Recombinant strain of high-yield terpenoids as well as construction method and application of recombinant strain

Abstract

The invention relates to the fields of genetic engineering and microbial fermentation, and discloses a recombinant strain for highly producing terpenoids as well as a construction method and application of the recombinant strain. The recombinant strain is obtained by genetic engineering modification of an original strain, compared with the original strain, the recombinant strain enhances the synthetic route of terpenoids, and the activity of pyruvate kinase, pyruvate dehydrogenase complex, glucose-6-phosphate dehydrogenase and key enzymes for synthesis of terpenoids is enhanced by using a CRISPR activation system; wherein the sgRNA expression promoters of the pyruvate kinase and the pyruvate dehydrogenase complex are respectively oleic acid induction type promoters, and the sgRNA expression promoters of the glucose-6-phosphate dehydrogenase and the terpene compound synthesis key enzyme are respectively copper ion induction type promoters. The recombinant strain can dynamically regulate and control an editing system of the terpenoids, precisely regulate and control the synthetic pathway flux of the terpenoids, and effectively improve the yield of the terpenoids (such as sclareol and bisabolol).

Description

Technical Field

[0001] The present invention relates to the fields of genetic engineering and microbial fermentation, and in particular to a recombinant strain with high yield of terpenoid compounds, a construction method thereof and an application thereof. Background Art

[0002] Sclareol, a sesquiterpenoid compound, is widely found in plant essential oils. It possesses antibacterial, antioxidant, and anti-inflammatory activities and is widely used in fragrances, medicine, and agriculture. However, since natural sclareol is primarily extracted from plants, it is difficult to meet industrial demand due to limitations in plant growth cycles, low yields, and complex extraction processes. Therefore, a microbial synthetic biology approach offers an alternative for the sustainable production of sclareol.

[0003] Yarrowia lipolytica is a non-traditional yeast widely used in biomanufacturing. Its excellent lipid metabolism, high-throughput precursor supply, and adaptability to industrial fermentation conditions have attracted widespread attention in synthetic biology and metabolic engineering. In recent years, the production of terpenoids using Yarrowia lipolytica has become a research hotspot. Its efficient acetyl-CoA supply provides an excellent foundation for the synthesis of sesquiterpenoids. Studies have investigated the introduction of genes encoding sclareol synthase and key enzymes in related metabolic pathways into Yarrowia lipolytica to increase sclareol production. While the sclareol synthesis pathway can be introduced into Yarrowia lipolytica through traditional overexpression of key enzymes, the precision of flux control in the sclareol synthesis pathway is limited, resulting in difficulties in achieving the desired sclareol yield, hindering the development of industrial sclareol production. Summary of the Invention

[0004] The purpose of the present invention is to overcome the problem of low precision in flux regulation of the synthesis pathway of sclareol in the prior art, and to provide a recombinant strain with high yield of terpenoid compounds and its construction method and application. The recombinant strain can dynamically regulate the editing system of terpenoid compounds, accurately regulate the flux of the synthesis pathway of terpenoid compounds, and effectively increase the yield of terpenoid compounds (such as sclareol).

[0005] To achieve the above objectives, the present invention provides, in a first aspect, a recombinant strain that produces high-yield terpenoid compounds. The recombinant strain is obtained by genetic engineering of a starting strain. Compared with the starting strain, the recombinant strain enhances the terpenoid synthesis pathway and utilizes a CRISPR activation system to enhance the activities of pyruvate kinase, pyruvate dehydrogenase complex, glucose-6-phosphate dehydrogenase, and key enzymes for terpenoid synthesis, respectively. The sgRNA expression promoters of the pyruvate kinase and pyruvate dehydrogenase complex are oleic acid-inducible promoters, and the sgRNA expression promoters of the glucose-6-phosphate dehydrogenase and key enzymes for terpenoid synthesis are copper ion-inducible promoters.

[0006] A second aspect of the present invention provides a method for constructing a recombinant strain that produces high-yield terpenoid compounds, the method comprising: genetically engineering a starting strain so that the starting strain enhances the terpenoid synthesis pathway, and utilizing a CRISPR activation system to enhance the activities of pyruvate kinase, pyruvate dehydrogenase complex, glucose-6-phosphate dehydrogenase, and key enzymes for terpenoid synthesis, respectively; wherein the sgRNA expression promoters of the pyruvate kinase and pyruvate dehydrogenase complex are respectively oleic acid-inducible promoters, and the sgRNA expression promoters of the glucose-6-phosphate dehydrogenase and key enzymes for terpenoid synthesis are respectively copper ion-inducible promoters.

[0007] The third aspect of the present invention provides the use of the aforementioned recombinant strain or the aforementioned method in preparing terpenoid compounds.

[0008] A fourth aspect of the present invention provides a method for fermenting terpenoid compounds, the method comprising: fermenting the aforementioned recombinant strain; or, constructing a recombinant strain according to the aforementioned method, and fermenting the obtained recombinant strain; wherein the fermentation process comprises a first fermentation stage and a second fermentation stage carried out in sequence, the carbon source in the culture medium used in the first fermentation stage being oleic acid, and glucose and copper ions being added in the second fermentation stage.

[0009] Through the above technical solution, the beneficial effects of the present invention are:

[0010] The recombinant strain provided by the present invention enhances the intracellular terpenoid synthesis pathway while enhancing the activity of intracellular pyruvate kinase, pyruvate dehydrogenase complex, glucose-6-phosphate dehydrogenase and key enzymes for terpenoid synthesis through the CRISPR activation system. By utilizing the cooperation of different enzymes with oleic acid-inducible promoters and copper ion-inducible promoters, the recombinant strain activates cell growth-related genes (pyruvate kinase, pyruvate dehydrogenase complex) in the early stage of fermentation to promote cell growth metabolism, inhibits cell growth-related genes in the late stage of fermentation, activates genes related to terpenoid synthesis, and promotes the production of terpenoids. This enables the fermentation process of the recombinant strain to dynamically regulate the editing system of terpenoids, achieves decoupling of cell growth and terpenoid synthesis, accurately regulates the flux of the terpenoid synthesis pathway, and effectively increases the yield of terpenoids (such as sclareol). BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is a structural diagram of the recombinant plasmid pUC-intC-HUH-SsTPS-SsLPPS in Example 1;

[0012] Figure 2 is a structural diagram of the recombinant plasmid pUC-HUH-B2-CRISPR-VPR in Example 1;

[0013] Figure 3 This is a structural diagram of the recombinant plasmid PMCS-CEN-LEU-sgRNA (pyruvate kinase) in Example 1. DETAILED DESCRIPTION

[0014] The endpoints of the ranges and any values disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0015] The first aspect of the present invention provides a recombinant strain that produces high-yield terpenoid compounds. The recombinant strain is obtained by genetic engineering of a starting strain. Compared with the starting strain, the recombinant strain enhances the terpenoid synthesis pathway and utilizes a CRISPR activation system to enhance the activities of pyruvate kinase, pyruvate dehydrogenase complex, glucose-6-phosphate dehydrogenase, and key enzymes for terpenoid synthesis. The sgRNA expression promoters of the pyruvate kinase and pyruvate dehydrogenase complex are oleic acid-inducible promoters, and the sgRNA expression promoters of the glucose-6-phosphate dehydrogenase and key enzymes for terpenoid synthesis are copper ion-inducible promoters.

[0016] During the research process, the inventors of the present invention discovered that by heterologously introducing the terpenoid synthesis pathway into the starting strain and simultaneously using the CRISPR activation (CRISPRa) system in combination with different promoters, the activity of pyruvate kinase, pyruvate dehydrogenase complex, glucose-6-phosphate dehydrogenase, and key enzymes for terpenoid synthesis in the starting strain was enhanced, thereby forming an editing system for dynamically regulating terpenoids in the recombinant strain. When oleic acid is used as a carbon source in the early stage of fermentation, the expression of the oleic acid-inducible promoter can be activated, thereby activating the gene expression of pyruvate kinase and pyruvate dehydrogenase complex, promoting cell growth and metabolism. In the late stage of fermentation, glucose and copper ions are introduced to activate the copper ion-inducible promoter, thereby activating the gene expression of glucose-6-phosphate dehydrogenase and key enzymes for terpenoid synthesis, and inhibiting growth-related genes such as pyruvate kinase and pyruvate dehydrogenase complex, promoting the production of terpenoids, accurately regulating the flux of the terpenoid synthesis pathway, and combining the metabolites and energy accumulated by cell growth in the early stage of fermentation to effectively improve the synthesis efficiency and yield of terpenoids (such as sclareol).

[0017] According to the present invention, the oleic acid-inducible promoter can be any promoter activated solely by oleic acid. Preferably, the oleic acid-inducible promoter is POX2, whose nucleotide sequence is shown in SEQ ID NO. 1. This preferred embodiment is advantageous in increasing the sensitivity of the oleic acid-inducible promoter to the presence of oleic acid, thereby promoting the dynamic regulation of cell growth in the early stages of fermentation of the recombinant strain.

[0018] According to the present invention, the copper ion-inducible promoter can be any promoter that is activated by copper ions and is not inhibited by glucose. Preferably, the copper ion-inducible promoter is PMT1 having a nucleotide sequence as shown in SEQ ID NO.2 and / or PMT2 having a nucleotide sequence as shown in SEQ ID NO.3. Further preferably, the copper ion-inducible promoter is PMT1 having a nucleotide sequence as shown in SEQ ID NO.2. Under this preferred embodiment, the copper ion-inducible promoter is sensitive to the presence of copper ions and is not affected by the culture medium components such as oleic acid and glucose used in the fermentation of the recombinant strain, such as the carbon source and nitrogen source. This can better promote the dynamic regulation of the synthesis of terpenoid compounds in the late fermentation period and increase the yield of terpenoid compounds.

[0019] According to the present invention, the terpenoid compound can be any compound whose molecular skeleton uses an isoprene unit (C5 unit) as a basic structural unit and its derivatives; preferably, the terpenoid compound is selected from at least one of sclareol, bisabolol, nerol, linalool and lavender alcohol, more preferably sclareol and / or bisabolol.

[0020] As a preferred embodiment, the terpene compound is sclareol, and the terpene compound synthesis pathway is enhanced by exogenously introducing sclareol synthase and sclareol heterologous lysine diol pyrophosphate synthase into the starting strain. The key enzymes for the synthesis of terpene compounds include sclareol synthase and sclareol heterologous lysine diol pyrophosphate synthase. At this time, the recombinant strain not only obtains the ability to synthesize sclareol by heterologously introducing two exogenous genes, sclareol synthase and sclareol heterologous lysine diol pyrophosphate synthase, but also constructs the corresponding CRISPRa editing system for sclareol synthase and sclareol heterologous lysine diol pyrophosphate synthase, and the corresponding sgRNA expression promoter adopts a copper ion inducible promoter, which can better promote the dynamic regulation of the synthesis of sclareol in the late fermentation stage and increase the yield of sclareol.

[0021] More preferably, the amino acid sequence of the sclareol synthase is shown in SEQ ID NO. 4, and the amino acid sequence of the sclareol heterologous lysine diol pyrophosphate synthase is shown in SEQ ID NO. 5. Further preferably, the nucleotide sequence of the sclareol synthase encoding gene sstps is shown in SEQ ID NO. 6, and the nucleotide sequence of the sclareol heterologous lysine diol pyrophosphate synthase encoding gene sslpps is shown in SEQ ID NO. 7. Under this preferred embodiment, it is beneficial to better promote the dynamic regulation of sclareol synthesis in the late fermentation stage and increase the yield of sclareol.

[0022] According to the present invention, preferably, the sgRNA nucleotide sequence of the sclareol synthase is shown as SEQ ID NO.8, and the sgRNA nucleotide sequence of the sclareol heterologous lysedil pyrophosphate synthase is shown as SEQ ID NO.9.

[0023] As another preferred embodiment, the terpene compound is bisabolol, and the terpene compound synthesis pathway is enhanced by exogenously introducing bisabolol synthase into the starting strain, and the key enzyme for the synthesis of the terpene compound includes bisabolol synthase; at this time, the recombinant strain not only improves the ability to synthesize bisabolol by heterologously introducing the coding gene for bisabolol synthase, but also constructs a CRISPRa editing system corresponding to bisabolol synthase, and the corresponding sgRNA expression promoter adopts a copper ion inducible promoter, which can better promote the dynamic regulation of bisabolol synthesis in the late fermentation stage and increase the yield of bisabolol.

[0024] More preferably, the amino acid sequence of the bisabolol synthase is shown in SEQ ID NO. 14; further preferably, the nucleotide sequence of the bisabolol synthase encoding gene BBS is shown in SEQ ID NO. 15. This preferred embodiment facilitates dynamic regulation of bisabolol synthesis in the late fermentation stage and increases bisabolol production.

[0025] According to the present invention, preferably, the sgRNA nucleotide sequence of the bisabolol synthase is shown as SEQ ID NO.16.

[0026] More preferably, the sgRNA nucleotide sequence for pyruvate kinase is shown in SEQ ID NO. 10, the sgRNA nucleotide sequence for the pyruvate dehydrogenase complex is shown in SEQ ID NO. 11, and the sgRNA nucleotide sequence for glucose-6-phosphate dehydrogenase is shown in SEQ ID NO. 12. This preferred embodiment facilitates enhanced expression of the various functional enzymes in the recombinant strain, further promoting the synthesis of terpenoids and increasing terpenoid yield. It also enables better coordination with oleic acid-inducible promoters and copper-ion-inducible promoters, improving the precision of flux regulation in the terpenoid synthesis pathway.

[0027] In the present invention, the pyruvate kinase, the pyruvate dehydrogenase complex and the glucose-6-phosphate dehydrogenase can adopt known sequences. For example, the NCBI number of the pyruvate kinase encoding gene Pyk is YALI1_F12842g, the NCBI number of the pyruvate dehydrogenase complex encoding gene pdh is YALI1_F27556g, and the NCBI number of the glucose-6-phosphate dehydrogenase encoding gene zwf is YALI1_E26811g.

[0028] According to the present invention, preferably, the starting strain is Yarrowia lipolytica; more preferably, it is the Yarrowia lipolytica strain MYA2613 purchased from the American Type Culture Collection (ATCC).

[0029] The inventors of the present invention found that, using Yarrowia lipolytica as a starting strain, the key enzymes of the terpenoid compound synthesis pathway, namely, sclareol synthase and sclareol heterologous pyrophosphate lysedil ester synthase, were exogenously introduced, and the corresponding CRISPR activation systems of pyruvate kinase, pyruvate dehydrogenase complex, glucose-6-phosphate dehydrogenase, sclareol synthase and sclareol heterologous pyrophosphate lysedil ester synthase were constructed in the cell respectively; wherein, the CRISPR activation systems of the pyruvate kinase and the pyruvate dehydrogenase complex The expression promoters of sgRNA in the system are oleic acid-inducible promoters, and the expression promoters of sgRNA in the CRISPR activation system of glucose-6-phosphate dehydrogenase, sclareol synthase and sclareol heterologous lysine diol pyrophosphate synthase are copper ion-inducible promoters; the obtained recombinant strain can more accurately regulate the flux of the sclareol synthesis pathway and more significantly improve the synthesis efficiency of sclareol, which is conducive to promoting the application of synthetic biology in the industrial production of natural products such as sclareol.

[0030] A second aspect of the present invention provides a method for constructing a recombinant strain that produces high-yield terpenoid compounds, the method comprising: genetically engineering a starting strain so that the starting strain enhances the terpenoid synthesis pathway, and utilizing a CRISPR activation system to enhance the activities of pyruvate kinase, pyruvate dehydrogenase complex, glucose-6-phosphate dehydrogenase, and key enzymes for terpenoid synthesis, respectively; wherein the sgRNA expression promoters of the pyruvate kinase and pyruvate dehydrogenase complex are respectively oleic acid-inducible promoters, and the sgRNA expression promoters of the glucose-6-phosphate dehydrogenase and key enzymes for terpenoid synthesis are respectively copper ion-inducible promoters.

[0031] According to the present invention, preferably, the oleic acid-inducible promoter is POX2 with a nucleotide sequence as shown in SEQ ID NO.1.

[0032] According to the present invention, preferably, the copper ion inducible promoter is PMT1 with a nucleotide sequence as shown in SEQ ID NO.2 and / or PMT2 with a nucleotide sequence as shown in SEQ ID NO.3.

[0033] According to the present invention, preferably, the terpenoid compound is selected from at least one of sclareol, bisabolol, nerol, linalool and lavenderol, more preferably sclareol and / or bisabolol.

[0034] According to the present invention, preferably, the terpene compound is sclareol, and the terpene compound synthesis pathway is enhanced by exogenously introducing sclareol synthase and sclareol heterologous lysine diol pyrophosphate synthase into the starting strain, and the key enzymes for the synthesis of terpene compounds include sclareol synthase and sclareol heterologous lysine diol pyrophosphate synthase; the amino acid sequence of the sclareol synthase is shown in SEQ ID NO.4, and the amino acid sequence of the sclareol heterologous lysine diol pyrophosphate synthase is shown in SEQ ID NO.5.

[0035] According to the present invention, preferably, the nucleotide sequence of the sstps gene encoding the sclareol synthase is shown as SEQ ID NO.6, and the nucleotide sequence of the sslpps gene encoding the sclareol pyrophosphate synthase is shown as SEQ ID NO.7.

[0036] According to the present invention, preferably, the sgRNA nucleotide sequence of the sclareol synthase is shown as SEQ ID NO.8, and the sgRNA nucleotide sequence of the sclareol heterologous lysedil pyrophosphate synthase is shown as SEQ ID NO.9.

[0037] According to the present invention, preferably, the terpenoid compound is bisabolol, and the terpenoid compound synthesis pathway is enhanced by exogenously introducing bisabolol synthase into the starting strain, wherein the key enzyme for terpenoid compound synthesis includes bisabolol synthase; the amino acid sequence of the bisabolol synthase is shown in SEQ ID NO. 14. More preferably, the nucleotide sequence of the gene encoding bisabolol synthase, BBS, is shown in SEQ ID NO. 15. Further preferably, the nucleotide sequence of the sgRNA encoding bisabolol synthase is shown in SEQ ID NO. 16.

[0038] According to the present invention, preferably, the sgRNA nucleotide sequence of the pyruvate kinase is shown as SEQ ID NO.10, the sgRNA nucleotide sequence of the pyruvate dehydrogenase complex is shown as SEQ ID NO.11, and the sgRNA nucleotide sequence of the glucose-6-phosphate dehydrogenase is shown as SEQ ID NO.12.

[0039] According to the present invention, preferably, the starting strain is Yarrowia lipolytica.

[0040] In the present invention, the CRISPR activation system (CRISPRa system) targets a specific promoter segment or enhancer region by connecting a transcriptional activation domain between the guide RNA (sgRNA) and the Cas9 protein, thereby upregulating the transcriptional activity of a specific gene and achieving activation of specific gene expression. The CRISPRa system is mainly composed of a Cas9-transcriptional activation domain fusion protein and a specific sgRNA. Among them, the CRISPRa system of pyruvate kinase, pyruvate dehydrogenase complex, glucose-6-phosphate dehydrogenase, sclareol synthase and sclareol heterologous pyrophosphate lysedil ester synthase can be sequentially connected to a vector plasmid to construct a recombinant vector that simultaneously expresses pyruvate kinase, pyruvate dehydrogenase complex, glucose-6-phosphate dehydrogenase, sclareol synthase and sclareol heterologous pyrophosphate lysedil ester synthase, and then introduced into a starting strain with an enhanced terpenoid synthesis pathway.

[0041] The corresponding CRISPRa system can be constructed using conventional methods in the art. Various methods for constructing recombinant vectors are known in the art for ligating target gene fragments (e.g., genes encoding the aforementioned enzymes) to expression vectors to prepare recombinant vectors, such as, but not limited to, the classic "enzyme digestion-ligation" method, the Gateway cloning system developed by Invitrogen, and the ClonExpress cloning system developed by Novozymes (e.g., the ClonExpress MultiS One Step Cloning Kit).

[0042] For example, the expression vector of the recombinant vector adopts PMCS-CEN-LEU-sgRNA vector, and the recombinant vector PMCS-CEN-LEU-sgRNA (pyruvate kinase)-sgRNA (pyruvate dehydrogenase complex)-sgRNA (glucose-6-phosphate dehydrogenase)-sgRNA (sstps)-sgRNA (sslpps) is obtained by designing primers and enzyme cutting-ligation method, but the present invention is not limited to this.

[0043] Subsequently, each recombinant vector can be introduced into the starting strain (such as Yarrowia lipolytica) using conventional methods in the art, such as but not limited to microinjection, gene gun, transformation (such as electroporation). The above-mentioned microinjection, gene gun or transformation are all routine operations in the art. For example, transformation refers to treating cells by some known methods in molecular biology and genetic engineering, so that the treated cells are in a competent state, and thereby contacted with exogenous DNA, so that the exogenous DNA enters the cells in the competent state. Common transformation methods include protoplast transformation, chemical transformation and electroporation transformation.

[0044] A third aspect of the present invention provides the use of the aforementioned recombinant strain or method for preparing terpenoids. By activating the expression activity of different functional enzymes in the recombinant strain at different times during fermentation, the flux of the terpenoid synthesis pathway can be more precisely regulated. Furthermore, by combining the metabolites and energy accumulated by cell growth during the early stages of fermentation, the synthesis efficiency and yield of terpenoids (e.g., sclareol) can be effectively improved.

[0045] In the present invention, preferably, the terpenoid compound is selected from at least one of sclareol, bisabolol, nerol, linalool and lavenderol, more preferably sclareol and / or bisabolol.

[0046] A fourth aspect of the present invention provides a method for fermenting terpenoid compounds, the method comprising: fermenting the aforementioned recombinant strain; or, constructing a recombinant strain according to the aforementioned method, and fermenting the obtained recombinant strain; wherein the fermentation process comprises a first fermentation stage and a second fermentation stage carried out in sequence, the carbon source in the culture medium used in the first fermentation stage being oleic acid, and glucose and copper ions being added in the second fermentation stage.

[0047] In the present invention, the recombinant strain uses oleic acid as the sole carbon source in the first fermentation stage, activates the gene expression of pyruvate kinase and pyruvate dehydrogenase complex related to cell growth, and can effectively promote cell growth metabolism; in the second fermentation stage, glucose and copper ions are introduced to activate the expression of the copper ion-inducible promoter and inhibit the expression of the oleic acid-inducible promoter, thereby activating the gene expression of glucose-6-phosphate dehydrogenase and key enzymes for terpenoid synthesis, increasing the flux of the terpenoid synthesis pathway in the cell, promoting the production of terpenoids, accurately regulating the flux of the terpenoid synthesis pathway, and combining the metabolites and energy accumulated by cell growth in the early stage of fermentation, effectively improving the synthesis efficiency and yield of terpenoids (such as sclareol).

[0048] According to the present invention, the parameters such as inoculum size, temperature, pH, rotation speed, time, etc. used in the fermentation culture can be conventional settings in the art. Preferably, according to the present invention, when the starting strain is Yarrowia lipolytica, preferably, the conditions of the first fermentation stage include: oleic acid content of 5-25g / L, temperature of 20-35°C, stirring rate of 200-250rpm, and time of 40-60h; the conditions of the second fermentation stage include: glucose addition of 20-80g / L, copper ion addition of 20-150mM, temperature of 20-35°C, stirring rate of 200-250rpm, and time of 40-60h. The inventors found that under this preferred embodiment, it is conducive to further promoting the growth of the recombinant strain, and accurately regulating the metabolic flux of the terpenoid synthesis pathway, so as to better improve the fermentation biomass and the yield of terpenoids.

[0049] The copper ions can be added in the form of any copper salt, such as copper sulfate, copper chloride, copper nitrate, etc., so as to form a corresponding concentration of copper ions in the culture medium.

[0050] In the present invention, the recombinant strain can be first prepared into a seed liquid, and then the seed liquid can be inoculated into the fermentation medium for fermentation to obtain a fermentation liquid containing terpenoid compounds.

[0051] In the present invention, preferably, the method for preparing the seed solution comprises: picking a single colony of the recombinant strain and inoculating it into a seed culture medium for seed culture to obtain the seed solution.

[0052] In the present invention, the seed culture medium is not particularly limited and can be any conventional medium used in the art for preparing Yarrowia lipolytica seed solution. Preferably, the seed culture medium contains a carbon source, a nitrogen source, and inorganic salts. Further preferably, the seed culture medium contains ammonium sulfate, YNB, and glucose. Exemplarily, the seed culture medium comprises, by mass / volume ratio, 4-6 g / L ammonium sulfate, 1-2 g / L yeast basal nitrogen source YNB, and 15-25 g / L glucose.

[0053] In the present invention, there is no particular limitation on the seed culture method. The parameters used in the seed culture, such as temperature, pH, rotation speed, and time, can be conventional settings in the art. Preferably, the conditions for the seed culture include: a temperature of 20-35°C, a rotation speed of 200-250 rpm, and a time of 20-28 hours. In the present invention, the seed culture can be inoculated with a single colony of the recombinant strain, for example, a colony selected from a streak culture of the recombinant strain on a solid plate. Exemplarily, the recombinant strain is inoculated and streaked onto a YNB solid plate, and cultured at a temperature of 20-35°C for 2-3 days to obtain a single colony of the recombinant strain. The YNB solid plate culture medium contains: 3-8 g / L ammonium sulfate, 15-25 g / L glucose, 1-2 g / L YNB, and 15-20 g / L agar.

[0054] In the present invention, the culture medium used for fermentation contains, in addition to the specified oleic acid, 3-8 g / L of ammonium sulfate and 1-2 g / L of YNB. In addition, it may also contain a carbon source such as glycerol that does not inhibit the oleic acid-inducible promoter. 40-60 hours after the start of fermentation, the corresponding glucose and copper ions are added to initiate the second stage of fermentation.

[0055] In the present invention, in order to increase the yield of terpenoid compounds, preferably, the inoculation amount of the seed liquid is 8-12 parts by volume relative to 100 parts by volume of the fermentation medium.

[0056] In the present invention, terpenoid compounds in the resulting fermentation broth can be isolated by known methods. For example, monoterpenes, sesquiterpenes, and diterpenes can be secreted extracellularly by the recombinant strain and can be isolated by removing cells from the fermentation broth and then concentrating the fermentation broth to crystallize the product, or performing ion exchange chromatography or the like. Triterpenes and tetraterpenes are intracellular products and can be extracted, isolated, and purified by cell disruption.

[0057] In the present invention, the terpenoid compounds in the fermentation broth can be detected by known methods, or the terpenoid compounds separated from the fermentation broth can be detected. For example, the terpenoid compounds can be detected by gas chromatography and the like.

[0058] The present invention will be described in detail below by way of examples. However, these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. In the following examples, unless otherwise stated, the experimental methods used are conventional methods well known to those skilled in the art, or according to the conditions recommended by the manufacturer.

[0059] In the following examples, unless otherwise specified, all reagents and culture media used were commercially available products, and all methods used were conventional methods; unless otherwise specified, the room temperature was 25±5°C.

[0060] In the following examples, the starting strain was Yarrowia lipolytica MYA2613 purchased from the American Type Culture Collection (ATCC).

[0061] In the following examples, YNB solid medium contains: ammonium sulfate 5 g / L, glucose 20 g / L, YNB 1.7 g / L, agar 15 g / L;

[0062] The seed culture medium is composed of 5 g / L ammonium sulfate, 1.7 g / L YNB, and 20 g / L glucose in a mass / volume ratio.

[0063] In the following examples, the preparation and transformation process of competent yeast Yarrowia lipolytica was as follows: spots on a plate were picked, inoculated into a seed culture medium, cultured overnight at 30°C and 220 rpm, and then competent cells were prepared and transformed using the Frozen-EZ Yeast Transformation II Kit produced by ZymoResearch.

[0064] In the following examples, the genome extraction process is as follows:

[0065] The transformants were picked and inoculated into seed culture medium. The culture was carried out at a temperature of 30°C and a rotation speed of 220 rpm for 24 h. Then, 50 mL of the bacterial solution was taken and centrifuged at 12000 rpm for 10 min to obtain the transformant bacteria. The genome of the transformant bacteria was extracted according to the operating instructions of the fungal genome extraction kit of Sangon Biotech (Shanghai) Co., Ltd.

[0066] In the following examples, the cloning process of genes and expression vectors is as follows:

[0067] The PCR enzyme used in PCR amplification was TAKARA's PrimeSTAR Max DNA polymerase; the PCR amplification system is shown in Table 1.

[0068] Table 1 PCR amplification system

[0069] Reagents Usage Final concentration PrimeSTAR Max(2×) 25 μl 1× Primer 1 10-15 pmol 0.2-0.3 μmol Primer 2 10-15 pmol 0.2-0.3 μmol Template <200ng Sterile distilled water Up to 50μL

[0070] The PCR amplification process was as follows: denaturation at 98°C for 10 s, annealing at 55°C for 10 s, and extension at 72°C. After 35 cycles, each fragment was purified and recovered using an AxyPrep™ DNA Gel Extraction Kit (purchased from Corning Life Sciences (Wujiang) Co., Ltd.). Extension time = target fragment length / 1 kb, unit: min.

[0071] One-step cloning was achieved using the ClonExpress MultiS One Step Cloning Kit from Nanjing Novozymes Biotechnology Co., Ltd. The reaction system is shown in Table 2. The reaction system was incubated at 50°C for 15 minutes to obtain a circular recombinant vector.

[0072] Table 2 One-step cloning system

[0073] Components Recombination reaction Linearized vector XμL N inserts <![CDATA[Y1+Y2+…Y n μL]]> 2×ClonExpress Mix 5μL <![CDATA[ddH2O]]> To 10μL

[0074] The circular recombinant vector was transformed into Escherichia coli DH5a competent cells, screened by ampicillin resistance plate and verified by colony PCR and sequencing to obtain positive recombinant plasmids, which were sent for sequencing.

[0075] Example 1

[0076] (1) Construction of the original plasmid

[0077] Based on the nucleotide sequences of the gene encoding sclareol synthase SsTPS, the gene encoding sclareol pyrophosphate lysedione synthase SsLPPS, and the VPR sequence provided on NCBI, after specific codon optimization, we commissioned Qingke Biotechnology Co., Ltd. to synthesize them; wherein, the amino acid sequence of sclareol synthase is shown in SEQ ID NO.4, the nucleotide sequence of the gene encoding SsTPS is shown in SEQ ID NO.6, the amino acid sequence of the gene encoding sclareol pyrophosphate lysedione synthase is shown in SEQ ID NO.5, the nucleotide sequence of the gene encoding SsLPPS is shown in SEQ ID NO.7, and the nucleotide sequence of the transcription activator VPR is shown in SEQ ID NO.13;

[0078] (2) Construction of recombinant plasmid pUC-HUH-intC-SsTPS-SsLPPS

[0079] The template plasmid pUC-HUH-intC was constructed, wherein URA3 is the orotic acid 5'-phosphate decarboxylase gene, intC-up and intC-up are the upstream and downstream homology arms of expression, and AmpR is the ampicillin resistance gene;

[0080] Using pUC-HUH-intC as the backbone, the circular plasmid was enzymatically cut into linear fragments using the endonuclease hindIII; then, primers were designed to amplify the endogenous promoter TDH1, the gene SsTPS, and the endogenous terminator mig1t. The pUC-HUH-intC backbone linear fragment and the aforementioned fragment were cloned in one step using the ClonExpress MultiS One Step Cloning Kit of Nanjing Novozymes Biotechnology Co., Ltd., and the recombinant plasmid pUC-HUH-intC-SsTPS was obtained after sequencing verification; then, using this plasmid as the backbone, the circular plasmid was enzymatically cut into linear fragments using the endonuclease EcoI, and the SsLPPS expression cassette (promoter: TEF, terminator: mig1t2) was inserted to obtain the recombinant plasmid pUC-HUH-intC-SsTPS-SsLPPS. The plasmid structure is as follows: Figure 1 The primer pairs for the above gene fragments are shown in Table 3.

[0081] Table 3 Primer sequences

[0082]

[0083] (3) Construction of recombinant plasmid pUC-HUH-B2-CRISPR-VPR

[0084] Using pUC-HUH-B2 as the backbone, intB2-up and intB2-dn as upstream and downstream homology arms, the circular plasmid was enzymatically cut into linear fragments using the endonuclease HindIII. Primers were designed to amplify the promoter TEFin, dCas9, VPR, and terminator xpr2t, respectively. dCas9 and VPR were connected with a GGGGS linker peptide. The puc-HUH-B2 backbone linear fragment and the aforementioned fragments were cloned in one step using a kit. After sequencing verification, the recombinant plasmid puc-HUH-B2-CRISPR-VPR was obtained. The plasmid structure is shown in the figure. Figure 2 The primer pairs for the above gene fragments are shown in Table 4.

[0085] Table 4 Primer sequences

[0086]

[0087]

[0088] (4) Construction of recombinant plasmid PMCS-CEN-LEU-sgRNA

[0089] The construction process of the recombinant vector PMCS-CEN-LEU-sgRNA (pyruvate kinase) is as follows: using PMCS-CEN-LEU as the backbone, cen as the replicon element, and LEU as the leucine selection marker, the circular plasmid was enzymatically cut into linear fragments using the endonuclease Hind III. Two sets of primer pairs were designed to amplify the expression cassette containing the promoter POX2 (nucleotide sequence shown in SEQ ID NO.1), the sgRNA corresponding to pyruvate kinase (nucleotide sequence shown in SEQ ID NO.10), and the terminator T. The PMCS-CEN-LEU backbone linear fragment and the aforementioned fragment were cloned in one step using a kit. After sequencing verification, the recombinant plasmid PMCS-CEN-LEU-sgRNA (pyruvate kinase) was obtained. The plasmid structure is shown in FIG. Figure 3 The primer pairs used are shown in Table 5.

[0090] Table 5

[0091]

[0092] The construction process of PMCS-CEN-LEU-sgRNA (pyruvate dehydrogenase complex) is similar to that of the recombinant vector PMCS-CEN-LEU-sgRNA (pyruvate kinase). The sgRNA nucleotide sequence corresponding to the pyruvate dehydrogenase complex is shown in SEQ ID NO.11; the primer pairs used are shown in Table 6.

[0093] Using the same method, the backbone linear fragment of the recombinant vector PMCS-CEN-LEU-sgRNA (pyruvate kinase) and the expression cassette containing the promoter POX2, the sgRNA corresponding to the pyruvate kinase, and the terminator T were cloned in one step using a kit to obtain the recombinant plasmid PMCS-CEN-LEU-sgRNA (pyruvate kinase)-sgRNA (pyruvate dehydrogenase complex).

[0094] Table 6

[0095]

[0096] The construction process of PMCS-CEN-LEU-sgRNA (glucose-6-phosphate dehydrogenase) is as follows: PMCS-CEN-LEU is used as the backbone, cen is the replicon element, and LEU is the leucine selection marker. The circular plasmid is enzymatically cut into linear fragments using the endonuclease hindIII. Two sets of primer pairs are designed to amplify the expression cassette containing the promoter PMT1 (nucleotide sequence shown in SEQ ID NO. 2), the sgRNA corresponding to glucose-6-phosphate dehydrogenase (nucleotide sequence shown in SEQ ID NO. 12), and the terminator T. The PMCS-CEN-LEU backbone linear fragment and the aforementioned fragment are cloned in one step using a kit. After sequencing verification, the recombinant plasmid PMCS-CEN-LEU-sgRNA (glucose-6-phosphate dehydrogenase) is obtained; the primer pairs used are shown in Table 7.

[0097] Using the same method, the backbone linear fragment of the recombinant vector PMCS-CEN-LEU-sgRNA (pyruvate kinase)-sgRNA (pyruvate dehydrogenase complex) and the expression cassette containing the promoter PMT1, sgRNA corresponding to glucose-6-phosphate dehydrogenase, and terminator T were cloned in one step using a kit to obtain the recombinant plasmid PMCS-CEN-LEU-sgRNA (pyruvate kinase)-sgRNA (pyruvate dehydrogenase complex)-sgRNA (glucose-6-phosphate dehydrogenase).

[0098] Table 7

[0099]

[0100] The construction process of PMCS-CEN-LEU-sgRNA (sstps) is similar to that of the recombinant vector PMCS-CEN-LEU-sgRNA (glucose-6-phosphate dehydrogenase), wherein the sgRNA nucleotide sequence corresponding to sstps is shown in SEQ ID NO. 8; the primer pairs used are shown in Table 8.

[0101] Using the same method, the backbone linear fragment of PMCS-CEN-LEU-sgRNA (pyruvate kinase)-sgRNA (pyruvate dehydrogenase complex)-sgRNA (glucose-6-phosphate dehydrogenase) and the expression cassette containing the promoter PMT1, sgRNA corresponding to sstps, and terminator T were cloned in one step using a kit to obtain the recombinant plasmid PMCS-CEN-LEU-sgRNA (pyruvate kinase)-sgRNA (pyruvate dehydrogenase complex)-sgRNA (glucose-6-phosphate dehydrogenase)-sgRNA (sstps).

[0102] Table 8

[0103]

[0104] The construction process of PMCS-CEN-LEU-sgRNA (sslpps) is similar to that of the recombinant vector PMCS-CEN-LEU-sgRNA (glucose-6-phosphate dehydrogenase), wherein the sgRNA nucleotide sequence corresponding to sslpps is shown in SEQ ID NO. 9; the primer pairs used are shown in Table 9.

[0105] Using the same method, the backbone linear fragment of PMCS-CEN-LEU-sgRNA (pyruvate kinase)-sgRNA (pyruvate dehydrogenase complex)-sgRNA (glucose-6-phosphate dehydrogenase)-sgRNA (sstps) and the expression cassette containing the promoter PMT1, the sgRNA corresponding to sslpps, and the terminator T were cloned in one step using a kit to obtain the recombinant plasmid PMCS-CEN-LEU-sgRNA (pyruvate kinase)-sgRNA (pyruvate dehydrogenase complex)-sgRNA (glucose-6-phosphate dehydrogenase)-sgRNA (sstps)-sgRNA (sslpps).

[0106] Table 9

[0107]

[0108]

[0109] Example 2

[0110] The recombinant plasmid pUC-HUH-intC-sstps-sslpps obtained in Example 1 was first transformed into the competent cells of the starting strain Yarrowia lipolytica to obtain recombinant strain I; then the recombinant plasmid pUC-HUH-B2-CRISPR-VPR obtained in Example 1 was transformed into the competent cells of the recombinant strain I to obtain recombinant strain II; finally, the recombinant plasmid PMCS-CEN-LEU-sgRNA (pyruvate kinase) -sgRNA (pyruvate dehydrogenase complex) -sgRNA (glucose-6-phosphate dehydrogenase) sgRNA (sstps) -sgRNA (sslpps) obtained in Example 1 was transformed into the competent cells of the recombinant strain II to obtain recombinant strain III.

[0111] Example 3

[0112] (1) The recombinant strain III obtained in Example 2 was streaked on a YNB solid plate. After culturing for 3 days, a single colony was selected and placed in a seed culture medium. The culture was carried out at a temperature of 28°C and a rotation speed of 200 rpm for 24 hours, and the well-cultured seed solution was selected.

[0113] (2) The culture medium (5 g / L ammonium sulfate, 1.7 g / L YNB, and 20 g / L oleic acid) was inoculated at a volume of 10%, and the mixture was fermented at 30°C and 200 rpm for 48 h. 50 g / L glucose and 60 mM copper sulfate were added, and the fermentation was continued at 30°C and 220 rpm for 48 h to obtain a fermentation broth.

[0114] Example 4

[0115] (1) The recombinant strain III obtained in Example 2 was streaked on a YNB solid plate. After culturing for 3 days, a single colony was selected and placed in a seed culture medium. The culture was carried out at a temperature of 28°C and a rotation speed of 200 rpm for 24 hours, and the well-cultured seed solution was selected.

[0116] (2) The culture medium (8 g / L ammonium sulfate, 2 g / L YNB, and 5 g / L oleic acid) was inoculated at a volume of 10%, and the mixture was fermented at a temperature of 35°C and a rotation speed of 200 rpm for 40 h. 30 g / L glucose and 30 mM copper sulfate were added, and the fermentation was continued at a temperature of 35°C and a rotation speed of 200 rpm for 40 h to obtain a fermentation broth.

[0117] Example 5

[0118] (1) The recombinant strain III obtained in Example 2 was streaked on a PDA solid plate. After culturing for 3 days, a single colony was selected and placed in a seed culture medium. The culture was carried out at a temperature of 28°C and a rotation speed of 200 rpm for 24 hours, and the well-cultured seed solution was selected.

[0119] (2) The inoculum was inoculated into a fermentation medium (ammonium sulfate 3 g / L, YNB 1 g / L, oleic acid 25 g / L) at an inoculum size of 10% by volume, and the fermentation was carried out at a temperature of 20°C and a rotation speed of 250 rpm for 60 h. 80 g / L glucose and 100 mM copper sulfate were added, and the fermentation was continued at a temperature of 20°C and a rotation speed of 250 rpm for 60 h to obtain a fermentation broth.

[0120] Example 6

[0121] (1) The recombinant strain III obtained in Example 2 was streaked on a PDA solid plate. After culturing for 3 days, a single colony was selected and placed in a seed culture medium. The culture was carried out at a temperature of 28°C and a rotation speed of 200 rpm for 24 hours, and the well-cultured seed solution was selected.

[0122] (2) The inoculum was inoculated into a fermentation medium (ammonium sulfate 3 g / L, YNB 1 g / L, oleic acid 30 g / L) at an inoculum size of 10% by volume, and the fermentation was carried out at a temperature of 20°C and a rotation speed of 250 rpm for 60 h. 15 g / L of glucose and 150 mM of copper sulfate were added, and the fermentation was continued at a temperature of 20°C and a rotation speed of 250 rpm for 60 h to obtain a fermentation broth.

[0123] Comparative Example 1

[0124] The recombinant strain I in Example 2 was fermented using the method of Example 3 to obtain a fermentation broth.

[0125] Comparative Example 2

[0126] The recombinant strain was constructed by the method of Example 2, except that the recombinant plasmid PMCS-CEN-LEU-sgRNA (pyruvate kinase)-sgRNA (pyruvate dehydrogenase complex)-sgRNA (glucose-6-phosphate dehydrogenase) sgRNA (sstps)-sgRNA (sslpps) were replaced with the recombinant plasmids PMCS-CEN-LEU-sgRNA (pyruvate kinase), PMCS-CEN-LEU-sgRNA (pyruvate dehydrogenase complex), PMCS-CEN-LEU-sgRNA (glucose-6-phosphate dehydrogenase), PMCS-CEN-LEU-sgRNA (sstps), and PMCS-CEN-LEU-sgRNA (sslpps) obtained in Example 1, respectively, to obtain recombinant strain IV to recombinant strain VIII.

[0127] The recombinant strain IV-recombinant strain VIII was fermented using the method of Example 3 to obtain a fermentation broth.

[0128] Test Case

[0129] 1. Take 4 mL of fermentation broth and centrifuge at 6000 rpm for 10 min to obtain the supernatant. Filter the supernatant through a 0.22 μm filter to remove the residue. Then, the filtrate is placed in a liquid phase injection bottle and the sclareol content is detected by HPLC. The results are shown in Table 4.

[0130] The HPLC detection conditions are as follows: injection port temperature 250°C, injection volume 1 μL, split ratio 20:1; chromatographic column: HP-5ms (30m×0.25mM); chromatographic conditions: initial temperature 60°C, increased to 150°C at a rate of 10°C / min, then increased to 280°C at 20°C / min, and maintained for 2 minutes; sclareol standards were used for qualitative and quantitative analysis.

[0131] 2. Qualitative and quantitative analysis of DCW (dry cell weight)

[0132] The cells of fermentation broths I to IV were collected and freeze-dried. The mass of the dried cells was weighed and the DCW was calculated. The results are shown in Table 10.

[0133] Table 10

[0134] serial number Sclareol content (mg / L fermentation broth) Biomass g / L Example 3 231 24 Example 4 187 23 Example 5 257 27 Example 6 129 16 Comparative Example 1 17.8 23 Recombinant strain IV 31.2 29 Recombinant strain V 25.3 28 Recombinant strain VI 35.3 22 Recombinant strain VII 53.2 24 Recombinant strain VIII 48.5 21

[0135] Example 7

[0136] The amino acid sequence of the gene encoding bisabolol synthase, BBS, is shown in SEQ ID NO.14, and the nucleotide sequence of the gene encoding BBS is shown in SEQ ID NO.15; the nucleotide sequence of the sgRNA corresponding to bisabolol synthase is shown in SEQ ID NO.16.

[0137] The recombinant plasmid pUC-HUH-intC-BBS, the recombinant plasmid pUC-HUH-B2-CRISPR-VPR and the recombinant vector PMCS-CEN-LEU-sgRNA (pyruvate kinase)-sgRNA (pyruvate dehydrogenase complex)-sgRNA (glucose-6-phosphate dehydrogenase)-sgRNA (BBS) were constructed using the method of Example 1 and sequentially transformed into Yarrowia lipolytica competent cells to obtain an engineered strain producing bisabolol.

[0138] The starting strain and the engineered strain for producing bisabolol obtained in Example 7 were fermented using the method of Example 3 to obtain a fermentation broth. Testing showed that the bisabolol yield in the fermentation broth of the starting strain was 15 mg / L, while the bisabolol yield in the fermentation broth of the engineered strain was increased to 96 mg / L.

[0139] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A recombinant strain for high production of terpenoids, characterized in that: The recombinant strain is obtained by genetically engineering the starting strain. Compared with the starting strain, the recombinant strain enhances the terpenoid synthesis pathway and utilizes a CRISPR activation system to enhance the activities of pyruvate kinase, pyruvate dehydrogenase complex, glucose-6-phosphate dehydrogenase, and key enzymes for terpenoid synthesis. The sgRNA expression promoters of the pyruvate kinase and the pyruvate dehydrogenase complex are oleic acid-inducible promoters, and the sgRNA expression promoters of the glucose-6-phosphate dehydrogenase and the key enzyme for terpenoid synthesis are copper ion-inducible promoters.

2. The recombinant strain according to claim 1, characterized in that The oleic acid-inducible promoter is POX2, whose nucleotide sequence is shown in SEQ ID NO.1; Preferably, the copper ion inducible promoter is PMT1 with a nucleotide sequence as shown in SEQ ID NO.2 and / or PMT2 with a nucleotide sequence as shown in SEQ ID NO.

3.

3. The recombinant strain according to claim 1 or 2, characterized in that The terpenoid compound is at least one selected from sclareol, bisabolol, nerol, linalool and lavender alcohol, preferably sclareol and / or bisabolol; Preferably, the terpenoid compound is sclareol, and the terpenoid compound synthesis pathway is enhanced by exogenously introducing sclareol synthase and sclareol heterologous lysedione pyrophosphate synthase into the starting strain, and the key enzymes for the synthesis of the terpenoid compound include sclareol synthase and sclareol heterologous lysedione pyrophosphate synthase; the amino acid sequence of the sclareol synthase is shown in SEQ ID NO.4, and the amino acid sequence of the sclareol heterologous lysedione pyrophosphate synthase is shown in SEQ ID NO.5; Preferably, the nucleotide sequence of the gene encoding sstps of the sclareol synthase is shown as SEQ ID NO.6, and the nucleotide sequence of the gene encoding sslpps of the sclareol heterologous lysinyl pyrophosphate synthase is shown as SEQ ID NO.7; Preferably, the sgRNA nucleotide sequence of the sclareol synthase is shown as SEQ ID NO.8, and the sgRNA nucleotide sequence of the sclareol heterologous lysedil pyrophosphate synthase is shown as SEQ ID NO.9; Preferably, the terpenoid compound is bisabolol, and the terpenoid compound synthesis pathway is enhanced by exogenously introducing bisabolol synthase into the starting strain, and the key enzyme for terpenoid compound synthesis includes bisabolol synthase; the amino acid sequence of the bisabolol synthase is shown in SEQ ID NO.14; Preferably, the nucleotide sequence of the bisabolol synthase encoding gene BBS is shown in SEQ ID NO.15; Preferably, the sgRNA nucleotide sequence of the bisabolol synthase is shown as SEQ ID NO.

16.

4. The recombinant strain according to claim 1 or 2, characterized in that The sgRNA nucleotide sequence of the pyruvate kinase is shown in SEQ ID NO.10, the sgRNA nucleotide sequence of the pyruvate dehydrogenase complex is shown in SEQ ID NO.11, and the sgRNA nucleotide sequence of the glucose-6-phosphate dehydrogenase is shown in SEQ ID NO.12; Preferably, the starting strain is Yarrowia lipolytica.

5. A method for constructing a recombinant strain with high terpenoid production, characterized in that: The method comprises: genetically engineering a starting strain so that the starting strain enhances the terpenoid synthesis pathway, and utilizing a CRISPR activation system to enhance the activities of pyruvate kinase, pyruvate dehydrogenase complex, glucose-6-phosphate dehydrogenase, and key enzymes for terpenoid synthesis; The sgRNA expression promoters of the pyruvate kinase and the pyruvate dehydrogenase complex are oleic acid-inducible promoters, and the sgRNA expression promoters of the glucose-6-phosphate dehydrogenase and the key enzyme for terpenoid synthesis are copper ion-inducible promoters.

6. The method according to claim 5, characterized in that The oleic acid-inducible promoter is POX2, whose nucleotide sequence is shown in SEQ ID NO.1; Preferably, the copper ion inducible promoter is PMT1 with a nucleotide sequence as shown in SEQ ID NO.2 and / or PMT2 with a nucleotide sequence as shown in SEQ ID NO.

3.

7. The method according to claim 5 or 6, characterized in that The terpenoid compound is selected from at least one of sclareol, bisabolol, nerol, linalool and lavender alcohol, more preferably sclareol and / or bisabolol; Preferably, the terpenoid compound is sclareol, and the terpenoid compound synthesis pathway is enhanced by exogenously introducing sclareol synthase and sclareol heterologous lysedione pyrophosphate synthase into the starting strain, and the key enzymes for the synthesis of the terpenoid compound include sclareol synthase and sclareol heterologous lysedione pyrophosphate synthase; the amino acid sequence of the sclareol synthase is shown in SEQ ID NO.4, and the amino acid sequence of the sclareol heterologous lysedione pyrophosphate synthase is shown in SEQ ID NO.5; Preferably, the nucleotide sequence of the gene encoding sstps of sclareol synthase is shown as SEQ ID NO.6, and the nucleotide sequence of the gene encoding sslpps of sclareol heterologous lysinyl pyrophosphate synthase is shown as SEQ ID NO.7; Preferably, the sgRNA nucleotide sequence of the sclareol synthase is shown as SEQ ID NO.8, and the sgRNA nucleotide sequence of the sclareol heterologous lysedil pyrophosphate synthase is shown as SEQ ID NO.9; Preferably, the terpenoid compound is bisabolol, and the terpenoid compound synthesis pathway is enhanced by exogenously introducing bisabolol synthase into the starting strain, and the key enzyme for terpenoid compound synthesis includes bisabolol synthase; the amino acid sequence of the bisabolol synthase is shown in SEQ ID NO.14; Preferably, the nucleotide sequence of the bisabolol synthase encoding gene BBS is shown in SEQ ID NO.15; Preferably, the sgRNA nucleotide sequence of the bisabolol synthase is shown as SEQ ID NO.16; Preferably, the sgRNA nucleotide sequence for pyruvate kinase is shown as SEQ ID NO.10, the sgRNA nucleotide sequence for pyruvate dehydrogenase complex is shown as SEQ ID NO.11, and the sgRNA nucleotide sequence for glucose-6-phosphate dehydrogenase is shown as SEQ ID NO.12; Preferably, the starting strain is Yarrowia lipolytica.

8. Use of the recombinant strain according to any one of claims 1 to 4 or the method according to any one of claims 5 to 7 in the preparation of terpenoid compounds; Preferably, the terpenoid compound is selected from at least one of sclareol, bisabolol, nerol, linalool and lavandulol, more preferably sclareol and / or bisabolol.

9. A method for producing terpenoid compounds by fermentation, characterized in that: The method comprises: fermenting the recombinant strain according to any one of claims 1 to 4; Alternatively, constructing a recombinant strain according to the method according to any one of claims 5 to 7, and fermenting the obtained recombinant strain; The fermentation process includes a first fermentation stage and a second fermentation stage, wherein the carbon source in the culture medium used in the first fermentation stage is oleic acid, and glucose and copper ions are added in the second fermentation stage.

10. The method according to claim 9, characterized in that The conditions of the first fermentation stage include: oleic acid content of 5-25 g / L, temperature of 20-35° C., stirring rate of 200-250 rpm, and time of 40-60 h; The conditions of the second fermentation stage include: glucose addition amount of 20-80 g / L, copper ion addition amount of 20-150 mM, temperature of 20-35° C., stirring rate of 200-250 rpm, and time of 40-60 h.

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