Engineered methanotroph with high gimalto a production and construction method and application thereof

Abstract

The application discloses high-yield gernien A engineering methanotrophs, a construction method and application thereof, and belongs to the technical field of metabolic engineering and synthetic biology. P mxaF expressing a gernien A synthase encoding gene gas overexpressing a 1-deoxy-xylose-5-phosphate synthase encoding gene in a gernien A synthesis pathway dxs1 and dxs2 a farnesyl pyrophosphate synthase encoding gene ispA a isopentenyl pyrophosphate isomerase encoding gene Bsidi simultaneously overexpressing 6-phosphogluconate dehydrogenase zwf1 and pfk1 and fba2 in an EMP pathway, to obtain gernien A engineering methanotrophs. The fermentation production of gernien A is carried out by using the engineering methanotrophs, and the yield of gernien A is obviously improved, so that the engineering methanotrophs have an industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the fields of metabolic engineering and synthetic biology, specifically relating to engineered methanogenic bacteria that produce high levels of gemmaene A, their construction methods, and applications. Background Technology

[0002] Gemarene A, a naturally occurring triterpenoid unsaturated hydrocarbon, possesses unique and excellent physiological activity and physicochemical properties, demonstrating irreplaceable application potential in several high-value fields. In the pharmaceutical field, it is a core precursor for the preparation of the broad-spectrum anticancer drug β-elemene, which has shown significant efficacy in the clinical treatment of various malignant tumors, making gemmarene A an indispensable key intermediate in the anticancer drug industry chain. Simultaneously, with the surge in demand for natural active ingredients in the functional food industry, the application scenarios of gemmarene A in functional dietary supplements and health food additives are constantly expanding, and market acceptance is continuously improving. However, with the rapid development of related industries globally, the market demand for gemmarene A is increasing daily. Traditional plant extraction methods have inherent drawbacks such as low extraction efficiency, high resource dependence, and high production costs, which are far from meeting the large-scale market supply demand. This supply-demand imbalance not only restricts the development of downstream high-value industries, but also forces researchers to accelerate technological breakthroughs and make every effort to develop a greener, more efficient, and sustainable new preparation process for gemmaene A. This is not only the core path to break the current supply bottleneck, but also an inevitable requirement to promote the high-quality and green development of related industries.

[0003] Currently, gemmaene A can be synthesized in cell factories using sugar as a raw material. However, the world's growing population and limited arable land pose challenges to cell factories using sugar-based raw materials, leading to potential competition between biomanufacturing and food supply. Therefore, there is a strong push to find alternative and more sustainable fermentation substrates.

[0004] Methane, a potent greenhouse gas, has become a promising carbon source for biomanufacturing applications. Utilizing microorganisms to convert methane into gemmaene A not only eliminates the need for glycosylated feedstocks in microbial manufacturing but also helps reduce carbon emissions and mitigate global climate change. Methanogenic bacteria can produce a range of chemicals, such as lactic acid and isobutyraldehyde, using methane as the sole carbon source. Although the natural methyl erythritol phosphate (MEP) pathway has been elucidated in type I methanogenic bacteria, the production of gemmaene A using this method still faces the following challenges: 1) Low yield. When producing gemmaene A using this method, the presence of a diversionary pathway in methanogenic bacteria results in only a small fraction of the carbon flux flowing to gemmaene A, and the production of numerous byproducts leads to carbon loss; 2) Demanding production requirements. The MEP pathway for the synthesis of gemmaene A involves eight different enzymes, and each gemmaene A molecule synthesized requires nine NADPH molecules, posing challenges to the supply of carbon flux and reducing equivalents. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the present invention aims to provide engineered methanogenic bacteria with high production of gemmaene A, its construction method and application, so as to solve the technical problems of low yield and demanding production requirements of the current gemmaene A production method.

[0006] To achieve the above objectives, the present invention employs the following technical solution: The first aspect of this invention discloses an engineered methanogenic bacterium that produces high levels of gemmaene A, using the methanogenic bacterium as the host bacterium and introducing a gemmaene A synthase encoding gene into the host bacterium. gas To construct the gemmaene A synthesis pathway, the gene encoding 1-deoxy-xylulose-5-phosphate synthase was overexpressed using an endogenous strong promoter. dxs1 , dxs2 Farnesyl pyrophosphate synthase encoding gene ispA Isopentenyl pyrophosphate isomerase encoding gene Bsidi To enhance metabolic flux, while overexpressing the gene encoding glucose-6-phosphate dehydrogenase. zwf1 To enhance reducing power, phosphofructokinase is overexpressed. pfk1 fructose-1,6-bisphosphate aldolase fba2, An engineered methanogenic bacterium that produces high levels of gemmaene A was obtained.

[0007] Preferably, the methanogenic bacteria are Methylotuvimicrobium buryatense 5GB1 Alkalicoccus glycogenes WONF2802 or Methylotuvibium sanxanigenens Both NG09 and methanogenic bacteria contain homologous recombination insertion sites in their genomes. ispAL and glgA1L Site.

[0008] Preferably, the gene encoding the gemmaene A synthase is... gas Source Nostoc parmelioides The NCBI number is WP_190566230.1.

[0009] Preferably, the gene encoding 1-deoxy-xylulose-5-phosphate synthase dxs1 , dxs2 Farnesyl pyrophosphate synthase encoding gene ispA The gene encoding glucose-6-phosphate dehydrogenase zwf1 Phosphofructokinase pfk1 fructose-1,6-bisphosphate aldolase fba2 All of them were derived from methanogenic bacteria, with NCBI numbers WP_017839846.1, WP_017842755.1, WP_017842756.1, WP_017841635.1, WP_341326251.1 and WP_341326872.1, respectively.

[0010] Preferably, the isopentenyl pyrophosphate isomerase encoding gene Bsidi It is derived from Bacillus subtilis, NCBI number WP_004399098.1.

[0011] A second aspect of the present invention discloses a method for constructing the above-mentioned engineered methanogenic bacteria that produce high levels of gemmaene A, comprising the following steps: 1) To the host bacteria ispAL DNA fragments transferred to the site Pc-Gm-P pqqA -zwf1-P tac -dxs1-P mxaF -ispA- dxs2 Engineered bacteria 1 was obtained; 2) Add the engineered bacteria 1 obtained in step 1) glgA1L Insertion site DNA fragment P 12965 -Bsidi-P tac -Bleo The engineered methanogenic bacteria IZDA were obtained. 3) Insertion into pAWP89 plasmid P mxaF Promoter linking to the gene encoding gemmaene A synthase gas Composition P mxaF - gas-pfk1-fba2 The expression cassette was then transferred into the engineered methanogenic bacteria IZDA to obtain an engineered methanogenic bacteria that produces high levels of gemmaene A. in, Pc-Gm-P pqqA-zwf1-P tac -dxs1-P mxaF -ispA-dxs2 The nucleotide sequence is shown in SEQ ID NO.1. P 12965 -Bsidi-P tac -Bleo The nucleotide sequence is shown in SEQ ID NO.2. P mxaF - gas-pfk1-fba2 The nucleotide sequence is shown in SEQ ID NO.4.

[0012] A third aspect of the present invention discloses the application of the above-mentioned engineered methanogenic bacteria with high production of gemmaene A in the production of gemmaene A.

[0013] In a fourth aspect, a method for producing gemmaene A is disclosed, wherein the seed culture of the above-mentioned engineered methanogenic bacteria that produces high gemmaene A is inoculated into a liquid inorganic salt culture medium, and fermentation is carried out under conditions containing methane to obtain gemmaene A.

[0014] Preferably, during the fermentation process, n-dodecane is added simultaneously at 10% to 20% of the volume of the liquid inorganic salt culture medium.

[0015] Preferably, the fermentation temperature is 25~30℃.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The engineered methanogenic bacteria that produce high levels of gemmaene A provided by this invention utilizes a strong promoter in the host aerobic methanogenic bacteria. P mxaF Expressing the gene encoding gemmaene A synthase gas This was achieved through the biotransformation of methane into gemmaene A. This was accomplished by overexpressing the gene encoding 1-deoxy-xylulose-5-phosphate synthase. dxs1 and dxs2 This enhances the ability of central metabolites to flow to terpenoid compounds; and by overexpressing the Fabrynylate pyrophosphate synthase-encoding gene... ispA This leads to the accumulation of the precursor compound farnesyl pyrophosphate; and through overexpression of the gene encoding isopentenyl pyrophosphate isomerase. Bsidi This is achieved by maintaining the balance between isopentenyl pyrophosphate and dimethylpropene pyrophosphate, thereby increasing the metabolic flux of the pathway containing the target product. This is achieved through overexpression of the gene encoding glucose-6-phosphate dehydrogenase. zwf1 To enhance the MEP pathway, which requires the consumption of reducing power, it is necessary to increase the supply of NADPH, thereby synergistically strengthening the flow of substances and reducing power. This is achieved by overexpressing the gene encoding phosphofructokinase. pfk1 Gene encoding fructose-1,6-bisphosphate aldolase fba2This led to the development of an engineered methanogenic bacterium that produces gemmaene A. Compared to the original methanogenic bacterium strain, this high-yield engineered methanogenic bacterium significantly increased the production of gemmaene A, opening up new raw materials for gemmaene A production and providing a reference for the development of biotransformation platforms for producing gemmaene A from methane. It has significant industrial application value and environmental significance, and provides a new pathway for the efficient synthesis of terpenoids through various metabolic optimization strategies.

[0017] The present invention provides a method for producing gemmaene A by introducing n-dodecane and methane, and fermenting engineered methanogenic bacteria that produce high-yield gemmaene A at 25-30°C. Under these conditions, the bacteria can better exert their catalytic activity, promote the biosynthesis of terpenoids, and significantly increase the yield of gemmaene A. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the method for constructing the engineered methanogenic bacteria that produce high levels of gemmaene A according to the present invention; Figure 2 This is a gas chromatography-mass spectrometry qualitative result of the engineered methanogenic bacteria that produces high levels of gemmaene A according to the present invention. Figure 3 The graphs show the yield growth curves and cell dry weight growth curves of gemmaene A obtained using the engineered methanogenic bacteria GA02. Figure 4 This is a comparison diagram of the production of gemmaene A by engineered methanogenic bacteria GA02 in Example 1 and engineered methanogenic bacteria GA01 in Comparative Example 1. Detailed Implementation

[0019] To enable those skilled in the art to understand the features and effects of the present invention, the following description and definitions are only general descriptions of the terms and expressions mentioned in the specification. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0020] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0021] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0022] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0023] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0024] The liquid inorganic salt culture medium used in this study consisted of 0.2–1 g / L MgSO4·7H2O, 0.008–0.012 g / L CaCl2·6H2O, 0.8–1.2 g / L KNO3, and 8–12 g / L NaCl, with the remainder being distilled water. The medium was sterilized at 121°C for 20 min. The solid inorganic salt culture medium was prepared by adding (10–15) g / L agar to the liquid inorganic salt culture medium. The LB medium consisted of 8–12 g / L tryptone, 4–6 g / L yeast extract, 8–12 g / L NaCl, and 10–15 g / L agar, with the remainder being distilled water, and the pH was adjusted to 7.0.

[0025] This invention provides an engineered methanogenic bacterium that produces high levels of gemmaene A. Using this methanogenic bacterium as the host bacterium, the invention achieves this by expressing a gemmaene A synthase-encoding gene within the host bacterium. gas Overexpression of the gene encoding 1-deoxy-xylulose-5-phosphate synthase dxs1 and dxs2 Farnesyl pyrophosphate synthase encoding gene ispA 6-phosphate dehydrogenase encoding gene zwf1 Isopentenyl pyrophosphate isomerase from Bacillus subtilis Bsidi Phosphofructokinase pfk1 fructose-1,6-bisphosphate aldolase fba2 get; Among them, the methanogenic bacteria are Methylotuvimicrobium buryatense 5GB1 Alkalicoccus glycogenes WONF2802 or Methylotuvibium sanxanigenens NG09, a methanogenic bacterium, contains both in its genome and in the genome. ispAL ( ispA (Left-side homologous recombination site of the gene) and glgA1L site ( glgA1 (the left homologous recombination site of the gene); the Alkalicoccus glycogenes WONF2802 andMethylotuvibium sanxanigenens NG09 has been deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession numbers CGMCC No. 28614 and CGMCC No. 30951, respectively; gene gas Source Nostoc parmelioides The NCBI accession number is WP_190566230.1, and its nucleotide sequence is shown in Table 1 as SEQ ID NO.11; gene dxs1 Derived from methanogenic bacteria, NCBI ID: WP_017839846.1; gene dxs2 Derived from methanogenic bacteria, NCBI ID: WP_017842755.1; gene ispA Derived from methanogenic bacteria, NCBI ID: WP_017842756.1; gene zwf1 Derived from methanogenic bacteria, NCBI ID: WP_017841635.1; gene Bsidi Derived from Bacillus subtilis, NCBI number WP_004399098.1; gene pfk1 Derived from methanogenic bacteria, NCBI ID: WP_341326251.1; gene fba2 Derived from methanogenic bacteria, NCBI ID WP_341326872.1; in this genetically engineered bacterium, gas Encodes gemmaene A synthase, which catalyzes the conversion of farnesyl pyrophosphate (FPP) to gemmaene A; dxs1 and dxs2 It participates in the synthesis of 1-deoxy-xylulose-5-phosphate and encodes the first rate-limiting enzyme in the MEP pathway; ispA It participates in the elongation of isopentenyl pyrophosphate (C5 unit); Bsidi Maintain the stability of the ratio of key intermediate products IPP and DMAPP; zwf1 Provides reducing power for the MEP pathway; pfk1 and fba2 Reduce the production of byproducts formic acid and carbon dioxide.

[0026] Table 1 Nucleotide Sequence List Name Sequence (5’-3’) Serial number <![CDATA[ Pc- Gm- P pqqA - zwf1- P tac - dxs1- P mxaF - ispA- dxs2 ]]> CTTCGACACCGGGAATACCCTGCTCGGGATGAATATCGACTGGCATTCCTCGACCATTGTCGCTAACTAATACAGAACCGTCTTTATATAATACGACAGTAATATTGTCGGCATGCCCTGCTATCGCTTCGTCGACGCTGTTGTCGACAACCTCTTGCACCAGATGGTTCGGTCGGGTCGTATCGGTATACATACCGGGCCGTTTCCGAACAGGTTCCAAGCCGCTTAATACCTCTATCGCGGCTGCATTGTAATCATTACTCATACTTCCTAATCACTCATTATCCAAATTCATATATAATGGCCGGTTTGATTCCATCGCCCAATTTCTACGTACTATTTTATCTACATGCCGAGAAAAAAAACCACGAATTTATTCGAAGAGTCCCTAGCCGAGCTTGAGCAACTCGTCGAGCAAATGGAACAAGGTGAGCTATCTTTAGAAGATTCGTTAAAATCATTTGAACGCGGCGTCGCTTTAACCCGAACCTGCCAGAAAGCCTTGCAGGAAGCCGAACAGAAAGTTCAGATTTTACTGGAAAAAAACGGCACTCAAACTCTGGAGCCCTTCACCGATGAGTAATTGACATAAGCCTGTTCGGTTCGTAAACTGTAATGCAAGTAGCGTATGCGCTCACGCAACTGGTCCAGAACCTTGACCGAACGCAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAGGTGGCTCAAGTATGGGCATCATTCGCACATGTAGGCTCGGCCCTGACCAAGTCAAATCCATGCGGGCTGCTCTTGATCTTTTCGGTCGTGAGTTCGGAGACGTAGCCACCTACTCCCAACATCAGCCGGACTCCGATTACCTCGGGAACTTGCTCCGTAGTAAGACATTCATCGCGCTTGCTGCCTTCGACCAAGAAGCGGTTGTTGGCGCTCTCGCGGCTTACGTTCTGCCCAGGTTTGAGCAGCCGCGTAGTGAGATCTATATCTATGATCTCGCAGTCTCCGGCGAGCACCGGAGGCAGGGCATTGCCACCGCGCTCATCAATCTCCTCAAGCATGAGGCCAACGCGCTTGGTGCTTATGTGATCTACGTGCAAGCAGATTACGGTGACGATCCCGCAGTGGCTCTCTATACAAAGTTGGGCATACGGGAAGAAGTGATGCACTTTGATATCGACCCAAGTACCGCCACCTAACTTGATTTGTCGCGACTTATACCCCTCTTAGATCAAAATTCGACGCCGGGCTGTCCGTCAAAGGGCGCCGTGAATACATCCCTATAGGCTCTATGCCGGCTCTATGCTGGCAAAGCCTTTGCCGAAAACCCCTGCGCCTCCTCAGGCGCTGCCGAAATTTGAAGTCCGAAAGGTATAAAGCCCAGCCCGATCGCTAAACCAATCGGTTGCTTTGCCTAAATTATCGTCGTATACTTCCGACCTAGCTTACTTGATCAAGCTGGCATAACACTTATAAACATACTTCTGGAGGTATTGATTATGTCTGCTGAACCCTGTACCTATGTCATTTTCGGAGCAACCGGCAATCTATCCCGAATTAAACTGATGCCGGCACTCTATCATTTGGAACTCGAAAACAAACTGCCGGAAGGCAGTCGTATTATCGGCATCGGCCGAAGGCCTTGGGATCAAAAAAAATGGCTAGAAGAGATCCGAGAGATGATCGGCACGAAGGTCAGTGAAGGTATCGATGAGGCCGTATTCGCACGGTTCAGCGAACGGCTTTCCTACCATCGAGGCAATCTCGATGAAGCTGAATGCTATAGAGGTCTGGCAGTTACCTTGAGCAAGAATGAGGATTTTCCCAAAAACATCGCATTTTATCTCGCGATCAGTCCTGAGGATTTCGGTAATGTCATCGAGTCGCTTAGCAAAGTCGAATTGTTGAATCAAGAATACGGCTGGAAACGCGTTATCATCGAAAAGCCTTTCGGTTACGACCTGGACAGCGCGCAATCGTTGCAAAAACGGATCGGACGCTTTTTAAGCGAAGAACAGATCTACCGTATCGATCATTATCTAGGCAAAGGCATGGTGCAGAATGTATTGGTATTCCGCTTCGCGAATGTCATGCTAGAGCCGCTGTGGAACCGCAACTACATAGACCATGTTCAGATTACCCATGCCGAAGATATCGGTATCGACACGCGCGGCGGCTATTACGATGGGGCAGGAGCTTTGCGCGACATGCTGCAAAGCCATTTACTACAACTGATGACACTGGTTGCGATGGAACCGCCCGCGTCGATGGAAGCGGAATCCTTGCGCGACGAGAAGGTCAAGGTCTTAAAATCGATTCGCTCAATTCCCAAATCGGCAGTACACGCGCATGCCTATCGCGGCCAATACGCCAAAGGCACGATCGGCAAGGAAAAGGTAAAGGGTTATTTGGAGGAAGAGAACATTCCGCCCAATAGCATTACAGAAACCTATGCTGCCGTGAAATTATTCATCGATAACTGGCGCTGGCGGGGCGTGCCGTTTTATCTACAGACCGGTAAACGACTGGCCAAAGGGCAATCGGTCGTCTCGATTTGCTTTCGTCATCCGCCGCTGCAGTTTTTTCGCGATACGCATGTGCAATGCATGAATCCGAATTGGGTGCTGTTGAGCATTCAACCCGAAGAACGCATTCGCATGGAAATGACCGTCAAAGAACCGGGCCTGGAAATGCGCACACGGACCAGCAGCCTGGATGCCGGTTTTCGAAACAGCGATGAAAAAGCGATCGACGCCTATGAAGATTTATTGCTCGATGTCATGAAAGGTGACAGCTCGTTGTTCCTGCGCTTCGATGAAGTCGAATATGCTTGGCGCATCGTCGACCCGATTCTGCAAACCTGGGCCGTAGAGCGCGACTTCATCCCGACTTATCCGGCCGGCAGTTGGGGCCCCGCCGAAAGCCGCCGTTTGTTCGAAAAAGAAGATCAATTCTGGCGTACCTCGTTGACGCCGGAGTGGTGCCAATAGCTCTGAAATGAGCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAGGTATTCACACAGGAAACAGCTATGAACCTAGCTAACGAGTATCCATTACTGAGCCTGATCGATAAACCGGCGGATTTACGAAAACTGACCAAAGCCAATCTGATTCCGCTTGCCAAAGAACTTCGCGAGTTTCTGACCCACACGGTCAGTATTTCCGGCGGGCATTTTTCGGCAGGACTCGGTACCGTGGAGTTGACCGTGGCACTGCATTACGTATTCGATACGCCGCGCGATCAATTGGTTTGGGATGTCGGCCATCAGGCTTATCCGCATAAGATCCTGACCGGACGCAAGGAGCGCATGACGACGATTCGCACGCGCGACGGCATTTGCGCGTTTCCGAATCGTTCCGAGAGCGAATACGATGCCTTCGGCGTCGGCCATTCGAGCACGTCGATCAGCGCGGCCTTAGGCATGGCGATCGCATCCGGTTTGCGCGGCGAGGACAAGCATTGCGTCGCGATTATCGGCGATGGCAGCATCACCGGCGGCATGGCCTTCGAAGCGATGAATCATGCCGGTGCGATCGACGCGAATTTGTTGGTGATCTTGAACGATAACGATATGTCGATTTCGCCGAATGTCGGCGCGTTGAATAATTATCTGACTAAGATTCTGTCGAGTAAGATTTATTCTTCGGTACGCGAAGAAAGCAAGAAGGCTCTCAGCAGCATGCCGAGCGTCTGGGAGCTGGCGCGCAAGACCGAGGAGCACATGAAAGGCATGATCGTGCCGGGCACCTTGTTCGAGGAAATGGGTTTCAATTATATCGGTCCGATCGACGGCCACGATCTGGACATGCTGGTGTCGACGCTGGAGAATCTGAAACACATTTCCGGCCCGCGTTTCTTGCATATCGTCACCAAGAAAGGCAAGGGCTATGCGCCGGCCGAGAAAGACCCGCTCGCCTATCACGGCGTACCGGCCTTCGACCCGAGCCGCGATTGCCTGCCGAAATCGGCCCCTTCTCCGCACCCGACGTATACGCAGGTATTCGGAGAATGGCTCTGCGACATGGCCGAGCAGGACGAGCGCTTATTGGGCATTACCCCGGCGATGCGCGAAGGCTCGGGCCTGGTCGCGTTTTCCGAACGCTTTCCGAAGCGCTATTTCGATGTCGCTATCGCCGAGCAGCATGCCGTAACCTTAGCGGCGGGCCTGGCCTGCGAGGGCGGCAAACCGGTCGTCGCGATCTATTCAACCTTCTTACAACGCGGCTACGATCAATTGATACACGATGTGGTCTTGCAGGACCTGGACGTATTGTTTGCGCTCGACCGCGCAGGTTTAGTCGGCCCGGACGGCCCGACCCATGCCGGCAGTTTCGATTACACCTACATGCGCTGCCTGCCGAACATGCTGATCATGGCGCCTGCCGATGAAAACGAATGCCGGCAGATGCTCTATACCGGTTACATGCATAAGGGTCCAGCCTCGGTACGCTATCCGCGCGGCAAAGGCCCGGGCGTGCCGGTCGATTGCACCATGACCGCGCTACCGATCGGCAAGGCCGAATTGCGCCATCAAGGCGGTCGCATCGCGATTCTGGCGTTCGGCAGCCTGGTGGCTCCGTCGGTCGAGGCCGGCAAGCAACTCGGCGCGACCGTAGTCAACATGCGTTTCGTCAAGCCGATCGATGAAGCATTGATTCTGGAACTGGCCAAGAGTCACGAAGTCATCGTCACGGTCGAGGAAAACGTCGTCGCCGGCGGCGCCGGCAGCGCAGTCAACGAATTTCTGCAGGCGCAAAGAATCGTGATGCCGGTGCTCAACATCGGCCTGCCCGACGCCTTCATCGAACAAGGCACGCGCGAGGAATTGCTGAGTCTGTGCGGCTTGGACTCACAAGGGATTCTGCAAGGCATCGAGCAGTTTTGCGGATAAAATTAAACCGGGAATGATGTCGGATATTTAACGGCAAAGCCATGGGAGCTTTTCCCGAATTTGAATGCCGACATACTCTCGGGATATTTTCCCTGTTTTTTCTTAGCGCTTTTCCCGTCATCTGGGTGCTGTATTCCGTAACGTCGCATCCCGCTCCTTCCGTATGATTACCGTCCGTGCGCTGCCCTCTATGAATGATTCGTTATGCGCCTTGATCAAGCTAAGCCGGTTGTAACAACAAACACCGCAATCAATAGGGGGCCGCGCCGACATTATGCGAAAAATCAATCTGGAGGAATTATGAGTAACGCACTGAAAGACTATCTGACCTTTTGTCAAAACCGAGTCGAAAGAGCCTTGGAAGCCCGACTGCCAAGCGAAACCCAAATACCGGCTAAATTGCACGAAGCGATGCGCTATTGCGTGTTGGACGGCGGTAAACGCATGCGTCCGATGCTAACCTACTGTACAGGAAAAGCCTTGGGCATTGCACCGGAAGATTTAGACGGAGCGGCCTGTGCGGTTGAATTCATTCATGTTTATTCATTGATACATGACGATTTGCCGGCCATGGACGACGACGATCTCAGACGCGGAAAACCGACCTGCCATATCGCTTATGATGAAGCGACCGCCATTCTGACCGGCGACGCACTACAAGCACTGGCATTCAAGGTCTTGGCGGACGACCCGACCATCCAAGCCGATGCCGAAAGCCGTCTAAAAATGATTACGATGCTGGCCAAAGCTAGCGGCTCTCAAGGCATGGTCGGCGGTCAAGCCATCGATTTAGAATCGGTCGGCACAATGCTGACGCTGCCTCAGCTTGAAAATATGCATATCCACAAGACCGGCGCGTTAATCCGAGCCAGCGTCAACATGGCAACATTAACCAAGCCCGATATCGACCCGAAACAAGCCGAAGGCCTCGATCATTACGCAAAATGCATCGGCCTATCCTTCCAAGTCAAGGACGATATTTTGGACGAAGAGAGCGATACTGCAACACTCGGCAAAACCCAAGGCAAGGACAAGGACAACGACAAGCCGACTTACCCTGCCTTACTCGGTTTGGCCGGGGCAAAACAAAAAGCCCAGGAACTTCATGAGCAAGCCATCGAAAGCTTGAGCGGATTTGGGTCCGAGGCCGACTTGCTACGCGACTTGTCGCTTTATATTATTCAGCGGGATCATTAACTCCCGACTTGAACACTAGGATATTAATCTGCATAATCAGTCGGTAACCGCGTCCGTGCCGATCAATAAGGAATCGATTTATTCATGAAAATCACAGGAAACTTCCCCATACTAGACAGTATCAAGCTCCCCTCCGACCTAAGAAAACTGCCGAAGGAACGATTAAAACCGCTTGCCAAAGAACTTCGCGAGTTTCTGACCCACACGGTCAGTATTTCCGGCGGGCATTTTTCGGCAGGCCTCGGTACCGTGGAGTTGACCGTGGCACTGCATTACGTATTCGATACGCCGCGCGATCAATTGGTTTGGGATGTCGGCCATCAGGCTTATCCGCATAAGATCCTGACCGGACGCAAGGAGCGCATGACGACGATTCGCACGCGCGACGGCATTTGCGCGTTTCCGAACCGTTCCGAGAGCGAATACGATGCCTTCGGCGTCGGCCATTCGAGCACGTCGATCAGCGCGGCCTTAGGCATGGCGATCGCATCCGGTTTGCGCGGCGAGGACAAGCATTGCGTCGCGATCATCGGCGATGGCAGCATCACCGGCGGCATGGCCTTCGAAGCGATGAATCATGCCGGTGCGATCGACGCGAATTTGTTGGTGATCTTGAACGATAACGATATGTCGATTTCGCCGAATGTCGGCGCGTTGAATAATTATCTGACTAAGATTCTGTCGAGTAAGATTTATTCTTCGGTA CGCGAAGAAAGCAAGAAGGTCCTCAGCAGCATGCCGAGCGTCTGGGAACTGGCGCGCAAGACCGAGGAGCACATGAAAGGCATGATCGTGCCGGGCACCTTGTTCGAGGAAATGGGTTTCAATTATATCGGTCCGATCGACGGCCACGATCTGGACATGCTGGTGTCGACGCTGGAGAATCTGAAACACATTTCCGGCCCGCGTTTCTTGCATATCGTCACCAAGAAAGGCAAGGGCTATGCGCCGGCCGAGAAAGACCCGCTCGCCTATCACGGCGTACCGGCCTTCGACCCGAGCCGCGATTGCCTGCCGAAATCGGCCCCTTCTCCGCACCCGACGTATACGCAGGTATTCGGAGAATGGCTCTGCGACATGGCCGAGCAGGACGAGCGCTTATTGGGCATTACCCCGGCGATGCGCGAAGGCTCGGGCCTGGTCGCGTTTTCCGAACGCTTTCCGAAGCGCTATTTCGATGTCGCTATCGCCGAGCAGCATGCCGTAACCTTAGCGGCGGGCCTGGCCTGCGAGGGCGGCAAACCGGTCGTCGCGATCTATTCAACCTTCTTACAACGCGGCTACGATCAATTGATACACGATGTGGTCTTGCAGGACCTGGACGTATTGTTTGCGCTCGACCGCGCAGGCTTAGTCGGCCCGGACGGCCCGACCCATGCAGGCAGTTTCGATTACACCTACATGCGCTGCCTGCCGAACATGCTGATCATGGCACCGGCCGACGAGAACGAATGCCGGCAGATGCTCTATACCGGTTATATTCATAAAGGCCCGGCTTCGGTCCGCTATCCACGCGGCAAAGGCCCGGGCGTGCCGGTCGATTGCACCATGACCGCACTGCCGATCGGCAAGGCCGAATTGCGCCATCAAGGCGGTCGCATCGCGATTCTGGCGTTCGGCAGCCTGGTGGCTCCGTCGGTCGAGGCCGGCAAGCAACTCGGCGCGACCGTAGTCAACATGCGTTTCATCAAGCCGATCGATGAAGCATTGATTCTGGAACTGGCCAAGAGTCACGACATTATCGTCACGGTCGAGGAAAACGTCGTCGCCGGCGGCGCCGGCAGCGCGGTCAACGAATTTCTGCAGGCGCAGAGAATCGTGATGCCGGTCTTGAATATCGGCCTGCCCGATGCCTTTATCGAACAAGGCACGCGCGAGGAATTACTGAGTTTTTGCGGATTGGATACACAAGGCATATTGCAGAGCATCGAGCAATTTTGCGCGTGA SEQ ID NO.1 <![CDATA[ P 12965 - Bsidi -P tac - Bleo ]]> GTTCGGGCAAGGTACAGAGTGGAATTTCAACCAAGCCTTGGATTGGTATGTATTGCAGCACCCTAGACACCAAGGTTTACATACTCTGGTCAAAGATCTTAATCATTTGTATAAAAACCACCCGGCACTTCACCAATACGACTTCAATCACAGCGGCTTTGACTGGATCGATTGCCATGATGTCGAACAATCGATTATCAGCTACCGCCGCAAAGGCACCAACGATGATTTGATCATCATACTTAACTTCACACCGATTGTTAGAGAAAACTATCATATCGGCGTGCCTTTCGAAGGGGTTTATTTTGAAATCTTTAATTCAGACTCTGCCTATTATGAAGGCAGCAATGTCGGCAACCGCGAGATACTATCGGAACCGGAGCCTTGGATGGGGCACCAGCAATCGATACACTTGACTTTACCTCCGTTAGGCGGAATCATCTTGACCCGGCAAACGAAAACCATAAACAGCTCTGCTCACTAATATAGGGACCTGCTTGAGAAAGTGAGCCCAGCGTGCCGTTGCGCACCGCAAATTAACTGACGACGGCCTAGCTTATAAAAAATTCGCTTCGATATTTATACGTAATAGGCGCAACTACGCGAGAAATGCCCGCAAAATAGAACTACCGCCGCCCCGAACGCAAACTTAAACTGATTTTTCAATAATGTCGGACGATCCTACTACAGCAGACAGTGAGCCAATAAACACTCAATCGGCTATTTTGGCGAAATCCACCTCGGTAAACTCAATTCTTTAATATACCTAGAACCGAATTTTGACTGCCTTTAAATTTAATTTGTCGAAGCGCACCAAAGACATGGTATTCCTCCTCAGAATACTCGCTTGGGAGGGCAAATCGGCATTCACGGCTTGGGGCGTGGCGATGAGAAAATTCATAGAACCATGAATTGGACCCATGGCTGTATTGCATTGACTAATAAGCAAATTGATCTTTTAAGTAAGTGGGTTGATAAAGAAATTACAGTGCAGATAAAATAGTTCTGGAATATTTTGAAAAAAAAGTTCAATATATTTCATCGAATGCTTTTGAAAATAATCTTAGTAACAACTCTTACAGTAAAAAAAGGAAAAATAATATGACTCGAGCAGAACGAAAAAGACAACACATCAATCATGCCTTGTCCATCGGCCAGAAGCGGGAAACAGGTCTTGATGATATTACGTTTGTTCACGTCAGTCTGCCCGATCTTGCATTAGAACAAGTAGATATTTCCACAAAAATCGGCGAACTTTCAAGCAGTTCGCCGATTTTTATCAATGCAATGACTGGCGGCGGCGGAAAACTTACATATGAGATTAATAAATCGCTTGCGCGAGCGGCTTCTCAGGCTGGAATTCCCCTTGCTGTGGGATCGCAAATGTCAGCATTAAAAGATCCATCAGAGCGTCTTTCCTATGAAATTGTTCGAAAGGAAAACCCAAACGGGCTGATTTTTGCCAACCTGGGAAGCGAGGCAACGGCTGCTCAGGCAAAGGAAGCCGTTGAGATGATTGGAGCAAACGCACTGCAGATCCACCTCAATGTGATTCAGGAAATTGTGATGCCTGAAGGGGACAGAAGCTTTAGCGGCGCATTGAAACGCATTGAACAAATTTGCAGCCGGGTCAGTGTACCGGTCATTGTGAAAGAAGTCGGCTTCGGTATGAGCAAAGCATCAGCAGGAAAGCTGTATGAAGCTGGTGCTGCAGCTGTTGACATTGGCGGTTACGGGGGAACAAATTTCTCGAAAATCGAAAATCTCCGAAGACAGCGGCAAATCTCCTTTTTTAATTCGTGGGGCATTTCGACAGCTGCAAGTTTGGCGGAAATCCGCTCTGAGTTTCCTGCAAGCACCATGATCGCCTCTGGCGGTCTGCAAGATGCGCTTGACGTGGCAAAGGCAATTGCGCTGGGGGCCTCTTGCACCGGAATGGCAGGGCATTTTTTAAAAGCGCTGACTGACAGCGGTGAGGAAGGACTGCTTGAGGAGATTCAGCTGATCCTTGAGGAATTAAAGTTGATTATGACCGTGCTGGGTGCCAGAACAATTGCCGATTTACAAAAGGCGCCCCTTGTGATCAAAGGTGAAACCCATCATTGGCTCACAGAGAGAGGGGTCAATACATCAAGCTATAGTGTGCGATAACTCTGAAATGAGCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAGGTATTCACACAGGAAACAGCTATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTGAATGAAACGAATTCTTTTTGTTACCAGTGAAGCACACCCTTTAATAAAAACCGGCGGCTTGGCGGATGTTTCAAGCAGCTTACCTAAGGCTTTGGCGGATCTAGGCCAGGATATCCGTATCATCATACCTAACTATCAGGCTATAAAAAAAACCGAAAACGTCCAACATCGGTGTACGCTGAGAATCAATAATTGCGATGTCAATATTCTCGAAACCCGCTTGCCGGAATCGAAAGTAATTGTATGGCTGATCGATTGTCCCCAGTTTTTTGACTACCCGGGCAATCCTTATCACGATGAATACGGTAATGCCTGGGCAAACAGTGCCGATCGCTTTTCGCTGTTCTGCCGCATAACCGTGGAAGTCGCGATGAACAGAGCCTACTTAGATTGGAAACCGGAAATCGTCCACTGCAACGACTGGCAAAGCGGCCTGGTTCCCGCCTTGTTAACGCTGGAATACAATCGCCCGGCAACCATTTTTACGATTCATAACATGGCCTATCAAGGGATCTTTCCCTATTCGACCTACAATGCGCTTAATCTTCCAAGACAACTTTGGAACCCAAATGTACTTGAGTATTACGGCAACATGTCGTTTTTAAAAGGCGGCATTGCTTGCTCCGATCGAGTAACAACGGTAAGTCCTACCTATGCCAAAGAAATTCAATCATCCGAGTTTGGTTACGGGCTAGAAGGCTTGTTAACCCATCGCAAGGAATTTCTATGCGGTATACTCAATGGAACCGACAATGACTGGAATCCCGAATTCGACAACAACATCGTCCAGCGCTACAGCTATAAAACG SEQ ID NO.2 ATGACTCGAGCAGAACGAAAAAGACAACACATCAATCATGCCTTGTCCATCGGCCAGAAGCGGGAAACAGGTCTTGATGATATTACGTTTGTTCACGTCAGTCTGCCCGATCTTGCATTAGAACAAGTAGATATTTCCACAAAAATCGGCGAACTTTCAAGCAGTTCGCCGATTTTTATCAATGCAATGACTGGCGGCGGCGGAAAACTTACATATGAGATTAATAAATCGCTTGCGCGAGCGGCTTCTCAGGCTGGAATTCCCCTTGCTGTGGGATCGCAAATGTCAGCATTAAAAGATCCATCAGAGCGTCTTTCCTATGAAATTGTTCGAAAGGAAAACCCAAACGGGCTGATTTTTGCCAACCTGGGAAGCGAGGCAACGGCTGCTCAGGCAAAGGAAGCCGTTGAGATGATTGGAGCAAACGCACTGCAGATCCACCTCAATGTGATTCAGGAAATTGTGATGCCTGAAGGGGACAGAAGCTTTAGCGGCGCATTGAAACGCATTGAACAAATTTGCAGCCGGGTCAGTGTACCGGTCATTGTGAAAGAAGTCGGCTTCGGTATGAGCAAAGCATCAGCAGGAAAGCTGTATGAAGCTGGTGCTGCAGCTGTTGACATTGGCGGTTACGGGGGAACAAATTTCTCGAAAATCGAAAATCTCCGAAGACAGCGGCAAATCTCCTTTTTTAATTCGTGGGGCATTTCGACAGCTGCAAGTTTGGCGGAAATCCGCTCTGAGTTTCCTGCAAGCACCATGATCGCCTCTGGCGGTCTGCAAGATGCGCTTGACGTGGCAAAGGCAATTGCGCTGGGGGCCTCTTGCACCGGAATGGCAGGGCATTTTTTAAAAGCGCTGACTGACAGCGGTGAGGAAGGACTGCTTGAGGAGATTCAGCTGATCCTTGAGGAATTAAAGTTGATTATGACCGTGCTGGGTGCCAGAACAATTGCCGATTTACAAAAGGCGCCCCTTGTGATCAAAGGTGAAACCCATCATTGGCTCACAGAGAGAGGGGTCAATACATCAAGCTATAGTGTGCGATAA SEQ ID NO.3 <![CDATA[ P mxaF - gas- pfk1- fba2 ]]> AATTAAACCGGGAATGATGTCGGATATTTAACGGCAAAGCCATGGGAGCTTTTCCCGAATTTGAATGCCGACATACTCTCGGGATATTTTCCCTGTTTTTTCTTAGCGCTTTTCCCGTCATCTGGGTGCTGTATTCCGTAACGTCGCATCCCGCTCCTTCCGTATGATTACCGTCCGTGCGCTGCCCTCTATGAATGATTCGTTATGCGCCTTGATCAAGCTAAGCCGGTTGTAACAACAAACACCGCAATCAATAGGGGGCCGCGCCGACATTATGCGAAAAATCAATCTGGAGGAATTATGGAAAAATTTACCTTTCCGAATTTGTATTGCCCGTTTCCGGAACGCAAAAATCCGTATTCGGAATTTTTGCAAGATTATGCCTTGCAATGGGTCATCCGCTTTAAATTGATCGATTCGGAATCGTTGTATCAACGCTTTTCGAAAGCCAAATTTTATTTGTTGACCGCCGGCGCCTATCCGCATTGCCAATTGGAAGAATTGAAGATTGCCAATGATGTCATCTCGTGGTTGTTTATCTGGGATGATCAATGCGATATCTCGGATTTGGGCAAAAAACCGGAATTGTTGAAAACCTGGTGCAATCGCTTTTTGGAAATCTTGAATGGCGCCGAATTGACCCCGGATGATTTGCCGTTGGGCTTTGCCTTGCGCGATATCCGCAATCGCATCATCAATCGCGGCGGCATCACCTTTTTTCATCATTTTGTCCGCAATTTTGAAGATTATTTTTATGGCTGCATCGAAGAAGCCCATAATCGCGTCAATGTCTCGGTCCCGGATGTCGAAGCCTATATCAAAATCCGCTCGGCCAATGCCGCGGCCGCCTTGTGTTTGAATTTGATCGAATTTTGCGATCGCGTCATGATCCCGTATTCGTTGCGCAATCATGAAACCTTGAAAAAATTGACCCAAATGACCATCAATATCTTGGCCTGGTCGAATGATATCTTTTCGGCCCCGCGCGAAATCGCCAACGGCGAAGTCCATAATTTGGTCTTTGTCATCCATCACCATCAAAAAATCCCGTTGGAAAAAGCCATGTTGGCCGCGGCCGCGATGCATAATCATGAAGTCCAAAAATTGGTCAATTTGGAATCGAAAATCGCCTCGTTTTCGGCCGAAACCGATGCCGAAATCACCAAATATATCTCGGGCTTGCATGCCTGGATCCGCGGCAATTTGGATTGGTATGCCCATTCGGGCCGCTATCAAATCACCGAAAAATTGGAATTGTTGGCCTCGTAATGTGGAGGTATTCACACAGGAAACAGCTATGAACAAACCAAAAAAAGTCGCAATTCTCACTGCGGGCGGTTTAGCGCCTTGCCTTAGTTCAGCCATCGGCAGTCTCATCGAGCGTTACACCGAAATCGACCCTTCGATCGAGATCATCTGCTATCGTAGCGGTTACAAAGGCCTGCTACTCGGCGATTCTTACGCCGTGACCCCGAAAATCCGCGAAAACGCCGCGTTGCTGCATAAGTTCGGCGGCTCGCCGATCGGCAACAGCCGGGTCAAACTGACCAACGTCAAAGACTGCATCAAGCGGGGGTTGGTTCAAGAAGGTCAGGACCCTCAAAAAGTGGCGGCCGATCAATTAGTCAAAGACGGCGTCGATGTTCTGCATACGATCGGCGGCGACGATACCAATACCGCAGCAGCCGATTTGGCGGCCTTCTTGGCGAAAAACGATTATGGATTGACGGTCATCGGTTTGCCGAAAACGATCGACAACGACGTATTCCCGATTAAACAATCCTTAGGTGCATGGACTGCAGCGGAGCAAGGCGCACATTATTTTCAAAATGTCGTGGCCGAGTATAACGCCAATCCACGCATGCTCATCGTTCATGAAGTCATGGGCCGCAATTGCGGATGGCTGACTGCCGCAACCGCAATGGAATACCGCAAATTGTTGGATCGCTCCGAATGGCTGCCTGAAATCGGTCTCGATCGCGCGGCATACGAAGTACACGGTGTCTTCGTTCCCGAAATGGAAATCGATCTGGCAGCCGAAGCGAAGCGCTTGCGCGAGGTGATGGATAAAGTCGATTGCGTCAATATATTCGTTTCGGAAGGCGCGGGTGTCGATGCGATCGTCGCCGAAATGCAGGCCAAGGGCCAAGAAGTTCCGCGCGATGCGTTCGGTCACATCAAGCTTGATGCGGTCAATCCGGGTAAATGGTTCGGCGAGCAATTCGCCGAAATGATCGGCGCGGAAAAAACCTTGATTCAAAAATCGGGATATTTCGCACGGGCATCGGCATCGAACGTCGATGATATTCGTTTGATCAAATCCTGTGCCGATTTAGCGGTCGAATGCGCATTACGCCGCGAGTCCGGCGTCATCGGTCATGATGAGGATAACGGTAACGTCTTGCGTGCGATCGAATTCCCGCGCATCAAAGGCGGCAAACCGTTCGATATCGACACGCCTTGGTTCGTGCAAATGCTTGGCGGAATCGGGCAAAGTAAAGGCGCGCGAGTCGAAGTGAGCCACTAAATTTTTTCGGTAACTAACACACAGGAGAAGTCAAATGGCACAAAAAATTTTAGATATTGTTAAACCAGGTGTCGTAACCGGTGAAGATGTACAAAAAGTCTTTGCGTTTTGTAAAGAACATAAGTTTGCACTTCCAGCTGTAAACGTCATCAGTACCGATACGATCAATGCAGTACTCGAAGGTGCGGCTAAAGCAAAATCAGCCGTTATCATTCAGTTTTCAAACGGCGGCGCTGCTTTTTTTGCCGGCAAAGGCGTCAGTTTAGAAGGTCAAATGCCTTCAATTTTGGGAGCGATCTCAGGCGCGCAGCACGTTCATCTTATGGCTGAGCATTACGGTGTACCGGTTATTTTACATACCGATCATGCCGCGAAAAAATTATTACCCTGGATCGATGGCTTATTAGATGCCGGGGAAAAGCATTTCGAAAAAACCGGAAAACCGTTATTCAGTTCTCATATGCTGGATCTTTCAGAAGAAAGTCTGGAAGAGAACATTGAAATTTGCGGTAAATATCTAGAGCGTATGTCCAAAATGGATATGACTCTTGAAATTGAACTGGGTTGCACGGGCGGCGAAGAAGACGGCGTAGATAATACCGGTATGGATCATTCTATGCTCTATACGCAGCCGGAAGATGTTGCCTATGCTTATGAGCATTTAAGCAAAATTAGCCACCGTTTTACGATCGCGGCATCTTTCGGTAATGTGCACGGTGTTTACAAACCCGGCAATGTTAAGCTAACTCCGACCATTTTGCTGAATTCGCAAAAATTCGTTTCCGAGAAATATGACTTACCGGAAAACAGCTTGACCTTCGTATTTCATGGCGGTTCAGGTTCCACTCCTGAAGAAATCAAGGAATCGATCAGCTACGGCGTCGTTAAAATGAATATCGACACCGATACTCAATGGGCAACCTGGGCTGGCGTCATGGAATTCTATAAGAAAAACGAAGGTTATTTGCAAGGCCAAATCGGTAACCCTGACGGCGACGACAAGCCGAATAAAAAATATTATGATCCACGCGTTTGGCAACGTGCCGGCCAAGTCGGCATGGTGACCCGCCTGCAGCAGGCTTTCCAAGACTTAAACGCAACTAATACGTTGTAA SEQ ID NO.4 ispAL-F GTATTGTTGCGACCCACCGAAC SEQ ID NO.5 ispAR-R TCCTGGGCTTTTTGTTTTGCC SEQ ID NO.6 glgA1L-F TTCGGGCAAGGTACAGAGTG SEQ ID NO.7 glgA1R-R TAGCTGTAGCGCTGGACG SEQ ID NO.8 PmxaF-F GCTGCTCTGAAATGAGCTGAATTAAACCGGGAATGATGTCGG SEQ ID NO.9 fba2-R ​ ​ ATGGAAAAATTTACCTTTCCGAATTTGTATTGCCCGTTTCCGGAACGCAAAAATCCGTATTCGGAATTTTTGCAAGATTATGCCTTGCAATGGGTCATCCGCTTTAAATTGATCGATTCGGAATCGTTGTATCAACGCTTTTCGAAAGCCAAATTTTATTTGTTGACCGCCGGCGCCTATCCGCATTGCCAATTGGAAGAATTGAAGATTGCCAATGATGTCATCTCGTGGTTGTTTATCTGGGATGATCAATGCGATATCTCGGATTTGGGCAAAAAACCGGAATTGTTGAAAACCTGGTGCAATCGCTTTTTGGAAATCTTGAATGGCGCCGAATTGACCCCGGATGATTTGCCGTTGGGCTTTGCCTTGCGCGATATCCGCAATCGCATCATCAATCGCGGCGGCATCACCTTTTTTCATCATTTTGTCCGCAATTTTGAAGATTATTTTTATGGCTGCATCGAAGAAGCCCATAATCGCGTCAATGTCTCGGTCCCGGATGTCGAAGCCTATATCAAAATCCGCTCGGCCAATGCCGCGGCCGCCTTGTGTTTGAATTTGATCGAATTTTGCGATCGCGTCATGATCCCGTATTCGTTGCGCAATCATGAAACCTTGAAAAAATTGACCCAAATGACCATCAATATCTTGGCCTGGTCGAATGATATCTTTTCGGCCCCGCGCGAAATCGCCAACGGCGAAGTCCATAATTTGGTCTTTGTCATCCATCACCATCAAAAAATCCCGTTGGAAAAAGCCATGTTGGCCGCGGCCGCGATGCATAATCATGAAGTCCAAAAATTGGTCAATTTGGAATCGAAAATCGCCTCGTTTTCGGCCGAAACCGATGCCGAAATCACCAAATATATCTCGGGCTTGCATGCCTGGATCCGCGGCAATTTGGATTGGTATGCCCATTCGGGCCGCTATCAAATCACCGAAAAATTGGAATTGTTGGCCTCGTAA SEQ IDNO.11 P89-F TAGTTGTCGGGAAGATGCGTG SEQ ID NO.12 P89-R CAGCTCATTTCAGAGCAGCTCAC SEQ ID NO.13 gas-F CACACAGGAAACAGCTATGGAAAAATTTACCTTTCCGAATTTGTATTGC SEQ ID NO.14 gas-R CACGCATCTTCCCGACAATTACGAGGCCAACAATTCCAATTTTTC SEQ ID NO.15 <![CDATA[P tac -R]]> AGCTGTTTCCTGTGTGAATACCTC SEQ ID NO.16 This invention provides a method for constructing the above-mentioned engineered methanogenic bacteria that produce high levels of gemmaene A, such as... Figure 1 As shown, it includes the following steps: (1) In the host bacteria ispAL The site inserts DNA fragments via electroporation. Pc-Gm-P pqqA -zwf1-P tac -dxs1- P mxaF -ispA-dxs2(The nucleotide sequence is shown in SEQ ID NO.1 in Table 1, derived from...) [[ID= ​ Then, liquid inorganic salt culture was carried out at 25-30℃, and the supernatant was discarded by centrifugation; the bacterial precipitate was spread on solid inorganic salt medium plates containing gentamicin and cultured at 25-30℃ for 4-7 days to obtain engineered bacteria 1; (2) The engineered bacteria 1 obtained in step (1) ​ Site, DNA fragment inserted via electroporation P 12965 - ​ tac ​ (The nucleotide sequence is shown in SEQ ID NO.2 in Table 1). After that, the bacterial cells were cultured in liquid inorganic salt at 25-30℃, centrifuged and the supernatant was discarded. The bacterial cell precipitate was spread on solid inorganic salt medium plates containing bleomycin and cultured at 25-30℃ for 4-7 days to obtain the engineered methanogenic bacteria IZDA. Among them, the aforementioned ​ Derived from Bacillus subtilis ​ Its NCBI number is WP_004399098.1. Its DNA sequence is shown in Table 1, SEQ ID NO.3. (3) Insertion into pAWP89 plasmid P mxaF Promoter linking to the gene encoding gemmaene A synthase ​ Phosphofructokinase encoding gene ​ Gene encoding fructose-1,6-bisphosphate aldolase ​ Composition P mxaF - ​ (The nucleotide sequence is shown in SEQ ID NO.4 in Table 1) The expression cassette was then transferred into the engineered methanogen IZDA obtained in step (2) by conjugation transfer to obtain an engineered methanogen that produces high levels of gemmaene A.

[0027] This invention provides a method for producing gemmaene A using the aforementioned engineered methanogenic bacteria that produce high levels of gemmaene A, comprising the following steps: (1) Activation of engineered bacteria: The engineered methanogenic bacteria that produce high levels of gemmaene A constructed by the above method were inoculated onto a solid inorganic salt culture medium and cultured statically at 25-30°C for 4-5 days to obtain activated test colonies; (2) Seed culture: The activated test colonies from step (1) were inoculated into an injection bottle containing 50 mL of liquid inorganic salt medium and cultured at 25~30℃ and 200 rpm for 48 h. During this period, the upper air was replaced every 24 h and 10%~30% methane was added to obtain the seed culture solution. The methane source is a dispersed methane source, including but not limited to: gas leaks from natural gas pipelines and storage facilities, coalbed methane extraction and emissions, landfill gas, anaerobic digestion gas from sewage treatment plants, fermentation gas from livestock and poultry manure, biogenic release gas from natural environments such as paddy fields and wetlands, associated gas from combustible ice extraction, and dispersed emissions from petrochemical production processes. The dispersed methane source is characterized by dispersed emission points, low single-point flux, difficulty in collection, or significant concentration fluctuations. (3) The seed culture obtained in step (2) is inoculated into a liquid inorganic salt culture medium with a pH of 9.0 and fermented for 25~30℃. The dissolved oxygen is controlled at 20% or more. At the same time, 10%~20% of the volume of the liquid inorganic salt culture medium is added with n-dodecane and methane is introduced. The fermentation culture is carried out for 144 h to obtain gemmaene A.

[0028] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0029] In the following embodiments, the following uses ​ NG09 is a publicly available strain, and its public information can be found in: Gao Z, Liu Y, Jiao S, et al. Biological valorization of methane and nitrogen gas-derived ammonia via methanotrophic bacteria for gut-beneficial nutrients[J]. Nature Communications, 2026.

[0030] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications in the art, unless otherwise stated.

[0031] I. Construction of engineered methanogenic bacteria for high production of gemmaene A Example 1: Construction of engineered methanogenic bacteria GA02 1. Constructing DNA fragments that can be electrically converted 1) Construction P c ​ pqqA​ tac ​ mxaF ​ Excerpt Using high-fidelity DNA polymerase to extract DNA fragments P c ​ pqqA ​ tac ​ mxaF ​ Using SEQ ID NO.1 (nucleotide sequence shown in Table 1) as a template, PCR amplification was performed under the guidance of upstream primer ispAL-F (nucleotide sequence shown in Table 1, SEQ ID NO.5) and downstream primer ispAR-R (nucleotide sequence shown in Table 1, SEQ ID NO.6) to obtain electroporable [product name]. P c ​ pqqA ​ tac ​ mxaF ​ Fragments; among them, ​ , ​ , ​ and ​ All were derived from the tested bacteria ​ NG09 corresponds to the NCBI numbers WP_017839846.1, WP_017842755.1, WP_017842756.1 and WP_017841635.1.

[0032] 2) Construction P 12965 ​ tac ​ Excerpt Using high-fidelity DNA polymerase to extract DNA fragments P 12965 ​ tac ​ Using SEQ ID NO.2 (nucleotide sequence shown in Table 1) as a template, PCR amplification was performed under the guidance of upstream primer glgA1L-F (nucleotide sequence shown in Table 1, SEQ ID NO.7) and downstream primer glgA1R-R (nucleotide sequence shown in Table 1, SEQ ID NO.8) to obtain electroporable [product name missing]. P 12965 ​ tac ​ Excerpt.

[0033] 2. Constructing engineered bacteria 1 by ​ NG09 was used as the test bacterium in its ​ left homologous recombination site of the gene ( ​ (site) insertion P c ​ pqqA ​ tac ​ mxaF ​ Fragment, construct engineered bacteria 1. Specific steps are as follows: 1) Test bacteria ​ NG09 was inoculated into liquid inorganic salt medium and cultured until the logarithmic growth phase, OD 600 2; Centrifuge the bacterial culture at 4℃ and 5000×g for 10 min, and discard the supernatant; Resuspend the bacterial cells in 50 mL of cold water, centrifuge at 4℃ and 5000×g for 10 min, and discard the supernatant; Resuspend the bacterial cells in 1 mL of distilled water to obtain competent cells; 2) Take 50 μL of competent cells obtained in step 1), and add 500 ng of the cells obtained in steps 1 and 1). P c ​ pqqA - ​ tac ​ mxaF ​ Fragments and gently mix to deliver to the host bacteria. ​ ​ NG09 ​ Site Insertion Fragment P c ​ pqqA ​ tac ​ mxaF ​ Then, the mixture was transferred to a low-temperature electroporation cup with a 1 mm gap and electroporated using an electroporator (conditions set at 1.5 kV, 25 μF, and 200 Ω). The cells were then revived and cultured in 10 mL of liquid inorganic salt medium at 25–30 °C for 24 h, centrifuged at 5000 × g for 10 min at room temperature, the supernatant was discarded, and the bacterial pellet was spread onto a solid inorganic salt medium plate containing 50 μg / mL gentamicin. The plate was incubated at 25–30 °C for 4 days, and recombinants were screened. The correct recombinants were designated as engineered bacteria 1.

[0034] 3. Construction of engineered methanogenic bacteria IZDA Using engineered bacteria 1 as the test bacteria, in its​ left homologous recombination site of the gene ( ​ (site) insertion P 12965 ​ tac ​ Fragments were used to construct an engineered methanogenic bacterium, IZDA, to enhance terpene synthesis capabilities. The specific steps are as follows: Referring to the construction method of engineered bacteria 1 in step 2, the host bacteria... ​ ​ Replace NG09 with engineered bacteria 1, and replace the DNA fragment with the one obtained in steps 1 and 2). P 12965 ​ tac - ​ The fragment can be injected into engineered bacteria 1 by replacing the antibiotic in the solid inorganic salt culture medium plate with 30 μg / mL bleomycin. ​ Site insertion P 12965 ​ tac ​ Fragments; if no base mutations are found in the recombinant after PCR identification, the engineered methanogenic bacteria IZDA is obtained.

[0035] 4. Activated engineered methanogenic bacteria IZDA The engineered methanogenic bacteria IZDA obtained in step 3 was spread on a solid inorganic salt medium containing 50 μg / mL gentamicin and 30 μg / mL bleomycin and cultured for 3 days to complete the expansion culture. The engineered methanogenic bacteria IZDA on the solid inorganic salt medium was transferred to a liquid inorganic salt medium containing 50 μg / mL gentamicin and 30 μg / mL bleomycin, with a liquid volume of 50 mL. The medium was cultured at 25-30℃ and 200 rpm until the logarithmic growth phase, and the strain was preserved.

[0036] 5. Construction of an engineered methanogenic bacterium GA02 that produces high levels of gemmaene A Using the engineered methanogenic bacterium IZDA as the test strain, the methane-containing bacteria were transferred via conjugation transfer. P mxaF - ​ The recombinant plasmid pAWP89 containing the target fragment was introduced into the engineered methanogenic bacterium IZDA as the test strain to construct the engineered methanogenic bacterium GA02, which produces gemmaene A. The specific steps are as follows: 1) with P mxaF ​Using SEQ ID NO.4 (nucleotide sequence shown in Table 1) as a template, PCR amplification was performed under the guidance of upstream primer PmxaF-F (nucleotide sequence shown in Table 1, SEQ ID NO.9) and downstream primer fba2-R (nucleotide sequence shown in Table 1, SEQ ID NO.10) to obtain... P mxaF - ​ Target fragment; among which, the gene encoding gemmaene A synthase. ​ The nucleotide sequence is shown in Table 1 as SEQ ID NO.11, derived from... ​ ​ The NCBI number is WP_190566230.1; 2) Primers P89-F (nucleotide sequence shown in SEQ ID NO.12 in Table 1) and P89-R (nucleotide sequence shown in SEQ ID NO.13 in Table 1) were designed based on the pAWP89 vector sequence. The vector was amplified by PCR and then digested with restriction endonuclease DpnI at 37°C for 2 h to obtain the linearized pAWP89 vector. 3) Use the plasmid construction kit to construct the plasmid obtained in step 1). P mxaF - ​ The target fragment and the pAWP89 linearized vector obtained in step 2) were used for recombination. The recombination system contained equimolar amounts of the linearized vector and the gene fragment, and the multi-fragment recombination conditions were 50°C for 45 min. After the recombination reaction, the product was transferred to Escherichia coli S17-1 using CaCl2 transformation. ​ S17). PCR confirmation showed the band size was correct and sequencing revealed no mutations, indicating successful construction of the recombinant plasmid. A sample containing the recombinant plasmid was obtained. ​ S17; 4) Take the recombinant plasmid obtained in step 3) ​ S17 was mixed in equal proportion with the engineered methanogenic bacteria IZDA obtained after activation on plates. After culturing on a solid inorganic salt medium containing 15% (v / v) LB medium for 48 h, the mixed bacteria were spread on a solid inorganic salt medium containing 50 mg / L kanamycin. Recombinants were screened to obtain the engineered methanogenic bacteria GA02.

[0037] Comparative Example 1: Construction of engineered methanogenic bacteria GA01 1. With ​ Using SEQ ID NO. 11 (nucleotide sequence shown in Table 1) as a template, PCR amplification was performed under the guidance of upstream primer gas-F (sequence shown in SEQ ID NO. 14) and downstream primer gas-R (nucleotide sequence shown in SEQ ID NO. 15) to obtain...​ Target segment.

[0038] 2. Using vector pAWP89 as a template, the upstream primer P89-F (nucleotide sequence shown as SEQ ID NO.12 in Table 1) and the downstream primer P... tac PCR amplification was performed under the guidance of -R (nucleotide sequence shown in SEQ ID NO.16 in Table 1), followed by restriction endonuclease DpnI digestion at 37°C for 2 h to obtain the pAWP89 linearized vector.

[0039] 3. Use a plasmid construction kit to process the plasmid obtained in step 1. ​ The target fragment and the linearized pAWP89 vector obtained in step 2 were subjected to a recombination reaction. The recombination system contained equimolar amounts of... ​ The target fragment and the pAWP89 linearized vector were used for multi-fragment recombination at 50°C for 45 min. After the recombination reaction, the product was transferred to CaCl2 conversion. ​ S17. PCR confirmation showed the band size was correct and sequencing revealed no mutations, indicating successful construction of the recombinant plasmid.

[0040] 4. The recombinant plasmid was transferred into the test bacteria via parental conjugation. ​ ​ NG09, which is the engineered methanogenic bacteria GA01.

[0041] II. Production of Gemmaene A using Engineered Methanogenic Bacteria with High Gemmaene A Production 1. Activation of engineered bacteria: The engineered methane oxidizing bacteria GA02 preserved in Example 1 was inoculated onto a solid inorganic salt culture medium, while the engineered methane oxidizing bacteria GA01 constructed in Comparative Example 1 was used as a control strain. The experimental strains were statically cultured at 25~30℃ for 4~5 days to obtain the activated test colonies. 2. Seed culture: The activated test colonies from step 1 were inoculated into an injection bottle containing 50 mL of liquid inorganic salt medium and cultured at 25-30℃ and 200 rpm for 48 h. During this period, the upper air was replaced every 24 h and 10%-30% of the gas phase volume of the culture system was replenished with methane to obtain the seed culture solution. 3. Inoculate 5 mL of the seed culture obtained in step 2 into a liquid inorganic salt medium with a pH of 9.0, and culture and ferment it by introducing methane at 25-30℃ and 200 rpm, keeping the dissolved oxygen above 20. 48 h after inoculation, add 10-20% n-dodecane of the total volume of the liquid inorganic salt medium, and continue to culture and ferment at 25-30℃ and 200 rpm. Among them, the engineered methane-oxidizing bacteria GA02 in Example 1 was cultured for 144 h, and the engineered methane-oxidizing bacteria GA01 constructed in Comparative Example 1 was cultured for 72 h. 4. Take 1 mL of the cultured bacterial solution, centrifuge at 4℃ and 10000 rpm for 5 min, take the upper organic phase, and perform qualitative and quantitative analysis of gemmaene A using gas chromatography-mass spectrometry.

[0042] The gas chromatography-mass spectrometry qualitative results of gemmaene A in the engineered methanogenic bacterium GA02 of Example 1 are as follows: ​ As shown in the figure, the fermentation product is gemmaene A; the yield and growth are as follows. ​ As shown, gemmaene A began to accumulate significantly from the 24th hour after the start of production, reaching its peak yield at 144 hours, ultimately achieving a gram-level production. The strain exhibited vigorous growth from 0 to 72 hours, ceasing growth after 72 hours. The results comparing the gemmaene A production of the strains in Example 1 and Comparative Example 1 are as follows... ​ As shown, the engineered methanogenic bacterium GA01 in Comparative Example 1 produced 22.35 mg / L of gemmaene A. However, after metabolic engineering of methanogenic bacteria to obtain an engineered methanogenic bacterium, and applying it to gemmaene A production, the gemmaene A yield increased to over 200 times that of engineered methanogenic bacterium GA01, reaching gram-level yields. These results indicate that the modified engineered methanogenic bacterium GA02 can significantly improve the yield of gemmaene A synthesized using methane as a carbon source.

[0043] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of this invention.

Claims

1. An engineered methanogenic bacterium that produces high levels of gemmaene A, characterized in that, Using methanogenic bacteria as the host bacteria, the gene encoding gemmaene A synthase was introduced into the host bacteria. gas To construct the gemmaene A synthesis pathway, the gene encoding 1-deoxy-xylulose-5-phosphate synthase was overexpressed using an endogenous strong promoter. dxs1 , dxs2 Farnesyl pyrophosphate synthase encoding gene ispA Isopentenyl pyrophosphate isomerase encoding gene Bsidi To enhance metabolic flux, while overexpressing the gene encoding glucose-6-phosphate dehydrogenase. zwf1 To enhance reducing power, phosphofructokinase is overexpressed. pfk1 fructose-1,6-bisphosphate aldolase fba2, An engineered methanogenic bacterium that produces high levels of gemmaene A was obtained.

2. The engineered methanogenic bacteria with high gemmaene A production according to claim 1, characterized in that, The methanogenic bacteria are Methylotuvimicrobium buryatense 5GB1 Alkalicoccus glycogenes WONF2802 or Methylotuvibium sanxanigenens Both NG09 and methanogenic bacteria contain homologous recombination insertion sites in their genomes. ispAL and glgA1L Site.

3. The engineered methanogenic bacteria with high gemmaene A production according to claim 1, characterized in that, The gene encoding gemmaene A synthase gas Source Nostoc parmelioides The NCBI number is WP_190566230.

1.

4. The engineered methanogenic bacteria with high gemmaene A production according to claim 1, characterized in that, 1-Deoxy-xylulose-5-phosphate synthase encoding gene dxs1 , dxs2 Farnesyl pyrophosphate synthase encoding gene ispA The gene encoding glucose-6-phosphate dehydrogenase zwf1 Phosphofructokinase pfk1 fructose-1,6-bisphosphate aldolase fba2 All of them were derived from methanogenic bacteria, with NCBI numbers WP_017839846.1, WP_017842755.1, WP_017842756.1, WP_017841635.1, WP_341326251.1 and WP_341326872.1, respectively.

5. The engineered methanogenic bacteria with high gemmaene A production according to claim 1, characterized in that, Isopentenyl pyrophosphate isomerase encoding gene Bsidi It is derived from Bacillus subtilis, NCBI number WP_004399098.

1.

6. The method for constructing the engineered methanogenic bacteria with high gemmaene A production according to any one of claims 1 to 5, characterized in that, Includes the following steps: 1) To the host bacteria ispAL DNA fragments transferred to the site Pc-Gm-P pqqA -zwf1-P tac -dxs1-P mxaF -ispA-dxs2 Engineered bacteria 1 was obtained; 2) Add the engineered bacteria 1 obtained in step 1) glgA1L Insertion site DNA fragment P 12965 -Bsidi-P tac -Bleo The engineered methanogenic bacteria IZDA were obtained. 3) Insertion into pAWP89 plasmid P mxaF Promoter linking to the gene encoding gemmaene A synthase gas Composition P mxaF - gas- pfk1-fba2 The expression cassette was then transferred into the engineered methanogenic bacteria IZDA to obtain an engineered methanogenic bacteria that produces high levels of gemmaene A. in, Pc-Gm-P pqqA -zwf1-P tac -dxs1-P mxaF -ispA-dxs2 The nucleotide sequence is shown in SEQ ID NO.

1. P 12965 -Bsidi-P tac -Bleo The nucleotide sequence is shown in SEQ ID NO.

2. P mxaF - gas-pfk1-fba2 The nucleotide sequence is shown in SEQ ID NO.

4.

7. The application of the engineered methanogenic bacteria with high gemmaene A production as described in any one of claims 1 to 5 in the production of gemmaene A.

8. A method for producing gemmaene A, characterized in that, The seed culture of the engineered methanogenic bacteria that produces high levels of gemmaene A according to any one of claims 1 to 5 is inoculated into a liquid inorganic salt culture medium and fermented under conditions containing methane to obtain gemmaene A.

9. A method for producing gemmaene A according to claim 8, characterized in that, During fermentation, n-dodecane is added at 10% to 20% of the volume of liquid inorganic salt culture medium.

10. A method for producing gemmaene A according to claim 9, characterized in that, The fermentation temperature is 25~30℃.

Images