Genetically engineered bacterium, preparation method thereof and application of genetically engineered bacterium in synthesis of sclareol

Abstract

The invention provides a genetically engineered bacterium, a preparation method thereof and application of the genetically engineered bacterium in synthesis of sclareol. The genetically engineered bacterium comprises a CcLPPS gene derived from Cistanus crenatus, and a CcLPPS gene derived from Cistanus crenatus, the genetically engineered bacterium is selected from yarrowia lipolytica. According to the invention, the stably expressed sclareol efficient de novo synthesis strain is constructed, and no organic reagent or any path intermediate substance is added in the whole fermentation process, so that the production cost is greatly reduced, and the subsequent separation and purification steps are simplified. The engineering yarrowia lipolytica strain provided by the invention is high in production intensity and simple in fermentation process, has a relatively good application prospect, and lays a foundation for producing sclareol by a microbial fermentation method.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a genetically engineered bacterium, a preparation method thereof and application thereof in synthesizing sclareol. Background Art

[0002] Sclareol, molecular formula is C 20 H 36 O 2 , is a plant-derived diterpene diterpene alcohol. As a natural flavor ingredient, it is widely used in perfumes, cosmetics, food seasoning and other fields. In addition, sclareol is the only practical raw material for the chemical synthesis of ambroxan (a natural ambergris substitute). At the same time, sclareol has strong antibacterial activity, regulates the growth of fungi and plants, and can also be used in the field of pesticides.

[0003] At present, sclareol is mainly extracted from plants, but the plant growth cycle is long and is easily affected by factors such as land, environment and climate. The sclareol content in plants is low and it is difficult to meet market demand. With the breakthrough of bioengineering technology, efficient production of sclareol through microbial fermentation is expected to meet market demand. Patent application CN 114989997 A discloses that a sclareol metabolic pathway was constructed by integrating key genes into the Yarrowia lipolytica Po1f host, and 817.65 mg / L and 12.9 g / L (240 h) of sclareol can be synthesized at the shake flask and fermentation tank levels, respectively, which is the highest yield reported so far. As a model strain of eukaryotic organisms, Saccharomyces cerevisiae is also widely used in the synthesis of high-value compounds. Patent application CN116262929 A discloses that Saccharomyces cerevisiae is used as a cell factory and its central metabolism is systematically transformed to achieve efficient synthesis of sclareol using glucose as a raw material, with 1.83 g / L and 11.4 g / L (192 h) at the shake flask and fermentation tank levels, respectively. However, the fermentation cycle of the above-mentioned synthetic sclareol is too long, and organic reagents need to be added during the fermentation process, which increases the production cost.

[0004] In summary, although de novo microbial synthesis of sclareol has been achieved, there are still problems such as low yield, high cost, complex process control and long fermentation cycle, which are not conducive to industrial production. Therefore, constructing a stable and efficient sclareol synthesis strain is crucial to realize the industrial production of sclareol by microorganisms. Summary of the invention

[0005] The main purpose of the present invention is to provide a genetically engineered bacterium, a preparation method thereof and an application thereof in synthesizing sclareol, so as to solve the problem of low sclareol yield in the prior art.

[0006] In order to achieve the above object, according to a first aspect of the present invention, a genetically engineered bacterium is provided, wherein the genetically engineered bacterium comprises a CcLPPS gene derived from Cistus creticus; and the genetically engineered bacterium is selected from Yarrowia lipolytica.

[0007] The amino acid sequence of the protein encoded by the CcLPPS gene from Cistus creticus includes any one of the following: SEQ ID NOs: 2-3.

[0008] Furthermore, the above genetically engineered bacteria also include: TP1132 gene derived from Salvia sclarea.

[0009] Furthermore, the amino acid sequence of the protein encoded by the TP1132 gene from Salvia sclarea includes any one of the following: SEQ ID NOs: 15-16.

[0010] Furthermore, the genetically engineered bacteria also include: a gene MvaS derived from Enterococcus faecalis and / or a gene MvaE derived from Enterococcus faecalis.

[0011] Furthermore, the protein encoded by the gene MvaS derived from Enterococcus faecalis has an amino acid sequence shown in SEQ ID NO: 5; and the protein encoded by the gene MvaE derived from Enterococcus faecalis has an amino acid sequence shown in SEQ ID NO: 6.

[0012] Furthermore, the genetically engineered bacteria also include a tHMG1 gene derived from Yarrowia lipolytica.

[0013] Furthermore, the protein encoded by the tHMG1 gene derived from Yarrowia lipolytica has an amino acid sequence shown in SEQ ID NO:7.

[0014] Furthermore, the genetically engineered bacteria also include the SsCrtE gene derived from the thermophilic cyanobacterium Synechococcus sp.

[0015] Furthermore, the protein encoded by the SsCrtE gene derived from the thermophilic cyanobacterium Synechococcus sp. has an amino acid sequence shown in SEQ ID NO:8.

[0016] Furthermore, the genetically engineered bacteria may include any one or more of the following genes: ERG12 from Yarrowia lipolytica, ERG20 from Yarrowia lipolytica, ERG8 from Yarrowia lipolytica, MVD1 from Yarrowia lipolytica or IDI1 from Yarrowia lipolytica.

[0017] Furthermore, the protein encoded by the gene ERG12 derived from Yarrowia lipolytica has the amino acid sequence shown in SEQ ID NO: 9; the protein encoded by the gene ERG20 derived from Yarrowia lipolytica has the amino acid sequence shown in SEQ ID NO: 10; the protein encoded by the gene ERG8 derived from Yarrowia lipolytica has the amino acid sequence shown in SEQ ID NO: 11; the protein encoded by the gene MVD1 derived from Yarrowia lipolytica has the amino acid sequence shown in SEQ ID NO: 12; and the protein encoded by the gene IDI1 derived from Yarrowia lipolytica has the amino acid sequence shown in SEQ ID NO: 13.

[0018] Furthermore, the genetically engineered bacteria also include the HpCrtE gene derived from Haematococcus pluvialis.

[0019] Furthermore, the protein encoded by the HpCrtE gene from Haematococcus pluvialis has an amino acid sequence shown in SEQ ID NO:14.

[0020] In order to achieve the above-mentioned object, according to the second aspect of the present invention, a method for preparing a genetically engineered bacterium is provided, the preparation method comprising: knocking in genes into a starting engineering bacterium to obtain the genetically engineered bacterium; the knocked-in genes at least include the CcLPPS gene derived from Cistus creticus, and the genetically engineered bacterium is selected from Yarrowia lipolytica.

[0021] The amino acid sequence of the protein encoded by the CcLPPS gene from Cistus creticus includes any one of the following: SEQ ID NOs: 2-3.

[0022] Furthermore, the knocked-in genes also include the TP1132 gene from Salvia sclarea.

[0023] Furthermore, the amino acid sequence of the protein encoded by the TP1132 gene from Salvia sclarea includes any one of the following: SEQ ID NOs: 15-16.

[0024] Furthermore, the knocked-in genes include: the gene MvaS derived from Enterococcus faecalis and / or the gene MvaE derived from Enterococcus faecalis.

[0025] Furthermore, the protein encoded by the gene MvaS derived from Enterococcus faecalis has an amino acid sequence shown in SEQ ID NO: 5; and the protein encoded by the gene MvaE derived from Enterococcus faecalis has an amino acid sequence shown in SEQ ID NO: 6.

[0026] Furthermore, the knocked-in genes also include: tHMG1 gene derived from Yarrowia lipolytica.

[0027] Furthermore, the protein encoded by the tHMG1 gene derived from Yarrowia lipolytica has an amino acid sequence shown in SEQ ID NO:7.

[0028] Furthermore, the knocked-in genes also include: SsCrtE gene derived from the thermophilic cyanobacterium Synechococcus sp.

[0029] Furthermore, the protein encoded by the SsCrtE gene derived from the thermophilic cyanobacterium Synechococcus sp. has an amino acid sequence shown in SEQ ID NO:8.

[0030] Furthermore, the knocked-in genes include any one or more of the following genes: ERG12 from Yarrowia lipolytica, ERG20 from Yarrowia lipolytica, ERG8 from Yarrowia lipolytica, MVD1 from Yarrowia lipolytica or IDI1 from Yarrowia lipolytica.

[0031] Furthermore, the protein encoded by the gene ERG12 derived from Yarrowia lipolytica has the amino acid sequence shown in SEQ ID NO: 9; the protein encoded by the gene ERG20 derived from Yarrowia lipolytica has the amino acid sequence shown in SEQ ID NO: 10; the protein encoded by the gene ERG8 derived from Yarrowia lipolytica has the amino acid sequence shown in SEQ ID NO: 11; the protein encoded by the gene MVD1 derived from Yarrowia lipolytica has the amino acid sequence shown in SEQ ID NO: 12; and the protein encoded by the gene IDI1 derived from Yarrowia lipolytica has the amino acid sequence shown in SEQ ID NO: 13.

[0032] Furthermore, the knocked-in genes also include: HpCrtE gene from Haematococcus pluvialis.

[0033] Furthermore, the protein encoded by the HpCrtE gene from Haematococcus pluvialis has an amino acid sequence shown in SEQ ID NO:14.

[0034] In order to achieve the above object, according to the third aspect of the present invention, a method for synthesizing sclareol is provided, the method comprising: using the above genetically engineered bacteria or the above genetically engineered bacteria prepared by the above method for preparing genetically engineered bacteria to ferment and synthesize the above sclareol in a fermentation medium.

[0035] Furthermore, the fermentation includes shake flask fermentation or fermentation tank fermentation.

[0036] Furthermore, the shake flask fermentation comprises: inoculating the genetically engineered bacteria cultured overnight into a fermentation medium for shake flask fermentation for expansion culture to obtain the sclareol.

[0037] Furthermore, the fermentation in the fermenter includes: inoculating the genetically engineered bacteria cultured overnight into a fermenter for expansion culture to obtain the sclareol.

[0038] The technical scheme of the present invention is applied, and the genetically engineered bacteria of the present invention contain the CcLPPS gene derived from Cistuscreticus; the genetically engineered bacteria are selected from Yarrowia lipolytica. Such genetically engineered bacteria can express lysinyl pyrophosphate synthase, so that the genetically engineered bacteria can convert geranylgeranyl pyrophosphate (GGPP) into the precursor lysinyl pyrophosphate (8OH-CPP), and 8OH-CPP can be spontaneously converted into sclareol. The use of the genetically engineered bacteria to synthesize sclareol has the beneficial effects of high production intensity, simple fermentation process and good application prospects, which lays a foundation for the production of sclareol by microbial fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0040] Figure 1 The fermentation results of LPPS gene-integrated strains from different sources according to an embodiment of the present invention are shown.

[0041] Figure 2 The diagram shows the results of truncating signal peptides of different lengths of the CcLPPS gene according to an embodiment of the present invention.

[0042] Figure 3 A diagram showing the CcLPPS gene mutation result according to an embodiment of the present invention is shown.

[0043] Figure 4 A diagram showing the fermentation results of TPS gene-integrated strains from different sources according to an embodiment of the present invention is shown.

[0044] Figure 5 The figure shows the fermentation results of integrating CrtE genes from different sources into strain Sc23 according to an embodiment of the present invention.

[0045] Figure 6 A synthetic pathway diagram of sclareol according to an embodiment of the present invention is shown.

[0046] Figure 7 The diagram shows the fermentation results of integrating TP1132 genes of different truncated lengths into strains according to an embodiment of the present invention. DETAILED DESCRIPTION

[0047] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0048] As mentioned in the background technology, sclareol is not only a natural flavor ingredient, but also has strong antibacterial activity and is widely used in the fields of cosmetics, food, and agriculture, with great application value and market prospects. However, the sclareol produced by microorganisms in the prior art has the problem of low yield.

[0049] The invention expresses lysoleyl pyrophosphate synthase by fermenting a genetically engineered bacterium containing an exogenously introduced lysoleyl pyrophosphate synthase encoding gene CcLPPS from Cistus creticus, thereby enabling the genetically engineered bacterium to convert geranylgeranyl pyrophosphate (GGPP) into a precursor lysoleyl pyrophosphate (8OH-CPP), and 8OH-CPP can be spontaneously converted into sclareol (see Figure 6 The sclareol produced by the genetically engineered bacteria has high yield and high efficiency, has good application prospects, and lays a foundation for the industrial production of sclareol by Yarrowia lipolytica fermentation. On this basis, the inventors proposed a series of protection schemes of the present invention.

[0050] In a first typical embodiment of the present invention, a genetically engineered bacterium is provided, wherein the genetically engineered bacterium includes a CcLPPS gene derived from Cistus creticus; the genetically engineered bacterium is selected from Yarrowia lipolytica. LPPS genes from some sources can be used to synthesize sclareol, and LPPS genes from different sources have different yields of sclareol.

[0051] In a more preferred embodiment of the present invention, the above-mentioned Yarrowia lipolytica includes: Yarrowia lipolytica Yl590 strain (classification name Yarrowia Lipolytica, deposit number CGMCCNO.30855, deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on June 4, 2024, and the deposit address is Beijing, China). The above-mentioned genetically engineered bacteria have the beneficial effects of fast growth rate, good activity and few by-products. The use of the above-mentioned genetically engineered bacteria to synthesize sclareol helps to improve the production efficiency of sclareol.

[0052] The present invention screens LPPS from different sources, including: Cistus creticus, Salvia sclarea, Capsicum annuum, Erythranthe guttata, Handroanthus impetiginosus, Lycium barbarum, Nicotiana sylvestris, Sesamum indicum and Solanum stenotomum, and finally screens out CcLPPS from Cistus creticus, which can be used for synthesizing sclareol and has a high yield of sclareol.

[0053] In addition, fermentation of the genetically engineered bacteria containing the CcLPPS gene with GGPP as a precursor was able to accumulate byproducts geranylgeraniol (GGOH) and (E)-lisbaden-13-ene-8,15-diol (LOH), indicating that the CcLPPS gene can effectively convert geranylgeranyl pyrophosphate (GGPP) into the precursor lisbaden-13-ene-8,15-diol pyrophosphate (8OH-CPP), and 8OH-CPP can be spontaneously converted into sclareol and the byproduct (E)-lisbaden-13-ene-8,15-diol (LOH).

[0054] The wild-type CcLPPS gene derived from rock rose Cistus creticus in the present invention has an amino acid sequence shown in SEQ ID NO: 1. Since the wild-type CcLPPS gene derived from rock rose Cistus creticus is of plant origin, it is found through protein structure simulation that the gene contains an unfolded polypeptide sequence, so it is speculated that it may be a signal peptide. The function of the signal peptide is to locate the protein after gene expression to a specific organelle. In the present invention, the signal peptide may locate the enzyme after CcLPPS gene expression to the organelle, thereby weakening the catalytic effect of the enzyme in the cytoplasm. Removing the signal peptide can make the enzyme expressed by the gene better participate in the reaction in the cytoplasm.

[0055] Therefore, the present invention performs amino acid truncation of different lengths starting from the N-terminus of the protein encoded by the wild-type CcLPPS gene, and the truncation lengths include 19, 29, 39, 49, 59, 69 and 79 amino acids. The yield of sclareol varies with different truncation lengths, and it is finally screened that when the truncation length is 49 amino acids (the amino acid sequence of the protein encoded by the truncated CcLPPS gene is shown in SEQ ID NO: 2), the yield of sclareol is the highest, which is 3 times higher than that of the non-truncated CcLPPS gene, indicating that the appropriate length of the truncation of the CcLPPS gene signal peptide can better exert the catalytic efficiency of the protein.

[0056] In order to improve the catalytic efficiency of the protein encoded by the CcLPPS gene, the protein encoded by the truncated CcLPPS gene was further mutated (the mutation sites include N210Y and D614A, and it should be noted that the number of the mutation site here refers to the site number of the amino acid sequence of the wild-type CcLPPS protein), and the protein encoded by the mutated CcLPPS gene has the amino acid sequence shown in SEQ ID NO: 3. The CcLPPS gene can be used to efficiently synthesize sclareol.

[0057] In a preferred embodiment of the present invention, the amino acid sequence of the protein encoded by the CcLPPS gene from Cistus creticus includes any one of the following: SEQ ID NOs: 1-3.

[0058] In order to further increase the yield of sclareol and reduce the accumulation of the byproduct (E)-lisdano-13-ene-8,15-diol LOH, in a preferred embodiment of the present invention, the genetically engineered bacteria further include: TP1132 gene derived from Salvia sclarea. The protein encoded by the TPS gene (TP1132 gene) has the function of catalyzing the synthesis of sclareol from the precursor lisdanodiol pyrophosphate ester 8OH-CPP.

[0059] The present invention screened 19 TPS genes from different sources (see Table 3), and finally screened out the TP1132 gene from Salvia sclarea, which can efficiently synthesize sclareol. In a preferred embodiment of the present invention, the amino acid sequence of the protein encoded by the TP1132 gene from Salvia sclarea includes any one of the following: SEQ ID NOs: 15-16.

[0060] Although the CcLPPS gene can effectively convert geranylgeranyl pyrophosphate (GGPP) into the precursor 8OH-CPP, 8OH-CPP can spontaneously convert to sclareol and the byproduct (E)-13-lysine-8,15-diol (LOH). However, when the TPS gene (TP1132 gene) is further introduced into the genetically engineered strain, 8OH-CPP is more inclined to synthesize sclareol, thereby increasing the accumulation of sclareol.

[0061] Generally, when the copy number of a gene in a genetically engineered bacterium increases, the effect of the gene in the genetically engineered bacterium may be enhanced. In a more preferred embodiment of the present invention, the genetically engineered bacterium comprises 2 copies of the TP1132 gene derived from Salvia sclarea.

[0062] In another more preferred embodiment of the present invention, the genetically engineered bacteria comprises three copies of the TP1132 gene from Salvia sclarea.

[0063] In a more preferred embodiment of the present invention, the genetically engineered bacteria comprises 4 copies of the TP1132 gene derived from Salvia sclarea.

[0064] To further enhance the supply of mevalonic acid MVA, a precursor of sclareol synthesis, in a preferred embodiment of the present invention, the genetically engineered bacteria further comprise: a gene MvaS derived from Enterococcus faecalis and / or a gene MvaE derived from Enterococcus faecalis.

[0065] In a preferred embodiment of the present invention, the protein encoded by the gene MvaS derived from Enterococcus faecalis has an amino acid sequence shown in SEQ ID NO: 5; the protein encoded by the gene MvaE derived from Enterococcus faecalis has an amino acid sequence shown in SEQ ID NO: 6.

[0066] In a more preferred embodiment of the present invention, the genetically engineered bacteria further comprises a tHMG1 gene derived from Yarrowia lipolytica. In a more preferred embodiment of the present invention, the genetically engineered bacteria further comprises two copies of the tHMG1 gene derived from Yarrowia lipolytica. In a preferred embodiment of the present invention, the protein encoded by the tHMG1 gene derived from Yarrowia lipolytica has an amino acid sequence as shown in SEQ ID NO: 7. Knocking in the MvaS, MvaE and tHMG1 genes into the genetically engineered bacteria helps increase the accumulation of the precursor mevalonic acid MVA, thereby further helping to increase the accumulation of sclareol.

[0067] In order to be able to convert the farnesyl pyrophosphate (FPP) produced in the body into geranylgeranyl pyrophosphate (GGPP), the above-mentioned genetically engineered bacteria also include the CrtE gene. The present invention screened 5 CrtE genes from different sources (see Table 4), and finally screened the SsCrtE gene derived from the thermophilic cyanobacterium Synechococcus sp., which can convert the farnesyl pyrophosphate (FPP) produced in the genetically engineered bacteria into geranylgeranyl pyrophosphate (GGPP), thereby increasing the yield of sclareol. At the same time, the accumulation of by-products is also significantly improved. Therefore, in a more preferred embodiment of the present invention, the above-mentioned genetically engineered bacteria also include the SsCrtE gene derived from the thermophilic cyanobacterium Synechococcus sp. In a preferred embodiment of the present invention, the protein encoded by the SsCrtE gene derived from the thermophilic cyanobacterium Synechococcus sp. has the amino acid sequence shown in SEQ ID NO: 8.

[0068] In order to further enhance MVA metabolism and improve the synthesis of precursor FPP, in a preferred embodiment of the present invention, the above-mentioned genetically engineered bacteria also include any one or more of the following genes: gene ERG12 derived from Yarrowia lipolytica, ERG20 derived from Yarrowia lipolytica, ERG8 derived from Yarrowia lipolytica, MVD1 derived from Yarrowia lipolytica or gene IDI1 derived from Yarrowia lipolytica.

[0069] In a more preferred embodiment of the present invention, the genetically engineered bacteria further comprise 2 copies of any one or more of the following genes: ERG12 derived from Yarrowia lipolytica, ERG20 derived from Yarrowia lipolytica, ERG8 derived from Yarrowia lipolytica, MVD1 derived from Yarrowia lipolytica or IDI1 derived from Yarrowia lipolytica.

[0070] In a preferred embodiment of the present invention, the protein encoded by the gene ERG12 derived from Yarrowia lipolytica has the amino acid sequence shown in SEQ ID NO: 9; the protein encoded by the gene ERG20 derived from Yarrowia lipolytica has the amino acid sequence shown in SEQ ID NO: 10; the protein encoded by the gene ERG8 derived from Yarrowia lipolytica has the amino acid sequence shown in SEQ ID NO: 11; the protein encoded by the gene MVD1 derived from Yarrowia lipolytica has the amino acid sequence shown in SEQ ID NO: 12; the protein encoded by the gene IDI1 derived from Yarrowia lipolytica has the amino acid sequence shown in SEQ ID NO: 13.

[0071] In a preferred embodiment of the present invention, the genetically engineered bacteria further include the HpCrtE gene derived from Haematococcus pluvialis. The HpCrtE gene can directly convert IPP and DMAPP into GGPP, achieve two-layer GGPP accumulation, and increase the yield of sclareol. In a preferred embodiment of the present invention, the protein encoded by the HpCrtE gene derived from Haematococcus pluvialis has the amino acid sequence shown in SEQ ID NO: 14.

[0072] In a second typical embodiment of the present invention, a method for preparing a genetically engineered bacterium is provided, the preparation method comprising: knocking in genes into a starting engineered bacterium to obtain the genetically engineered bacterium; the genes at least include the CcLPPS gene derived from Cistus creticus, and the genetically engineered bacterium is selected from Yarrowia lipolytica.

[0073] In a more preferred embodiment of the present invention, the above-mentioned Yarrowia lipolytica includes: Yarrowia lipolytica Y1590 strain (classification name Yarrowia Lipolytica, deposit number CGMCC NO.30855, deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on June 4, 2024, and the deposit address is Beijing, China). The above-mentioned genetically engineered bacteria have the beneficial effect of fast growth rate. The use of the above-mentioned genetically engineered bacteria to synthesize sclareol helps to improve the production efficiency of sclareol.

[0074] In a preferred embodiment of the present invention, the amino acid sequence of the protein encoded by the CcLPPS gene from Cistus creticus includes any one of the following: SEQ ID NOs: 1-3.

[0075] In a preferred embodiment of the present invention, the above-mentioned gene further comprises: TP1132 gene derived from Salvia sclarea.

[0076] In a more preferred embodiment of the present invention: the above gene also includes 2 copies of TP1132 gene derived from Salvia sclarea.

[0077] In another more preferred embodiment of the present invention, the genetically engineered bacteria comprises three copies of the TP1132 gene from Salvia sclarea.

[0078] In a more preferred embodiment of the present invention, the genetically engineered bacteria comprises 4 copies of the TP1132 gene derived from Salvia sclarea.

[0079] In a preferred embodiment of the present invention, the amino acid sequence of the protein encoded by the TP1132 gene from Salvia sclarea includes any one of the following: SEQ ID NOs: 15-16.

[0080] In a preferred embodiment of the present invention, the above-mentioned gene further comprises: gene MvaS derived from Enterococcus faecalis and / or gene MvaE derived from Enterococcus faecalis. In a preferred embodiment of the present invention, the protein encoded by gene MvaS derived from Enterococcus faecalis has the amino acid sequence shown in SEQ ID NO: 5; the protein encoded by gene MvaE derived from Enterococcus faecalis has the amino acid sequence shown in SEQ ID NO: 6.

[0081] In a preferred embodiment of the present invention, the above-mentioned gene further comprises: a tHMG1 gene derived from Yarrowia lipolytica. In a more preferred embodiment of the present invention, the above-mentioned genetically engineered bacteria further comprises 2 copies of the tHMG1 gene derived from Yarrowia lipolytica. In a preferred embodiment of the present invention, the protein encoded by the above-mentioned tHMG1 gene derived from Yarrowia lipolytica has the amino acid sequence shown in SEQ ID NO: 7.

[0082] In a preferred embodiment of the present invention, the above gene further comprises: SsCrtE gene derived from thermophilic cyanobacterium Synechococcus sp. In a preferred embodiment of the present invention, the protein encoded by the SsCrtE gene derived from thermophilic cyanobacterium Synechococcus sp. has an amino acid sequence shown in SEQ ID NO:8.

[0083] In a preferred embodiment of the present invention, the above-mentioned gene further comprises any one or more of the following genes: ERG12 derived from Yarrowia lipolytica, ERG20 derived from Yarrowia lipolytica, ERG8 derived from Yarrowia lipolytica, MVD1 derived from Yarrowia lipolytica or IDI1 derived from Yarrowia lipolytica.

[0084] In a more preferred embodiment of the present invention, the genetically engineered bacteria further comprise 2 copies of any one or more of the following genes: ERG12 derived from Yarrowia lipolytica, ERG20 derived from Yarrowia lipolytica, ERG8 derived from Yarrowia lipolytica, MVD1 derived from Yarrowia lipolytica or IDI1 derived from Yarrowia lipolytica.

[0085] In a preferred embodiment of the present invention, the protein encoded by the gene ERG12 derived from Yarrowia lipolytica has the amino acid sequence shown in SEQ ID NO: 9; the protein encoded by the gene ERG20 derived from Yarrowia lipolytica has the amino acid sequence shown in SEQ ID NO: 10; the protein encoded by the gene ERG8 derived from Yarrowia lipolytica has the amino acid sequence shown in SEQ ID NO: 11; the protein encoded by the gene MVD1 derived from Yarrowia lipolytica has the amino acid sequence shown in SEQ ID NO: 12; the protein encoded by the gene IDI1 derived from Yarrowia lipolytica has the amino acid sequence shown in SEQ ID NO: 13.

[0086] In a preferred embodiment of the present invention, the above gene further comprises: HpCrtE gene derived from Haematococcus pluvialis. In a preferred embodiment of the present invention, the protein encoded by the HpCrtE gene derived from Haematococcus pluvialis has an amino acid sequence shown in SEQ ID NO:14.

[0087] Chassis microbial modification is an important method to increase the production of microbial target metabolites, which involves gene knock-in or knock-out. The purpose of gene knock-out is to reduce the accumulation of byproducts in the metabolic pathway, while gene knock-in is to increase raw materials and power for the synthesis of target products. Among them, knock-in gene refers to the direct integration of genes into the bacterial genome, and knock-out gene refers to the knock-out of genes on the bacterial genome.

[0088] The knock-in order of the gene of the present invention is not specifically limited, and the timing of knock-in genes can be selected according to specific needs. The knock-in number of the same gene can also be selected according to actual needs. When a certain gene is knocked into multiple copies of a certain gene in a genetically engineered bacterium, the effect of this gene in the genetically engineered bacterium will be enhanced, and the knock-in timing of multiple copies of the gene can be the same or different.

[0089] It should be noted that the above-mentioned "introduction", "transfer" and "knock-in" in the present invention mean that the gene is integrated into the genome of the genetically engineered bacteria, rather than existing in the genetically engineered bacteria in the form of a plasmid. After the gene is transferred into the genetically engineered bacteria in the form of a plasmid, the gene is not integrated into the genome of the genetically engineered bacteria. In a preferred embodiment of the present invention, the knock-in of the gene of the present invention is performed by a gene editing method.

[0090] When integrating genes into Yarrowia lipolytica by gene editing, a gene in the genome needs to be knocked out as a screening marker gene. In a preferred embodiment of the present invention, the URA3 gene, LEU gene, HIS gene or TRP gene in the genetically engineered bacteria is knocked out.

[0091] In a third typical embodiment of the present invention, a method for synthesizing sclareol is provided, the method comprising: fermenting the sclareol in a fermentation medium using the genetically engineered bacteria or the genetically engineered bacteria prepared by the method for preparing the genetically engineered bacteria.

[0092] In a preferred embodiment of the present invention, the fermentation comprises shake flask fermentation or fermentation tank fermentation.

[0093] In a preferred embodiment of the present invention, the shake flask fermentation comprises: inoculating the genetically engineered bacteria cultured overnight into a fermentation medium of the shake flask fermentation for expansion culture to obtain the sclareol.

[0094] In a preferred embodiment of the present invention, the fermentation in a fermenter comprises: inoculating the genetically engineered bacteria cultured overnight into a fermenter for expansion culture to obtain the sclareol.

[0095] In a preferred embodiment of the present invention, the conditions for the overnight culture are 28-32°C, 180-220rpm; the conditions for the expanded culture are 28-32°C, 180-220rpm.

[0096] In a preferred embodiment of the present invention, the fermentation medium of the shake flask fermentation comprises: 15-25 g / L glucose, 10-20 g / L yeast powder and 10-20 g / L peptone. Under such conditions, sclareol is synthesized, which has the beneficial effect of high sclareol yield.

[0097] In a preferred embodiment of the present invention, the fermentation in the fermenter includes: inoculating the genetically engineered bacteria cultured overnight into the fermenter for expansion culture to obtain the sclareol. In a preferred embodiment of the present invention, the conditions for the overnight culture are 28-32°C, 180-220rpm; the conditions for the expansion culture are DO 5%-20%, pH 3.0-6.0, 28-32°C; and the inoculation amount of the inoculation is 5%-20%.

[0098] In a preferred embodiment of the present invention, the fermentation medium of the fermentation tank fermentation comprises: 2-4 g / L ammonium sulfate, 15-20 g / L potassium dihydrogen phosphate, 0.5-2 g / L magnesium sulfate heptahydrate, 200-400 g / L glucose, 5-20 g / L yeast powder, 5-15 mL / L trace element stock solution, 10-20 mL / L vitamin stock solution, and 1-2 mL / L biotin;

[0099] The vitamin stock solution includes 1 g / L calcium pantothenate, 1 g / L niacin, 25 g / L inositol, 1 g / L thiamine hydrochloride, 1 g / L pyridoxine hydrochloride and 0.2 g / L p-aminobenzoic acid;

[0100] The above-mentioned trace element stock solution includes 6 g / L zinc sulfate heptahydrate, 0.3 g / L manganese chloride tetrahydrate, 0.3 g / L copper sulfate pentahydrate, 0.5 g / L cobalt chloride hexahydrate, 0.5 g / L sodium molybdate dihydrate, 3 g / L calcium chloride dihydrate, 3 g / L ferrous sulfate heptahydrate and 12 g / L ethylenediaminetetraacetic acid. Under this condition, the synthesis of sclareol has high yield, short production cycle and no need to add any organic reagents and any pathway precursors in the entire fermentation process, which has great economic value and good industrialization prospects.

[0101] It should be noted that in order to avoid excessive concentration of glucose in the initial fermentation medium, the 200-400 g / L glucose of the present invention is preferably added in two batches, wherein 20 g / L of glucose is first added to the initial medium, and the remaining glucose is added to the fermentation medium after the fermentation starts. During the addition process, the concentration of glucose in the fermentation medium needs to be controlled to be lower than 5 g / L.

[0102] The present invention is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed by the present invention.

[0103] Example 1: Transformation of strain Y1590

[0104] The wild-type Yarrowia lipolytica Yl590 strain (accession number: CGMCC NO.30855, deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on June 4, 2024, with the deposit address in Beijing, China) was modified, and the URA3 selection marker gene was knocked out to facilitate the subsequent CRISPR gene editing for key gene integration. The genome of Yarrowia lipolytica Yl590 strain was used as a template, and URA-Up-F / R and URA-Down-F / R were used as primers. PCR was used to obtain the upstream and downstream homologous arm fragments of the gene URA3, and then the two fragments were fused by PCR to obtain the fragment URA3-Up+Down. The fusion fragment was transferred into the Yl590 strain by the lithium acetate (LiAC) method, and the resuscitated bacterial liquid was spread on 5-FOA plates. After culturing at 30°C for 2 days, single clones were picked and spotted on SD-URA and YPD solid plates for preliminary screening. The mutant strains obtained were verified by colony PCR again, and the URA3-deficient strain was successfully obtained.

[0105] Table 1 Primers used for URA3 gene knockout

[0106]

[0107] Example 2: LPPS gene screening

[0108] Screening of LPPS gene sources: Using Yl590 strain as the host, 9 LPPS genes from different sources were compared (see Table 2), and Sc01-Sc10 strains were constructed. Among them, the CcLPPS gene from Cistus spathulatus was expressed, and the sclareol production was the highest, which was 0.35 mg / L. The SsLPPS gene was second, producing 0.31 mg / L sclareol. The expression of the remaining genes did not produce the target product ( Figure 1 ).

[0109] Table 2

[0110]

[0111] Protein structure simulation revealed that the CcLPPS gene contained a signal peptide sequence. Therefore, the CcLPPS gene was truncated to different lengths of amino acids starting from the N-terminus, with the truncation lengths being 19, 19, 39, 49, 59, 69, and 79 amino acids, respectively. The strain Yl590 was used as the parent strain for integration, and the Sc11-Sc17 strains were constructed. The results are shown in the figure. Figure 2It showed that truncation of signal peptides of different lengths in the CcLPPS gene could increase the yield of sclareol. Among them, the CcLPPS gene truncated by 49 amino acids had the best integration effect, producing 1.03 mg / L of sclareol, which was 3 times higher than that of the non-truncated CcLPPS gene. This showed that truncation of the CcLPPS gene signal peptide to an appropriate length could better exert the catalytic efficiency of the protein.

[0112] The CcLPPS gene was further mutated, and two effective mutation sites, N210Y and D614A, were screened. The CcLPPS* mutant gene was expressed in the Yl590 strain, and the Sc18-Sc20 strain was constructed. The sclareol production was increased to 1.35 mg / L ( Figure 3 ); Among them, the mutation site of CcLPPS in the Sc18 strain is N210Y; the mutation site of CcLPPS in the Sc19 strain is D614A; the mutation site of CcLPPS in the Sc20 strain is N210Y+D614A.

[0113] The fermentation method of the recombinant Yarrowia lipolytica strain and the detection method of sclareol are as follows: the recombinant Yarrowia lipolytica strains Sc01-Sc20 are inoculated into YPD medium, cultured overnight at 30°C and 220rpm, and the YPD medium includes 20 g / L glucose, 10 g / L yeast powder, and 20 g / L peptone. The conditions for detecting the yield of sclareol by gas chromatography at different time points during the fermentation process are as follows: chromatographic column Agilent HP-5 30m×0.32mm, 0.25 μm, initial column temperature 100°C maintained for 2 min, heating rate 20°C / min heating to 280°C maintained for 10 min, injector temperature 300°C, detector temperature 320°C, carrier gas flow rate 2 mL / min, injection volume 1 μL.

[0114] Example 3: TPS gene screening

[0115] 19 TPS genes from different sources were integrated into the Sc20 strain to construct the Sc20a-Sc20s strains (see Table 3). The experimental results are shown in Figure 4 It was shown that the sclareol production increased to 3.21 mg / L after the integration of TP1132 gene.

[0116] Table 3

[0117]

[0118] Protein structure simulation revealed that the TP1132 gene contained a signal peptide sequence. Therefore, the TP1132 gene was truncated to different lengths of amino acids starting from the N-terminus, with the truncation lengths being 19, 29, 39, 49, 59, 69, and 79 amino acids, respectively. The strain Sc20 was used as the parent strain for integration, and the Sc20t-Sc20z strain was constructed. The results are shown in the figure. Figure 7 The integration effect of the TP1132 gene truncated by 39 amino acids was the best, producing 5.31 mg / L of clarysol, which was 1.6 times higher than that of the non-truncated TP1132 gene, indicating that truncation of the TP1132 gene signal peptide to an appropriate length can better exert the catalytic efficiency of the protein.

[0119] The fermentation method of the above-mentioned recombinant Yarrowia lipolytica strains Sc20a-Sc20z and the detection method of sclareol are the same as those in Example 2.

[0120] Example 4: Construction of Sc38 strain for efficient supply of sclareol precursor

[0121] The Sc20 strain was constructed by introducing the CcLPPS mutant gene from Cistus creticus into strain Yl590, which was able to synthesize 1.35 mg / L sclareol from scratch. The TP1132 gene (with the amino acid sequence shown in SEQ ID NO: 16) with 39 amino acids truncated was further introduced into the Sc20 strain to construct the Sc20v strain, and the sclareol production was increased to 3.21 mg / L. Among them, the by-products geranylgeraniol (GGOH) and (E)-lisbaden-13-ene-8,15-diol (LOH) also accumulated in small amounts, indicating that the CcLPPS gene can effectively convert GGPP into the precursor lisbaden-13-ene-8,15-diol pyrophosphate (8OH-CPP), and 8OH-CPP can spontaneously convert into sclareol and the by-product (E)-lisbaden-13-ene-8,15-diol (LOH). After the TP1132 gene with a 39-amino acid truncation (having the amino acid sequence shown in SEQ ID NO: 16) was introduced, 8OH-CPP was more inclined to synthesize sclareol, thereby increasing the accumulation of sclareol.

[0122] In order to further enhance the supply of mevalonic acid (MVA), a precursor of sclareol synthesis, the gene MvaS (ID: WP_002357756.1) from Enterococcus faecalis was introduced into Sc20v to successfully construct strain Sc21, with a sclareol production of 9.21 mg / L. Furthermore, the gene MvaE (ID: WP_002357755.1) from Enterococcus faecalis was integrated into strain Sc21 to successfully construct strain Sc22, with the sclareol production increased to 15.41 mg / L.

[0123] Using the genome of Yarrowia lipolytica Yl590 strain as a template, the tHMG1 gene was amplified and integrated into the Sc22 strain to construct the Sc23 strain. The sclareol production was increased to 17.94 mg / L, indicating that the expression of MvaS, MvaE and tHMG1 genes can increase the accumulation of the precursor mevalonic acid MVA, thereby increasing the accumulation of sclareol.

[0124] Using Sc23 as the host, we integrated five different CrtE genes to construct Sc24-Sc28 strains. Among them, SsCrtE gene had the best expression effect ( Figure 5 ), which can convert farnesyl pyrophosphate (FPP) produced in the body into geranylgeranyl pyrophosphate (GGPP), thereby increasing the production of claryol to 55.23 mg / L and significantly increasing the accumulation of by-products.

[0125] Table 4

[0126]

[0127] To further enhance MVA metabolism and improve the synthesis of precursor FPP, the ERG12, ERG20, ERG8, MVD1 and IDI1 genes were amplified using the genome of Yarrowia lipolytica Yl590 as a template and sequentially integrated into strain Sc27 to obtain Sc29-Sc33 (see Table 5). Strain Sc33 produced 369.62 mg / L clarysol after 96 h of fermentation.

[0128] Table 5

[0129]

[0130] In addition, the TP1132 gene (having the amino acid sequence shown in SEQ ID NO: 16) truncated by 39 amino acids was introduced into the Sc33 chassis strain to construct the Sc34 strain, which synthesized 720.04 mg / L clarysol after 96 h of fermentation.

[0131] Then, the HpCrtE (ID: QHF16627.1) gene from Haematococcus pluvialis was integrated into the Sc34 strain to construct the Sc35 strain. The HpCrtE gene can directly convert IPP and DMAPP into GGPP, achieving two-layer GGPP accumulation. The sclareol production was increased to 1093.22 mg / L, which is higher than the currently reported highest shake flask yield of 817.65 mg / L.

[0132] In order to further enhance the conversion of precursor GGPP to sclareol and reduce the accumulation of byproducts GGOH and LOH, a copy of the gene CcLPPS* was first integrated into the Sc35 strain to obtain the strain Sc36, and the sclareol production increased to 1.45 g / L. Then, a copy of the TP1132 gene (with the amino acid sequence shown in SEQ ID NO: 16) with 39 amino acids truncated was integrated into the strain Sc36 to obtain the strain Sc37, and the sclareol production increased to 2.24 g / L. On the basis of Sc37, a copy of the TP1132 gene (with the amino acid sequence shown in SEQ ID NO: 16) with 39 amino acids truncated was further integrated to construct the Sc38 strain. After fermentation at 30℃ for 120 h, the sclareol production was 2.61 g / L, and the byproducts GGOH and LOH were 102.53 mg / L and 257.24 mg / L, respectively.

[0133] The fermentation method of the recombinant Yarrowia lipolytica strain and the detection method of sclareol are the same as those in Example 2.

[0134] Example 5: Production of sclareol in a 5 L fermenter

[0135] The engineered strains Sc35-Sc38 were fermented to amplify the production of sclareol. The recombinant Yarrowia lipolytica strains Sc35-Sc38 were inoculated into YPD medium, cultured overnight at 30℃, and transferred to the fermenter at a 10% inoculation rate. The fermentation process controlled pH 5.0, temperature 30℃, initial dissolved oxygen (DO) 10%, and the rotation speed and air volume were adjusted according to the dissolved oxygen. After 120 h of fermentation, the Sc35 strain synthesized 13.5 g / L sclareol, which was higher than the highest yield of 12.9 g / L reported so far. The fermentation sclareol yields of Sc36 and Sc37 strains increased to 15.25 g / L and 18.92 g / L, respectively, and the engineered strain Sc38 finally obtained could synthesize 20.25 g / L sclareol.

[0136] The fermentation method of the recombinant Yarrowia lipolytica strain and the detection method of sclareol are as follows: the recombinant strains are transferred to the fermentation medium (2.5 g / L ammonium sulfate, 14.4 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, 300 g / L glucose, 10 g / L yeast powder, 10 ml / L trace element stock solution, 12 ml / L vitamin stock solution, and 1.2 ml / L biotin) at a 10% inoculation rate, and the pH, temperature, and initial dissolved oxygen (DO) are controlled at 5.0, 30°C, and 10% during the fermentation process. The rotation speed and air volume are adjusted in conjunction with the dissolved oxygen, and the glucose concentration is controlled to be lower than 5 g / L by adding 800 g / L glucose during the fermentation process. The above-mentioned trace element stock solution includes 6 g / L zinc sulfate heptahydrate, 0.3 g / L manganese chloride tetrahydrate, 0.3 g / L copper sulfate pentahydrate, 0.5 g / L cobalt chloride hexahydrate, 0.5 g / L sodium molybdate dihydrate, 3 g / L calcium chloride dihydrate, 3 g / L ferrous sulfate heptahydrate and 12 g / L ethylenediaminetetraacetic acid. The above-mentioned vitamin stock solution includes 1 g / L calcium pantothenate, 1 g / L nicotinic acid, 25 g / L inositol, 1 g / L thiamine hydrochloride, 1 g / L pyridoxine hydrochloride and 0.2 g / L p-aminobenzoic acid.

[0137] The conditions for detecting the production of sclareol using gas chromatography at different time points during the fermentation process were as follows: chromatographic column Agilent HP-5 30m×0.32mm, 0.25 μm, initial column temperature 100℃ maintained for 2 min, heating rate 20℃ / min to 280℃ maintained for 10 min, injector temperature 300℃, detector temperature 320℃, carrier gas flow rate 2 mL / min, injection volume 1μL.

[0138] The above-mentioned embodiments of the present invention achieve the following technical effects: the present invention uses the self-screened wild-type lipolytic yeast strain to construct an effective gene editing system, and improves the sclareol production of the strain by optimizing the expression intensity of key genes in the sclareol synthesis pathway, enhancing the precursor supply, and combining gene screening, mutation and protein truncation. A 5 L fermentation tank can reach 20 g / L, which is the highest yield reported so far. The strain of the present invention not only has a yield that exceeds the highest level reported so far, but also has a production cycle that is half as short as reported, and the entire fermentation process does not require the addition of any organic reagents and any pathway precursors, and has great economic value and good industrial prospects.

[0139] The present invention integrates the relevant genes into the genome of Yarrowia lipolytica Y1590 strain, constructs a stably expressed sclareol efficient de novo synthesis strain, and does not require the addition of any organic reagents or any intermediate substances in the entire fermentation process, greatly reducing the production cost and simplifying the subsequent separation and purification steps. The engineered Yarrowia lipolytica strain provided by the present invention has high production intensity and simple fermentation process, has good application prospects, and lays a foundation for the production of sclareol by microbial fermentation.

[0140] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria include the CcLPPS gene derived from Cistus creticus; The genetically engineered bacteria is selected from Yarrowia lipolytica; The amino acid sequence of the protein encoded by the CcLPPS gene from Cistus creticus includes any one of the following: SEQ ID NOs: 2-3.

2. The genetically engineered bacterium according to claim 1, characterized in that: The genetically engineered bacteria also include: TP1132 gene derived from Salvia sclarea.

3. The genetically engineered bacterium according to claim 2, characterized in that: The amino acid sequence of the protein encoded by the TP1132 gene from Salvia sclarea includes any one of the following: SEQ ID NOs: 15-16.

4. The genetically engineered bacterium according to claim 3, characterized in that: The genetically engineered bacteria further include: a gene MvaS derived from Enterococcus faecalis and / or a gene MvaE derived from Enterococcus faecalis.

5. The genetically engineered bacterium according to claim 4, characterized in that: The protein encoded by the gene MvaS derived from Enterococcus faecalis has an amino acid sequence shown in SEQ ID NO: 5; the protein encoded by the gene MvaE derived from Enterococcus faecalis has an amino acid sequence shown in SEQ ID NO:

6.

6. The genetically engineered bacterium according to any one of claims 5, characterized in that The genetically engineered bacteria also include a tHMG1 gene derived from Yarrowia lipolytica.

7. The genetically engineered bacterium according to claim 6, characterized in that: The protein encoded by the tHMG1 gene derived from Yarrowia lipolytica has an amino acid sequence shown in SEQ ID NO:

7.

8. The genetically engineered bacterium according to claim 7, characterized in that: The genetically engineered bacteria also include the SsCrtE gene derived from the thermophilic cyanobacterium Synechococcus sp.

9. The genetically engineered bacterium according to claim 8, characterized in that: The protein encoded by the SsCrtE gene derived from the thermophilic cyanobacterium Synechococcus sp. has an amino acid sequence shown in SEQ ID NO:

8.

10. The genetically engineered bacterium according to claim 9, characterized in that: The genetically engineered bacteria further include any one or more of the following genes: ERG12 from Yarrowia lipolytica, ERG20 from Yarrowia lipolytica, ERG8 from Yarrowia lipolytica, MVD1 from Yarrowia lipolytica or IDI1 from Yarrowia lipolytica.

11. The genetically engineered bacterium according to claim 10, characterized in that: The protein encoded by the gene ERG12 from Yarrowia lipolytica has an amino acid sequence shown in SEQ ID NO: 9; The protein encoded by the gene ERG20 from Yarrowia lipolytica has an amino acid sequence shown in SEQ ID NO: 10; The protein encoded by the gene ERG8 derived from Yarrowia lipolytica has an amino acid sequence shown in SEQ ID NO: 11; The protein encoded by the gene MVD1 derived from Yarrowia lipolytica has an amino acid sequence shown in SEQ ID NO: 12; The protein encoded by the gene IDI1 derived from Yarrowia lipolytica has an amino acid sequence shown in SEQ ID NO:

13.

12. The genetically engineered bacterium according to claim 11, characterized in that: The genetically engineered bacteria also include the HpCrtE gene derived from Haematococcus pluvialis.

13. The genetically engineered bacterium according to claim 12, characterized in that: The protein encoded by the HpCrtE gene from Haematococcus pluvialis has an amino acid sequence shown in SEQ ID NO:

14.

14. A method for preparing a genetically engineered bacterium, characterized in that: The preparation method comprises: knocking in a gene into a starting engineering bacterium to obtain the genetically engineered bacterium; The knocked-in gene at least includes the CcLPPS gene derived from Cistus creticus, and the genetically engineered bacteria is selected from Yarrowia lipolytica; The amino acid sequence of the protein encoded by the CcLPPS gene from Cistus creticus includes any one of the following: SEQ ID NOs: 2-3.

15. The preparation method according to claim 14, characterized in that: The knocked-in genes also include the TP1132 gene from Salvia sclarea.

16. The preparation method according to claim 15, characterized in that: The amino acid sequence of the protein encoded by the TP1132 gene from Salvia sclarea includes any one of the following: SEQ ID NOs: 15-16.

17. The preparation method according to claim 16, characterized in that: The knocked-in genes also include: the gene MvaS derived from Enterococcus faecalis and / or the gene MvaE derived from Enterococcus faecalis.

18. The preparation method according to claim 17, characterized in that: The protein encoded by the gene MvaS derived from Enterococcus faecalis has an amino acid sequence shown in SEQ ID NO: 5; the protein encoded by the gene MvaE derived from Enterococcus faecalis has an amino acid sequence shown in SEQ ID NO:

6.

19. The preparation method according to claim 18, characterized in that: The knocked-in genes also include: tHMG1 gene derived from Yarrowia lipolytica.

20. The preparation method according to claim 19, characterized in that: The protein encoded by the tHMG1 gene derived from Yarrowia lipolytica has an amino acid sequence shown in SEQ ID NO:

7.

21. The preparation method according to claim 20, characterized in that: The knocked-in genes also include: SsCrtE gene derived from thermophilic cyanobacteria Synechococcus sp.

22. The preparation method according to claim 21, characterized in that: The protein encoded by the SsCrtE gene derived from the thermophilic cyanobacterium Synechococcus sp. has an amino acid sequence shown in SEQ ID NO:

8.

23. The preparation method according to claim 22, characterized in that: The knocked-in gene also includes any one or more of the following genes: ERG12 from Yarrowia lipolytica, ERG20 from Yarrowia lipolytica, ERG8 from Yarrowia lipolytica, MVD1 from Yarrowia lipolytica or IDI1 from Yarrowia lipolytica.

24. The preparation method according to claim 23, characterized in that: The protein encoded by the gene ERG12 from Yarrowia lipolytica has an amino acid sequence shown in SEQ ID NO: 9; The protein encoded by the gene ERG20 from Yarrowia lipolytica has an amino acid sequence shown in SEQ ID NO: 10; The protein encoded by the gene ERG8 derived from Yarrowia lipolytica has an amino acid sequence shown in SEQ ID NO: 11; The protein encoded by the gene MVD1 derived from Yarrowia lipolytica has an amino acid sequence shown in SEQ ID NO: 12; The protein encoded by the gene IDI1 derived from Yarrowia lipolytica has an amino acid sequence shown in SEQ ID NO:

13.

25. The preparation method according to claim 24, characterized in that: The knocked-in genes also include: HpCrtE gene derived from Haematococcus pluvialis.

26. The preparation method according to claim 25, characterized in that: The protein encoded by the HpCrtE gene from Haematococcus pluvialis has an amino acid sequence shown in SEQ ID NO:

14.

27. A method for synthesizing sclareol, characterized in that: The method comprises: fermenting and synthesizing the sclareol in a fermentation medium using the genetically engineered bacteria prepared by the genetically engineered bacteria described in any one of claims 1 to 13 or the method for preparing the genetically engineered bacteria described in any one of claims 14 to 26.

28. The method according to claim 27, characterized in that The fermentation includes shake flask fermentation or fermenter fermentation.

29. The method according to claim 28, characterized in that The shake flask fermentation comprises: inoculating the genetically engineered bacteria cultured overnight into a fermentation medium of the shake flask fermentation for expansion culture to obtain the sclareol.

30. The method according to claim 28, characterized in that The fermentation in a fermenter comprises: inoculating the genetically engineered bacteria cultured overnight into a fermenter for expansion culture to obtain the sclareol.

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