Microbial chassis cell capable of producing terpenoids at high yield and application of microbial chassis cell

Abstract

The invention discloses a microbial chassis cell capable of producing terpenoids at high yield and application of the microbial chassis cell. Based on gene homologous recombination of a specific site mediated by a conjugational transfer technology, a key rate-limiting enzyme gene of an MEP pathway is overexpressed in an integrated manner at a gene high expression site in a genome, streptomyces chassis cells with high yield of terpenes are established, and establishment of an efficient production system of sesquiterpenes and diterpenes is realized. By utilizing the microorganism provided by the embodiment of the invention, the yields of bisabolol, 18-carboxyl labdate, kaurene and atelonic acid are obviously improved, the production period is short, and the production efficiency is high.

Description

Technical Field

[0001] The present invention relates to the technical field of natural product biosynthesis, and specifically refers to the construction of a high-yield terpene chassis of Streptomyces and the efficient production of different terpene compounds based on the chassis strain. Background Art

[0002] Terpenoids are a general term for compounds containing isoprene units. They are widely found in nature. So far, more than 120,000 terpenoids have been found in animals, plants and microorganisms. This type of compound has many physiological activities and is widely used in food, cosmetics and pharmaceutical industries. Terpenoids are mainly extracted by natural products, chemical synthesis, microbial fermentation and other methods. Natural product extraction is mainly to obtain the corresponding terpenoids by extracting, purifying and separating specific plants. However, this production method is affected by many uncontrollable factors such as climate, variety, geographical location, maturity, etc., has obvious seasonality, unstable content, and the cost of large-scale planting and breeding is high, and the content is usually low. In addition, it is technically very difficult to purify and separate specific terpenoids from extracts containing multiple components. These together make many compounds with good physiological activity expensive. The chemical synthesis method is limited by the characteristics of many steps in the synthesis process, high difficulty in synthesis, and pollution. The quality, safety and scope of use of its products are limited. The microbial fermentation method mainly utilizes the biological metabolism of microorganisms to convert basic metabolites such as glucose, starch, soybean cake powder and other cheap raw materials into corresponding terpenoid compounds. This method is not affected by factors such as season, region, climate, etc. The raw materials are easy to obtain, the production cycle is short, the process operation is simple, the cost is low, the product quality is controllable, the product is easy to purify, the safety is high, and there is less environmental pollution. It not only solves the problem of occupying a large amount of arable land due to planting plants, but also solves the disadvantages of chemical synthesis that is not environmentally friendly. Most importantly, the terpenoid compounds produced by the fermentation method are natural products, and their activity is consistent with the active ingredients extracted from natural plants. They are considered to be the most promising method for the production of terpenoid compounds. The present invention takes terpenoid products as the research target, uses white Streptomyces (Streptomyces albus) as the heterologous expression host, creates a white Streptomyces chassis with high yield of terpenes, establishes an actinomycete terpene high-yield chassis that increases the yield of terpene natural products, and provides a method for producing terpenoid compounds with Streptomyces.

[0003] The terpene compound-producing bacteria that are currently being studied the most are yeast and Escherichia coli. However, yeast is a eukaryotic organism, and its culture conditions and genetic manipulation are more complicated than those of bacteria, and other substances are produced during the fermentation process. Escherichia coli has a simple regulatory system and poor ability to produce secondary metabolites, making it difficult to mass-produce terpene compounds. It also has phage contamination and poor resistance to heterologous substances, making it more difficult to produce terpenes. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a microbial chassis cell with high terpene production, specifically involving the modification of a Streptomyces chassis with high terpene production and the efficient synthesis of the sesquiterpene compound bisabolol and the diterpene andrographolide with anti-inflammatory activity, the precursor of the kaurenane skeleton, as well as the kaurenane and athisanane skeletons.

[0005] To this end, another object of the present invention is to provide a microbial chassis cell, which has high production yield of terpenoid compounds, short production cycle and high production efficiency.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] In the first aspect of the present invention, the present invention provides a microbial chassis cell, which contains a gene that improves the ability of the chassis cell to produce terpenoid compound precursors, and the gene is a gene of the MEP pathway derived from Streptomyces; the gene is derived from the 1813M1, 1813M2, and 1813M3 genes of Streptomyces.s.1813, and the nucleotide sequence of the 1813M1 gene is shown in SEQ ID NO:1; the nucleotide sequence of the 1813M2 gene is shown in SEQ ID NO:2; and the nucleotide sequence of the 1813M3 gene is shown in SEQ ID NO:3.

[0008] As a preferred embodiment of the present invention, the microbial chassis cells are eukaryotic cells or prokaryotic cells; preferably, the microorganism is Streptomyces, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Bacillus, Aspergillus nidulans, Aspergillus niger, Neurospora crassa, Alternaria alternata or Fusarium; more preferably, the microorganism is Streptomyces albus, Streptomyces avermitilis, Streptomyces lividans, Streptomyces coelicolor; more preferably, the microorganism is Streptomyces albus.

[0009] As a preferred embodiment of the present invention, the terpenoid compound is selected from sesquiterpenoid compounds and diterpenoid compounds.

[0010] In the second aspect of the present invention, the present invention provides a use of a gene fragment, wherein the gene fragment is composed of 1813M1, 1813M2, and 1813M3 genes derived from Streptomyces.s.1813 connected in sequence, and the gene fragment is used to improve the production of terpene compound precursors by chassis cells.

[0011] The microbial chassis cells of the present invention are developed by the following steps:

[0012] Step 1): Establish a microbial chassis strain promoter library: screen promoters from different sources, and characterize the optimal expression system by regulating the expression of the introduced gene;

[0013] Step 2): Using the MEP pathway from Streptomyces as a reference, the original MEP pathway synthase genes 1813M1, 1813M2, and 1813M3 were screened from the genome database of Streptomyces.s.1813 through sequence homology analysis.

[0014] Step 3): The three key pathway synthases of the MEP pathway from Streptomyces.s.1813 screened in step 2) are constructed onto different plasmids under the regulation of promoters of different strengths characterized in step 1) to obtain the chassis cells.

[0015] Therefore, the method for preparing the microbial chassis cells described in the present invention includes adopting a conjugation transfer strategy to integrate the 1813M1, 1813M2, and 1813M3 genes derived from Streptomyces.s.1813 into any site of the Streptomyces albus genome to obtain the microbial chassis cells.

[0016] In the third aspect of the present invention, the present invention provides a microbial strain with a higher yield of sesquiterpene precursors. The microbial chassis cells of the present invention are used as the starting strain, and the polyisopentenyl synthase gene IspA from Escherichia coli or the polyisopentenyl synthase gene PtlB from Streptomyces albus is selectively overexpressed to obtain a microbial strain with a higher yield of sesquiterpene precursors. Preferably, the nucleotide sequence of the gene PtlB is shown in SEQ ID NO:4; the nucleotide sequence of the gene IspA is shown in SEQ ID NO:5,

[0017] In the fourth aspect of the present invention, a microbial strain with a higher production of diterpene precursors is obtained by using the microbial chassis cells of the present invention as the starting strain and overexpressing the polyisopentenyl synthase gene 1813T4 from Streptomyces.s.1813, i.e., obtaining a microbial strain with a higher production of diterpene precursors. Preferably, the nucleotide sequence of gene 1813T4 is shown in SEQ ID NO:7.

[0018] In the fifth aspect of the present invention, the present invention provides a microbial strain with a higher yield of sesquiterpenes (bisabolol), using the microbial strain with a higher yield of sesquiterpenes precursors described in the third aspect of the present invention as a starting strain, overexpressing the bisabolol synthase gene (MCTC1) from Streptomyces citrinum that has been codon-optimized by Streptomyces, thereby obtaining a microbial strain with a higher yield of sesquiterpenes. The nucleotide sequence of the bisabolol synthase gene from Streptomyces citrinum is shown in SEQ ID NO:6.

[0019] In the sixth aspect of the present invention, the present invention provides a microbial strain with high diterpenoid production, which uses the microbial strain with higher diterpene precursor production described in the fourth aspect of the present invention as a starting strain, and respectively combines and expresses diterpene synthase genes 1813T2 (SEQ ID NO: 8), 1813T1 (SEQ ID NO: 11), bjks (SEQ ID NO: 10) and cytochrome P450 enzyme gene 1813O2 (SEQ ID NO: 9) derived from Streptomyces.s.1813, thereby obtaining the microbial strain with high diterpenoid production.

[0020] In a seventh aspect of the present invention, the present invention provides a method for obtaining a crude terpenoid compound produced in a microbial production process. According to an embodiment of the present invention, the method comprises: subjecting the aforementioned microorganism to a fermentation treatment; and subjecting the fermentation treatment product to an extraction treatment to obtain the crude terpenoid compound. By obtaining the terpenoid compound produced by the microorganism according to an embodiment of the present invention, all of the terpenoid compounds can be obtained as efficiently as possible.

[0021] According to an embodiment of the present invention, the method for obtaining a crude microbial terpenoid compound may further include at least one of the following additional technical features:

[0022] According to an embodiment of the present invention, the method for obtaining a crude microbial terpenoid compound according to an embodiment of the present invention has high acquisition efficiency.

[0023] According to an embodiment of the present invention, the fermentation treatment is achieved in the following manner: the microorganism is subjected to basic fermentation treatment, and the basic fermentation treatment is carried out in a basic fermentation medium, and the basic fermentation medium is the A medium: soluble starch 20g / L, glucose 10g / L, peptone 5g / L, yeast extract 5g / L, NaCl4g / L, K2HPO40.5 g / L, MgSO4·7H2O 0.5g / L, CaCO32 g / L, PH=7.0.

[0024] The B culture medium includes 40 g / L dextrin, 7.5 g / L tomato paste, 2.5 g / L hydrolyzed casein, 5 g / L yeast extract, and pH = 7.0. The D culture medium includes 20 g / L glucose, 60 g / L soluble starch, 15 g / L soybean powder, 5 g / L yeast extract, 5 g / L malt extract, 2 g / L calcium carbonate, 2 g / L NaCl, and pH = 7.2.

[0025] The E culture medium includes: 4 g / L yeast extract, 10 g / L malt extract, 10 g / L glucose, and pH=7.2.

[0026] The F medium: sucrose 20g / L, glucose 10g / L, casamino acid 0.1g / L, yeast extract 5g / L, MOPS 5g / L, K2SO4 0.25 g / L, MgCl·6H2O 1g / L, trace element salt solution: 10ml / L, PH=7.2. Trace element salt solution: ZnCl2 40 mg / L, FeCl3·6H2O 200mg / L, CuCl2 10 mg / L, MnCl2·4H2O 10mg / L, Na2B4O7·10H2O 10mg / L, (NH4)6Mo7O 24 4H2O 10mg / L.

[0027] The G culture medium includes: 31 g / L glucose, 22 g / L soybean powder, 1.6 g / L K2HPO4·3H2O, and PH=7.2.

[0028] The GYE medium contains: 15 g / L glucose, 5 g / L yeast extract, 0.2 g / L CaCl2·2H2O, and PH=7.2.

[0029] The XTM culture medium includes: 10 g / L yeast extract, 10 g / L malt extract, 10 g / L maltose, 10 ml / L trace element salt solution, and PH=7.2.

[0030] The PTMM culture medium includes: 40 g / L dextrin, 40 g / L lactose, 5 g / L yeast extract, 20 g / L MOPS, 10 ml / L trace element salt solution, and PH=7.3.

[0031] The MS culture medium includes: 20 g / L soybean powder, 20 g / L mannitol, 3 g / L CaCO3, and PH=7.2.

[0032] According to an embodiment of the present invention, the extraction process includes: centrifuging the fermentation product, ultrasonic crushing and organic extraction. The inventors found that centrifuging the fermentation product and then separating the supernatant and the bacterial cells can reduce the emulsification phenomenon in the extraction process and obtain the corresponding product as completely as possible.

[0033] In the eighth aspect of the present invention, the present invention proposes the use of the aforementioned microorganism in high production of terpenoid compounds.

[0034] Beneficial effects:

[0035] The present invention is based on the gene homologous recombination at a specific site mediated by the conjugation transfer technology, and integrates and overexpresses the key rate-limiting enzyme gene of the MEP pathway at any gene site in the genome, establishes a high-yield terpene Streptomyces chassis cell, and realizes the establishment of an efficient production system for sesquiterpenes and diterpenes. Using the microorganisms of the embodiments of the present invention, the production yield of bisabolol, 18-carboxyhelidinone, atisan acid, and kaurene is significantly improved, the production cycle is short, and the production efficiency is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is a schematic diagram of the structure of the plasmid pLDL0001 constructed according to Example 2 of the present invention;

[0037] Figure 2 is a schematic diagram of the structure of the constructed plasmid pLDL0002 according to Example 2 of the present invention;

[0038] Figure 3 is a schematic diagram of the structure of the plasmid pLDL0003 constructed according to Example 2 of the present invention;

[0039] Figure 4 is a schematic diagram of the structure of the plasmid pLDL0004 constructed according to Example 2 of the present invention;

[0040] Figure 5 is a schematic diagram of the structure of the constructed plasmid pLDL0009 according to Example 2 of the present invention;

[0041] Figure 6 is a schematic diagram of the structure of the plasmid pLDL0010 constructed according to Example 2 of the present invention;

[0042] Figure 7 is a schematic diagram of the structure of the plasmid pLDL0011 constructed according to Example 2 of the present invention;

[0043] Figure 8 is a schematic diagram of the structure of the plasmid pLDL0012 constructed according to Example 2 of the present invention;

[0044] Fig. 9is a schematic diagram of the structure of the constructed plasmid pLDL0013 according to Example 2 of the present invention;

[0045] Fig.10 is a schematic diagram of the structure of the constructed plasmid pLDL0014 according to Example 2 of the present invention;

[0046] Fig.11 is a schematic diagram of the structure of the constructed plasmid pLDL0015 according to Example 2 of the present invention;

[0047] Fig.12 is a schematic diagram of the structure of the constructed plasmid pLDL0016 according to Example 2 of the present invention;

[0048] Fig.13 is a schematic diagram of the structure of the constructed plasmid pLDL0017 according to Example 2 of the present invention;

[0049] Fig.14 Schematic diagram of the construction process of the high-yield chassis for terpenoid substrates and some experimental results according to an embodiment of the present invention.

[0050] Fig.15 An optimized yield diagram of atesinic acid according to one embodiment of the present invention.

[0051] The blue T4-M1M2M3 represents the recombinant strain containing the pLDL0004 plasmid, the orange T2O2T1 represents the recombinant strain containing the pLDL0016 plasmid, and the gray T2O2T1+T4-M1M2M3 represents the recombinant strain containing the pLDL0013 plasmid. The horizontal axis represents different fermentation media, and the vertical axis represents the yield (mg / L).

[0052] Fig.16 A yield diagram of optimized helicin according to one embodiment of the present invention.

[0053] Among them, the blue fpps-M1M2M3 represents the recombinant strain containing the pLDL0003 plasmid, the orange MCTC1 represents the recombinant strain containing the pLDL0017 plasmid, the gray MCTC1+ispA-M1M2M3 represents the recombinant strain containing the pLDL0010 plasmid, and the yellow MCTC1+ptlB-M1M2M3 represents the recombinant strain containing the pLDL0009 plasmid. The horizontal axis represents different fermentation media, and the vertical axis represents the yield (mg / L).

[0054] Fig.17 A diagram showing the optimized yield of bisabolol according to one embodiment of the present invention.

[0055] The blue T4-M1M2M3 represents the recombinant strain containing the pLDL0004 plasmid, the orange T2O2 represents the recombinant strain containing the pLDL0014 plasmid, and the gray T2O2+T4-M1M2M3 represents the recombinant strain containing the pLDL0011 plasmid. The horizontal axis represents different fermentation media, and the vertical axis represents the yield (mg / L).

[0056] Fig.18 A yield diagram of kaurene optimization according to an embodiment of the present invention.

[0057] The blue T4-M1M2M3 represents the recombinant strain containing the pLDL0004 plasmid, the orange BJKS represents the recombinant strain containing the pLDL0015 plasmid, and the gray BJKS+T4-M1M2M3 represents the recombinant strain containing the pLDL0012 plasmid. The horizontal axis represents different fermentation media, and the vertical axis represents the yield (mg / L).

[0058] Fig.19 The NMR spectrum of atesinic acid according to one embodiment of the present invention.

[0059] Fig. 20 The NMR spectrum of schizonepeta tenuifolia according to one embodiment of the present invention.

[0060] Fig.21 The NMR spectrum of bisabolol according to one embodiment of the present invention.

[0061] Fig. 22 The NMR spectrum of kaurene according to one embodiment of the present invention. DETAILED DESCRIPTION

[0062] The scheme of the present invention will be explained below in conjunction with the examples. Those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. Where specific techniques or conditions are not indicated in the examples, the techniques or conditions described in the literature in the art or in accordance with the product instructions are carried out. The methods used are conventional methods known in the art unless otherwise specified, and the consumables and reagents used are commercially available unless otherwise specified. Unless otherwise specified, the professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the recorded content may also be applied to the present invention. Wherein it represents gene connection, for example 1813M1 (nucleotide sequence as shown in SEQ ID NO: 1)-1813M2 (nucleotide sequence as shown in SEQ ID NO: 2)-1813M3 (nucleotide sequence as shown in SEQ ID NO: 3) means that the gene 1813M1 with a nucleotide sequence as shown in SEQ ID NO: 1, the gene 1813M2 with a nucleotide sequence as shown in SEQ ID NO: 2, and the gene 1813M3 with a nucleotide sequence as shown in SEQ ID NO: 3 are connected in sequence (the full nucleotide sequence of the connection method is shown in SEQ ID NO: 16).

[0063] The present invention provides a microbial chassis cell, and according to an embodiment of the present invention, the microorganism overexpresses 1813M1, 1813M2, and 1813M3 genes. Using the microorganism according to the embodiment of the present invention, the yield of terpene precursors is significantly increased.

[0064] On this basis, the present invention proposes several microorganisms for producing sesquiterpenes and diterpenes. According to an embodiment of the present invention, further overexpression includes at least one selected from IspA, PtlB, 1813T4, 1813T2, 1813T1, S1813O2, bisabolol synthase gene, and bjks gene. Using the microorganisms according to the embodiments of the present invention, the yield of terpenes is significantly improved.

[0065] The microorganisms described in the present application are used to produce bisabolol, atessenic acid, helicin and kaurene, and their yields are significantly improved compared to the prior art, all reaching grams per liter.

[0066] The inventors have found that by further regulating the expression intensity of at least one of the above genes, the yield of terpenes produced by the microorganism is further significantly improved.

[0067] It should be noted that experiments have shown that further introduction of the expression of 1813M1, 1813M2, and 1813M3 genes can achieve the efficient synthesis of terpene precursor five-carbon units in the enhanced MEP pathway of Streptomyces; introduction of the expression of part or all of 1813T4, PtlB, and IspA can achieve the further efficient production of sesquiterpene precursors and diterpene precursors, thereby achieving the purpose of constructing a terpene chassis for microbial cells; further introduction of the expression of 1813T2, 1813O2, 1813T1, bjks, and bisabolol synthase genes can enable the further conversion of sesquiterpene and diterpene precursors, thereby achieving the purpose of efficient production of bisabolol, atessenic acid, hemipteratin, and kaurene.

[0068] The present invention provides a method for obtaining a crude product of terpenoid compounds produced by microorganisms. According to an embodiment of the present invention, the method comprises: subjecting the aforementioned microorganisms to fermentation treatment; and subjecting the fermentation treatment product to extraction treatment to obtain the target terpenoid compound extract. By using the method for obtaining a fermentation terpenoid product according to an embodiment of the present invention, terpenoid compounds produced by microorganisms with a high recovery rate can be obtained.

[0069] According to a specific embodiment of the present invention, the fermentation process is carried out through two parts: seed liquid fermentation process and microbial production fermentation process, as described below:

[0070] The positive monoclonal colonies screened by conjugation transfer were transferred to new MS solid medium for culture. After 5-7 days of spore growth, the fresh spores were inoculated into a 250ml triangular shaking bottle containing glass beads and 50ml tryptone soy broth liquid medium, and 50μL apramycin was added. The culture was cultured at 28°C and 250rpm constant temperature shaking incubator for 48h. After 48h, the cultured bacterial liquid was transferred to a sterilized basal medium (the transfer volume was 4%), and cultured again at 28°C and 250rpm constant temperature shaking incubator for 7 days to complete the fermentation.

[0071] According to another specific embodiment of the present invention, the extraction and collection of bacteria treatment includes: centrifuging the fermented broth after fermentation, ultrasonic crushing treatment and organic extraction treatment. The inventor optimized and screened the extraction process after the fermentation treatment. The inventor found that soaking in acetone before ultrasonic treatment helps the lysis of the bacteria and greatly improves the dissolution rate of the substrate. In addition, different terpenoid compounds have different concentration differences in the intracellular and extracellular culture media, which are affected by the preference of their own transport proteins. It should be noted that the "organic extraction treatment" described in this application refers to the use of organic solvents to extract the ultrasonic crushing product. The method of organic extraction is not particularly limited. For example, according to a specific embodiment of the present invention, ethyl acetate solvent can be used to treat the terpenoid compounds released in the cell after ultrasonic crushing with acetone solvent.

[0072] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. If no specific techniques or conditions are specified in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used that do not specify the manufacturer are all conventional products that can be obtained commercially.

[0073] Example 1 Identification of MEP Pathway Synthase in Strepttomyces.s.1813

[0074] Using the MEP pathway of Escherichia coli as a reference, the original MEP pathway synthase 1813M1,

[0075] 1813M2, 1813M3.

[0076] Example 2 Construction of expression vector

[0077] All genes were obtained by PCR amplification, and the primers used are shown in Sequence Table 1:

[0078] Table 1 Primer sequences used in the examples

[0079]

[0080]

[0081] Plasmid construction mainly adopts PCR amplification of target fragments, restriction enzyme cutting of the vector to be inserted, and homologous recombination. The specific construction method is as follows: In this embodiment, the inventors introduce the construction process of each plasmid in detail.

[0082] (1) Construction of plasmids corresponding to diterpene high-yield chassis

[0083] Construction of pLDLL0001 plasmid: Using the genome of Streptomyces.s.1813 as a template, primers 1-F / 1-R (primer sequences are shown in Table 1) were used to amplify 1813M1 (nucleotide sequence is shown in SEQ ID NO: 1), primers 2-F / 2-R were used to amplify 1813M2 (nucleotide sequence is shown in SEQ ID NO: 2), and primers 3-F / 3-R were used to amplify 1813M3 (nucleotide sequence is shown in SEQ ID NO: 3). NO: 3), the amplification conditions are: 95℃, 3min pre-denaturation, then 30 PCR cycles at 95℃, 15s: 60℃, 15s; 72℃, 1min, and finally 72℃ for full extension for 10min. The plasmid pSET-152-kasO with kasO type promoter is digested with NdeI and EcoRI, and the digestion is carried out at 37℃ for 1.5h. After gel recovery and product purification, the DNA concentration is determined. The three fragments amplified above are assembled with the linear plasmid fragment of pSET-152-kasO (nucleotide sequence as shown in SEQ ID NO: 17) after digestion by homologous recombination method, and the reaction conditions are 37℃ for 30min. Escherichia coli DH5a is then transformed and the plasmid is extracted to obtain the plasmid pLDL0001 containing the 1813M1-1813M2-1813M3 fragment (the full nucleotide sequence of its connection method is shown in SEQ ID NO: 16). The schematic diagram of the plasmid is shown in Figure 1 As shown, the plasmid controls gene expression through the kasO promoter (nucleotide sequence as SEQ ID NO: 12), and the plasmid replicon is the P15A replicon.

[0084] Construction of pLDL0004 plasmid: Using the genome of Streptomyces.s.1813 as a template, the nucleotide sequence 1813T4 and the preceding sequence shown in SEQ ID NO:7 were amplified by primers 4-F / 4-R, the constructed pLDL0001 plasmid was digested with NdeI, and after gel recovery and purification, homologous recombination was completed using the C112 homologous recombination kit, and then Escherichia coli DH5a was transformed to extract the plasmid to obtain the pLDL0004 plasmid. The schematic diagram of the plasmid is shown in the figure Figure 4 As shown, the plasmid controls gene expression with the kasO promoter and the plasmid replicon is the P15A replicon. The above two plasmids serve as the basis for the construction of pLDL0011, pLDL0012, and pLDL0013 in the next step.

[0085] Construction of pLDL0011 plasmid: Using the genome of Streptomyces.s.1813 as a template, the nucleotide sequence 1813T2 and the preceding sequence as shown in SEQ ID NO:8 were amplified by primers 5-F / 5-R, and the nucleotide sequence 1813O2 as shown in SEQ ID NO:9 was amplified by primers 6-F / 6-R, and the constructed pLDL0004 plasmid was digested with EcoRV, and after gel recovery and purification, the above three fragments were homologously recombined using the C115 homologous recombination kit, and then transformed into Escherichia coli DH5a to extract the plasmid to obtain the pLDL0011 plasmid. The schematic diagram of the plasmid is shown in Figure 7 As shown, the plasmid controls gene expression through the kasO promoter, and the plasmid replicon is the P15A replicon.

[0086] Construction of pLDL0014 plasmid: pSET-152-kasO was digested with NdeI and EcoRI, and the 1813T2-1813O2 fragment constructed in pLDL0011 was amplified with primers 6-F / 14-R and assembled with the linear plasmid fragment of pSET-152-kasO after digestion by homologous recombination method, and homologous recombination was completed using C115 homologous recombination kit, and then Escherichia coli DH5a was transformed to extract the plasmid to obtain pLDL0014 plasmid. The schematic diagram of the plasmid is shown in the figure below. Fig.10 As shown, the plasmid controls gene expression through the kasO promoter, and the plasmid replicon is the P15A replicon.

[0087] Construction of pLDL0012 plasmid: Using the genome of Streptomyces.s.1813 as a template, 1813T2 and the preceding sequence (T2 promoter, nucleotide sequence SEQ ID NO: 14) were amplified by primers 5-F / 5-R, and the codon-optimized bjks fragment of the synthesized nucleotide sequence as shown in SEQ ID NO: 10 was amplified by primers 7-F / 7-R, and the constructed pLDL0004 plasmid was digested with EcoRV, and after gel recovery and purification, the above three fragments were homologously recombined using the C115 homologous recombination kit, and then transformed into Escherichia coli DH5a to extract the plasmid to obtain pLDL0012. The schematic diagram of the plasmid is shown in Figure 8 As shown, the plasmid controls gene expression through the kasO promoter, and the plasmid replicon is the P15A replicon.

[0088] Construction of pLDL0015 plasmid: pSET-152-kasO was digested with NdeI and EcoRI, and the 1813T2-bjks fragment constructed in pLDL0012 was amplified with primers 7-F / 15-R and the linear plasmid fragment of pSET-152-kasO after digestion was assembled by homologous recombination method, and homologous recombination was completed using C112 homologous recombination kit, and then Escherichia coli DH5a was transformed to extract the plasmid to obtain pLDL0015 plasmid. The schematic diagram of the plasmid is shown in the figure below. Fig.11 As shown, the plasmid controls gene expression through the kasO promoter, and the plasmid replicon is the P15A replicon.

[0089] Construction of pLDL0013 plasmid: Using the genome of Streptomyces.s.1813 as a template, the nucleotide sequence 1813T2 and the preceding sequence as shown in SEQ ID NO:8 were amplified by primers 5-F / 5-R, the nucleotide sequence 1813O2 as shown in SEQ ID NO:9 was amplified by primers 6-F / 6-R, and the nucleotide sequence 1813T1 sequence as shown in SEQ ID NO:11 was amplified by primers 8-F / 8-R, the constructed pLDL0004 plasmid was digested with EcoRV, and after gel recovery and purification, the above three fragments were homologously recombined using the C115 homologous recombination kit, and then transformed into Escherichia coli DH5a to extract the plasmid to obtain the pLDL00013 plasmid. The schematic diagram of the plasmid is shown in Fig. 9 As shown, the plasmid controls gene expression through the kasO promoter, and the plasmid replicon is the P15A replicon.

[0090] Construction of pLDL0016 plasmid: pSET-152-kasO was digested with NdeI and EcoRI, and the 1813T2, 1813O2 and 1813T1 in the amplified pLDL0013 were homologously recombined with the linear plasmid fragment of pSET-152-kasO after digestion using primers 8-F / 16-R. Homologous recombination was completed using the C112 homologous recombination kit, and then Escherichia coli DH5a was transformed to extract the plasmid to obtain the pLDL0016 plasmid. The schematic diagram of the plasmid is shown in the figure. Fig.12 As shown, the plasmid controls gene expression through the kasO promoter, and the plasmid replicon is the P15A replicon.

[0091] (2) Construction of plasmids corresponding to the high-yield sesquiterpene chassis

[0092] Construction of pLDL0002 plasmid: Using the FPP synthase gene from the literature (CN Tetzla, Z. You, DECane, S. Takamatsu, S. Omura and H. Ikeda, Biochemistry, 2006, 45, 6179-6186.) as a template, the nucleotide sequence PtlB shown in SEQ ID NO: 4 and its preceding promoter (GAPDHp (SEQ ID NO: 13)) were amplified by primers 10-F / 10-R, and an EcoRV restriction site was introduced by the front primer. The constructed pLDL0001 plasmid was digested with NdeI, and after gel recovery and purification, the PCR fragment PtlB and the restriction vector were homologously recombined, and then Escherichia coli DH5a was transformed to extract the plasmid to obtain the pLDL0002 plasmid. The schematic diagram of the plasmid is shown in the following sequence: Figure 2 shown.

[0093] Construction of pLDL0003 plasmid: Using the genome of Escherichia coli K-12MG1655 as a template, primers 9-F / 9-R were used to amplify the nucleotide sequence of IspA shown in SEQ ID NO: 5 and its preceding promoter (P9R7 (SEQ ID NO: 15)), and an EcoRV restriction site was introduced by the front primer. The constructed pLDL0001 plasmid was digested with NdeI, and after gel recovery and purification, the PCR fragment IspA was homologously recombined with the restriction vector, and then Escherichia coli DH5a was transformed to extract the plasmid to obtain the pLDL0003 plasmid. The schematic diagram of the plasmid is shown in the following sequence: Figure 3 The above two plasmids were used as the basis for the construction of pLDL0010 plasmid in the next step.

[0094] Construction of pLDL0010 plasmid: The bisabolol synthase gene from Streptomyces citrinum synthesized by chemical codon optimization was named MCTC1. The nucleotide sequence of the MCTC1 gene of Streptomyces citrinum synthesized by codon optimization as shown in SEQ ID NO:6 was used as a template, and the gene fragment of the bisabolol synthase gene with the nucleotide sequence shown in SEQ ID NO:6 was amplified by primers 11-F / 11-R. The constructed pLDL0003 plasmid was digested with EcoRV, and after gel recovery and purification, the PCR fragment of the bisabolol synthase gene was digested with the vector for homologous recombination, and then transformed into Escherichia coli DH5a to extract the plasmid, and then the construction of the pLDL0010 plasmid was completed. The schematic diagram of the plasmid is shown in the figure. Figure 6 shown.

[0095] Construction of pLDL0009 plasmid: The nucleotide sequence of the codon-optimized bisabolol synthase gene MCTC1 from Streptomyces citrinum was obtained by PCR amplification according to the above method, and the constructed pLDL0002 plasmid was digested with EcoRV. The PCR fragment of the bisabolol synthase gene was purified by gel recovery and the vector was digested for homologous recombination. Then, Escherichia coli DH5a was transformed to obtain the plasmid, and the pLDL0009 plasmid was obtained. The schematic diagram of the plasmid is shown in the following sequence: Figure 5 shown.

[0096] Construction of pLDL0017 plasmid: pSET-152-kasO was digested with NdeI and EcoRI, and the constructed MCTC1 in the amplified pLDL0009 and the linear plasmid fragment of pSET-152-kasO after digestion were assembled by homologous recombination using primers 8-F / 17-R. Homologous recombination was completed using the C112 homologous recombination kit, and then Escherichia coli DH5a was transformed to extract the plasmid to obtain the pLDL0017 plasmid. The schematic diagram of the plasmid is shown in the figure below. Fig.13 As shown, the plasmid controls gene expression through the kasO promoter, and the plasmid replicon is the P15A replicon.

[0097] Table 2 Plasmid correspondence table

[0098]

[0099] Example 2 Construction of recombinant strains that produce high yields of different types of terpenes

[0100] (1) The constructed recombinant plasmid was transformed into E. coli ET12567 (pUZ8002) competent cells, and the E. coli ET12567 (pUZ8002) containing the verified recombinant plasmid was inoculated into 5 mL of LB liquid culture medium containing 0.1% apramycin, kanamycin and chloramphenicol, and cultured in a constant temperature shaking incubator at 37°C and 225 rpm for 12 h to 18 h.

[0101] (2) Take 50uLE.coil ET12567 (pUZ8002) cultured bacterial solution and add it to 10mL LB liquid medium containing 0.1% apramycin, kanamycin and chloramphenicol, and culture it in a constant temperature shaking incubator at 37°C and 225rpm until

[0102] OD600 = 0.3-0.5. Transfer the cultured bacterial solution to a sterilized 15 ml centrifuge tube and centrifuge at 3500 rpm at 4°C for 10 min to collect the bacteria and discard the supernatant. Resuspend the bacteria in 1 mL of LB liquid medium and centrifuge again. Repeat the operation three times to remove the antibiotics on the surface of E. coli. Finally, add 400 μL of LB liquid medium to resuspend for subsequent operations.

[0103] (3) During the culture of E.coil ET12567 (pUZ8002) to the corresponding OD600, perform the Streptomyces treatment operation. In a clean bench, use a cotton swab or toothpick to transfer the well-growing white Streptomyces spores from the solid culture medium plate to a sterilized 1.5 mL centrifuge tube, centrifuge, and resuspend in sterile water. Repeat three times to remove the mycelium. Finally, add 500 μL

[0104] 2×YT medium, resuspend evenly and heat shock at 50℃ for 10min. After the heat shock, let the Streptomyces spores stand and cool to room temperature, and then pre-germinate in a constant temperature shaking incubator at 28℃ for 1-2h.

[0105] (4) After the pre-germination of Streptomyces spores, centrifuge and resuspend in 500 μL 2×YT medium, mix well with the treated E. coli added with 400 μL LB liquid medium, and spread evenly on ISP4 solid medium containing 20 mM MgCl2. After the medium is dry, culture in a constant temperature incubator at 28°C for 18-20 hours.

[0106] (5) After 18-20 hours, add 25 μL of nalidixic acid and 30 μL of apramycin to 600 μL of sterile water, mix well, and evenly spread on the plate. After drying, continue to culture in a 28°C constant temperature incubator for 4-6 days.

[0107] (6) After 4-6 days, the Streptomyces monoclonal colonies on the plate were picked with a toothpick onto a solid culture medium containing 0.1% apramycin (3-4 colonies of each monoclonal colony were picked) and cultured in a 28°C constant temperature incubator for 3-4 days.

[0108] (7) After 3-4 days, the colonies on the solid culture medium containing resistance were picked into a tryptone soy broth liquid culture medium containing 0.1% apramycin and cultured in a constant temperature shaking incubator at 28°C for 2-3 days.

[0109] (8) After centrifuging the Streptomyces cultured in the test tube, resuspend it in 1 mL of ultrapure water and boil it at 100°C for 10 min to release the genome. Then, the bacterial solution was verified by PCR using it as a template, and positive monoclonal colonies were screened for fermentation experiments. The strain with the positive verification result obtained is the recombinant strain obtained by successfully integrating the plasmid we constructed into the Streptomyces. That is, strains L1-L4 that produce different types of terpenes are obtained. The schematic diagram of obtaining strains L1-L4 is shown in Table 3.

[0110] Table 3 Detailed information of each strain

[0111]

[0112]

[0113] Example 3 Fermentation method of strains L1-L4 and product identification

[0114] The positive monoclonal colonies screened by conjugation transfer were transferred to a new solid culture medium for culture. After spores were grown for 3-4 days, the recombinant strain containing the target plasmid was obtained. Then, the fresh spores were inoculated into a 250ml triangular shaking bottle containing glass beads and 50ml tryptone soy broth liquid culture medium, and 50μL apramycin was added. The culture was cultured at 28°C and 250rpm constant temperature shaking shaker for 48h. After 48h, the cultured bacterial liquid was transferred to the sterilized basic culture medium (the transfer volume was 4%, and the culture medium was the above-mentioned A, B, D, E, F, G, GYE, XTM, PTMM culture medium), and cultured again at 28°C and 250rpm constant temperature shaking shaker for 7 days to complete the fermentation. Subsequently, the fermentation broth was centrifuged at 3750rpm, 4℃, for 20min to obtain the supernatant and bacterial precipitate, respectively. The supernatant was extracted three times with ethyl acetate to obtain a crude extract. The precipitate was soaked in an equal volume of acetone in a glass beaker and placed in a KQ5200DE CNC ultrasonic cleaner for three times, each time for 2-4 hours. The supernatant was obtained by centrifugation and the acetone was blown dry. The aqueous phase was extracted three times with ethyl acetate to obtain a crude extract. Finally, the supernatant and the precipitate were combined and separated by silica gel column and preparative high performance liquid phase to obtain the target product, which was identified by nuclear magnetic resonance ( Figure 19-22 ), whose output is Figure 19-22 shown.

[0115] The strain (L1) producing hyaluronic acid was fermented with A, B, D, E, F, G, GYE, XTM, PTMM medium, and the crude product containing the product was obtained by post-treatment according to the above steps. After separation and determination of the target molecular structure, the maximum yield was 1204 mg / L (B medium).

[0116] The kaurene-producing strain (L2) was fermented with A, B, D, E, F, G, GYE, XTM, and PTMM culture media, and the crude product containing the product was obtained by post-processing according to the above steps. After separation and determination of the target molecular structure, the maximum yield was 1353 mg / L (E culture medium).

[0117] The bisabolol-producing strain (L3) was fermented with A, B, D, E, F, G, GYE, XTM, and PTMM culture media, and the crude product containing the product was obtained by post-treatment according to the above steps. After separation and determination of the target molecular structure, the maximum yield was 1153 mg / L (B culture medium);

[0118] The atetic acid-producing strain (L4) was fermented with A, B, D, E, F, G, GYE, XTM, and PTMM culture media, and the crude product containing the product was obtained by post-processing according to the above steps. After separation and determination of the target molecular structure, the maximum yield was quantified to be 1052 mg / L (PTMM culture medium).

[0119] Culture medium A: soluble starch 20 g / L, glucose 10 g / L, peptone 5 g / L, yeast extract 5 g / L, NaCl 4 g / L, K2HPO4 0.5 g / L, MgSO4·7H2O 0.5 g / L, CaCO3 2 g / L, PH=7.0.

[0120] Culture medium B: dextrin 40 g / L, tomato paste 7.5 g / L, hydrolyzed casein 2.5 g / L, yeast extract 5 g / L, PH=7.0.

[0121] D culture medium: glucose 20 g / L, soluble starch 60 g / L, soybean powder 15 g / L, yeast extract 5 g / L, malt extract 5 g / L, calcium carbonate 2 g / L, NaCl 2 g / L, PH = 7.2.

[0122] E culture medium: yeast extract 4 g / L, malt extract 10 g / L, glucose 10 g / L, PH=7.2.

[0123] F medium: sucrose 20g / L, glucose 10g / L, casamino acid 0.1g / L, yeast extract 5g / L, MOPS 5g / L, K2SO4 0.25 g / L, MgCl·6H2O 1g / L, trace element salt solution: 10ml / L, PH = 7.2. Trace element salt solution: ZnCl2 40mg / L, FeCl3·6H2O 200mg / L, CuCl2 10 mg / L, MnCl2·4H2O 10mg / L, Na2B4O7·10H2O 10mg / L, (NH4)6Mo7O 24 4H2O 10mg / L.

[0124] G culture medium: glucose 31 g / L, soybean powder 22 g / L, K2HPO4·3H2O 1.6 g / L, PH=7.2.

[0125] GYE medium: glucose 15 g / L, yeast extract 5 g / L, CaCl2·2H2O 0.2 g / L, PH=7.2.

[0126] XTM medium: yeast extract 10g / L, malt extract 10g / L, maltose 10g / L, trace element salt solution:

[0127] 10ml / L,PH=7.2.

[0128] PTMM culture medium: dextrin 40 g / L, lactose 40 g / L, yeast extract 5 g / L, MOPS 20 g / L, trace element salt solution: 10 ml / L, PH=7.3.

[0129] MS culture medium: soybean powder 20 g / L, mannitol 20 g / L, CaCO3 3 g / L, PH=7.2.

Claims

1. A microbial chassis cell producing terpenoid compounds, It is characterized in that The chassis cells contain 1813M1 gene, 1813M2 gene and 1813M3 gene of the MEP pathway gene module derived from Streptomyces.s.1813; the nucleotide sequence of the 1813M1 gene is shown in SEQ ID NO:1; the nucleotide sequence of the 1813M2 gene is shown in SEQ ID NO:2; and the nucleotide sequence of the 1813M3 gene is shown in SEQ ID NO:

3.

2. The microbial chassis cell according to claim 1, It is characterized in that The microorganism is a eukaryotic cell or a prokaryotic cell; preferably, the microorganism is Streptomyces, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Bacillus, Aspergillus nidulans, Aspergillus niger, Neurospora crassa, Alternaria alternata or Fusarium; further preferably, the microorganism is Streptomyces albus, Streptomyces avermitilis, Streptomyces lividans, Streptomyces coelicolor; more preferably, the microorganism is Streptomyces albus.

3. The microbial chassis cell according to claim 1, It is characterized in that The terpenoid compound is selected from one or more of sesquiterpenoid compounds, diterpenoid compounds and polyterpenoid compounds.

4. A method for preparing the microbial chassis cells according to claim 1 or 2, It is characterized in that The method comprises adopting a conjugation transfer strategy to transfer 1813M1, 1813M2 and 1813M3 genes derived from Streptomyces.s.1813 to any site of the Streptomyces albus genome respectively, so as to obtain the microbial chassis cells.

5. A microbial strain capable of producing a higher yield of sesquiterpene precursors, It is characterized in that The microbial chassis cell according to claim 1 or 2 is used as a starting strain, and the polyisopentenyl synthase gene IspA from Escherichia coli or the polyisopentenyl synthase gene PtlB from Streptomyces albus is selectively overexpressed, preferably the polyisopentenyl synthase gene IspA from Escherichia coli is overexpressed to obtain a microbial strain with a higher yield of sesquiterpene precursors. The polyisopentenyl synthase gene PtlB from Streptomyces albus is shown in SEQ ID NO: 4, and the polyisopentenyl synthase gene IspA from Escherichia coli is shown in SEQ ID NO:

3.

6. A microbial strain that produces a higher yield of diterpene precursors, It is characterized in that The microbial chassis cell of claim 1 or 2 is used as the starting strain, and the polyisopentenyl synthase gene 1813T4 from Streptomyces.s.1813 is overexpressed to obtain a microbial strain with a higher diterpene precursor production. The sequence of the polyisopentenyl synthase gene 1813T4 from Streptomyces.s.1813 is shown in SEQ ID NO:

7.

7. A microbial strain that produces high yields of sesquiterpenoid compounds, It is characterized in that The microbial chassis strain described in claim 5 is used as the starting strain, and the codon-optimized bisabolol synthase gene MCTC1 from Streptomyces citrinum is overexpressed to obtain a microbial strain with high yield of sesquiterpene compounds; the sequence of the codon-optimized bisabolol synthase gene MCTC1 from Streptomyces citrinum is shown in SEQ ID NO:

6.

8. A microbial strain that produces high yields of diterpenoid compounds, It is characterized in that The microbial chassis strain described in claim 6 is used as the starting strain, and the diterpene synthase gene 1813T2 and cytochrome P450 enzyme genes 1813O2, bjks, 1813T1, etc. derived from Streptomyces.s.1813 are co-expressed to obtain the microbial strain with high diterpenoid production; the sequence of gene 1813T2 derived from Streptomyces.s.1813 is shown in SEQ ID NO:8, the sequence of gene 1813O2 is shown in SEQ ID NO:9, the sequence of gene 1813T1 is shown in SEQ ID NO:11, and the sequence of gene bjks derived from soybean rhizobium is shown in SEQID NO:

10.

9. A method for obtaining a crude terpenoid compound, It is characterized in that The method comprises: subjecting the microbial strain according to any one of claims 7 to 8 to fermentation treatment, and subjecting the fermentation treatment product to extraction treatment to obtain the crude terpenoid compound; the fermentation treatment is preferably achieved by the following method: the fermentation is carried out in different basic culture media, wherein the different basic culture media are respectively A culture media, B culture media, D culture media, E culture media, F culture media, G culture media, GYE culture media, XTM culture media, PTMM culture media, and MS culture media: The A culture medium: soluble starch 20g / L, glucose 10g / L, peptone 5g / L, yeast extract 5g / L, NaCl 4g / L, K 2 HPO 4 0.5 g / L,MgSO 4 7H 2 O 0.5g / L,CaCO 3 2 g / L, PH=7.0; The B culture medium: dextrin 40g / L, tomato paste 7.5g / L, hydrolyzed casein 2.5g / L, yeast extract 5g / L, pH=7.0; the D culture medium: glucose 20g / L, soluble starch 60g / L, soybean powder 15g / L, yeast extract 5g / L, malt extract 5g / L, calcium carbonate 2g / L, NaCl 2g / L, pH=7.2; The E medium: 4 g / L yeast extract, 10 g / L malt extract, 10 g / L glucose, pH = 7.2; the F medium: 20 g / L sucrose, 10 g / L glucose, 0.1 g / L casamino acid, 5 g / L yeast extract, 5 g / L MOPS, K 2 SO40.25 g / L,MgCl·6H 2 O 1g / L, trace element salt solution: 10ml / L, PH=7.

2. Trace element salt solution: ZnCl 2 40 mg / L,FeCl 3 6H 2 O 200mg / L,CuCl 2 10 mg / L,MnCl 2 ·4H 2 O10mg / L,Na 2 B 4 O 7 10H 2 O 10mg / L, (NH 4 ) 6 Mo 7 O 24 ·4H 2 O 10 mg / L; The G medium: glucose 31g / L, soybean powder 22g / L, K 2 HPO 4 ·3H 2 O 1.6g / L, PH = 7.2; the GYE medium: glucose 15g / L, yeast extract 5g / L, CaCl 2 ·2H 2 O 0.2g / L, PH=7.2; The XTM medium: yeast extract 10 g / L, malt extract 10 g / L, maltose 10 g / L, trace element salt solution: 10 ml / L, pH = 7.2; The PTMM culture medium: dextrin 40 g / L, lactose 40 g / L, yeast extract 5 g / L, MOPS 20 g / L, trace element salt solution: 10 ml / L, PH = 7.3; The MS medium: soybean powder 20g / L, mannitol 20g / L, CaCO 3 3 g / L, PH=7.

2.

10. Application of 1813M1, 1813M2 and 1813M3 genes from Streptomyces.s.1813 in constructing microbial chassis cells with high production of terpenoid compounds.

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