Genetically engineered bacterium, preparation method therefor, and use thereof in synthesizing sclareol

Abstract

Provided in the present invention are a genetically engineered bacterium, a preparation method therefor, and use thereof in synthesizing sclareol. The described genetically engineered bacterium comprises a CcLPPS gene derived from Cistus creticus. The described genetically engineered bacterium is selected from Yarrowia lipolytica. The present invention constructs a strain with stable expression for efficient de novo synthesis of sclareol, and no organic reagents or pathway intermediate substances need to be added during the entire fermentation process, which greatly reduces production costs while simplifying subsequent separation and purification steps. The engineered Yarrowia lipolytica strain provided in the present invention has high production intensity and a simple fermentation process, exhibits promising application prospects, and lays a foundation for the production of sclareol by means of microbial fermentation.

Description

Genetically engineered bacteria, their preparation methods, and their application in the synthesis of perillaldehyde.

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese Patent Application No. 202510356375.X, filed on March 25, 2025, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] This invention relates to the field of bioengineering technology, and more specifically, to a genetically engineered bacterium, its preparation method, and its application in the synthesis of perillaldehyde. Background Technology

[0004] Sclareol, with the molecular formula C 20 H 36 O2 is a plant-derived hemispheryl diterpenoid diterpenol, widely used as a natural fragrance ingredient in perfumes, cosmetics, and food flavorings. Furthermore, perillyl alcohol is the only practical chemically synthesized raw material for ambroxol (a natural ambergris substitute). Perillyl alcohol also possesses strong antibacterial activity, regulating the growth of fungi and plants, and can be used in the pesticide field.

[0005] Currently, perillaldehyde is mainly extracted from plants. However, plants have long growth cycles and are easily affected by factors such as soil, environment, and climate, resulting in low perillaldehyde content in plants, which is difficult to meet market demand. With breakthroughs in bioengineering technology, efficient production of perillaldehyde through microbial fermentation is expected to meet market demand. Patent application CN 114989997 A discloses the construction of a perillaldehyde metabolic pathway by integrating key genes into the *Yersinia lipolytica* Po1f host, achieving yields of 817.65 mg / L and 12.9 g / L (240 h) of perillaldehyde in shake flasks and fermenters, respectively, which is the highest reported yield to date. *Saccharomyces cerevisiae*, as a model strain of eukaryotes, is also widely used in the synthesis of high-value compounds. Patent application CN 116262929 A discloses the use of *Saccharomyces cerevisiae* as a cell factory, systematically modifying its central metabolism to achieve efficient synthesis of perillaldehyde from glucose, with yields of 1.83 g / L in shake flasks and 11.4 g / L (192 h) in fermenters. However, the fermentation cycle of the above-mentioned synthetic perillaldehyde is too long, and the addition of organic reagents is required during the fermentation process, which increases the production cost.

[0006] In summary, although de novo microbial synthesis of perillaldehyde has been achieved, problems such as low yield, high cost, complex process control, and long fermentation cycle still exist, which are not conducive to industrial production. Therefore, constructing stable and efficient perillaldehyde synthesis strains is crucial for realizing the industrial production of perillaldehyde by microorganisms. Summary of the Invention

[0007] The main objective of this invention is to provide a genetically engineered bacterium, its preparation method, and its application in the synthesis of perillaldehyde, so as to solve the problem of low perillaldehyde yield in the prior art.

[0008] To achieve the above objectives, according to a first aspect of the present invention, a genetically engineered bacterium is provided, the genetically engineered bacterium comprising the CcLPPS gene derived from *Cistus creticus*; the genetically engineered bacterium is selected from *Yarrowia lipolytica*.

[0009] 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.

[0010] Furthermore, the aforementioned genetically engineered bacteria also include the TP1132 gene derived from Salvia sclarea.

[0011] 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.

[0012] Furthermore, the aforementioned genetically engineered bacteria also include: the gene MvaS derived from Enterococcus faecalis and / or the gene MvaE derived from Enterococcus faecalis.

[0013] Furthermore, the protein encoded by the gene MvaS from Enterococcus faecalis has the amino acid sequence shown in SEQ ID NO: 5; the protein encoded by the gene MvaE from Enterococcus faecalis has the amino acid sequence shown in SEQ ID NO: 6.

[0014] Furthermore, the aforementioned genetically engineered bacteria also include the tHMG1 gene derived from Yersinia lipolytica.

[0015] Furthermore, the protein encoded by the tHMG1 gene from Yersinia lipolytica has the amino acid sequence shown in SEQ ID NO: 7.

[0016] Furthermore, the aforementioned genetically engineered bacteria also include the SsCrtE gene derived from the thermophilic cyanobacterium Synechococcus sp.

[0017] Furthermore, the protein encoded by the SsCrtE gene from the thermophilic cyanobacterium Synechococcus sp. has the amino acid sequence shown in SEQ ID NO: 8.

[0018] Furthermore, the aforementioned genetically engineered bacteria also include any one or more of the following genes: ERG12 derived from Yersinia lipolytica, ERG20 derived from Yersinia lipolytica, ERG8 derived from Yersinia lipolytica, MVD1 derived from Yersinia lipolytica, or IDI1 derived from Yersinia lipolytica.

[0019] Furthermore, the protein encoded by the gene ERG12 from *Yersinia lipolytica* has the amino acid sequence shown in SEQ ID NO: 9; the protein encoded by the gene ERG20 from *Yersinia lipolytica* has the amino acid sequence shown in SEQ ID NO: 10; the protein encoded by the gene ERG8 from *Yersinia lipolytica* has the amino acid sequence shown in SEQ ID NO: 11; the protein encoded by the gene MVD1 from *Yersinia lipolytica* has the amino acid sequence shown in SEQ ID NO: 12; and the protein encoded by the gene IDI1 from *Yersinia lipolytica* has the amino acid sequence shown in SEQ ID NO: 13.

[0020] Furthermore, the aforementioned genetically engineered bacteria also include the HpCrtE gene derived from Haematococcus pluvialis.

[0021] Furthermore, the protein encoded by the HpCrtE gene from Haematococcus pluvialis has the amino acid sequence shown in SEQ ID NO: 14.

[0022] To achieve the above objective, according to a second aspect of the present invention, a method for preparing a genetically engineered bacterium is provided, the method comprising: knocking in a gene into a starting engineered bacterium to obtain the genetically engineered bacterium; wherein the knocked-in gene includes at least the CcLPPS gene derived from *Cistus creticus*, and the genetically engineered bacterium is selected from *Yarrowia lipolytica*.

[0023] 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.

[0024] Furthermore, the knock-in genes also include the TP1132 gene from Salvia sclarea.

[0025] 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.

[0026] Furthermore, the knock-in genes also include: the MvaS gene from Enterococcus faecalis and / or the MvaE gene from Enterococcus faecalis.

[0027] Furthermore, the protein encoded by the gene MvaS from Enterococcus faecalis has the amino acid sequence shown in SEQ ID NO: 5; the protein encoded by the gene MvaE from Enterococcus faecalis has the amino acid sequence shown in SEQ ID NO: 6.

[0028] Furthermore, the knock-in genes also include the tHMG1 gene derived from Yeastia lipolytica.

[0029] Furthermore, the protein encoded by the tHMG1 gene from Yersinia lipolytica has the amino acid sequence shown in SEQ ID NO: 7.

[0030] Furthermore, the knock-in genes also include the SsCrtE gene from the thermophilic cyanobacterium Synechococcus sp.

[0031] Furthermore, the protein encoded by the SsCrtE gene from the thermophilic cyanobacterium Synechococcus sp. has the amino acid sequence shown in SEQ ID NO: 8.

[0032] Furthermore, the knock-in genes also include any one or more of the following genes: ERG12 from Yersinia lipolytica, ERG20 from Yersinia lipolytica, ERG8 from Yersinia lipolytica, MVD1 from Yersinia lipolytica, or IDI1 from Yersinia lipolytica.

[0033] Furthermore, the protein encoded by the gene ERG12 from *Yersinia lipolytica* has the amino acid sequence shown in SEQ ID NO: 9; the protein encoded by the gene ERG20 from *Yersinia lipolytica* has the amino acid sequence shown in SEQ ID NO: 10; the protein encoded by the gene ERG8 from *Yersinia lipolytica* has the amino acid sequence shown in SEQ ID NO: 11; the protein encoded by the gene MVD1 from *Yersinia lipolytica* has the amino acid sequence shown in SEQ ID NO: 12; and the protein encoded by the gene IDI1 from *Yersinia lipolytica* has the amino acid sequence shown in SEQ ID NO: 13.

[0034] Furthermore, the knock-in genes also include the HpCrtE gene from Haematococcus pluvialis.

[0035] Furthermore, the protein encoded by the HpCrtE gene from Haematococcus pluvialis has the amino acid sequence shown in SEQ ID NO: 14.

[0036] To achieve the above objective, according to a third aspect of the present invention, a method for synthesizing perillyl is provided, the method comprising: fermenting the above-mentioned genetically engineered bacteria or the genetically engineered bacteria prepared by the above-mentioned method in a fermentation medium to synthesize the above-mentioned perillyl.

[0037] Furthermore, the above fermentation includes shake flask fermentation or fermenter fermentation.

[0038] Furthermore, the above-mentioned shake-flask fermentation includes: inoculating the above-mentioned genetically engineered bacteria cultured overnight into the fermentation medium for shake-flask fermentation for expansion culture to obtain the above-mentioned perillaldehyde.

[0039] Furthermore, the fermentation in the above-mentioned fermenter includes: inoculating the above-mentioned genetically engineered bacteria that have been cultured overnight into the fermenter for scale-up culture to obtain the above-mentioned perillaldehyde.

[0040] Applying the technical solution of this invention, the genetically engineered bacteria of this invention contain the CcLPPS gene derived from *Cistus creticus*; the aforementioned genetically engineered bacteria are selected from *Yarrowia lipolytica*. Such genetically engineered bacteria can express lysine pyrophosphate diol synthase, thereby enabling the bacteria to convert geranyl geranyl pyrophosphate (GGPP) into the precursor lysine pyrophosphate diol (8OH-CPP), which can then spontaneously convert into perillyl alcohol. The synthesis of perillyl alcohol using this genetically engineered bacteria offers advantages such as high production intensity, a simple fermentation process, and promising application prospects, laying the foundation for the microbial fermentation production of perillyl alcohol. Attached Figure Description

[0041] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0042] Figure 1 shows the fermentation results of LPPS gene-integrating strains from different sources according to an embodiment of the present invention.

[0043] Figure 2 shows the results of truncation of signal peptides of different lengths in the CcLPPS gene according to an embodiment of the present invention.

[0044] Figure 3 shows the CcLPPS gene mutation results according to an embodiment of the present invention.

[0045] Figure 4 shows the fermentation results of TPS gene-integrated strains from different sources according to an embodiment of the present invention.

[0046] Figure 5 shows the fermentation results of CrtE genes from different sources integrated into strain Sc23 according to an embodiment of the present invention.

[0047] Figure 6 shows a synthetic route diagram of perillaldehyde according to an embodiment of the present invention.

[0048] Figure 7 shows the fermentation results of TP1132 gene of different truncated lengths integrated into the strain according to an embodiment of the present invention. Detailed Implementation

[0049] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0050] As mentioned in the background section, perillyl alcohol is not only a natural fragrance ingredient but also possesses strong antibacterial activity, making it widely used in cosmetics, food, and agriculture, and possessing significant application value and market potential. However, existing technologies for producing perillyl alcohol through microorganisms suffer from low yields.

[0051] This invention expresses lysandrindiol pyrophosphate synthase by fermenting a genetically engineered bacterium containing the exogenously introduced gene encoding lysandrindiol pyrophosphate synthase CcLPPS from *Cistus creticus*. This allows the engineered bacterium to convert geranyl-geranyl pyrophosphate (GGPP) into the precursor lysandrindiol pyrophosphate (8OH-CPP), which can then spontaneously convert to perillyl alcohol (see Figure 6). The perillyl alcohol produced using this genetically engineered bacterium is produced in high yield and with high efficiency, showing promising application prospects and laying the foundation for the industrial-scale production of perillyl alcohol using *Yersinia lipolytica* fermentation. Based on this, the inventors have proposed a series of protection schemes for this invention.

[0052] In a first typical embodiment of the present invention, a genetically engineered bacterium is provided, comprising the CcLPPS gene derived from *Cistus creticus*; the genetically engineered bacterium is selected from *Yarrowia lipolytica*. LPPS genes from certain sources can be used to synthesize perillaldehyde, and the yield of perillaldehyde varies depending on the source of the LPPS gene.

[0053] In a more preferred embodiment of the present invention, the above-mentioned *Yarrowia lipolytica* comprises: *Yarrowia lipolytica* strain Yl590 (classification name *Yarrowia lipolytica*, accession number CGMCC NO.30855, deposited on June 4, 2024 at the China General Microbiological Culture Collection Center, Beijing, China). The above-mentioned genetically engineered bacteria have the beneficial effects of rapid growth, high activity, and few byproducts. Using the above-mentioned genetically engineered bacteria to synthesize perillaldehyde helps to improve the production efficiency of perillaldehyde.

[0054] This invention screened LPPS from different sources, including: rockrose (Cistus creticus), sclarea (Salvia sclarea), capsicum annuum (Capsicum annuum), Erythranthe guttata (Erythranthe guttata), Handroanthus impetiginosus (Handroanthus impetiginosus), Lycium barbarum (Lycium barbarum), Nicotiana sylvestris (Nicotiana sylvestris), sesame (Sesamum indicum), and Solanum stenotomum (Solanum stenotomum). Ultimately, CcLPPS from rockrose (Cistus creticus) was selected as suitable for synthesizing sclarea alcohol with a high yield.

[0055] Furthermore, using GGPP as a precursor, fermentation of the genetically engineered bacteria containing the CcLPPS gene can accumulate the byproducts geraniol (GGOH) and (E)-lysanthan-13-en-8,15-diol (LOH), indicating that the CcLPPS gene can effectively convert geraniol pyrophosphate (GGPP) into the precursor lysanthan-13-en-8,15-diol pyrophosphate (8OH-CPP), and 8OH-CPP can spontaneously convert into geraniol and the byproduct (E)-lysanthan-13-en-8,15-diol (LOH).

[0056] The wild-type CcLPPS gene from *Cistus creticus* in this invention has the amino acid sequence shown in SEQ ID NO: 1. Since the wild-type CcLPPS gene from *Cistus creticus* is plant-derived, protein structure simulation revealed that the gene contains an unfolded polypeptide sequence, which is speculated to be a signal peptide. The function of a signal peptide is to localize the expressed protein to specific organelles. In this invention, the signal peptide may localize the enzyme expressed by the CcLPPS gene to organelles, thereby weakening the catalytic activity of the enzyme in the cytoplasm. Removing the signal peptide allows the enzyme expressed by the gene to participate in reactions more effectively in the cytoplasm.

[0057] Therefore, this invention truncates the protein encoded by the wild-type CcLPPS gene to different lengths of amino acids, including 19, 29, 39, 49, 59, 69, and 79 amino acids. The yield of perillaldehyde varies with different truncation lengths. Ultimately, the highest perillaldehyde yield was found when the truncation length was 49 amino acids (the amino acid sequence of the protein encoded by the truncated CcLPPS gene is shown in SEQ ID NO: 2), which was 3 times higher than that of the untrunculated CcLPPS gene. This indicates that appropriately truncating the CcLPPS gene signal peptide can better enhance the catalytic efficiency of the protein.

[0058] To improve the catalytic efficiency of the protein encoded by the CcLPPS gene, the truncated CcLPPS gene was further mutated (mutation sites included N210Y and D614A; it should be noted that the mutation site numbers here refer to the amino acid sequence numbers of the wild-type CcLPPS protein). The mutated CcLPPS gene encoded a protein with the amino acid sequence shown in SEQ ID NO: 3. The CcLPPS gene can be used to efficiently synthesize perillyl alcohol.

[0059] 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.

[0060] To further increase the yield of perillaldehyde and reduce the accumulation of the byproduct (E)-lysantho-13-ene-8,15-diol LOH, in a preferred embodiment of the present invention, the genetically engineered bacteria further includes the TP1132 gene derived from Salvia sclarea. The protein encoded by the TPS gene (TP1132 gene) catalyzes the synthesis of perillaldehyde from the precursor lysantho-13-ene-8,15-diol pyrophosphate 8OH-CPP.

[0061] This invention screened 19 TPS genes from different sources (see Table 3) and ultimately identified the TP1132 gene from Salvia sclarea as capable of efficiently synthesizing perillaldehyde. In a preferred embodiment of this 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.

[0062] Although the CcLPPS gene can effectively convert geranyl-geranyl pyrophosphate (GGPP) into the precursor lysine pyrophosphate diol ester (8OH-CPP), and 8OH-CPP can spontaneously convert into perillyl alcohol and the byproduct (E)-lysine-13-en-8,15-diol (LOH), when the TPS gene (TP1132 gene) is further introduced into the genetically engineered strain, 8OH-CPP is more inclined to synthesize perillyl alcohol, thereby increasing the accumulation of perillyl alcohol.

[0063] Generally, when the copy number of a gene in genetically engineered bacteria increases, the function of that gene in the genetically engineered bacteria may be enhanced. In a more preferred embodiment of the present invention, the genetically engineered bacteria include two copies of the TP1132 gene derived from Salvia sclarea.

[0064] In another preferred embodiment of the present invention, the above-mentioned genetically engineered bacteria include three copies of the TP1132 gene derived from Salvia sclarea.

[0065] In a more preferred embodiment of the present invention, the above-mentioned genetically engineered bacteria includes four copies of the TP1132 gene derived from Salvia sclarea.

[0066] To further enhance the supply of mevalonic acid (MVA), a precursor for the synthesis of perillaldehyde, in a preferred embodiment of the present invention, the above-mentioned genetically engineered bacteria further includes: the gene MvaS derived from Enterococcus faecalis and / or the gene MvaE derived from Enterococcus faecalis.

[0067] In a preferred embodiment of the present invention, the protein encoded by the gene MvaS from Enterococcus faecalis has the amino acid sequence shown in SEQ ID NO: 5; and the protein encoded by the gene MvaE from Enterococcus faecalis has the amino acid sequence shown in SEQ ID NO: 6.

[0068] In a more preferred embodiment of the present invention, the above-mentioned genetically engineered bacteria further includes the tHMG1 gene derived from *Yarrowia lipolytica*. In a more preferred embodiment of the present invention, the above-mentioned genetically engineered bacteria further includes two 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. Knocking the above-mentioned MvaS, MvaE and tHMG1 genes into the genetically engineered bacteria helps to increase the accumulation of the precursor mevalonate (MVA), thereby further helping to increase the accumulation of perillaldehyde.

[0069] To convert farnesyl pyrophosphate (FPP) produced in vivo into gerany-gerany-gerany-phosphate (GGPP), the aforementioned genetically engineered bacteria also include the CrtE gene. This invention screened five CrtE genes from different sources (see Table 4) and ultimately selected the SsCrtE gene from the thermophilic cyanobacterium *Synechococcus* sp., which can convert farnesyl pyrophosphate (FPP) produced in the genetically engineered bacteria into gerany-gerany-gerany-phosphate (GGPP), thereby increasing the yield of gerany-gerany-phosphate. Simultaneously, the accumulation of byproducts was also significantly increased. Therefore, in a more preferred embodiment of this invention, the aforementioned genetically engineered bacteria also include the SsCrtE gene from the thermophilic cyanobacterium *Synechococcus* sp. In a preferred embodiment of this invention, the protein encoded by the SsCrtE gene from the thermophilic cyanobacterium *Synechococcus* sp. has the amino acid sequence shown in SEQ ID NO: 8.

[0070] To further enhance MVA metabolism and improve precursor FPP synthesis, in a preferred embodiment of the present invention, the above-mentioned genetically engineered bacteria further includes any one or more of the following genes: ERG12 from Yersinia lipolytica, ERG20 from Yersinia lipolytica, ERG8 from Yersinia lipolytica, MVD1 from Yersinia lipolytica, or IDI1 from Yersinia lipolytica.

[0071] In a more preferred embodiment of the present invention, the above-mentioned genetically engineered bacteria further includes two copies of any one or more of the following genes: ERG12 from Yersinia lipolytica, ERG20 from Yersinia lipolytica, ERG8 from Yersinia lipolytica, MVD1 from Yersinia lipolytica, or IDI1 from Yersinia lipolytica.

[0072] In a preferred embodiment of the present invention, the protein encoded by the gene ERG12 of *Yersinia lipolytica* has the amino acid sequence shown in SEQ ID NO: 9; the protein encoded by the gene ERG20 of *Yersinia lipolytica* has the amino acid sequence shown in SEQ ID NO: 10; the protein encoded by the gene ERG8 of *Yersinia lipolytica* has the amino acid sequence shown in SEQ ID NO: 11; the protein encoded by the gene MVD1 of *Yersinia lipolytica* has the amino acid sequence shown in SEQ ID NO: 12; and the protein encoded by the gene IDI1 of *Yersinia lipolytica* has the amino acid sequence shown in SEQ ID NO: 13.

[0073] In a preferred embodiment of the present invention, the above-mentioned genetically engineered bacteria further includes the HpCrtE gene derived from Haematococcus pluvialis. The HpCrtE gene can directly convert IPP and DMAPP into GGPP, achieving the accumulation of two layers of GGPP and increasing the yield of styrosine. 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.

[0074] In a second typical embodiment of the present invention, a method for preparing genetically engineered bacteria is provided. The preparation method includes: knocking in a gene into a starting engineered bacterium to obtain the genetically engineered bacteria; the gene includes at least the CcLPPS gene from Cistus creticus, and the genetically engineered bacteria is selected from Yersinia lipolytica.

[0075] In a more preferred embodiment of the present invention, the above-mentioned *Yarrowia lipolytica* comprises: *Yarrowia lipolytica* strain Yl590 (taxonomic name: *Yarrowia lipolytica*, accession number: CGMCC NO.30855, deposited on June 4, 2024 at the China General Microbiological Culture Collection Center, Beijing, China). The above-mentioned genetically engineered bacteria has the beneficial effect of rapid growth. Using the above-mentioned genetically engineered bacteria to synthesize perillyl alcohol helps to improve the production efficiency of perillyl alcohol.

[0076] 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.

[0077] In a preferred embodiment of the present invention, the above-mentioned gene further includes: the TP1132 gene derived from Salvia sclarea.

[0078] In a more preferred embodiment of the present invention: the above-mentioned gene also includes two copies of the TP1132 gene derived from Salvia sclarea.

[0079] In another preferred embodiment of the present invention, the above-mentioned genetically engineered bacteria include three copies of the TP1132 gene derived from Salvia sclarea.

[0080] In a more preferred embodiment of the present invention, the above-mentioned genetically engineered bacteria includes four copies of the TP1132 gene derived from Salvia sclarea.

[0081] 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.

[0082] In a preferred embodiment of the present invention, the above-mentioned genes further include: the gene MvaS derived from Enterococcus faecalis and / or the gene MvaE derived from Enterococcus faecalis. In a preferred embodiment of the present invention, the protein encoded by the gene MvaS derived from Enterococcus faecalis has the amino acid sequence shown in SEQ ID NO: 5; the protein encoded by the gene MvaE derived from Enterococcus faecalis has the amino acid sequence shown in SEQ ID NO: 6.

[0083] In a preferred embodiment of the present invention, the gene further includes the tHMG1 gene derived from *Yarrowia lipolytica*. In a more preferred embodiment of the present invention, the genetically engineered bacteria further includes 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 the amino acid sequence shown in SEQ ID NO: 7.

[0084] In a preferred embodiment of the present invention, the gene further includes 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 Synechococcus sp. has the amino acid sequence shown in SEQ ID NO: 8.

[0085] In a preferred embodiment of the present invention, the above-mentioned gene further includes any one or more of the following genes: ERG12 derived from Yersinia lipolytica, ERG20 derived from Yersinia lipolytica, ERG8 derived from Yersinia lipolytica, MVD1 derived from Yersinia lipolytica, or IDI1 derived from Yersinia lipolytica.

[0086] In a more preferred embodiment of the present invention, the above-mentioned genetically engineered bacteria further includes two copies of any one or more of the following genes: ERG12 from Yersinia lipolytica, ERG20 from Yersinia lipolytica, ERG8 from Yersinia lipolytica, MVD1 from Yersinia lipolytica, or IDI1 from Yersinia lipolytica.

[0087] In a preferred embodiment of the present invention, the protein encoded by the gene ERG12 of *Yersinia lipolytica* has the amino acid sequence shown in SEQ ID NO: 9; the protein encoded by the gene ERG20 of *Yersinia lipolytica* has the amino acid sequence shown in SEQ ID NO: 10; the protein encoded by the gene ERG8 of *Yersinia lipolytica* has the amino acid sequence shown in SEQ ID NO: 11; the protein encoded by the gene MVD1 of *Yersinia lipolytica* has the amino acid sequence shown in SEQ ID NO: 12; and the protein encoded by the gene IDI1 of *Yersinia lipolytica* has the amino acid sequence shown in SEQ ID NO: 13.

[0088] In a preferred embodiment of the present invention, the above-mentioned gene further includes the 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 the amino acid sequence shown in SEQ ID NO: 14.

[0089] Chassis microbial modification is an important method for increasing the yield of target microbial metabolites, involving gene knock-in or knock-out. Gene knock-out aims to reduce the accumulation of byproducts in metabolic pathways, while gene knock-in provides the raw materials and impetus for the synthesis of the target product. Specifically, gene knock-in refers to the direct integration of a gene into the bacterial genome, while gene knock-out refers to the removal of a gene from the bacterial genome.

[0090] The gene knock-in order of this invention is not specifically limited, and the timing of gene knock-in can be selected according to specific needs. The number of knock-ins of the same gene can also be selected according to actual needs. When multiple copies of a certain gene are knocked into a genetically engineered bacterium, the function of that gene in the genetically engineered bacterium will be enhanced. The knock-in timing of multiple copies of the gene can be the same or different.

[0091] It should be noted that in this invention, the terms "import," "transfer," and "knock-in" refer to integrating the gene into the genome of the genetically engineered bacteria, rather than the gene existing in the genetically engineered bacteria in plasmid form. When the gene is transferred into the genetically engineered bacteria in plasmid form, the gene is not integrated into the genome of the genetically engineered bacteria. In a preferred embodiment of this invention, gene knock-in is performed using gene editing methods.

[0092] When integrating genes into *Yarrowia lipophila* using gene editing, a specific gene in the genome needs to be knocked out as a selection 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.

[0093] In a third typical embodiment of the present invention, a method for synthesizing perillyl alcohol is provided, the method comprising: fermenting the above-mentioned genetically engineered bacteria or the above-mentioned genetically engineered bacteria prepared by the above-mentioned method in a fermentation culture medium to synthesize the above-mentioned perillyl alcohol.

[0094] In a preferred embodiment of the present invention, the above fermentation includes shake flask fermentation or fermenter fermentation.

[0095] In a preferred embodiment of the present invention, shake-flask fermentation includes: inoculating the above-mentioned genetically engineered bacteria cultured overnight into the fermentation medium for shake-flask fermentation for expansion culture to obtain the above-mentioned perillaldehyde.

[0096] 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 scale-up culture to obtain the above-mentioned perillaldehyde.

[0097] In a preferred embodiment of the present invention, the overnight culture conditions are 28-32°C and 180-220 rpm; the scale-up culture conditions are 28-32°C and 180-220 rpm.

[0098] In a preferred embodiment of the present invention, the fermentation medium for the shake-flask fermentation comprises: 15-25 g / L glucose, 10-20 g / L yeast extract, and 10-20 g / L peptone. Under these conditions, perillaldehyde is synthesized, resulting in a high yield of perillaldehyde.

[0099] In a preferred embodiment of the present invention, the fermentation in the fermenter includes: inoculating the genetically engineered bacteria cultured overnight into a fermenter for scale-up culture to obtain the above-mentioned perillaldehyde. In a preferred embodiment of the present invention, the overnight culture conditions are 28-32°C and 180-220 rpm; the scale-up culture conditions are DO 5%-20%, pH 3.0-6.0, and 28-32°C; and the inoculum size is 5%-20%.

[0100] In a preferred embodiment of the present invention, the fermentation medium for the above-mentioned fermenter includes: 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.

[0101] The vitamin reservoir contains 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 para-aminobenzoic acid.

[0102] The aforementioned trace element stock solution comprises 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 these conditions, the synthesis of perillaldehyde exhibits high yield, a short production cycle, and requires no addition of any organic reagents or precursors during the entire fermentation process, demonstrating significant economic value and promising industrialization prospects.

[0103] It should be noted that, in order to avoid excessive glucose concentration in the initial fermentation medium, the 200-400 g / L glucose of this invention is preferably added in two batches. First, 20 g / L of glucose is added to the initial medium. After fermentation begins, the remaining glucose is added to the fermentation medium. During the addition process, the glucose concentration in the fermentation medium needs to be controlled to be below 5 g / L.

[0104] The present invention will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed by the present invention.

[0105] Example 1: Modification of strain Yl590

[0106] Wild-type *Yerovia lipolytica* strain Yl590 (CGMCC NO.30855, deposited on June 4, 2024 at the China General Microbiological Culture Collection Center, Beijing, China) was modified to knock out the URA3 selection marker gene, facilitating subsequent CRISPR gene editing for key gene integration. Using the genome of *Yerovia lipolytica* strain Yl590 as a template, and with URA-Up-F / R and URA-Down-F / R primers, PCR was used to obtain the upstream and downstream homologous arm fragments of the URA3 gene. The two fragments were then fused by PCR to obtain the URA3-Up+Down fragment. The fusion fragment was transformed into strain Yl590 using the lithium acetate (LiAC) method. The recovered bacterial culture was plated on 5-FOA plates and incubated at 30°C for 2 days. Single colonies were then picked and spot-patched onto SD-URA and YPD solid plates for preliminary screening. The obtained mutant strains were then verified by colony PCR, and a URA3-deficient strain was successfully obtained.

[0107] Table 1 Primers used for URA3 gene knockout

[0108] Example 2: LPPS gene screening

[0109] Screening of LPPS gene sources: Using strain Yl590 as the host, LPPS genes from 9 different sources were compared (see Table 2). Strains Sc01-Sc10 were constructed. Among them, the expression of the CcLPPS gene from Rosa rockosa produced the highest yield of perillaldehyde, 0.35 mg / L, followed by the SsLPPS gene, which produced 0.31 mg / L of perillaldehyde. The expression of the other genes did not produce the target product (Figure 1).

[0110] Table 2

[0111] Protein structure simulation revealed the presence of a signal peptide sequence in the CcLPPS gene. Therefore, the CcLPPS gene was truncated to different lengths (19, 19, 39, 49, 59, 69, and 79 amino acids) starting from the N-terminus. Using strain Yl590 as the parent strain, strains Sc11-Sc17 were constructed. Figure 2 shows that truncating the CcLPPS gene signal peptide of different lengths increased the yield of perillaldehyde. The CcLPPS gene truncated by 49 amino acids showed the best integration effect, producing 1.03 mg / L of perillaldehyde, a three-fold increase compared to the untruncated CcLPPS gene. This indicates that appropriately truncating the CcLPPS gene signal peptide can better enhance the catalytic efficiency of the protein.

[0112] Further mutations were performed on the CcLPPS gene, and two effective mutation sites, N210Y and D614A, were screened. The CcLPPS* mutant gene was expressed in strain Yl590, and strains Sc18-Sc20 were constructed, increasing the yield of perillaldehyde to 1.35 mg / L (Figure 3). Among them, the mutation site of CcLPPS in strain Sc18 was N210Y; the mutation site of CcLPPS in strain Sc19 was D614A; and the mutation site of CcLPPS in strain Sc20 was N210Y+D614A.

[0113] The fermentation method of the above-mentioned recombinant *Yarrowia lipolytica* strain and the detection method of perillaldehyde were as follows: Recombinant *Yarrowia lipolytica* strains Sc01-Sc20 were inoculated into YPD medium and cultured overnight at 30℃ and 220 rpm. The YPD medium contained 20 g / L glucose, 10 g / L yeast extract, and 20 g / L peptone. The conditions for detecting perillaldehyde yield at different time points during fermentation using gas chromatography were as follows: Agilent HP-5 column 30 m × 0.32 mm, 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 1 μL.

[0114] Example 3: TPS gene screening

[0115] Nineteen TPS genes from different sources were integrated into the Sc20 strain to construct Sc20a-Sc20s strains (see Table 3). The experimental results are shown in Figure 4. After integrating the TP1132 gene, the yield of perillaldehyde increased to 3.21 mg / L.

[0116] Table 3

[0117] Protein structure simulation revealed the presence of a signal peptide sequence in the TP1132 gene. Therefore, the TP1132 gene was truncated to different lengths of amino acids, starting from the N-terminus, with truncated lengths of 19, 29, 39, 49, 59, 69, and 79 amino acids. Using strain Sc20 as the parent strain, the Sc20t-Sc20z strain was constructed. The results, as shown in Figure 7, indicate that the TP1132 gene truncated by 39 amino acids showed the best integration effect, producing 5.31 mg / L of perillyl alcohol, which is 1.6 times higher than that of the untruncated TP1132 gene. This suggests that truncating the TP1132 gene signal peptide to an appropriate length can better enhance the catalytic efficiency of the protein.

[0118] The fermentation method and the detection method of styraxol for the above-mentioned recombinant Yersinia lipophila strain Sc20a-Sc20z are the same as in Example 2.

[0119] Example 4: Construction of a highly efficient chassis Sc38 strain for supplying perillyl alcohol precursor

[0120] A CcLPPS mutant gene from *Cistus creticus* was introduced into strain Yl590 to construct strain Sc20, which was able to synthesize 1.35 mg / L of perillaldehyde de novo. Further introduction of the TP1132 gene (with the amino acid sequence shown in SEQ ID NO: 16), which was truncated by 39 amino acids, into strain Sc20v increased the perillaldehyde yield to 3.21 mg / L. Among them, the byproducts geraniol (GGOH) and (E)-lysanthan-13-en-8,15-diol (LOH) were also accumulated in small amounts, indicating that the CcLPPS gene can effectively convert GGPP into the precursor lysanthan-13-en-8,15-diol pyrophosphate (8OH-CPP). 8OH-CPP can spontaneously convert into perillyl alcohol and the byproduct (E)-lysanthan-13-en-8,15-diol (LOH). When the TP1132 gene (with the amino acid sequence shown in SEQ ID NO: 16) which is truncated by 39 amino acids is introduced, 8OH-CPP is more likely to synthesize perillyl alcohol, thereby increasing the accumulation of perillyl alcohol.

[0121] To further enhance the supply of mevalonic acid (MVA), a precursor for perillaldehyde synthesis, the gene MvaS (ID: WP_002357756.1) from Enterococcus faecalis was introduced into Sc20v to successfully construct strain Sc21, which yielded 9.21 mg / L of perillaldehyde. Furthermore, the gene MvaE (ID: WP_002357755.1) from Enterococcus faecalis was integrated into strain Sc21 to successfully construct strain Sc22, which increased the perillaldehyde yield to 15.41 mg / L.

[0122] Using the genome of Yersinia lipolytica strain Yl590 as a template, the tHMG1 gene was amplified and integrated into strain Sc22 to construct strain Sc23. The yield of perillaldehyde was increased to 17.94 mg / L, indicating that the expression of MvaS, MvaE and tHMG1 genes can increase the accumulation of the precursor mevalonate MVA, thereby increasing the accumulation of perillaldehyde.

[0123] Five different CrtE genes were integrated into the Sc23 strain to construct strains Sc24-Sc28. Among them, the SsCrtE gene showed the best expression effect (Figure 5), which could convert farnesyl pyrophosphate (FPP) produced in vivo into geraniol pyrophosphate (GGPP), thereby increasing the yield of geraniol to 55.23 mg / L and significantly increasing the accumulation of by-products.

[0124] Table 4

[0125] To further enhance MVA metabolism and increase precursor FPP synthesis, the ERG12, ERG20, ERG8, MVD1, and IDI1 genes were amplified using the genome of *Yerovia lipolytica* strain Yl590 as a template. These genes were then sequentially superimposed and integrated into strain Sc27, resulting in Sc29-Sc33 (see Table 5). Strain Sc33 synthesized 369.62 mg / L of perillyl alcohol after 96 h of fermentation.

[0126] Table 5

[0127] In addition, the TP1132 gene (with 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 of perillyl alcohol after fermentation for 96 h.

[0128] Next, the HpCrtE (ID: QHF16627.1) gene from Haematococcus pluvialis was integrated into strain Sc34 to construct strain Sc35. The HpCrtE gene can directly convert IPP and DMAPP into GGPP, achieving the accumulation of two layers of GGPP. The yield of styracilol was increased to 1093.22 mg / L, which is higher than the highest reported yield of 817.65 mg / L in shake flasks.

[0129] To further enhance the conversion of the precursor GGPP to perillaldehyde and reduce the accumulation of byproducts GGOH and LOH, a single copy of the gene CcLPPS* was integrated into strain Sc35 to obtain strain Sc36, increasing perillaldehyde yield to 1.45 g / L. Subsequently, a single copy of the 39-amino acid truncated TP1132 gene (with the amino acid sequence shown in SEQ ID NO: 16) was integrated into strain Sc36 to obtain strain Sc37, increasing perillaldehyde yield to 2.24 g / L. Further integration of a single copy of the 39-amino acid truncated TP1132 gene (with the amino acid sequence shown in SEQ ID NO: 16) into Sc37 constructed strain Sc38. Fermentation at 30℃ for 120 h yielded 2.61 g / L of perillaldehyde, with byproducts GGOH and LOH at 102.53 mg / L and 257.24 mg / L, respectively.

[0130] The fermentation method for the above-mentioned recombinant Yersinia lipophila strain and the detection method for perillaldehyde are the same as in Example 2.

[0131] Example 5: Production of perillaldehyde in a 5L fermenter

[0132] The engineered strains Sc35-Sc38 were used to scale up the production of perillaldehyde in a fermenter. Recombinant Yersinia lipolytica strains Sc35-Sc38 were inoculated into YPD medium and cultured overnight at 30°C, then transferred to the fermenter at a 10% inoculation rate. The fermentation process was controlled at pH 5.0, temperature 30°C, and initial dissolved oxygen (DO) of 10%, with the turbine speed and aeration rate adjusted in conjunction with dissolved oxygen levels. After 120 hours of fermentation, strain Sc35 synthesized 13.5 g / L of perillaldehyde, higher than the highest reported yield of 12.9 g / L. The perillaldehyde yields of strains Sc36 and Sc37 were increased to 15.25 g / L and 18.92 g / L, respectively, while the final engineered strain Sc38 could synthesize 20.25 g / L of perillaldehyde.

[0133] The fermentation method of the above-mentioned recombinant Yersinia lipolytica strain and the detection method of perillaldehyde are as follows: The above-mentioned recombinant strain is 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 extract, 10 ml / L trace element stock solution, 12 ml / L vitamin stock solution, 1.2 ml / L biotin) at a 10% inoculation rate during fermentation. The pH is controlled at 5.0, the temperature at 30℃, and the initial dissolved oxygen (DO) at 10%. The rotation speed and air volume are adjusted according to the dissolved oxygen. During the fermentation process, glucose is fed in at a concentration of 800 g / L and the glucose concentration is controlled to be below 5 g / L. The aforementioned trace element stock solution comprises 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 aforementioned vitamin stock solution comprises 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 para-aminobenzoic acid.

[0134] The conditions for detecting perillaldehyde yield at different time points during fermentation using gas chromatography were as follows: Agilent HP-530m×0.32mm, 0.25μm column; 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 2mL / min; and injection volume 1μL.

[0135] The above embodiments of the present invention achieve the following technical effects: The present invention utilizes a self-selected wild-type lipolytic yeast strain to construct an effective gene editing system. By optimizing the expression intensity of key genes in the perillaldehyde synthesis pathway and enhancing precursor supply, combined with gene screening, mutation, and protein truncation, the perillaldehyde yield of the strain is increased. A yield of 20 g / L can be achieved in a 5L fermenter, which is the highest reported yield to date. The strain of the present invention not only exceeds the highest reported yield but also shortens the production cycle by half compared to previous reports. Furthermore, the entire fermentation process requires no addition of any organic reagents or precursors, demonstrating significant economic value and promising industrialization prospects.

[0136] This invention integrates relevant genes into the genome of *Yerobacterium lipolytica* strain Yl590, constructing a stable, high-efficiency de novo synthesis strain for perillaldehyde. The entire fermentation process requires no added organic reagents or intermediate substances, significantly reducing production costs and simplifying subsequent separation and purification steps. The engineered *Yerobacterium lipolytica* strain provided by this invention exhibits high production intensity and a simple fermentation process, demonstrating promising application prospects and laying the foundation for the microbial fermentation production of perillaldehyde.

[0137] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection 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 were selected from Yersinia 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 the TP1132 gene derived from Salvia sclarea.

3. The genetically engineered bacteria 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 bacteria according to claim 3, characterized in that, The genetically engineered bacteria also include: the gene MvaS derived from Enterococcus faecalis and / or the 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 from Enterococcus faecalis has the amino acid sequence shown in SEQ ID NO: 5; the protein encoded by the gene MvaE from Enterococcus faecalis has the 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 the tHMG1 gene derived from Yersinia lipolytica.

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

7.

8. The genetically engineered bacteria 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 from the thermophilic cyanobacterium Synechococcus sp. has the 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 also include any one or more of the following genes: ERG12 from Yersinia lipolytica, ERG20 from Yersinia lipolytica, ERG8 from Yersinia lipolytica, MVD1 from Yersinia lipolytica, or IDI1 from Yersinia lipolytica.

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

14.

14. A method for preparing genetically engineered bacteria, characterized in that, The preparation method includes: knocking in a gene into a starting engineered bacterium to obtain the genetically engineered bacterium; The knock-in gene includes at least the CcLPPS gene from Cistus creticus, and the genetically engineered bacteria is selected from Yersinia lipophila. 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 knock-in gene also includes the TP1132 gene derived 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 knock-in genes also include: the MvaS gene from Enterococcus faecalis and / or the MvaE gene from Enterococcus faecalis.

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

6.

19. The preparation method according to claim 18, characterized in that, The knock-in gene also includes the tHMG1 gene derived from Yersinia lipolytica.

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

7.

21. The preparation method according to claim 20, characterized in that, The knock-in gene also includes the SsCrtE gene from the thermophilic cyanobacterium Synechococcus sp.

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

8.

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

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

13.

25. The preparation method according to claim 24, characterized in that, The knock-in gene also includes the 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 the amino acid sequence shown in SEQ ID NO:

14.

27. A method for synthesizing perillyl alcohol, characterized in that, The method comprises: fermenting the genetically engineered bacteria prepared by the preparation method of any one of claims 1-13 or any one of claims 14-26 in a fermentation medium to synthesize the perillol.

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 includes: inoculating the genetically engineered bacteria cultured overnight into the fermentation medium for shake-flask fermentation for expansion culture to obtain the perillaldehyde.

30. The method according to claim 28, characterized in that, The fermentation process in the fermenter includes: inoculating the genetically engineered bacteria that have been cultured overnight into the fermenter for expansion culture to obtain the perillaldehyde.

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