Engineered methanotroph with high squalene production and construction method and application thereof

Abstract

This invention discloses an engineered methanogenic bacterium that produces high levels of squalene, its construction method, and its applications, belonging to the fields of metabolic engineering and synthetic biology. This is achieved by using a strong promoter in the host bacterium (methanogenic bacterium). P mxaF Expression of squalene synthase encoding gene sqs Overexpression of the gene encoding 1-deoxy-xylulose-5-phosphate synthase in the squalene synthesis pathway dxs1 and dxs2 Farnesyl pyrophosphate synthase encoding gene ispA Isopentenyl pyrophosphate isomerase encoding gene My friends Simultaneously overexpressing glucose-6-phosphate dehydrogenase zwf1 and the EMP pathway pfk1 and fba2 An engineered methanogenic bacterium producing squalene was obtained. Using this engineered methanogenic bacterium for squalene fermentation production can significantly increase squalene yield and has excellent prospects for industrial application.

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 squalene, their construction methods, and applications. Background Technology

[0002] Squalene, also known as tricarboxyhexaene (chemical formula C360), is a squalene-3- 30 H 50 Squalene (Squalene) is a polyunsaturated hydrocarbon produced during cholesterol synthesis and other metabolic processes. It contains six isoprene double bonds and belongs to the terpenoid class of compounds. Squalene is a key intermediate in cholesterol synthesis and possesses various physiological functions, including strong antioxidant and anticancer activity. It can inhibit cancer cells, treat leukopenia caused by radiotherapy and chemotherapy, improve hypoxia in cardiovascular and cerebrovascular diseases, enhance the body's immunity, and has anti-aging effects. It is widely used in medicine, food, cosmetics, and other fields, serving as a moisturizer in cosmetics and a lubricant in food processing machinery. Market demand remains strong.

[0003] Currently, existing methods for synthesizing squalene are mainly divided into three categories: natural extraction, chemical synthesis, and biosynthesis. Natural extraction is the traditional method, initially extracted from deep-sea shark liver oil, and later expanded to include plant-based raw materials such as deodorized olive oil distillate and deodorized soybean oil distillate. Squalene is obtained through organic solvent extraction and freeze crystallization. However, this method is limited by the availability of raw materials. Deep-sea shark resources are increasingly scarce, and the squalene content in plant-based raw materials is low (often less than 5% of the unsaponifiable matter in vegetable oils). Furthermore, the extraction process requires complex separation and purification steps, resulting in high costs and making it difficult to meet the needs of large-scale production. In addition, deep-sea shark extraction also presents serious ecological protection problems. Since the first successful synthesis of squalene in 1931, chemical synthesis has developed various process routes. Representative methods include synthesis from succinaldehyde via Claisen rearrangement and Wittig reaction, and synthesis from geranylacetone via coupling reaction. However, existing chemical synthesis routes generally suffer from problems such as excessively long routes, harsh reaction conditions (e.g., requiring extremely low temperatures), use of expensive reagents or flammable and explosive raw materials, risks of polymerization explosions, numerous byproducts, and unstable yields, hindering industrial-scale production. Some processes also generate environmental pollution. Biosynthesis has become a research hotspot in recent years. Its core is the conversion of acetyl-CoA into squalene through enzymatic reactions. It is mainly divided into the mevalonate pathway (MVA pathway, primarily in eukaryotes) and the methyl erythritol phosphate pathway (MEP pathway, primarily in prokaryotes). Squalene yield can be increased by modifying microbial strains and optimizing enzyme activity. However, this method faces bottlenecks such as limited enzyme sources, difficulty in regulating strain metabolism, low synergistic efficiency, and difficulty in industrialization, and has not yet achieved large-scale industrial application.

[0004] Methane (CH4) is a colorless, odorless, and flammable gas. It is a major component of natural gas, biogas, and pit gas, and is abundant in nature. It is an inexpensive and widely available renewable carbon source, as well as an important chemical raw material and fuel. It can be used to produce various chemical products such as acetylene, hydrogen, and syngas, and has wide applications in the chemical and energy sectors. Furthermore, methane emissions exacerbate the greenhouse effect; its rational utilization can not only reduce environmental pollution risks but also achieve efficient resource conversion, possessing significant economic value and environmental significance.

[0005] Based on the resource advantages of methane and the market demand for squalene, researchers have developed methods for preparing squalene from methane in recent years. Currently, the mainstream technology is the metabolic engineering of methanogenic bacteria, using type I methanogenic bacteria such as *Methylmonas DH-1* as host strains. This method leverages the inherent MEP pathway and hopane pathway of these bacteria to naturally synthesize squalene, and through genome engineering, constructs highly efficient engineered strains to achieve the conversion of methane to squalene. However, this method still faces the following problems: 1) Low yield. When producing squalene using this method, due to the existence of a diversion pathway in the methanogenic bacteria's MEP pathway, only a small portion of the carbon flux reaches squalene, and a large amount of byproducts are produced, resulting in carbon loss; 2) Stringent production requirements. The MEP pathway for squalene synthesis involves eight different enzymes, and each synthesized squalene molecule requires 18 NADPH molecules, posing challenges to the supply of carbon flux and reducing equivalent. Therefore, it is urgent to improve the carbon yield and productivity of long-chain terpenoids in methane and to develop corresponding high-yield fermentation processes. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, the present invention aims to provide engineered methanogenic bacteria with high squalene production, their construction method and application, so as to solve the technical problems of low yield and demanding production requirements of current squalene production methods.

[0007] To achieve the above objectives, the present invention employs the following technical solution: In a first aspect, the present invention discloses an engineered methanogenic bacterium that produces high levels of squalene, using the methanogenic bacterium as a host bacterium and introducing a squalene synthase encoding gene into the host bacterium. sqs To construct a squalene 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, the gene encoding phosphofructokinase is overexpressed. pfk1Gene encoding fructose-1,6-bisphosphate aldolase fba2 An engineered methanogenic bacterium that produces high levels of squalene was obtained.

[0008] Preferably, the genomes of the methanogenic bacteria all contain... ispAL and glgA1L Site.

[0009] Preferably, the methanogenic bacteria are Methylotuvimicrobium buryatense 5GB1 Alkalicoccus glycogenes WONF2802 or Methylotuvibium sanxanigenens NG09.

[0010] Preferably, the squalene synthase encoding gene sqs Source Saccharomyces cerevisiae S288C, 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 encoding gene pfk1 Gene encoding fructose-1,6-bisphosphate aldolase fba2 All are derived from methanogenic bacteria, and the isopentenyl pyrophosphate isomerase encoding gene. Bsidi It originates from Bacillus subtilis.

[0011] A second aspect of the present invention discloses a method for constructing the above-mentioned engineered methanogenic bacteria that produce high levels of squalene, 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 squalene synthase encoding gene sqs Composition P mxaF - sqs-pfk1-fba2The expression cassette was then transferred into the engineered methanogenic bacteria IZDA to obtain squalene-producing engineered methanogenic bacteria; 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 - sqs-pfk1-fba2 The nucleotide sequence is shown in SEQ ID NO.4.

[0012] Preferably, the host bacteria and DNA fragments Pc-Gm-P pqqA -zwf1-P tac -dxs1-P mxaF -ispA-dxs2 The dosage ratio of engineered bacteria 1 to DNA fragments P 12965 -Bsidi-P tac -Bleo The dosage ratio was 1 μL:10 ng.

[0013] Preferably, in step 3), pAWP89 plasmid in an equimolar ratio is used with... P mxaF Promoter linking to squalene synthase encoding gene sqs Composition P mxaF - sqs-pfk1-fba2 The expression cassette undergoes a recombination reaction.

[0014] A third aspect of the present invention discloses the application of the above-mentioned engineered methanogenic bacteria with high squalene production in the production of squalene.

[0015] A fourth aspect of the present invention discloses a method for producing squalene, comprising the steps of culturing the aforementioned engineered methanogenic bacteria that produce high levels of squalene, and collecting the squalene produced.

[0016] Preferably, the seed culture of engineered methanogenic bacteria that produce high levels of squalene is inoculated into a liquid inorganic salt culture medium and fermented under conditions containing n-dodecane and methane to obtain squalene.

[0017] Preferably, the seed culture of the engineered methanogenic bacteria that produce high squalene is prepared by inoculating the activated colonies of the engineered methanogenic bacteria that produce high squalene into a liquid inorganic salt culture medium and culturing it at 25-30°C for 48 h, during which the upper air is renewed and 10%-30% methane is added every 24 h to obtain the seed culture.

[0018] Preferably, the fermentation culture temperature is 25~30℃.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The engineered methanogenic bacteria that produce high levels of squalene provided by this invention utilizes a strong promoter in the host bacterium, methanogenic bacteria. P mxaF Expression of squalene synthase encoding gene sqs This was achieved through the biotransformation of methane into squalene. 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 basilyl 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 reduced energy required for the MEP pathway, the supply of NADPH is increased, thereby synergistically strengthening both material flow and reduced energy. This is achieved by overexpressing the phosphofructokinase encoding gene in the EMP pathway. pfk1 Gene encoding fructose-1,6-bisphosphate aldolase fba2 This led to the development of an engineered methanogenic bacterium that produces squalene. Compared to the original methanogenic bacterium strain, this high-yield engineered methanogenic bacterium significantly increased squalene production, opening up new raw materials for squalene production and providing a reference for the development of biotransformation platforms for squalene production 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.

[0020] The present invention provides a method for producing squalene, which utilizes engineered methanogenic bacteria that produce high squalene, effectively utilizing methane, promoting the biosynthesis of terpenoids, and significantly increasing the yield of squalene. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the method for constructing the engineered methanogenic bacteria that produce high levels of squalene according to the present invention; Figure 2This is a gas chromatography-mass spectrometry qualitative result of the engineered methanogenic bacteria that produces high levels of squalene according to the present invention. Figure 3 This is a graph showing the yield growth of squalene obtained by the engineered methanogenic bacteria SQ02, which produces high levels of squalene, according to the present invention. Figure 4 This is a graph showing the cell dry weight growth of the engineered methanogenic bacterium SQ02, which produces high levels of squalene, in Example 1 of this invention. Figure 5 This is a comparison diagram of the squalene production of engineered methanogenic bacteria SQ02 (high squalene production) in Example 1 of the present invention and engineered methanogenic bacteria SQ01 (comparative example 1). Detailed Implementation

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.”

[0026] 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.

[0027] Based on the teachings of this invention and common knowledge in the field, those skilled in the art will know and can utilize genes from various sources to construct the above-mentioned engineered methanogenic bacteria.

[0028] In this article, the squalene synthase encoding gene is described. sqs , originating from Saccharomyces cerevisiae S288C, NCBI number NP_012060.1; the gene encoding the 1-deoxy-xylulose-5-phosphate synthase. dxs1 1-Deoxy-xylulose-5-phosphate synthase encoding gene dxs2 Farnesyl pyrophosphate synthase encoding gene ispA 6-phosphate glucose dehydrogenase encoding gene, phosphofructokinase encoding gene pfk1 genes encoding fructose-1 and 6-bisphosphate aldolases fba2 All are derived from methanogenic bacteria, and the isopentenyl pyrophosphate isomerase encoding gene. Bsidi It originates from Bacillus subtilis.

[0029] The culture medium formulations used in this paper are as follows: Liquid inorganic salt medium formulation: 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. Sterilization was performed at 121°C for 20 min. Solid inorganic salt medium formulation: (10–15) g / L agar was added to the liquid inorganic salt medium. LB medium formulation: 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, adjusted to pH 7.0.

[0030] In this article, methanogenic bacteria include, but are not limited to, […]. Methylotuvimicrobium buryatense 5GB1 Alkalicoccus glycogenes WONF2802 or Methylotuvibium sanxanigenens NG09, the genomes of the methanogenic bacteria all contain simultaneously ispAL ( ispA (Left-side homologous recombination site of the gene) and glgA1L site ( glgA1 (Left-side homologous recombination site of the gene). Used in this invention Alkalicoccus glycogenes WONF2802 and Methylotuvibium sanxanigenens All NG09 strains have been deposited at the China General Microbiological Culture Collection Center (CGMCC), with preservation numbers CGMCC No. 28614 and CGMCC No. 30951, respectively.

[0031] In this article, the methane referred to is a dispersed methane source, which includes, but is not limited to: gas leaking from natural gas pipelines and storage facilities, coalbed methane extraction and emission gas, 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 emission gas 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.

[0032] This invention provides an engineered methanogenic bacterium that produces high levels of squalene, using the methanogenic bacterium as the host bacterium, and expressing a squalene synthase-encoding gene in the host bacterium. sqs 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 The isopentenyl pyrophosphate isomerase encoding gene from Bacillus subtilis Bsidi Phosphofructokinase encoding gene pfk1 Gene encoding fructose-1,6-bisphosphate aldolase fba2 Obtained. In this genetically engineered bacterium, sqs Encodes squalene synthase, which catalyzes the conversion of farnesyl pyrophosphate (FPP) to squalene; 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.

[0033] Table 1 Nucleotide sequence list Name Sequence (5’-3’) Serial number <![CDATA[ Pc-Gm- P pqqA -zwf1- P tac -dxs1- P mxaF -ispA- dxs2 ]]> CTTCGACACCGGGAATACCCTGCTCGGGATGAATATCGACTGGCATTCCTCGACCATTGTCGCTAACTAATACAGAACCGTCTTTATATAATACGACAGTAATATTGTCGGCATGCCCTGCTATCGCTTCGTCGACGCTGTTGTCGACAACCTCTTGCACCAGATGGTTCGGTCGGGTCGTATCGGTATACATACCGGGCCGTTTCCGAACAGGTTCCAAGCCGCTTAATACCTCTATCGCGGCTGCATTGTAATCATTACTCATACTTCCTAATCACTCATTATCCAAATTCATATATAATGGCCGGTTTGATTCCATCGCCCAATTTCTACGTACTATTTTATCTACATGCCGAGAAAAAAAACCACGAATTTATTCGAAGAGTCCCTAGCCGAGCTTGAGCAACTCGTCGAGCAAATGGAACAAGGTGAGCTATCTTTAGAAGATTCGTTAAAATCATTTGAACGCGGCGTCGCTTTAACCCGAACCTGCCAGAAAGCCTTGCAGGAAGCCGAACAGAAAGTTCAGATTTTACTGGAAAAAAACGGCACTCAAACTCTGGAGCCCTTCACCGATGAGTAATTGACATAAGCCTGTTCGGTTCGTAAACTGTAATGCAAGTAGCGTATGCGCTCACGCAACTGGTCCAGAACCTTGACCGAACGCAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAGGTGGCTCAAGTATGGGCATCATTCGCACATGTAGGCTCGGCCCTGACCAAGTCAAATCCATGCGGGCTGCTCTTGATCTTTTCGGTCGTGAGTTCGGAGACGTAGCCACCTACTCCCAACATCAGCCGGACTCCGATTACCTCGGGAACTTGCTCCGTAGTAAGACATTCATCGCGCTTGCTGCCTTCGACCAAGAAGCGGTTGTTGGCGCTCTCGCGGCTTACGTTCTGCCCAGGTTTGAGCAGCCGCGTAGTGAGATCTATATCTATGATCTCGCAGTCTCCGGCGAGCACCGGAGGCAGGGCATTGCCACCGCGCTCATCAATCTCCTCAAGCATGAGGCCAACGCGCTTGGTGCTTATGTGATCTACGTGCAAGCAGATTACGGTGACGATCCCGCAGTGGCTCTCTATACAAAGTTGGGCATACGGGAAGAAGTGATGCACTTTGATATCGACCCAAGTACCGCCACCTAACTTGATTTGTCGCGACTTATACCCCTCTTAGATCAAAATTCGACGCCGGGCTGTCCGTCAAAGGGCGCCGTGAATACATCCCTATAGGCTCTATGCCGGCTCTATGCTGGCAAAGCCTTTGCCGAAAACCCCTGCGCCTCCTCAGGCGCTGCCGAAATTTGAAGTCCGAAAGGTATAAAGCCCAGCCCGATCGCTAAACCAATCGGTTGCTTTGCCTAAATTATCGTCGTATACTTCCGACCTAGCTTACTTGATCAAGCTGGCATAACACTTATAAACATACTTCTGGAGGTATTGATTATGTCTGCTGAACCCTGTACCTATGTCATTTTCGGAGCAACCGGCAATCTATCCCGAATTAAACTGATGCCGGCACTCTATCATTTGGAACTCGAAAACAAACTGCCGGAAGGCAGTCGTATTATCGGCATCGGCCGAAGGCCTTGGGATCAAAAAAAATGGCTAGAAGAGATCCGAGAGATGATCGGCACGAAGGTCAGTGAAGGTATCGATGAGGCCGTATTCGCACGGTTCAGCGAACGGCTTTCCTACCATCGAGGCAATCTCGATGAAGCTGAATGCTATAGAGGTCTGGCAGTTACCTTGAGCAAGAATGAGGATTTTCCCAAAAACATCGCATTTTATCTCGCGATCAGTCCTGAGGATTTCGGTAATGTCATCGAGTCGCTTAGCAAAGTCGAATTGTTGAATCAAGAATACGGCTGGAAACGCGTTATCATCGAAAAGCCTTTCGGTTACGACCTGGACAGCGCGCAATCGTTGCAAAAACGGATCGGACGCTTTTTAAGCG AAGAACAGATCTACCGTATCGATCATTATCTAGGCAAAGGCATGGTGCAGAATGTATTGGTATTCCGCTTCGCGAATGTCATGCTAGAGCCGCTGTGGAACCGCAACTACATAGACCATGTTCAGATTACCCATGCCGAAGATATCGGTATCGACACGCGCGGCGGCTATTACGATGGGGCAGGAGCTTTGCGCGACATGCTGCAAAGCCATTTACTACAACTGATGACACTGGTTGCGATGGAACCGCCCGCGTCGATGGAAGCGGAATCCTTGCGCGACGAGAAGGTCAAGGTCTTAAAATCGATTCGCTCAATTCCCAAATCGGCAGTACACGCGCATGCCTATCGCGGCCAATACGCCAAAGGCACGATCGGCAAGGAAAAGGTAAAGGGTTATTTGGAGGAAGAGAACATTCCGCCCAATAGCATTACAGAAACCTATGCTGCCGTGAAATTATTCATCGATAACTGGCGCTGGCGGGGCGTGCCGTTTTATCTACAGACCGGTAAACGACTGGCCAAAGGGCAATCGGTCGTCTCGATTTGCTTTCGTCATCCGCCGCTGCAGTTTTTTCGCGATACGCATGTGCAATGCATGAATCCGAATTGGGTGCTGTTGAGCATTCAACCCGAAGAACGCATTCGCATGGAAATGACCGTCAAAGAACCGGGCCTGGAAATGCGCACACGGACCAGCAGCCTGGATGCCGGTTTTCGAAACAGCGATGAAAAAGCGATCGACGCCTATGAAGATTTATTGCTCGATGTCATGAAAGGTGACAGCTCGTTGTTCCTGCGCTTCGATGAAGTCGAATATGCTTGGCGCATCGTCGACCCGATTCTGCAAACCTGGGCCGTAGAGCGCGACTTCATCCCGACTTATCCGGCCGGCAGTTGGGGCCCCGCCGAAAGCCGCCGTTTGTTCGAAAAAGAAGATCAATTCTGGCGTACCTCGTTGACGCCGGAGTGGTGCCAATAGCTCTGAAATGAGCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAGGTATTCACACAGGAAACAGCTATGAACCTAGCTAACGAGTATCCATTACTGAGCCTGATCGATAAACCGGCGGATTTACGAAAACTGACCAAAGCCAATCTGATTCCGCTTGCCAAAGAACTTCGCGAGTTTCTGACCCACACGGTCAGTATTTCCGGCGGGCATTTTTCGGCAGGACTCGGTACCGTGGAGTTGACCGTGGCACTGCATTACGTATTCGATACGCCGCGCGATCAATTGGTTTGGGATGTCGGCCATCAGGCTTATCCGCATAAGATCCTGACCGGACGCAAGGAGCGCATGACGACGATTCGCACGCGCGACGGCATTTGCGCGTTTCCGAATCGTTCCGAGAGCGAATACGATGCCTTCGGCGTCGGCCATTCGAGCACGTCGATCAGCGCGGCCTTAGGCATGGCGATCGCATCCGGTTTGCGCGGCGAGGACAAGCATTGCGTCGCGATTATCGGCGATGGCAGCATCACCGGCGGCATGGCCTTCGAAGCGATGAATCATGCCGGTGCGATCGACGCGAATTTGTTGGTGATCTTGAACGATAACGATATGTCGATTTCGCCGAATGTCGGCGCGTTGAATAATTATCTGACTAAGATTCTGTCGAGTAAGATTTATTCTTCGGTACGCGAAGAAAGCAAGAAGGCTCTCAGCAGCATGCCGAGCGTCTGGGAGCTGGCGCGCAAGACCGAGGAGCACATGAAAGGCATGATCGTGCCGGGCACCTTGTTCGAGGAAATGGGTTTCAATTATATCGGTCCGATCGACGGCCACGATCTGGACATGCTGGTGTCGACGCTGGAGAATCTGAAACACATTTCCGGCCCGCGTTTCTTGCATATCGTCACCAAGAAAGGCAAGGGCTATGCGCCGGCCGAGAAAGACCCGCTCGCCTATCACGGCGTACCGGCCTTCGACCCGAGCCGCGATTGCCTGCCGAAATCGGCCCCTTCTCCGCACCCGACGTATACGCAGGTATTCGGAGAATGGCTCTGCGACATGGCCGAGCAGGACGAGCGCTTATTGGGCATTACCCCGGCGATGCGCGAAGGCTCGGGCCTGGTCGCGTTTTCCGAACGCTTTCCGAAGCGCTATTTCG ATGTCGCTATCGCCGAGCAGCATGCCGTAACCTTAGCGGCGGGCCTGGCCTGCGAGGGCGGCAAACCGGTCGTCGCGATCTATTCAACCTTCTTACAACGCGGCTACGATCAATTGATACACGATGTGGTCTTGCAGGACCTGGACGTATTGTTTGCGCTCGACCGCGCAGGTTTAGTCGGCCCGGACGGCCCGACCCATGCCGGCAGTTTCGATTACACCTACATGCGCTGCCTGCCGAACATGCTGATCATGGCGCCTGCCGATGAAAACGAATGCCGGCAGATGCTCTATACCGGTTACATGCATAAGGGTCCAGCCTCGGTACGCTATCCGCGCGGCAAAGGCCCGGGCGTGCCGGTCGATTGCACCATGACCGCGCTACCGATCGGCAAGGCCGAATTGCGCCATCAAGGCGGTCGCATCGCGATTCTGGCGTTCGGCAGCCTGGTGGCTCCGTCGGTCGAGGCCGGCAAGCAACTCGGCGCGACCGTAGTCAACATGCGTTTCGTCAAGCCGATCGATGAAGCATTGATTCTGGAACTGGCCAAGAGTCACGAAGTCATCGTCACGGTCGAGGAAAACGTCGTCGCCGGCGGCGCCGGCAGCGCAGTCAACGAATTTCTGCAGGCGCAAAGAATCGTGATGCCGGTGCTCAACATCGGCCTGCCCGACGCCTTCATCGAACAAGGCACGCGCGAGGAATTGCTGAGTCTGTGCGGCTTGGACTCACAAGGGATTCTGCAAGGCATCGAGCAGTTTTGCGGATAAAATTAAACCGGGAATGATGTCGGATATTTAACGGCAAAGCCATGGGAGCTTTTCCCGAATTTGAATGCCGACATACTCTCGGGATATTTTCCCTGTTTTTTCTTAGCGCTTTTCCCGTCATCTGGGTGCTGTATTCCGTAACGTCGCATCCCGCTCCTTCCGTATGATTACCGTCCGTGCGCTGCCCTCTATGAATGATTCGTTATGCGCCTTGATCAAGCTAAGCCGGTTGTAACAACAAACACCGCAATCAATAGGGGGCCGCGCCGACATTATGCGAAAAATCAATCTGGAGGAATTATGAGTAACGCACTGAAAGACTATCTGACCTTTTGTCAAAACCGAGTCGAAAGAGCCTTGGAAGCCCGACTGCCAAGCGAAACCCAAATACCGGCTAAATTGCACGAAGCGATGCGCTATTGCGTGTTGGACGGCGGTAAACGCATGCGTCCGATGCTAACCTACTGTACAGGAAAAGCCTTGGGCATTGCACCGGAAGATTTAGACGGAGCGGCCTGTGCGGTTGAATTCATTCATGTTTATTCATTGATACATGACGATTTGCCGGCCATGGACGACGACGATCTCAGACGCGGAAAACCGACCTGCCATATCGCTTATGATGAAGCGACCGCCATTCTGACCGGCGACGCACTACAAGCACTGGCATTCAAGGTCTTGGCGGACGACCCGACCATCCAAGCCGATGCCGAAAGCCGTCTAAAAATGATTACGATGCTGGCCAAAGCTAGCGGCTCTCAAGGCATGGTCGGCGGTCAAGCCATCGATTTAGAATCGGTCGGCACAATGCTGACGCTGCCTCAGCTTGAAAATATGCATATCCACAAGACCGGCGCGTTAATCCGAGCCAGCGTCAACATGGCAACATTAACCAAGCCCGATATCGACCCGAAACAAGCCGAAGGCCTCGATCATTACGCAAAATGCATCGGCCTATCCTTCCAAGTCAAGGACGATATTTTGGACGAAGAGAGCGATACTGCAACACTCGGCAAAACCCAAGGCAAGGACAAGGACAACGACAAGCCGACTTACCCTGCCTTACTCGGTTTGGCCGGGGCAAAACAAAAAGCCCAGGAACTTCATGAGCAAGCCATCGAAAGCTTGAGCGGATTTGGGTCCGAGGCCGACTTGCTACGCGACTTGTCGCTTTATATTATTCAGCGGGATCATTAACTCCCGACTTGAACACTAGGATATTAATCTGCATAATCAGTCGGTAACCGCGTCCGTGCCGATCAATAAGGAATCGATTTATTCATGAAAATCACAGGAAACTTCCCCATACTAGACAGTATCAAGCTCCCCTCCGACCTAAGAAAACTGCCGAAGGAACGATTAAAACCGCTTGCCA AAGAACTTCGCGAGTTTCTGACCCACACGGTCAGTATTTCCGGCGGGCATTTTTCGGCAGGCCTCGGTACCGTGGAGTTGACCGTGGCACTGCATTACGTATTCGATACGCCGCGCGATCAATTGGTTTGGGATGTCGGCCATCAGGCTTATCCGCATAAGATCCTGACCGGACGCAAGGAGCGCATGACGACGATTCGCACGCGCGACGGCATTTGCGCGTTTCCGAACCGTTCCGAGAGCGAATACGATGCCTTCGGCGTCGGCCATTCGAGCACGTCGATCAGCGCGGCCTTAGGCATGGCGATCGCATCCGGTTTGCGCGGCGAGGACAAGCATTGCGTCGCGATCATCGGCGATGGCAGCATCACCGGCGGCATGGCCTTCGAAGCGATGAATCATGCCGGTGCGATCGACGCGAATTTGTTGGTGATCTTGAACGATAACGATATGTCGATTTCGCCGAATGTCGGCGCGTTGAATAATTATCTGACTAAGATTCTGTCGAGTAAGATTTATTCTTCGGTACGCGAAGAAAGCAAGAAGGTCCTCAGCAGCATGCCGAGCGTCTGGGAACTGGCGCGCAAGACCGAGGAGCACATGAAAGGCATGATCGTGCCGGGCACCTTGTTCGAGGAAATGGGTTTCAATTATATCGGTCCGATCGACGGCCACGATCTGGACATGCTGGTGTCGACGCTGGAGAATCTGAAACACATTTCCGGCCCGCGTTTCTTGCATATCGTCACCAAGAAAGGCAAGGGCTATGCGCCGGCCGAGAAAGACCCGCTCGCCTATCACGGCGTACCGGCCTTCGACCCGAGCCGCGATTGCCTGCCGAAATCGGCCCCTTCTCCGCACCCGACGTATACGCAGGTATTCGGAGAATGGCTCTGCGACATGGCCGAGCAGGACGAGCGCTTATTGGGCATTACCCCGGCGATGCGCGAAGGCTCGGGCCTGGTCGCGTTTTCCGAACGCTTTCCGAAGCGCTATTTCGATGTCGCTATCGCCGAGCAGCATGCCGTAACCTTAGCGGCGGGCCTGGCCTGCGAGGGCGGCAAACCGGTCGTCGCGATCTATTCAACCTTCTTACAACGCGGCTACGATCAATTGATACACGATGTGGTCTTGCAGGACCTGGACGTATTGTTTGCGCTCGACCGCGCAGGCTTAGTCGGCCCGGACGGCCCGACCCATGCAGGCAGTTTCGATTACACCTACATGCGCTGCCTGCCGAACATGCTGATCATGGCACCGGCCGACGAGAACGAATGCCGGCAGATGCTCTATACCGGTTATATTCATAAAGGCCCGGCTTCGGTCCGCTATCCACGCGGCAAAGGCCCGGGCGTGCCGGTCGATTGCACCATGACCGCACTGCCGATCGGCAAGGCCGAATTGCGCCATCAAGGCGGTCGCATCGCGATTCTGGCGTTCGGCAGCCTGGTGGCTCCGTCGGTCGAGGCCGGCAAGCAACTCGGCGCGACCGTAGTCAACATGCGTTTCATCAAGCCGATCGATGAAGCATTGATTCTGGAACTGGCCAAGAGTCACGACATTATCGTCACGGTCGAGGAAAACGTCGTCGCCGGCGGCGCCGGCAGCGCGGTCAACGAATTTCTGCAGGCGCAGAGAATCGTGATGCCGGTCTTGAATATCGGCCTGCCCGATGCCTTTATCGAACAAGGCACGCGCGAGGAATTACTGAGTTTTTGCGGATTGGATACACAAGGCATATTGCAGAGCATCGAGCAATTTTGCGCGTGA SEQ ID NO.1 <![CDATA[ P 12965 - Bsidi- P tac - Bleo ]]> GTTCGGGCAAGGTACAGAGTGGAATTTCAACCAAGCCTTGGATTGGTATGTATTGCAGCACCCTAGACACCAAGGTTTACATACTCTGGTCAAAGATCTTAATCATTTGTATAAAAACCACCCGGCACTTCACCAATACGACTTCAATCACAGCGGCTTTGACTGGATCGATTGCCATGATGTCGAACAATCGATTATCAGCTACCGCCGCAAAGGCACCAACGATGATTTGATCATCATACTTAACTTCACACCGATTGTTAGAGAAAACTATCATATCGGCGTGCCTTTCGAAGGGGTTTATTTTGAAATCTTTAATTCAGACTCTGCCTATTATGAAGGCAGCAATGTCGGCAACCGCGAGATACTATCGGAACCGGAGCCTTGGATGGGGCACCAGCAATCGATACACTTGACTTTACCTCCGTTAGGCGGAATCATCTTGACCCGGCAAACGAAAACCATAAACAGCTCTGCTCACTAATATAGGGACCTGCTTGAGAAAGTGAGCCCAGCGTGCCGTTGCGCACCGCAAATTAACTGACGACGGCCTAGCTTATAAAAAATTCGCTTCGATATTTATACGTAATAGGCGCAACTACGCGAGAAATGCCCGCAAAATAGAACTACCGCCGCCCCGAACGCAAACTTAAACTGATTTTTCAATAATGTCGGACGATCCTACTACAGCAGACAGTGAGCCAATAAACACTCAATCGGCTATTTTGGCGAAATCCACCTCGGTAAACTCAATTCTTTAATATACCTAGAACCGAATTTTGACTGCCTTTAAATTTAATTTGTCGAAGCGCACCAAAGACATGGTATTCCTCCTCAGAATACTCGCTTGGGAGGGCAAATCGGCATTCACGGCTTGGGGCGTGGCGATGAGAAAATTCATAGAACCATGAATTGGACCCATGGCTGTATTGCATTGACTAATAAGCAAATTGATCTTTTAAGTAAGTGGGTTGATAAAGAAATTACAGTGCAGATAAAATAGTTCTGGAATATTTTGAAAAAAAAGTTCAATATATTTCATCGAATGCTTTTGAAAATAATCTTAGTAACAACTCTTACAGTAAAAAAAGGAAAAATAATATGACTCGAGCAGAACGAAAAAGACAACACATCAATCATGCCTTGTCCATCGGCCAGAAGCGGGAAACAGGTCTTGATGATATTACGTTTGTTCACGTCAGTCTGCCCGATCTTGCATTAGAACAAGTAGATATTTCCACAAAAATCGGCGAACTTTCAAGCAGTTCGCCGATTTTTATCAATGCAATGACTGGCGGCGGCGGAAAACTTACATATGAGATTAATAAATCGCTTGCGCGAGCGGCTTCTCAGGCTGGAATTCCCCTTGCTGTGGGATCGCAAATGTCAGCATTAAAAGATCCATCAGAGCGTCTTTCCTATGAAATTGTTCGAAAGGAAAACCCAAACGGGCTGATTTTTGCCAACCTGGGAAGCGAGGCAACGGCTGCTCAGGCAAAGGAAGCCGTTGAGATGATTGGAGCAAACGCACTGCAGATCCACCTCAATGTGATTCAGGAAATTGTGATGCCTGAAGGGGACAGAAGCTTTAGCGGCGCATTGAAACGCATTGAACAAATTTGCAGCCGGGTCAGTGTACCGGTCATTGTGAAAGAAGTCGGCTTCGGTATGAGCAAAGCATCAGCAGGAAAGCTGTATGAAGCTGGTGCTGCAGCTGTTGACATTGGCGGTTACGGGGGAACAAATTTCTCGAAAATCGAAAATCTCCGAAGACAGCGGCAAATCTCCTTTTTTAATTCGTGGGGCATTTCGACAGCTGCAAGTTTGGCGGAAATCCGCTCTGAGTTTCCTGCAAGCACCATGATCGCCTCTGGCGGTCTGCAAGATGCGCTTGACGTGGCAAAGGCAATTGCGCTGGGGGCCTCTTGCACCGGAATGGCAGGGCATTTTTTAAAAGCGCTGACTGACAGCGGTGAGGAAGGACTGCTTGAGGAGATTCAGCTGATCCTTGAGGAATTAAAGTTGATTATGACCGTGCTGGGTGCCAGAACAATTGCCGATTTACAAAAGGCGCCCCTTGTGATCAAAGGTGAAACCCATCATTGGCTCACAGAGAGAGGGGTCAATACATCAAGCTATAGTGTG CGATAACTCTGAAATGAGCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAGGTATTCACACAGGAAACAGCTATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTGAATGAAACGAATTCTTTTTGTTACCAGTGAAGCACACCCTTTAATAAAAACCGGCGGCTTGGCGGATGTTTCAAGCAGCTTACCTAAGGCTTTGGCGGATCTAGGCCAGGATATCCGTATCATCATACCTAACTATCAGGCTATAAAAAAAACCGAAAACGTCCAACATCGGTGTACGCTGAGAATCAATAATTGCGATGTCAATATTCTCGAAACCCGCTTGCCGGAATCGAAAGTAATTGTATGGCTGATCGATTGTCCCCAGTTTTTTGACTACCCGGGCAATCCTTATCACGATGAATACGGTAATGCCTGGGCAAACAGTGCCGATCGCTTTTCGCTGTTCTGCCGCATAACCGTGGAAGTCGCGATGAACAGAGCCTACTTAGATTGGAAACCGGAAATCGTCCACTGCAACGACTGGCAAAGCGGCCTGGTTCCCGCCTTGTTAACGCTGGAATACAATCGCCCGGCAACCATTTTTACGATTCATAACATGGCCTATCAAGGGATCTTTCCCTATTCGACCTACAATGCGCTTAATCTTCCAAGACAACTTTGGAACCCAAATGTACTTGAGTATTACGGCAACATGTCGTTTTTAAAAGGCGGCATTGCTTGCTCCGATCGAGTAACAACGGTAAGTCCTACCTATGCCAAAGAAATTCAATCATCCGAGTTTGGTTACGGGCTAGAAGGCTTGTTAACCCATCGCAAGGAATTTCTATGCGGTATACTCAATGGAACCGACAATGACTGGAATCCCGAATTCGACAACAACATCGTCCAGCGCTACAGCTATAAAACG SEQ ID NO.2 ATGACTCGAGCAGAACGAAAAAGACAACACATCAATCATGCCTTGTCCATCGGCCAGAAGCGGGAAACAGGTCTTGATGATATTACGTTTGTTCACGTCAGTCTGCCCGATCTTGCATTAGAACAAGTAGATATTTCCACAAAAATCGGCGAACTTTCAAGCAGTTCGCCGATTTTTATCAATGCAATGACTGGCGGCGGCGGAAAACTTACATATGAGATTAATAAATCGCTTGCGCGAGCGGCTTCTCAGGCTGGAATTCCCCTTGCTGTGGGATCGCAAATGTCAGCATTAAAAGATCCATCAGAGCGTCTTTCCTATGAAATTGTTCGAAAGGAAAACCCAAACGGGCTGATTTTTGCCAACCTGGGAAGCGAGGCAACGGCTGCTCAGGCAAAGGAAGCCGTTGAGATGATTGGAGCAAACGCACTGCAGATCCACCTCAATGTGATTCAGGAAATTGTGATGCCTGAAGGGGACAGAAGCTTTAGCGGCGCATTGAAACGCATTGAACAAATTTGCAGCCGGGTCAGTGTACCGGTCATTGTGAAAGAAGTCGGCTTCGGTATGAGCAAAGCATCAGCAGGAAAGCTGTATGAAGCTGGTGCTGCAGCTGTTGACATTGGCGGTTACGGGGGAACAAATTTCTCGAAAATCGAAAATCTCCGAAGACAGCGGCAAATCTCCTTTTTTAATTCGTGGGGCATTTCGACAGCTGCAAGTTTGGCGGAAATCCGCTCTGAGTTTCCTGCAAGCACCATGATCGCCTCTGGCGGTCTGCAAGATGCGCTTGACGTGGCAAAGGCAATTGCGCTGGGGGCCTCTTGCACCGGAATGGCAGGGCATTTTTTAAAAGCGCTGACTGACAGCGGTGAGGAAGGACTGCTTGAGGAGATTCAGCTGATCCTTGAGGAATTAAAGTTGATTATGACCGTGCTGGGTGCCAGAACAATTGCCGATTTACAAAAGGCGCCCCTTGTGATCAAAGGTGAAACCCATCATTGGCTCACAGAGAGAGGGGTCAATACATCAAGCTATAGTGTGCGATAA SEQ ID NO.3 <![CDATA[ P mxaF - sqs- pfk1- fba2 ]]> AATTAAACCGGGAATGATGTCGGATATTTAACGGCAAAGCCATGGGAGCTTTTCCCGAATTTGAATGCCGACATACTCTCGGGATATTTTCCCTGTTTTTTCTTAGCGCTTTTCCCGTCATCTGGGTGCTGTATTCCGTAACGTCGCATCCCGCTCCTTCCGTATGATTACCGTCCGTGCGCTGCCCTCTATGAATGATTCGTTATGCGCCTTGATCAAGCTAAGCCGGTTGTAACAACAAACACCGCAATCAATAGGGGGCCGCGCCGACATTATGCGAAAAATCAATCTGGAGGAATTATGGGAAAGCTATTACAATTGGCATTGCATCCGGTCGAGATGAAGGCAGCTTTGAAGCTGAAGTTTTGCAGAACACCGCTATTCTCCATCTATGATCAGTCCACGTCTCCATATCTCTTGCACTGTTTCGAACTGTTGAACTTGACCTCCAGATCGTTTGCTGCTGTGATCAGAGAGCTGCATCCAGAATTGAGAAACTGTGTTACTCTCTTTTATTTGATTTTAAGGGCTTTGGATACCATCGAAGACGATATGTCCATCGAACACGATTTGAAAATTGACTTGTTGCGTCACTTCCACGAGAAATTGTTGTTAACTAAATGGAGTTTCGACGGAAATGCCCCCGATGTGAAGGACAGAGCCGTTTTGACAGATTTCGAATCGATTCTTATTGAATTCCACAAATTGAAACCAGAATATCAAGAAGTCATCAAGGAGATCACCGAGAAAATGGGTAATGGTATGGCCGACTACATCTTAGATGAAAATTACAACTTGAATGGGTTGCAAACCGTCCACGACTACGACGTGTACTGTCACTACGTAGCTGGTTTGGTCGGTGATGGTTTGACCCGTTTGATTGTCATTGCCAAGTTTGCCAACGAATCTTTGTATTCTAATGAGCAATTGTATGAAAGCATGGGTCTTTTCCTACAAAAAACCAACATCATCAGAGATTACAATGAAGATTTGGTCGATGGTAGATCCTTCTGGCCCAAGGAAATCTGGTCACAATACGCTCCTCAGTTGAAGGACTTCATGAAACCTGAAAACGAACAACTGGGGTTGGACTGTATAAACCACCTCGTCTTAAACGCATTGAGTCATGTTATCGATGTGTTGACTTATTTGGCCGGTATCCACGAGCAATCCACTTTCCAATTTTGTGCCATTCCCCAAGTTATGGCCATTGCAACCTTGGCTTTGGTATTCAACAACCGTGAAGTGCTACATGGCAATGTAAAGATTCGTAAGGGTACTACCTGCTATTTAATTTTGAAATCAAGGACTTTGCGTGGCTGTGTCGAGATTTTTGACTATTACTTACGTGATATCAAATCTAAATTGGCTGTGCAAGATCCAAATTTCTTAAAATTGAACATTCAAATCTCCAAGATCGAACAGTTTATGGAAGAAATGTACCAGGATAAATTACCTCCTAACGTGAAGCCAAATGAAACTCCAATTTTCTTGAAAGTTAAAGAAAGATCCAGATACGATGATGAATTGGTTCCAACCCAACAAGAAGAAGAGTACAAGTTCAATATGGTTTTATCTATCATCTTGTCCGTTCTTCTTGGGTTTTATTATATATACACTTTACACAGAGCGTGATGTGGAGGTATTCACACAGGAAACAGCTATGAACAAACCAAAAAAAGTCGCAATTCTCACTGCGGGCGGTTTAGCGCCTTGCCTTAGTTCAGCCATCGGCAGTCTCATCGAGCGTTACACCGAAATCGACCCTTCGATCGAGATCATCTGCTATCGTAGCGGTTACAAAGGCCTGCTACTCGGCGATTCTTACGCCGTGACCCCGAAAATCCGCGAAAACGCCGCGTTGCTGCATAAGTTCGGCGGCTCGCCGATCGGCAACAGCCGGGTCAAACTGACCAACGTCAAAGACTGCATCAAGCGGGGGTTGGTTCAAGAAGGTCAGGACCCTCAAAAAGTGGCGGCCGATCAATTAGTCAAAGACGGCGTCGATGTTCTGCATACGATCGGCGGCGACGATACCAATACCGCAGCAGCCGATTTGGCGGCCTTCTTGGCGAAAAACGATTATGGATTGACGGTCATCGGTTTGCCGAAAACGATCGACAACGACGTATTCCCGATTAAACA ATCCTTAGGTGCATGGACTGCAGCGGAGCAAGGCGCACATTATTTTCAAAATGTCGTGGCCGAGTATAACGCCAATCCACGCATGCTCATCGTTCATGAAGTCATGGGCCGCAATTGCGGATGGCTGACTGCCGCAACCGCAATGGAATACCGCAAATTGTTGGATCGCTCCGAATGGCTGCCTGAAATCGGTCTCGATCGCGCGGCATACGAAGTACACGGTGTCTTCGTTCCCGAAATGGAAATCGATCTGGCAGCCGAAGCGAAGCGCTTGCGCGAGGTGATGGATAAAGTCGATTGCGTCAATATATTCGTTTCGGAAGGCGCGGGTGTCGATGCGATCGTCGCCGAAATGCAGGCCAAGGGCCAAGAAGTTCCGCGCGATGCGTTCGGTCACATCAAGCTTGATGCGGTCAATCCGGGTAAATGGTTCGGCGAGCAATTCGCCGAAATGATCGGCGCGGAAAAAACCTTGATTCAAAAATCGGGATATTTCGCACGGGCATCGGCATCGAACGTCGATGATATTCGTTTGATCAAATCCTGTGCCGATTTAGCGGTCGAATGCGCATTACGCCGCGAGTCCGGCGTCATCGGTCATGATGAGGATAACGGTAACGTCTTGCGTGCGATCGAATTCCCGCGCATCAAAGGCGGCAAACCGTTCGATATCGACACGCCTTGGTTCGTGCAAATGCTTGGCGGAATCGGGCAAAGTAAAGGCGCGCGAGTCGAAGTGAGCCACTAAATTTTTTCGGTAACTAACACACAGGAGAAGTCAAATGGCACAAAAAATTTTAGATATTGTTAAACCAGGTGTCGTAACCGGTGAAGATGTACAAAAAGTCTTTGCGTTTTGTAAAGAACATAAGTTTGCACTTCCAGCTGTAAACGTCATCAGTACCGATACGATCAATGCAGTACTCGAAGGTGCGGCTAAAGCAAAATCAGCCGTTATCATTCAGTTTTCAAACGGCGGCGCTGCTTTTTTTGCCGGCAAAGGCGTCAGTTTAGAAGGTCAAATGCCTTCAATTTTGGGAGCGATCTCAGGCGCGCAGCACGTTCATCTTATGGCTGAGCATTACGGTGTACCGGTTATTTTACATACCGATCATGCCGCGAAAAAATTATTACCCTGGATCGATGGCTTATTAGATGCCGGGGAAAAGCATTTCGAAAAAACCGGAAAACCGTTATTCAGTTCTCATATGCTGGATCTTTCAGAAGAAAGTCTGGAAGAGAACATTGAAATTTGCGGTAAATATCTAGAGCGTATGTCCAAAATGGATATGACTCTTGAAATTGAACTGGGTTGCACGGGCGGCGAAGAAGACGGCGTAGATAATACCGGTATGGATCATTCTATGCTCTATACGCAGCCGGAAGATGTTGCCTATGCTTATGAGCATTTAAGCAAAATTAGCCACCGTTTTACGATCGCGGCATCTTTCGGTAATGTGCACGGTGTTTACAAACCCGGCAATGTTAAGCTAACTCCGACCATTTTGCTGAATTCGCAAAAATTCGTTTCCGAGAAATATGACTTACCGGAAAACAGCTTGACCTTCGTATTTCATGGCGGTTCAGGTTCCACTCCTGAAGAAATCAAGGAATCGATCAGCTACGGCGTCGTTAAAATGAATATCGACACCGATACTCAATGGGCAACCTGGGCTGGCGTCATGGAATTCTATAAGAAAAACGAAGGTTATTTGCAAGGCCAAATCGGTAACCCTGACGGCGACGACAAGCCGAATAAAAAATATTATGATCCACGCGTTTGGCAACGTGCCGGCCAAGTCGGCATGGTGACCCGCCTGCAGCAGGCTTTCCAAGACTTAAACGCAACTAATACGTTGTAA 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 GCATCTTCCCGACAACTATTACAACGTATTAGTTGCGTTTAAGTCTTG SEQ IDNO.10 ATGGGAAAGCTATTACAATTGGCATTGCATCCGGTCGAGATGAAGGCAGCTTTGAAGCTGAAGTTTTGCAGAACACCGCTATTCTCCATCTATGATCAGTCCACGTCTCCATATCTCTTGCACTGTTTCGAACTGTTGAACTTGACCTCCAGATCGTTTGCTGCTGTGATCAGAGAGCTGCATCCAGAATTGAGAAACTGTGTTACTCTCTTTTATTTGATTTTAAGGGCTTTGGATACCATCGAAGACGATATGTCCATCGAACACGATTTGAAAATTGACTTGTTGCGTCACTTCCACGAGAAATTGTTGTTAACTAAATGGAGTTTCGACGGAAATGCCCCCGATGTGAAGGACAGAGCCGTTTTGACAGATTTCGAATCGATTCTTATTGAATTCCACAAATTGAAACCAGAATATCAAGAAGTCATCAAGGAGATCACCGAGAAAATGGGTAATGGTATGGCCGACTACATCTTAGATGAAAATTACAACTTGAATGGGTTGCAAACCGTCCACGACTACGACGTGTACTGTCACTACGTAGCTGGTTTGGTCGGTGATGGTTTGACCCGTTTGATTGTCATTGCCAAGTTTGCCAACGAATCTTTGTATTCTAATGAGCAATTGTATGAAAGCATGGGTCTTTTCCTACAAAAAACCAACATCATCAGAGATTACAATGAAGATTTGGTCGATGGTAGATCCTTCTGGCCCAAGGAAATCTGGTCACAATACGCTCCTCAGTTGAAGGACTTCATGAAACCTGAAAACGAACAACTGGGGTTGGACTGTATAAACCACCTCGTCTTAAACGCATTGAGTCATGTTATCGATGTGTTGACTTATTTGGCCGGTATCCACGAGCAATCCACTTTCCAATTTTGTGCCATTCCCCAAGTTATGGCCATTGCAACCTTGGCTTTGGTATTCAACAACCGTGAAGTGCTACATGGCAATGTAAAGATTCGTAAGGGTACTACCTGCTATTTAATTTTGAAATCAAGGACTTTGCGTGGCTGTGTCGAGATTTTTGACTATTACTTACGTGATATCAAATCTAAATTGGCTGTGCAAGATCCAAATTTCTTAAAATTGAACATTCAAATCTCCAAGATCGAACAGTTTATGGAAGAAATGTACCAGGATAAATTACCTCCTAACGTGAAGCCAAATGAAACTCCAATTTTCTTGAAAGTTAAAGAAAGATCCAGATACGATGATGAATTGGTTCCAACCCAACAAGAAGAAGAGTACAAGTTCAATATGGTTTTATCTATCATCTTGTCCGTTCTTCTTGGGTTTTATTATATATACACTTTACACAGAGCGTGA SEQ IDNO.11 P89-F TAGTTGTCGGGAAGATGCGTG SEQ IDNO.12 P89-R CAGCTCATTTCAGAGCAGCTCAC SEQ IDNO.13 sqs-F CACACAGGAAACAGCTATGGGAAAGCTATTACAATTGGCATTG SEQ IDNO.14 sqs-R CACGCATCTTCCCGACAATCACGCTCTGTGTAAAGTGTATATATAATAAAACC SEQ IDNO.15 <![CDATA[P tac -R]]> AGCTGTTTCCTGTGTGAATACCTC SEQ IDNO.16 This invention provides a method for constructing the above-mentioned engineered methanogenic bacteria that produce high levels of squalene, 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...) Methylotuvimicrobium buryatense 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) glgA1L DNA fragments inserted at the site via electroporation P 12965 -Bsidi- P tac -Bleo (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 pellet 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. (3) Insertion into pAWP89 plasmid P mxaF Promoter linking to squalene synthase encoding gene sqs Phosphofructokinase encoding gene pfk1 and fructose-1,6-bisphosphate aldolase encoding gene fba2 Composition P mxaF - sqs-pfk1-fba2 (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 squalene.

[0034] This invention provides a method for producing squalene, comprising the following steps: (1) Activation of engineered bacteria: The engineered methanogenic bacteria that produce high squalene constructed by the above method were inoculated onto a solid inorganic salt culture medium and cultured statically at 25-30℃ 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 vol%~30 vol% methane was added to obtain the seed culture solution. (3) The seed culture obtained in step (2) is inoculated into a liquid inorganic salt culture medium with a pH of 8.5~9.5 and fermented for 25~30℃. The dissolved oxygen is controlled above 20. 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. Fermentation culture is carried out for 144 h.

[0035] The following is based on Methylotuvibium sanxanigenens Using NG09 as the test bacterium, and in conjunction with specific embodiments, the present invention is further illustrated. 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.

[0036] In the following examples, the squalene synthase encoding gene sqs , originating from Saccharomyces cerevisiae S288C, NCBI ID NP_012060.1; gene encoding 1-deoxy-xylulose-5-phosphate synthase. dxs1 Source Methylotuvibium sanxanigenens NG09, NCBI ID WP_017839846.1; gene encoding 1-deoxy-xylulose-5-phosphate synthase. dxs2 Source Methylotuvibium sanxanigenens NG09, NCBI ID WP_017842755.1; farnesyl pyrophosphate synthase encoding gene ispA Source Methylotuvibium sanxanigenens NG09, NCBI ID WP_017842756.1; gene encoding glucose-6-phosphate dehydrogenase. zwf1 Source Methylotuvibium sanxanigenens NG09, NCBI ID WP_017841635.1; isopentenyl pyrophosphate isomerase encoding gene. Bsidi Derived from Bacillus subtilis Bacillus subtilis NCBI ID: WP_004399098.1; phosphofructokinase encoding gene pfk1 Source Methylotuvibium sanxanigenens NG09, NCBI ID WP_341326251.1; gene encoding fructose-1,6-bisphosphate aldolase. fba2 Source Methylotuvibium sanxanigenens NG09, NCBI ID: WP_341326872.1.

[0037] In a specific implementation, the squalene synthase encoding gene sqs The nucleotide sequence is shown in SEQ ID NO. 11 in Table 1; the isopentenyl pyrophosphate isomerase encoding gene Bsidi The nucleotide sequence is shown in Table 1 as SEQ ID NO.3.

[0038] In the following examples, the liquid inorganic salt culture medium was formulated as follows: 0.6 g / L MgSO4·7H2O, 0.01 g / L CaCl2·6H2O, 1 g / L KNO3, and 10 g / L NaCl, with the balance being distilled water. It was sterilized at 121°C for 20 min. The solid inorganic salt culture medium was formulated by adding 13 g / L agar to the liquid inorganic salt culture medium. The LB medium was formulated as follows: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and 15 g / L agar, with the balance being distilled water, and the pH was adjusted to 7.0.

[0039] In the following examples, conventional instruments and equipment in the art were used. Experimental methods in the following examples, unless otherwise specified, were 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.

[0040] In the following embodiments, the following uses Methylotuvibium sanxanigenens 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.

[0041] Example 1 I. Construction of an engineered methanogenic bacterium SQ02 that produces high levels of squalene 1. Constructing DNA fragments that can be electrically converted (1) Using high-fidelity DNA polymerase to extract DNA fragments P c -Gm-P pqqA -zwf1-P tac -dxs1-P mxaF -ispA- dxs2 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-Gm-P pqqA -zwf1-P tac -dxs1-P mxaF -ispA-dxs2 Fragment; (2) Using high-fidelity DNA polymerase to extract DNA fragments P 12965 -Bsidi-P tac -Bleo 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]. P 12965 -Bsidi-P tac -Bleo Excerpt.

[0042] 2. Preparation of competent cells Test bacteria Methylotuvibium sanxanigenens 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.

[0043] 3. With Methylotuvibium sanxanigenens NG09 was used as the test bacterium to construct engineered bacterium 1. Take 50 μL of competent cells obtained in step 2 and add 500 ng of the cells obtained in step 1. P c -Gm-P pqqA -zwf1-P tac - dxs1-P mxaF -ispA-dxs2 Fragments and gently mix to introduce into the host bacteria. Methylotuvibium sanxanigenens NG09 ispAL Site Insertion Fragment P c -Gm-P pqqA -zwf1-P tac -dxs1-P mxaF -ispA-dxs2Then, 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 30°C for 24 h. After centrifugation 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 30°C for 4 days, and recombinants were screened. The correct recombinants were designated as engineered bacteria 1.

[0044] 4. Using engineered strain 1 as the test strain, construct the engineered methanogenic bacteria IZDA. (1) Referring to the construction methods in steps 2 and 3, the host bacteria are prepared. Methylotuvibium sanxanigenens Replace NG09 with engineered bacteria 1, and replace the DNA fragment with the one obtained in step 1. P 12965 -Bsidi-P tac -Bleo 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. glgA1L Site insertion P 12965 -Bsidi-P tac -Bleo Fragments; if no base mutations are found in the recombinant after PCR identification, the engineered methanogenic bacteria IZDA is obtained.

[0045] (2) The engineered methanogenic bacteria IZDA 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 and cultured at 30℃ and 200 rpm until the logarithmic growth phase was reached and the strain was preserved.

[0046] 5. Using engineered methanogenic bacteria IZDA as the test strain, a high-squalene-producing engineered methanogenic bacteria SQ02 was constructed. (1) with P mxaF -sqs-pfk1-fba2 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 - sqs-pfk1-fba2 Target segment; (2) Primers P89-F (nucleotide sequence as shown in SEQ ID NO.12 in Table 1) and P89-R (nucleotide sequence as shown in SEQ ID NO.13 in Table 1) were designed based on the pAWP89 sequence. The pAWP89 linearized vector was obtained by PCR amplification and then digested with restriction endonuclease DpnI at 37°C for 2 h. (3) Use a plasmid construction kit to process the plasmid obtained in step (1). P mxaF - sqs-pfk1-fba2 The target fragment and the pAWP89 linearized vector obtained in step (2) were subjected to a recombination reaction. The recombination system contained equimolar amounts of the linearized vector and the gene fragment. The multi-fragment recombination conditions were 50°C for 45 min. After the recombination reaction, the product was transferred to CaCl2 using the CaCl2 conversion method. E. coli 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. E. coli S17.

[0047] (4) Take the recombinant plasmid obtained in step (3) E. coli 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% LB medium for 48 h, the mixed bacteria were spread on a solid inorganic salt medium containing 50 mg / L kanamycin, and recombinants were screened to obtain the engineered methanogenic bacteria SQ02.

[0048] II. Squalene Production Using the High-Yielding Engineered Methanogenic Bacteria SQ02 1. Activation of engineered bacteria: The engineered methanogenic bacteria SQ02, which produces high levels of squalene and was preserved in step one, was inoculated onto a solid inorganic salt culture medium and incubated at 30°C 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 30℃ and 200 rpm for 48 h. During this period, the upper air was replaced and 20 vol% methane was added every 24 h to obtain the seed culture solution. 3. The seed culture obtained in step 2 was inoculated into a liquid inorganic salt medium with a pH of 9.0 for continued fermentation. The dissolved oxygen was controlled at 30°C and above 20. At the same time, 15% of the total volume of the liquid inorganic salt medium was added with n-dodecane and methane was introduced. The fermentation culture was carried out for 144 h. 4. Take 1 mL of the fermented bacterial culture, centrifuge at 4℃ and 10000 rpm for 5 min, take the upper organic phase, and perform qualitative and quantitative analysis of squalene using gas chromatography-mass spectrometry.

[0049] Gas chromatography-mass spectrometry qualitative results of squalene production by the engineered methanogenic bacterium SQ02, which produces high-yield squalene, are as follows: Figure 2 As shown in the figure, the fermentation product is squalene; the yield is as follows. Figure 3 As shown, squalene began to accumulate significantly from the 24th hour of fermentation until it essentially stopped accumulating after 120 hours, ultimately reaching gram-level yields. The growth of strain SQ02 is shown below. Figure 4 As shown, the strain grows vigorously from 0 to 48 hours, and basically stops growing after 96 hours.

[0050] Comparative Example 1 I. Construction of engineered methanogenic bacteria SQ01 1. With sqs (The nucleotide sequence is shown in Table 1 as SEQ ID NO.11, derived from...) Saccharomyces cerevisiae Using sqs-F as a template, PCR amplification was performed under the guidance of upstream primer sqs-F (nucleotide sequence shown in SEQ ID NO.14 in Table 1) and downstream primer sqs-R (nucleotide sequence shown in SEQ ID NO.15 in Table 1) to obtain... sqs Target segment.

[0051] 2. Using 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.

[0052] 3. Use the Novizan one-step cloning kit to clone the sample obtained in step 1. sqs 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 sqs 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. E. coli S17. PCR confirmation showed the band size was correct and sequencing revealed no mutations, indicating successful construction of the recombinant plasmid.

[0053] 4. The recombinant plasmid was transferred into the test bacteria via parental conjugation. Methylotuvibium sanxanigenensNG09, which is the engineered methanogenic bacteria SQ01.

[0054] II. Production of Squalene using Engineered Methanogenic Bacteria SQ01 1. Activation of engineered bacteria: The engineered methanogenic bacteria SQ01 preserved in step one is inoculated onto a solid inorganic salt culture medium (formula as in Example 1) and incubated at 30°C for 4-5 days to obtain the activated test colonies. 2. Take one loopful of engineered bacteria from the solid inorganic salt culture medium and inoculate it into a 300 mL shake flask containing 50 mL of liquid inorganic salt culture medium (formulation same as in Example 1). Incubate at 30℃ and 200 rpm for 48 h to obtain the seed culture. The OD of the seed culture... 600 The value is 2.0~3.5. The culture system adopts a sealable gas-liquid two-phase system. Methane is added to the culture system at the beginning and during the culture process. The amount of methane added is 20% of the gas phase volume of the culture system.

[0055] 3. Take 5 mL of the seed culture from step 2 and inoculate it into a 300 mL shake flask containing liquid inorganic salt medium. The liquid volume after inoculation should be 50 mL. Incubate at 30°C and 200 rpm by passing methane through the flask. Add 5-10 mL of n-dodecane at 48 h after inoculation and continue incubating at 30°C and 200 rpm for another 72 h.

[0056] 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 squalene using gas chromatography-mass spectrometry.

[0057] Comparing Example 1 and Comparative Example 1, the results are as follows: Figure 5 As shown, quantitative analysis revealed a squalene yield of 4.22 mg / L. After metabolic engineering of methanogenic bacteria to obtain engineered methanogenic bacteria, their application in squalene production increased the yield to over 100 times that of the initial strain, reaching gram-level yields. These results demonstrate that the engineered methanogenic bacteria SQ02, which produces high squalene, can significantly improve the yield of squalene synthesized using methane as a carbon source.

[0058] 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 squalene, characterized in that, Using methanogenic bacteria as the host bacteria, the squalene synthase encoding gene was introduced into the host bacteria. sqs To construct a squalene 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, the gene encoding phosphofructokinase is overexpressed. pfk1 Gene encoding fructose-1,6-bisphosphate aldolase fba2 An engineered methanogenic bacterium that produces high levels of squalene was obtained.

2. The engineered methanogenic bacteria with high squalene production according to claim 1, characterized in that, The genomes of the methanogenic bacteria all contain ispAL and glgA1L Site.

3. The engineered methanogenic bacteria with high squalene production according to claim 1, characterized in that, The methanogenic bacteria are Methylotuvimicrobium buryatense 5GB1 Alkalicoccus glycogenes WONF2802 or Methylotuvibium sanxanigenens NG09.

4. The engineered methanogenic bacteria with high squalene production according to claim 1, characterized in that, The squalene synthase encoding gene sqs Source Saccharomyces cerevisiae S288C, 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 encoding gene pfk1 Gene encoding fructose-1,6-bisphosphate aldolase fba2 All are derived from methanogenic bacteria, and the isopentenyl pyrophosphate isomerase encoding gene. Bsidi It originates from Bacillus subtilis.

5. The method for constructing the engineered methanogenic bacteria with high squalene production according to any one of claims 1 to 4, 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 squalene synthase encoding gene sqs Composition P mxaF - sqs- 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 squalene. 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 - sqs-pfk1-fba2 The nucleotide sequence is shown in SEQ ID NO.

4.

6. The method for constructing a high-squalene-producing engineered methanogenic bacterium according to claim 5, characterized in that, Host bacteria and DNA fragments Pc-Gm-P pqqA -zwf1-P tac -dxs1-P mxaF -ispA-dxs2 The dosage ratio of engineered bacteria 1 to DNA fragments P 12965 -Bsidi-P tac -Bleo The dosage ratio was 1 μL:10 ng.

7. The method for constructing engineered methanogenic bacteria with high squalene production according to claim 5, characterized in that, In step 3), pAWP89 plasmid in an equimolar ratio is used with... P mxaF Promoter linking to squalene synthase encoding gene sqs Composition P mxaF - sqs-pfk1-fba2 The expression cassette undergoes a recombination reaction.

8. The application of the engineered methanogenic bacteria with high squalene production as described in any one of claims 1 to 4 in the production of squalene.

9. A method for producing squalene, characterized in that, The method includes the steps of cultivating engineered methanogenic bacteria that produce high levels of squalene as described in any one of claims 1 to 4, and collecting the squalene produced.

10. A method for producing squalene according to claim 9, characterized in that, The seed culture of engineered methanogenic bacteria that produce high levels of squalene was inoculated into a liquid inorganic salt culture medium and fermented under conditions containing n-dodecane and methane to obtain squalene.

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