeleton of taxadiene. At present, taxadiene can be synthesized in cell factories using sugar as raw materials.

[0003] 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. Methane, a potent greenhouse gas, has emerged as a promising carbon source for biomanufacturing applications. Using microorganisms to convert methane to taxadiene would not only eliminate the need for sugar-based feedstocks for microbial manufacturing, but also help reduce carbon emissions and mitigate global climate change.

[0004] Methanophiles are able to 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, there is still a need to improve the carbon yield and productivity of long-chain terpenoids in methane. The MEP pathway for the synthesis of taxadiene involves eight different enzymes, and each taxadiene molecule synthesized requires 12 NADPH molecules, such as the scheme disclosed in "The Methylerythritol Phosphate Pathway to Isoprenoids" from "Chemical Reviews", which poses challenges to the supply of carbon flux and reducing equivalents. In addition, taxadiene synthase has poor thermal stability and can better exert catalytic activity at low temperatures. Therefore, in order to promote the biosynthesis of terpenoids, the culture conditions also need to be optimized.

[0005] At present, taxadiene has been synthesized in microorganisms such as Saccharomyces cerevisiae and Escherichia coli, for example, the synthesis method disclosed in "Metabolic engineering of taxadiene biosynthesis in yeast as a first step towards Taxol (Paclitaxel) production" from "Metabolic Engineering". However, most of these model microorganisms use expensive glucose as raw material, which will also cause the problem of microorganisms competing with humans for food. Therefore, how to use aerobic methanophiles to synthesize taxadiene to solve the above problems and avoid the dependence of microorganisms on glucose is very worthy of study. 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 taxadiene, and a construction method and application thereof, to improve the expression level of taxadiene synthase in aerobic methanophilic bacteria, to strengthen the material flow and energy flow of the engineered bacteria participating in product synthesis, and to increase the yield of taxadiene; the present invention improves the production efficiency of taxadiene, opens up new raw materials for the production of taxadiene, and has important industrial application value and environmental significance.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] An aerobic methanophilic engineered bacterium producing taxadiene, wherein a taxadiene synthase gene TXS is expressed in a host bacterium, and 1-deoxy-xylulose-5-phosphate synthase genes dxs1 and dxs2, a geranyl pyrophosphate synthase gene ispA, a 6-phosphoglucose dehydrogenase gene zwf1, and an isopentenyl pyrophosphate isomerase Bsidi are overexpressed;

[0009] The host bacteria include aerobic methanophilic bacteria Methylotuvimicrobium buryatense5GB1, Alkalicoccus glycogenes WONF2802 or Methylotuvibium sanxanigenens NG09. The Alkalicoccusglycogenes WONF2802 and Methylotuvibium sanxanigenens NG09 have been deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration, and their deposit numbers are CGMCC No.28614 and CGMCC No.30951, respectively.

[0010] The taxadiene synthase gene TXS is derived from Taxus brevifolia, its NCBI number is Q41594.1, and its DNA sequence is shown in SEQ ID NO:1.

[0011] The dxs1, dxs2, ispA and 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] The Bsidi is derived from Bacillus subtilis, its NCBI number is WP_004399098.1, and its DNA sequence is shown in SEQ ID NO:2.

[0013] The method for constructing the above-mentioned aerobic methanophilic engineered bacteria producing taxadiene comprises the following steps:

[0014] (1) Insert P into pAWP89 plasmid 16200 The promoter is connected to the taxadiene synthase gene TXS. 16200 -TXS expression cassette, the plasmid is transferred into the host bacteria by conjugation to obtain the engineered bacteria;

[0015] (2) Inserting the DNA fragment Pc-Gm-P into the ispAL site of the engineered bacteria obtained in step (1) by electroporation pqqA -zwf1-P tac -dxs1-P mxaF -ispA-dxs2;

[0016] (3) Inserting the DNA fragment P into the glgA1L site of the engineered bacteria obtained in step (2) by electroporation 12965 -Bsidi-P tac -Bleo.

[0017] The ispAL site is inserted into -Gm-P pqqA -zwf1-P tac -dxs1-P mxaF -ispA-dxs2, the sequence of which is shown in SEQ ID NO:3.

[0018] The insertion of P at the glgA1L site 12965 -Bsidi-P tac -Bleo, the sequence of which is shown in SEQ ID NO:4.

[0019] The pAWP89 plasmid was inserted into 16200 The promoter is connected to the taxadiene synthase gene TXS.16200 -TXS expression cassette, DNA fragment P 16200 The sequence of -TXS is shown in SEQ ID NO:5.

[0020] The application of the aerobic methanophilic engineered bacteria for producing taxadiene is to apply the aerobic methanophilic engineered bacteria to produce taxadiene by fermentation.

[0021] The aerobic methanophilic engineered bacteria are used in fermentation to produce taxadiene, specifically comprising the following steps:

[0022] (1) Seed culture: Genetically engineered bacteria producing taxadiene are inoculated into a culture medium, the upper air is renewed every 24 hours, and methane equivalent to the volume of the culture medium is supplemented. The culture is carried out for 48 hours or more to obtain a seed culture solution.

[0023] The culture medium comprises: MgSO 4 7H 2 O 0.2-1g, CaCl 2 6H 2 O 0.008-0.012g, KNO 3 0.8-1.2g, NaCl 6-10g, make up to 1L with distilled water.

[0024] (2) Fermentation production of taxadiene: The seed liquid is inoculated into a fermentation medium, and n-dodecane accounting for 8%-15% of the total volume of the fermentation medium is added to the medium. The ventilation rate per liter of the medium is (700-900 mL of air + 150-250 mL of methane) / min. The maximum solubility of oxygen is controlled within 20% by adjusting the stirring speed, the pH is controlled at 8.5-9.5, and the fermentation is carried out at 22°C-30°C for 120-144 hours.

[0025] The fermentation medium includes: MgSO 4 7H 2 O 0.2-1g, CaCl 2 6H 2 O0.008-0.012g, KNO 3 6-10 g, NaCl 6-10 g, make up to 1 L with distilled water.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] 1. The present invention provides a methanophilic engineered bacterium for producing taxadiene by using a strong promoter P 16200The expression of TXS realizes the biotransformation of methane to taxadiene, increases the expression of taxadiene synthase, and thus promotes the conversion of GGPP to taxadiene; overexpression of 1-deoxy-xylulose-5-phosphate synthase genes dxs1 and dxs2 promotes the condensation of pyruvate and 3-phosphoglyceraldehyde, thereby enhancing the ability of central metabolites to flow to the terpene synthesis pathway; overexpression of geranyl pyrophosphate synthase gene ispA promotes further extension of isoprene units and causes a large accumulation of geranyl pyrophosphate, a precursor of diterpenoid compounds; overexpression of isopentenyl pyrophosphate isomerase gene Bsidi maintains the balance between isopentenyl pyrophosphate and dimethylpropylene pyrophosphate, providing sufficient precursor compounds for the synthesis of taxadiene; overexpression of 6-phosphoglucose dehydrogenase gene zwf1 increases the proportion of intracellular NADPH to supplement the reducing power consumed by the MEP pathway, further increasing the yield of taxadiene.

[0028] 2. The present invention provides a method for constructing a taxadiene-producing methanogenic engineering bacterium and its application, wherein a strong promoter P 16200 The use of dxs1 solved the problem of insufficient expression of taxadiene synthase; the overexpression of dxs1, dxs2, ispA and Bsidi in the MEP pathway solved the problem of insufficient carbon flow supply; the overexpression of zwf1 in the pentose phosphate pathway solved the problem of imbalance of reducing power in cells and increased the production of taxadiene. Compared with the original methanophilic strain, the taxadiene production in the methanophilic engineered strain was significantly increased, providing a reference for the development of a bioconversion platform for producing taxadiene from methane.

[0029] In summary, the expression level of taxadiene synthase is increased in aerobic methanophilic bacteria of the present invention, the material flow and energy flow of the engineered bacteria participating in product synthesis are strengthened, the yield and production efficiency of taxadiene are increased, new raw materials are developed for the production of taxadiene, and the present invention has important industrial application value and environmental significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the logarithmic growth curve of the engineered bacteria in Example 3 of the present invention.

[0031] Figure 2 This is the production curve of taxadiene in Example 3 of the present invention. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. The embodiments are intended to enable those skilled in the art to better understand the technical solutions of the present invention, but are not intended to limit the scope of protection of the present invention.

[0033] An aerobic methanophilic engineered bacterium producing taxadiene, wherein a taxadiene synthase gene TXS is expressed in a host bacterium, and 1-deoxy-xylulose-5-phosphate synthase genes dxs1 and dxs2, a geranyl pyrophosphate synthase gene ispA, a 6-phosphoglucose dehydrogenase gene zwf1, and an isopentenyl pyrophosphate isomerase Bsidi are overexpressed;

[0034] Wherein, the host bacteria include aerobic methanophilic bacteria Methylotuvimicrobium buryatense5GB1, Alkalicoccus glycogenes WONF2802 or Methylotuvibium sanxanigenens NG09; Alkalicoccus glycogenes WONF2802 and Methylotuvibium sanxanigenensNG09 used in the present invention have been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, and their deposited bacteria numbers are CGMCC No.28614 and CGMCC No.30951 respectively.

[0035] The method for constructing an aerobic methanophilic engineered bacterium producing taxadiene comprises the following steps:

[0036] (1) Insert P into pAWP89 plasmid 16200 The promoter is connected to the taxadiene synthase gene TXS. 16200 -TXS expression cassette, and the plasmid is transferred into the host bacteria by conjugation transfer, as shown in Example 1. Example 1 takes the taxadiene-producing bacteria TD01 as an example.

[0037] (2) Inserting the DNA fragment Pc-Gm-P into the ispAL site of the engineered bacteria obtained in step (1) by electroporation pqqA -zwf1-P tac -dxs1-P mxaF -ispA-dxs2.

[0038] (3) Inserting the DNA fragment P into the glgA1L site of the engineered bacteria obtained in step (2) by electroporation 12965 -Bsidi-P tac -Bleo, see Example 2 for details. Example 2 takes the construction of the methanophilic engineering bacteria TD02 as an example to introduce steps (2) and (3) in detail.

[0039] The taxadiene synthase gene TXS is derived from Taxus brevifolia, its NCBI number is Q41594.1, and its DNA sequence is shown in SEQ ID NO:1.

[0040] Among them, the dxs1, dxs2, ispA and 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.

[0041] The Bsidi is derived from Bacillus subtilis, and its NCBI number is WP_004399098.1. Its DNA sequence is shown in SEQ ID NO:2.

[0042] The ispAL site is inserted into -Gm-P pqqA -zwf1-P tac -dxs1-P mxaF -ispA-dxs2, the sequence of which is shown in SEQ ID NO:3.

[0043] The insertion of P at the glgA1L site 12965 -Bsidi-P tac -Bleo, the sequence of which is shown in SEQ ID NO:4.

[0044] The pAWP89 plasmid was inserted into 16200 The promoter is connected to the taxadiene synthase gene TXS. 16200 -TXS expression cassette, DNA fragment P 16200 The sequence of -TXS is shown in SEQ ID NO:5.

[0045] Example 1: Construction of Taxadiene-producing strain TD01 and detection of Taxadiene

[0046] Test bacteria: Alkalicoccusglycogenes WONF2802.

[0047] Liquid medium: MgSO 4 7H 2 O 0.2g, CaCl 2 ·2H 2 O 0.02g, KNO 3 0.8-1.2g, NaCl 6-10g, make up to 1L with distilled water.

[0048] Solid medium: MgSO 4 7H 2 O 0.2g, CaCl 2 ·2H 2 O 0.02g, KNO 30.8-1.2g, NaCl 6-10g, 15g agar powder, make up to 1L with distilled water.

[0049] (1) According to P 16200 -TXS DNA sequence to design a pair of primers, amplify the DNA fragment by PCR method, and design a pair of primers as follows:

[0050] P 16200 -F:5'-CCGCCTTTGAGTGAGCTGcaaaaaaaattacatagcttgctatgccc-3'

[0051] TXS-R:5'-CACGCATCTTCCCGACAAttagacttggatcggatcgatatagac-3'

[0052] (2) Design a pair of primers based on the pAWP89 sequence, obtain the pAWP89 linearized vector by PCR, and design a pair of primers as follows:

[0053] P89-F:5'-TTGTCGGGAAGATGCGTG-3'

[0054] P89-R:5'-CAGCTCACTCAAAGGCGG-3'

[0055] (3) The pAWP89 linearized vector obtained in step (2) was digested with restriction endonuclease DpnI at 37°C for 2 h, and the resulting digestion product was the pAWP89 linearized vector;

[0056] (4) The DNA fragments obtained in step (1) and step (3) and the pAWP89 linearized vector were recombined using the Novozymes One-Step Cloning Kit. The recombination system contained an equimolar ratio of the linearized vector and the 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.

[0057] (5) The recombinant plasmid was transferred into the test bacteria through biparental conjugation to obtain the methanophilic engineered bacteria TD01.

[0058] Example 2: Construction of engineered methanophilic bacteria TD02 with enhanced terpene synthesis ability

[0059] Test bacteria: the methanophilic engineered bacteria TD01 in Example 1.

[0060] Liquid culture medium: the same as that of Example 1.

[0061] Solid culture medium: the same as that of Example 1.

[0062] (1) ispAL site insertion fragment

[0063] P c -Gm-P pqqA -zwf1-P tac -dxs1-P mxaF -ispA-dxs2, PCR primers are as follows: ispAL-F:5'-gtattgttgcgacccaccgaac-3'

[0064] ispAR-R:5'-tcctgggctttttgttttgcc-3'

[0065] (2) Insertion of P at the glgA1L site 12965 -Bsidi-P tac -Bleo, PCR primers are as follows: glgA1L-F: 5'-ttcgggcaaggtacagagtg-3'

[0066] glgA1R-R:5'-tagctgtagcgctggacg-3'

[0067] (3) Using 2×PhantaMax MasterMix high-fidelity DNA polymerase, the DNA fragments described in (1) and (2) are amplified using corresponding primers to obtain DNA fragments that can be electrically converted.

[0068] (4) The test bacteria were inoculated into liquid culture medium and cultured until the logarithmic growth phase. 600 2; centrifuge the bacterial solution at 4°C and 5000×g for 10 min, discard the supernatant; resuspend the bacteria in 50mL of cold water, centrifuge at 4°C and 5000×g for 10 min, discard the supernatant; resuspend the bacteria in 1mL of distilled water to obtain competent cells.

[0069] (5) Take 50 μL of the competent cells obtained in step (4), add 500 ng of the gene fragment obtained in step (3) and mix gently; then, transfer the mixture to a low-temperature electric shock cup with a gap of 1 mm, and use an electroporator for electroporation (the conditions are set to 1.5 kV, 25 μF and 200 Ω); then, use 10 mL of liquid culture medium to resuscitate and culture for 24 hours; centrifuge at room temperature and 5000×g for 10 min, discard the supernatant, and spread the bacterial precipitate on a solid culture medium plate containing 50 μg / mL gentamicin and 30 μg / mL bleomycin, culture for 4 to 7 days, and screen for recombinants.

[0070] (6) After the recombinant is identified by PCR, if no base mutation is found in the sequencing, the methanophilic engineered bacteria TD02 is obtained.

[0071] (7) The methanotrophic engineered bacteria TD02 was spread on a solid culture medium containing 50 μg / mL gentamicin and 30 μg / mL bleomycin and cultured for 3 to 5 days to complete the expansion culture. The methanotrophic engineered bacteria TD02 on the solid culture medium was transferred to a liquid culture medium containing 50 μg / mL gentamicin and 30 μg / mL bleomycin, wherein the liquid volume was 50 mL. The culture was cultured until the logarithmic growth phase, and the strain was preserved.

[0072] Example 3: Fermentation and detection of taxadiene by methanotrophic engineered bacteria TD02

[0073] Test bacteria: Methanophilic engineered bacteria TD02.

[0074] Fermentation medium: Liquid medium: MgSO 4 7H 2 O 0.2g, CaCl 2 ·2H 2 O 0.02g, KNO 3 6-10 g, NaCl 6-10 g, make up to 1 L with distilled water.

[0075] (1) Seed culture: Genetically engineered bacteria producing taxadiene are inoculated into a culture medium, the upper air is renewed every 24 hours, and methane equivalent to the volume of the culture medium is supplemented. The culture is carried out for 48 hours or more to obtain a seed culture solution.

[0076] The culture medium comprises: MgSO 4 7H 2 O 0.2-1g, CaCl 2 6H 2 O 0.008-0.012g, KNO 3 0.8-1.2g, NaCl 6-10g, make up to 1L with distilled water.

[0077] (2) Fermentation culture of TD02: The seed culture solution was inoculated with OD 600 An inoculum size of 0.5 was inoculated into the fermentation medium, the ventilation volume was (800 mL air + 200 mL methane) / L·min, the temperature was controlled at 22°C-30°C, the dissolved oxygen was controlled above 20 by adjusting the stirring speed, the pH was controlled at 9.0, and 10% (v / v) n-dodecane of the total volume of the fermentation liquid was added, and the fermentation culture was carried out for 132 hours.

[0078] (3) 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 to quantitatively analyze taxadiene. The gas chromatograph is Agilent 7820A, the chromatographic column is HP-5, and the carrier gas flow rate is 2 mL / min. The oven temperature is set to 80°C for 1 minute, increase to 250°C at a rate of 10°C / min, and maintain for 1 minute.

[0079] Depend on Figure 1 It can be seen that the engineered bacteria TD02 grew rapidly in the 0-48h period and basically stopped growing after 48h. Figure 2 It can be seen that taxadiene began to accumulate at 36 hours and reached a peak of 249.51 mg / L at 144 hours.

[0080] The results show that by metabolically engineering the methanophilic bacteria and using the engineered methanophilic bacteria of the present invention, the yield of taxadiene synthesized using methane as a carbon source is greatly increased.

Claims

1. An aerobic methanophilic engineered bacterium producing taxadiene, characterized in that: The taxadiene synthase gene TXS was expressed in the host bacteria, and the 1-deoxy-xylulose-5-phosphate synthase genes dxs1 and dxs2, the geranyl pyrophosphate synthase gene ispA, the 6-phosphoglucose dehydrogenase gene zwf1 and the isopentenyl pyrophosphate isomerase Bsidi were overexpressed; The host bacteria include aerobic methanophilic bacteria Methylotuvimicrobium buryatense 5GB1, Alkalicoccus glycogenes WONF2802 or Methylotuvibium sanxanigenens NG09. The Alkalicoccusglycogenes WONF2802 and Methylotuvibium sanxanigenens NG09 have been deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration, and their deposit numbers are CGMCC No. 28614 and CGMCC No. 30951, respectively.

2. The aerobic methanophilic engineered bacteria producing taxadiene according to claim 1, characterized in that: The taxadiene synthase gene TXS is derived from Taxus brevifolia, its NCBI number is Q41594.1, and its DNA sequence is shown in SEQ ID NO:

1.

3. The aerobic methanophilic engineered bacteria producing taxadiene according to claim 1, characterized in that: The dxs1, dxs2, ispA and 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.

4. The aerobic methanophilic engineered bacteria producing taxadiene according to claim 1, characterized in that: The Bsidi is derived from Bacillus subtilis, its NCBI number is WP_004399098.1, and its DNA sequence is shown in SEQ ID NO:

2.

5. A method for constructing an aerobic methanophilic engineered bacterium producing taxadiene, characterized in that: The steps include: (1) Insert P into pAWP89 plasmid 16200 The promoter is connected to the taxadiene synthase gene TXS. 16200 -TXS expression cassette, the plasmid is transferred into the host bacteria by conjugation to obtain an engineered bacterium; (2) Inserting the DNA fragment Pc-Gm-P into the ispAL site of the engineered bacteria obtained in step (1) by electroporation pqqA -zwf1-P tac -dxs1-P mxaF -ispA-dxs2; (3) Inserting the DNA fragment P into the glgA1L site of the engineered bacteria obtained in step (2) by electroporation 12965 -Bsidi-P tac -Bleo.

6. The method for constructing an aerobic methanophilic engineered bacterium producing taxadiene according to claim 5, characterized in that: The ispAL site is inserted -Gm-P pqqA -zwf1-P tac -dxs1-P mxaF -ispA-dxs2, the sequence of which is shown in SEQ ID NO:

3.

7. The method for constructing an aerobic methanophilic engineered bacterium producing taxadiene according to claim 5, characterized in that: The insertion of P at the glgA1L site 12965 -Bsidi-P tac -Bleo, the sequence of which is shown in SEQ ID NO:

4.

8. The method for constructing an aerobic methanophilic engineered bacterium producing taxadiene according to claim 5, characterized in that: The pAWP89 plasmid was inserted into 16200 The promoter is connected to the taxadiene synthase gene TXS. 16200 -TXS expression cassette, DNA fragment P 16200 The sequence of -TXS is shown in SEQ ID NO:

5.

9. The use of the aerobic methanotrophic engineered bacteria for producing taxadiene, wherein the aerobic methanotrophic engineered bacteria is used in fermentation to produce taxadiene.

10. The use of an aerobic methanophilic engineered bacterium producing taxadiene according to claim 9, characterized in that: The aerobic methanophilic engineered bacteria are used in fermentation to produce taxadiene, specifically comprising the following steps: (1) Seed culture: Genetically engineered bacteria producing taxadiene are inoculated into a culture medium, the upper air is renewed every 24 hours and methane is supplemented in an amount equivalent to the volume of the culture medium, and the culture is carried out for 48 hours or more to obtain a seed culture solution; The culture medium comprises: MgSO4·7H2O 0.2-1g, CaCl2·6H2O 0.008-0.012g, KNO3 0.8-1.2g, NaCl 6-10g, and is supplemented to 1L with distilled water; (2) Fermentation production of taxadiene: The seed liquid is inoculated into a fermentation medium, and n-dodecane accounting for 8% to 15% of the total volume of the fermentation medium is added to the medium. The ventilation rate per liter of the medium is (700-900 mL of air + 150-250 mL of methane) / min. The maximum solubility of oxygen is controlled within 20% by adjusting the stirring speed, the pH is controlled at 8.5-9.5, and the fermentation is carried out at 22° C. to 30° C. for 120-144 hours. The fermentation medium comprises: MgSO4·7H2O 0.2-1g, CaCl2·6H2O 0.008-0.012g, KNO3 6-10g, NaCl 6-10g, and is supplemented to 1L with distilled water.

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