A gene, recombinant vector, engineered bacteria and their applications

Abstract

This application discloses a gene, a recombinant vector, an engineered bacterium, and their applications. A limonene synthase (LS) gene is provided, wherein the limonene synthase (LS) gene is selected from the following DNA fragments: a1) or a2); a1) comprises a DNA fragment whose coding sequence is the DNA fragment represented by SEQ ID NO. 1; and a2) comprises a DNA fragment that is 90% or greater identical to the DNA fragment defined in a1) and encodes a protein having the same function. The limonene synthase (LS) gene is optimized and can be successfully expressed in host cells.

Description

Technical Field

[0001] The present application relates to a gene, a recombinant vector, an engineered bacterium and their applications, and belongs to the field of genetic engineering. Background Art

[0002] Limonene is a natural, functional monoterpene. It is ubiquitous in plants and is a major component of certain plant flavors, resins, and pigments. Furthermore, limonene is a precursor to the biosynthesis of linalool, carveol, perillyl alcohol, and menthol. Due to their pleasant aroma, unique biological activity, and favorable physical and chemical properties, limonene and its derivatives are widely used in high-value-added fine chemical industries, including food, beverages, health supplements, pharmaceuticals, cosmetics, biomaterials, and biofuels.

[0003] There are two main methods for producing limonene: plant extraction and chemical synthesis. Limonene isolated from plant sources has low abundance or yield, and this method has low extraction efficiency, harsh reaction conditions, and expensive equipment, making it economically unfeasible. Due to limitations in equipment and raw materials, chemical synthesis of limonene is not only inefficient, costly, and difficult to obtain a single enantiomer, but also poses environmental problems due to energy consumption. Microbial production of limonene has inherent advantages. Currently, the most important issue is overcoming the toxicity of limonene to microorganisms in the environment. With the rapid development of biotechnology, metabolic engineering of microorganisms to produce limonene has become a potential alternative.

[0004] Previous studies have mainly focused on the transformation of two conventional strains of Escherichia coli and Saccharomyces cerevisiae and the unconventional strain Yarrowia lipolytica, but the yields were not very high. For example, in 2019, the Dudley research group overexpressed the limonene biosynthesis pathway in Escherichia coli, lysed the cells until the pathway enzymes were obtained, used glucose as a substrate for cell-free biosynthesis of limonene, and adjusted the cofactor levels, ultimately obtaining 90.2 mg / L of limonene (Dudley QM, Nash CJ, Jewett MC. Cell-free biosynthesis of limonene using enzyme-enriched Escherichia colilysates. Synth Biol, 2019, 4(1): ysz003.); the Rolf research group introduced the limonene synthase and mevalonate pathway from spearmint into Escherichia coli, and used diisononyl phthalate as an in situ extractant for fed-batch fermentation, obtaining 7.3 g / L orgThe Hu research group introduced d-limonene synthase from lemon and overexpressed the endogenous geranyl diphosphate synthase gene (ERG20) and its variant ERG20 into Saccharomyces cerevisiae. F96W-N127WBy increasing the metabolic flux to limonene and optimizing the fermentation conditions, 23.7 mg / L d-limonene was obtained (Hu Z, Lin L, Li H, et al. Engineering Saccharomyces cerevisiae for production of the valuable monoterpene d-limonene during Chinese Baijiu fermentation. J Ino Microbiol Biot, 2020, 47(1).); the Dusseaux research group located the mevalonate pathway and the limonene synthesis pathway on the peroxisome, and used the partitioning effect of the organelle to isolate the biosynthesis of limonene, so that more metabolic flux flowed to the target product. The constructed platform strain can also effectively synthesize other terpenoids (such as geraniol, sabinene, monoindole alkaloids, etc.), and finally obtained 2.58 g / L limonene by batch fermentation (Dusseaux S, Wajn WT, Liu Y, et al. Transforming yeast peroxisomes into microfactories for the efficient production of high-value isoprenoids. Proc Natl Acad Sci USA, 2020, 117(50): 31789-31799.). Cheng's research group introduced the limonene synthesis pathway into Yarrowia lipolytica and optimized the limonene synthesis pathway, mevalonate pathway and culture medium. The resulting high-yield strain was subjected to batch fed-batch fermentation to produce 165.3 mg / L of limonene (Cheng BQ, Wei LJ, Lv YB, et al. Elevating Limonene Production in Oleaginous Yeast Yarrowia lipolytica via Genetic Engineering of Limonene Biosynthesis Pathway and Optimization of Medium Composition. Biotechnology Bioproc E, 2019, 24(3): 500-506.). Although Escherichia coli has a high production of limonene, the plasmid in E. coli exists in a free form, resulting in poor robustness of the strain and making it unsuitable for industrial use; the gene can be integrated into the yeast genome to improve its stability, but the ability of the yeast strain modified in this way to biosynthesize limonene is still relatively low, which limits the industrial production of limonene. Currently, there is an urgent need for genetically engineered bacteria with high limonene production. Summary of the Invention

[0005] According to one aspect of the present application, a limonene synthase LS gene is provided. The limonene synthase LS gene is an optimized limonene synthase LS gene that can be successfully expressed in a host cell.

[0006] A limonene synthase LS gene, wherein the limonene synthase LS gene is selected from the DNA fragment shown in a1) or a2) below;

[0007] a1) the coding sequence of the coding strand is the DNA fragment shown in SEQ ID NO.1;

[0008] a2) A DNA fragment that is 90% or more identical to the DNA fragment defined in a1) and encodes a protein having the same function.

[0009] Optionally, a1) is:

[0010] a1) The sequence is the DNA fragment shown in SEQ ID NO.1.

[0011] According to one aspect of the present application, a nerolyl pyrophosphate synthase NPPS gene is provided, wherein the nerolyl pyrophosphate synthase NPPS gene is selected from the DNA fragment shown in the following A1) or A2);

[0012] A1) The coding sequence of the coding strand is the DNA fragment shown in SEQ ID NO. 2;

[0013] A2) A DNA fragment that is 90% or more identical to the DNA fragment defined in A1) and encodes a protein having the same function.

[0014] Optionally, said A1) is:

[0015] A1) The sequence is the DNA fragment shown in SEQ ID NO.2.

[0016] According to one aspect of the present application, a recombinant vector I is provided, wherein the recombinant vector I is an integrative backbone vector I into which a fragment I is inserted;

[0017] The fragment I contains target gene module I;

[0018] The nucleotide sequence of the target gene module I contains the nucleotide sequence of the limonene synthase LS gene described in any one of the above items and the nucleotide sequence of the nerolyl pyrophosphate synthase NPPS gene described in any one of the above items.

[0019] Optionally, along the expression direction, the nucleotide sequence of the target gene module I sequentially contains the nucleotide sequence of the limonene synthase LS gene and the nucleotide sequence of the nerol pyrophosphate synthase NPPS gene; the nucleotide sequence of the limonene synthase LS gene and the nucleotide sequence of the nerol pyrophosphate synthase NPPS gene are connected by a connecting peptide gene;

[0020] Preferably, the nucleotide sequence of the target gene module I further contains the nucleotide sequence of the hydroxymethylglutaryl-CoA reductase HMGR gene with a truncated N-terminal transmembrane region;

[0021] Optionally, along the expression direction, the nucleotide sequence of the target gene module I sequentially contains the nucleotide sequence of the limonene synthase LS gene, the nucleotide sequence of the nerol pyrophosphate synthase NPPS gene, and the nucleotide sequence of the N-truncated hydroxymethylglutaryl-CoA reductase HMGR gene;

[0022] The nucleotide sequence of the limonene synthase LS gene and the nucleotide sequence of the nerol pyrophosphate synthase NPPS gene, and the nucleotide sequence of the nerol pyrophosphate synthase NPPS gene and the nucleotide sequence of the N-truncated hydroxymethylglutaryl-CoA reductase HMGR gene are respectively connected by connecting peptide genes;

[0023] Optionally, the nucleotide sequence of the target gene module I is as shown in SEQ ID No. 3 or as shown in bases 1 to 4128 of SEQ ID No. 4;

[0024] Optionally, the fragment I further contains a nucleotide sequence of a promoter I upstream of the nucleotide sequence of the target gene module I;

[0025] Optionally, the promoter 1 is selected from any one of ARA, GPD, TEF2, and XYL;

[0026] Optionally, the insertion is to replace a smaller fragment between the ECoRV recognition site and the SpeI recognition site of the integrative vector I with the fragment I, while keeping other nucleotide sequences of the integrative vector I unchanged;

[0027] Optionally, the integrative backbone vector I is an Agrobacterium-mediated binary expression vector;

[0028] Optionally, the integrative backbone vector I is selected from any one of Gateway Vector pB4GWnY and Takara pRI.

[0029] According to one aspect of the present application, an engineered bacterium is provided, wherein the engineered bacterium is obtained by transforming a recombinant vector A into a host cell;

[0030] The recombinant vector A includes any of the recombinant vectors I described above.

[0031] Optionally, the recombinant vector A further comprises a recombinant vector II;

[0032] The recombinant vector II is an insertion fragment II into the integration backbone vector II;

[0033] The fragment II contains the target gene module II;

[0034] The nucleotide sequence of the target gene module II contains the nucleotide sequence of at least one of the MVA pathway genes;

[0035] Optionally, the MVA pathway gene includes at least one of the mevalonate kinase MmMK gene of Methanosarcina mazei, the mevalonate synthase EfMvaS gene of Enterococcus faecalis, and the acetoacetyl-CoA thiolase / 3-hydroxy-3-methylglutaryl-CoA reductase EfMvaE gene of Enterococcus faecalis;

[0036] Optionally, along the expression direction, the nucleotide sequence of the target gene module II sequentially contains the nucleotide sequence of the mevalonate kinase MmMK gene of Methanosarcina mazei, the nucleotide sequence of the mevalonate synthase EfMvaS gene of Enterococcus faecalis, and the nucleotide sequence of the acetoacetyl-CoA thiolase / 3-hydroxy-3-methylglutaryl-CoA reductase EfMvaE gene of Enterococcus faecalis;

[0037] The MVA pathway genes include a mevalonate kinase MmMK gene nucleotide sequence of Methanosarcina mazei and a mevalonate synthase EfMvaS gene nucleotide sequence of Enterococcus faecalis, and a mevalonate synthase EfMvaS gene nucleotide sequence of Enterococcus faecalis and an acetoacetyl-CoA thiolase / 3-hydroxy-3-methylglutaryl-CoA reductase EfMvaE gene nucleotide sequence of Enterococcus faecalis, respectively connected by a connecting peptide;

[0038] Optionally, the nucleotide sequence of the target gene module II is shown in SEQ ID No. 5 in the sequence listing;

[0039] Optionally, the fragment II further contains a nucleotide sequence of a promoter II upstream of the nucleotide sequence of the target gene module II;

[0040] Preferably, the promoter II is selected from any one of ARA, GPD, TEF2, and XYL;

[0041] Optionally, the insertion is to replace a smaller fragment between the ECoRV recognition site and the SpeI recognition site of the integrative backbone vector II with the fragment II, while keeping other nucleotide sequences of the integrative backbone vector II unchanged;

[0042] Optionally, the integrative backbone vector II is an Agrobacterium-mediated binary expression vector, and the transformation is carried out by Agrobacterium-mediated transformation;

[0043] Optionally, the integrative backbone vector II is selected from any one of Gateway Vector pB4GWnY and Takara pRI.

[0044] Optionally, the host cell comprises at least one fungus of the genus Rhodotorula;

[0045] Optionally, the Rhodotorula fungus includes any one of Rhodotorula toruloides, Rhodotorula glutinis, Rhodotorula acheni, and Rhodotorula graminis;

[0046] Optionally, the Rhodotorula fungus is a Rhodotorula fungus in which the carotenoid synthesis pathway is knocked out.

[0047] Optionally, the hydroxymethylglutaryl-CoA reductase HMGR with a truncated N-terminal transmembrane region refers to the hydroxymethylglutaryl-CoA reductase HMGR having amino acids 1 to 798 truncated.

[0048] Optionally, the nucleotide sequence of the hydroxymethylglutaryl-CoA reductase HMGR with a truncated N-terminal transmembrane region is shown as bases 2578 to 4128 of SEQ ID No. 4.

[0049] According to one aspect of the present application, a method for producing limonene is provided, comprising the following steps: culturing any of the above-mentioned engineered bacteria to obtain the limonene.

[0050] Optionally, the method comprises the following steps:

[0051] fermenting the engineered bacteria in a culture medium I to obtain a fermentation liquid I, adding dodecane I, and continuing the fermentation to obtain the limonene;

[0052] Optionally, the conditions of the fermentation I include:

[0053] Temperature is 15-35℃;

[0054] The speed is 100-200 rpm;

[0055] The duration is 8 to 144 hours;

[0056] Preferably, the conditions for continued fermentation are:

[0057] Temperature is 15-35℃;

[0058] The speed is 100-200 rpm;

[0059] The duration is 4 to 7 days;

[0060] Optionally, the volume ratio of the dodecane I to the culture medium I is 1:2-5.

[0061] Optionally, the volume ratio of the dodecane I to the culture medium I is 1:3-5.

[0062] Optionally, the fermentation liquid obtained by further fermentation is subjected to secondary extraction with n-hexane to obtain the limonene;

[0063] Optionally, the method comprises the following steps:

[0064] adding the seed liquid and dodecane II to culture medium II, fermenting II to obtain the limonene;

[0065] Optionally, the volume ratio of the seed solution to the culture medium II is 1-15:85-99;

[0066] The volume ratio of the dodecane II to the culture medium II is 1:2-5.

[0067] Optionally, the volume ratio of the dodecane II to the culture medium II is 1:3-5.

[0068] The conditions of the fermentation II include:

[0069] pH 3.0-6.5;

[0070] Ventilation volume is 0.5 to 3 L / min;

[0071] The stirring speed is 50-350 rpm;

[0072] Glucose concentration is higher than 2g / L;

[0073] Optionally, during fermentation II, when the glucose concentration drops below 2 g / L, the following feeding is performed:

[0074] Supplement glucose 20-50g / L, yeast extract powder 10-20g / L, and ammonium sulfate 2-10g / L.

[0075] Optionally, during fermentation II, when the glucose concentration drops below 2 g / L, the following feeding is performed:

[0076] Supplement 20-30g / L of glucose, 15-20g / L of yeast extract powder, and 5-10g / L of ammonium sulfate.

[0077] Optionally, during fermentation II, when the glucose concentration drops below 2 g / L, the following feeding is performed:

[0078] Supplement 20-25g / L of glucose, 18-20g / L of yeast extract powder, and 8-10g / L of ammonium sulfate.

[0079] After 5 to 10 feedings, fermentation II is terminated;

[0080] Optionally, the seed solution is obtained through the following steps:

[0081] A single colony is cultured in culture medium A to obtain a first seed solution, and the first seed solution is cultured in culture medium B to obtain the seed solution;

[0082] Optionally, the volume ratio of the first seed solution to the culture medium B is 1-10:50-100;

[0083] Optionally, the conditions for culturing A and culturing B independently include:

[0084] Temperature is 15-35℃;

[0085] The speed is 100-200 rpm;

[0086] The time is 12 to 48 hours.

[0087] As an embodiment, the present application provides a genetically engineered bacterium of red yeast that produces limonene, which is constructed by connecting the optimized genes LS and NPPS to a vector carrying an antibiotic to form a recombinant vector, and then transforming the red yeast (Rhodotorula / Rhodosporidium toruloides) in which carotenoids are knocked out. The nucleotide sequence of the optimized gene limonene synthase LS is shown in the sequence listing SEQ ID No. 1; the nucleotide sequence of the optimized nerol pyrophosphate synthase gene NPPS is shown in the sequence listing SEQ ID No. 2.

[0088] Optionally, the recombinant vector is a plasmid Xyl-LN-Ntc, wherein XYL is a promoter, LN is a gene module formed by connecting limonene synthase LS and nerol pyrophosphate synthase gene NPPS using a 2A peptide, and the sequence of the target gene module of the plasmid Xyl-LN-Ntc is shown in the sequence listing as SEQ ID No. 3; the carotenoid-knockout red yeast is red yeast np11 prepared according to the preparation method in Sun W, Yang X, Wang X, et al. Homologous gene targeting of acarotenoids biosynthetic gene in Rhodosporidium toruloides by Agrobacterium-mediated transformation. Biotechnol Lett, 2017, 39: 1001-1007.

[0089] Alternatively, it is constructed by transforming XYL-20190821-Ble into carotenoid-knockout red yeast (R. toruloides). The plasmid XYL-20190821-Ble, XYL is a promoter, 20190821 is a gene module formed by connecting limonene synthase LS, nerolyl pyrophosphate synthase gene NPPS and hydroxymethylglutaryl coenzyme A reductase HMGR using a 2A peptide, and the sequence obtained by fusion of the target gene module of plasmid XYL-20190821-Ble and the his tag is shown in the sequence listing SEQ ID No. 4.

[0090] Optionally, it is constructed by transforming transformant A with XYL-KSE-Ntc, wherein the target gene module of the plasmid XYL-KSE-Ntc is shown in SEQ ID No. 5 in the sequence listing;

[0091] Optionally, the transformant A is constructed by transforming the plasmid XYL-20190821-Ble into carotenoid-knockout red yeast (R. toruloides).

[0092] As another embodiment, the present application provides a method for producing limonene, which comprises the following steps: culturing the limonene-producing carotenoid knockout red yeast genetically engineered bacteria described in any of the above items, adding dodecane for two-phase in situ extraction and fermentation to obtain a fermentation broth and a dodecane phase, and after fermentation, extracting and centrifuging with n-hexane to obtain an organic phase.

[0093] Optionally, the ratio of the volume of dodecane to the volume of the fermentation broth is 1:2-5, preferably 1:3, and the volume ratio is the volume ratio of dodecane to the fermentation broth before adding dodecane.

[0094] As another embodiment, the present application provides the use of any of the above-mentioned genetically engineered limonene-producing red yeast bacteria in the preparation of limonene.

[0095] As another embodiment, the present application provides a method for preparing a genetically engineered red yeast strain that produces limonene, comprising the following steps:

[0096] (1) Constructing a recombinant vector containing the optimized gene LS and the optimized gene NPPS, wherein the nucleotide sequence of the optimized gene LS is shown in the sequence listing SEQ ID No. 1; the nucleotide sequence of the optimized gene NPPS is shown in the sequence listing SEQ ID No. 2.

[0097] (2) The red yeast np11 with the carotenoid biosynthetic pathway knocked out was prepared according to the preparation method in Sun W, Yang X, Wang X, et al. Homologous gene targeting of acarotenoids biosynthetic gene in Rhodosporidium toruloides by Agrobacterium-mediated transformation. Biotechnol Lett, 2017, 39: 1001-1007.

[0098] (3) The Xyl-LN-Ntc recombinant vector prepared in step (1) is transformed into the carotenoid-knockout red yeast (R. toruloides) prepared in step (2) to obtain a limonene-producing transformant.

[0099] Optionally, it further includes the following steps:

[0100] (4) Constructing plasmid XYL-20190821-Ble, transforming the red yeast np11 with the carotenoid synthesis pathway knocked out obtained in step (2) with the plasmid XYL-20190821-Ble, the sequence obtained by fusion of the target gene nucleotide sequence of the plasmid XYL-20190821-Ble and the his tag is shown in SEQ ID No. 4 in the sequence listing.

[0101] (5) Constructing plasmid XYL-KSE-Ntc, and transforming the transformant obtained in step (4) with the plasmid XYL-KSE-Ntc. The nucleotide sequence of the target gene of the plasmid XYL-KSE-Ntc is shown in SEQ ID No. 5 in the sequence listing.

[0102] As another embodiment, the present application provides a recombinant vector for preparing a genetically engineered strain of red yeast producing limonene as described in any of the above items, which contains an optimized gene LS and an optimized gene NPPS, the nucleotide sequence of the optimized gene LS is shown in the sequence listing SEQ ID No. 1; the nucleotide sequence of the optimized gene NPPS is shown in the sequence listing SEQ ID No. 2; the recombinant vector is a plasmid Xyl-LN-Ntc obtained by introducing the optimized gene LS and the optimized gene NPPS into the plasmid pZPK, and the sequence of the target gene module of the plasmid Xyl-LN-Ntc is shown in the list SEQ ID No. 3.

[0103] As another embodiment, the present application provides a genetically engineered limonene-producing red yeast and its application. This genetically engineered bacterium is constructed by ligating codon-optimized limonene synthase (LS) and nerolyl pyrophosphate synthase (NPPS) genes to vectors carrying antibiotics to form recombinant vectors, which are then transformed into carotenoid-deleted red yeast (Rhodotorula / Rhodosporidium toruloides). The nucleotide sequences of the optimized LS and NPPS genes are shown in SEQ ID Nos. 1 and 2, respectively. Based on this, the engineered strain was engineered to simultaneously overexpress the endogenous N-truncated hydroxymethylglutaryl-CoA reductase (HMGR) and introduce heterologous key enzymes involved in the mevalonate (MVA) pathway (abbreviated as KSE: mevalonate kinase (MmMK) from Methanosarcina mazei; mevalonate synthase (EfMvaS) from Enterococcus faecalis; and acetoacetyl-CoA thiolase / 3-hydroxy-3-methylglutaryl-CoA reductase (EfMvaE) from Enterococcus faecalis). A two-phase fermentation was performed on the engineered strain to eliminate limonene toxicity, enhance its robustness, and further increase limonene production. In situ extraction of limonene was performed by adding dodecane, resulting in an optimized yield of 5 g / L. This engineered strain is suitable for large-scale commercial production and holds great promise.

[0104] The beneficial effects of this application include:

[0105] (1) The limonene synthase LS gene / neryl pyrophosphate synthase NPPS gene provided in this application is an optimized gene that can be heterologously expressed in host cells to achieve the purpose of producing limonene.

[0106] (2) The recombinant vector provided in this application can integrate the genes of enzymes related to limonene synthesis into host cells, thereby improving the stability and yield of limonene-producing engineered bacteria.

[0107] (3) The engineered bacteria provided in this application have genes for enzymes related to limonene synthesis integrated into their genomes, which not only have high yields but also have good robustness and are suitable for industrial use.

[0108] (4) The method for producing limonene provided in this application has a simple process, high yield, and is suitable for scale-up production. BRIEF DESCRIPTION OF THE DRAWINGS

[0109] Figure 1 The present invention discloses the metabolic pathway for limonene synthesis by red yeast after the limonene synthesis gene is introduced into the present invention.

[0110] Figure 2 The plasmid structure diagram of plasmid Xyl-LN-Ntc containing the limonene synthesis genes LS and NPPS.

[0111] Figure 3 The plasmid structure diagram of plasmid XYL-20190821-Ble containing the overexpression HMGR gene.

[0112] Figure 4 Figure 2 is a plasmid structure diagram of the plasmid XYL-KSE-Ntc containing the overexpression of heterologous MVA pathway genes.

[0113] Figure 5 For strains Xyl-LN-Ntc-np11-9, pZPK-20190821-Ntc-ΔCrt-np11-16 and pZPK-201908212-KSE-

[0114] The results of Ntc-ΔCrt-np11-6 fermentation production of limonene. DETAILED DESCRIPTION

[0115] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0116] Among them, the starting strain red yeast (Rhodosporidium toruloides R. toruloides) in the embodiment was obtained from the Dalian Institute of Chemical Physics, which is a conventional red yeast np11 in the field and has been published in Fems Yeast Research (Lin X, Wang Y, Zhang S, et al. Functional integration of multiple genes into the genome of the oleaginous yeast Rhodosporidium toruloides. Fems Yeast Research, 2014 (4): 547-555.).

[0117] Although the strain np11 is used as the starting strain in the examples of the present invention, conventional red yeast (R. toruloides) can be used as the starting strain for transformation to carry out the experiments in the examples.

[0118] In addition, Rhodotorula glutinis, Rhodotorula acheniorum and Rhodotorula graminis of the genus Rhodotorula can also be used as starting strains.

[0119] The red yeast np11 with the carotenoid biosynthesis pathway knocked out was prepared according to the preparation method in Sun W, Yang X, Wang X, et al. Homologous gene targeting of a carotenoids biosynthetic gene in Rhodosporidium toruloides by Agrobacterium-mediated transformation. Biotechnol Lett, 2017, 39: 1001-1007.

[0120] The recombinant vector and engineered bacteria of the present application can be obtained by repeating the method disclosed in the present application.

[0121] The nucleotide sequence of the optimized gene LS described in the present application is shown in the sequence listing SEQ ID No. 1; the nucleotide sequence of the optimized gene NPPS is shown in the sequence listing SEQ ID No. 2.

[0122] ECoRV endonuclease was purchased from Takara;

[0123] SpeI endonuclease was purchased from Takara;

[0124] The backbone vector used in the construction of the recombinant vector in this application is Gateway Vector pB4GWnY.

[0125] In this application, the inoculation ratio refers to the volume of the bacterial seed liquid: the sum of the volumes of the fermentation medium and the bacterial seed liquid. For example, an inoculation ratio of 15% means that the volume of the bacterial seed liquid is 15, the volume of the fermentation medium is 85, and the sum of the volumes of the fermentation medium and the bacterial seed liquid is 100.

[0126] Figure 1The present invention discloses a metabolic pathway for the synthesis of limonene by red yeast after the introduction of the limonene synthesis gene. The pathway comprises the following: glucose, acetyl-coA, acetyl-coenzyme A, HMG-CoA, hydroxymethylglutaryl-coenzyme A, mevalonate, mevalonate-5P, 5-phosphomevalonate, IPP, isopentenyl pyrophosphate, DMAPP, dimethyl propylene pyrophosphate, NPP, neryl pyrophosphate, GPP, geranyl pyrophosphate, FPP, farnesyl pyrophosphate, carotenoids, limonene, EfMvaE, acetoacetyl-CoA thiolase / 3-hydroxy-3-methylglutaryl-CoA reductase from Enterococcus faecalis, EfMvaS, mevalonate synthase from Enterococcus faecalis, MmMK, mevalonate kinase from Methanosarcina mazei, and ACCT, acetyl-CoA. C-acyltransferase, HMGS is hydroxymethylglutaryl-CoA synthase, HMG-R is hydroxymethylglutaryl-CoA reductase, MK is mevalonate kinase, IDI isopentenyl diphosphate delta isomerase, NPPS is neryl diphosphate synthase, FPPS is farnesyl diphosphate synthase, CrtI is carotenoid desaturase, and LS1 is limonene synthase 1.

[0127] The technical problem to be solved by the present invention is to overcome the deficiency of the prior art in lacking a genetically engineered bacterium that can produce high limonene production, and to provide a genetically engineered red yeast bacterium that can produce limonene and its application.

[0128] After creative work, the inventors discovered that by introducing genes related to limonene synthesis into red yeast, they optimized two genes related to limonene synthesis: the LS gene, which is derived from lemon and can convert neryl diphosphate into limonene, and the NPPS gene, which is derived from tomato and can condense isopentenyl pyrophosphate and dimethylallyl pyrophosphate into neryl diphosphate. These two optimized genes were then transformed into red yeast with the carotenoid pathway knocked out, enabling the yeast to produce limonene. The inventors further discovered that, based on the optimization of these two genes, overexpressing the gene HMGR, which reduces hydroxymethylglutaryl-CoA (HMG-CoA) to mevalonate, can increase limonene production (see [1]). Figure 1The inventors also discovered that, based on the optimization of two genes, the introduction of heterologous MVA pathway-related genes (abbreviated as KSE)—the bifunctional enzyme acetoacetyl-CoA thiolase / 3-hydroxy-3-methylglutaryl-CoA reductase (EfMvaE) from Enterococcus faecalis, which can reduce acetyl-CoA to acetoacetyl-CoA and HMG-CoA to mevalonate; the mevalonate synthase (EfMvaS) from Enterococcus faecalis, which converts acetoacetyl-CoA to HMG-CoA; and the mevalonate kinase (MmMK) from Methanosarcina mazei, which converts mevalonate to phosphomevalonate—can significantly increase limonene production. Furthermore, the inventors discovered that adding dodecane as a blanket during fermentation can better promote the synthesis of limonene in genetically engineered strains.

[0129] The technical solutions provided by the present invention are as follows:

[0130] One of the technical solutions of the present invention is: a genetically engineered red yeast that produces limonene, which is constructed by transforming a recombinant vector containing an optimized gene LS and an optimized gene NPPS into a red yeast (R. toruloides) in which the carotenoid pathway has been knocked out, wherein the nucleotide sequence of the optimized gene LS is shown in the sequence listing as SEQ ID No. 1; the nucleotide sequence of the optimized gene NPPS is shown in the sequence listing as SEQ ID No. 2.

[0131] In the present invention, the wild-type red yeast (R. toruloides) is a conventional red yeast np11 in the art, and the red yeast np11 with the carotenoid synthesis pathway knocked out was prepared according to the preparation method in Sun W, Yang X, Wang X, et al. Homologous gene targeting of a carotenoids biosynthetic gene in Rhodosporidium toruloides by Agrobacterium-mediated transformation. Biotechnol Lett, 2017, 39: 1001-1007, and was obtained from the Dalian Institute of Chemical Physics.

[0132] The optimized gene LS is obtained by optimizing the nucleotide sequence of the LS gene from lemon; the optimized gene NPPS is obtained by optimizing the nucleotide sequence of the NPPS gene from tomato.

[0133] In the present invention, preferably, the recombinant vector contains restriction sites for ECoRV and SpeI. Preferably, the recombinant vector further contains a promoter and / or terminator sequence. More preferably, the recombinant vector further contains the strong promoter XYL.

[0134] The backbone vector used in the recombinant vector of the present invention is a conventional backbone vector in the art, which can transform red yeast and contains the above-mentioned optimized gene LS and gene NPPS.

[0135] The recombinant vector is a recombinant plasmid Xyl-LN-Ntc obtained by introducing the above-mentioned optimized gene LS and optimized gene NPPS into the backbone vector Gateway Vector pB4GWnY. The sequence of the target gene module of the plasmid Xyl-LN-Ntc is shown in the sequence listing SEQ ID No. 3. The Ntc is a nourseothricin resistance marker used for screening transformants.

[0136] In the present invention, the plasmid Xyl-LN-Ntc was transformed into the red yeast (R. toruloides) to obtain a genetically engineered strain capable of producing limonene, which was named Xyl-LN-Ntc-np11-9.

[0137] The second technical solution of the present invention is: a genetically engineered strain of red yeast that produces limonene. More preferably, the genetically engineered strain of red yeast that produces limonene is obtained by transforming the plasmid XYL-20190821-Ble into the red yeast (R. toruloides) with the above-mentioned knockout of the carotenoid biosynthesis pathway to obtain a genetically engineered strain capable of producing limonene, which is named pZPK-20190821-Ntc-ΔCrt-np11-16. The sequence obtained by fusion of the target gene module of the plasmid XYL-20190821-Ble and the his tag is shown in the sequence listing as SEQ ID No. 4. The Ble is a bleomycin resistance marker used for screening transformants.

[0138] The plasmid XYL-20190821-Ble contains the endogenous gene HMGR from red yeast (accession number in NCBI is XP_016270872.1, July 12, 2012).

[0139] In the present invention, the red yeast genetic engineering strain pZPK-20190821-Ntc-ΔCrt-np11-16 constructed by transforming the above-mentioned plasmid XYL-20190821-Ble into the above-mentioned red yeast (R. toruloides) can produce more limonene.

[0140] A third technical solution of the present invention is a genetically engineered strain of red yeast that produces limonene. Preferably, the genetically engineered strain of red yeast that produces limonene is a strain transformed with the plasmid XYL-KSE-Ntc to generate a strain with increased limonene production, designated pZPK-201908212-KSE-Ntc-ΔCrt-np11-6. The sequence of the target gene module of the plasmid XYL-KSE-Ntc is shown in SEQ ID No. 5 in the sequence listing.

[0141] The plasmid XYL-KSE-Ntc contains mevalonate kinase (MmMK) from Methanosarcina mazei, mevalonate synthase (EfMvaS) from Enterococcus faecalis, and acetyl-CoA thiolase / 3-hydroxy-3-methylglutaryl-CoA reductase (EfMvaE) from Enterococcus faecalis.

[0142] In the present invention, the red yeast genetically engineered strain pZPK-201908212-KSE-Ntc-ΔCrt-np11-6 constructed by transforming the above-mentioned plasmid XYL-KSE-Ntc can produce more limonene.

[0143] A fourth technical solution of the present invention is: a method for producing limonene, comprising the following steps: subjecting the above-mentioned genetically engineered red yeast strain producing limonene to two-phase fermentation.

[0144] In the present invention, the culture medium is a culture medium commonly used in the art, preferably a seed culture medium and a fermentation culture medium is a YPD culture medium. The YPD culture medium is composed of 20 g / L glucose, 20 g / L peptone and 10 g / L yeast extract.

[0145] In the present invention, due to the high volatility and significant toxicity of limonene to the basement cells, dodecane is added to the culture medium during fermentation for in situ extraction. Extracting the limonene released by the cells into the culture medium into the organic phase not only mitigates its toxicity to the cells but also reduces limonene volatilization. This increases cell robustness and yields more limonene. Preferably, dodecane is added for 8 hours of fermentation. The concentration of dodecane added is 1:2-5, preferably 1:3, where the ratio is the ratio of added dodecane to culture medium.

[0146] The fifth technical solution of the present invention is: use of the above-mentioned genetically engineered limonene-producing red yeast in the preparation of limonene.

[0147] Based on the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain various preferred embodiments of the present invention.

[0148] Unless otherwise specified, all reagents and raw materials used in the present invention are commercially available.

[0149] The present invention demonstrates a significant improvement in its effectiveness by introducing optimized nucleotide sequences of the lemon-derived LS gene and the tomato-derived NPPS gene into a Rhodotorula strain with a carotenoid pathway knockout. This then overexpresses the HMGR gene, resulting in a genetically engineered Rhodotorula strain that significantly increases limonene production. Furthermore, heterologous overexpression of MVA pathway genes further enhances limonene production. This limonene-producing Rhodotorula strain can achieve a limonene yield of 407.23 mg / L, exceeding the yield of a strain incorporating only the LS and NPPS genes by 78.3 times, and exceeding the yield of a strain overexpressing HMGR by 10.8 times.

[0150] By comparing fermentation methods, a fermentation method suitable for producing limonene using a genetically engineered red yeast strain was identified, further increasing limonene production. Furthermore, the method for producing limonene using the genetically engineered red yeast strain provided by the present invention is simple to operate, has good compatibility with fermentation equipment, and exhibits excellent strain stability, making it suitable for commercial production. The resulting limonene can be safely used in food, pharmaceuticals, cosmetics, and other fields, demonstrating its promising prospects.

[0151] Plasmids Xyl-LN-Ntc, XYL-20190821-Ble, and XYL-KSE-Ntc were prepared using the double enzyme digestion and ligation method.

[0152] Example 1 Construction of strain Xyl-LN-Ntc-np11-9

[0153] (1) The optimized sequences of two genes involved in limonene biosynthesis, LS1 and NPPS (whose nucleotide sequences are shown in the sequence listing SEQ ID NO. 1 and 2, respectively) were connected using the gene sequence of P2A to form a gene module LS-NPPS. The nucleotide sequence is shown in the sequence listing SEQ ID No. 3, wherein bases 1 to 1668 of SEQ ID No. 3 are LS, bases 1669 to 1734 are P2A, and bases 1735 to 2514 are NPPS.

[0154] The nucleotide sequence of SEQ ID No.3, promoter pPGK, Ntc resistance gene, terminator Tnos, promoter pXYL, terminator Thsp were cloned into the Takara In-fusion seamless cloning kit. Figure 2 The sequence shown was connected and inserted into the backbone vector Gateway Vector pB4GWnY to obtain plasmid Xyl-LN-Ntc. The plasmid structure is shown in Figure 2(Kan is included in the backbone vector.) Ntc is the bleomycin resistance gene, and the amino acid sequence of its encoded protein is GenBank: AAS47018.1; LB is the left arm of the integration vector pB4GWnY, and RB is its right arm, and its nucleic acid sequence is the sequence on the backbone vector; the promoters (pPGK, pXYL) and terminators (Tnos, Thsp) are reported sequences (Lin XP, Wang YN, Zhang SF, Zhu ZW, Zhou YJ, Yang F, Sun WY, Wang XY, Zhao ZK. Functional integration of multiple genes into the genome of the oleaginous yeast Rhodosporidium toruloides. FEMS Yeast Res. 2014, 14(6), 547–555.)

[0155] (2) The plasmid Xyl-LN-Ntc obtained in step (1) was integrated into the genome of red yeast (R. toruloides) with the carotenoid synthesis gene knocked out by Agrobacterium-mediated transformation (ATMT) (for specific operation methods, refer to Lin X, Gao N, Liu S, et al. Characterization of the carotenoid productions and profiles of three Rhodosporidium toruloides mutants from Agrobacterium tumefaciens-mediated transformation. Yeast, 2017, 34(8): 335-342.) to obtain the engineered strain Xyl-LN-Ntc-np11-9 capable of producing limonene.

[0156] Example 2 Construction of strain pZPK-20190821-Ntc-ΔCrt-np11-16

[0157] (1) The truncated HMGR gene from Rhodosporidium toruloides (NCBI accession number XP_016270872.1) was linked to the gene module LS1-NPPS via the gene sequence of T2A to obtain the gene module LS1-NPPS-HMGR. The nucleotide sequence is shown in bases 1 to 4128 of SEQ ID No. 4 in the sequence listing, wherein bases 1 to 1668 of SEQ ID No. 4 are LS, bases 1669 to 1734 are P2A, bases 1735 to 2514 are NPPS, bases 2515 to 2577 are T2A, bases 2578 to 4128 are HMGR, and bases 4129 to 4146 are His tag genes.

[0158] The nucleotide sequence of SEQ ID No.4, promoter pPGK, Ble resistance gene, terminator Tnos, promoter pXYL, terminator Thsp were cloned into the Takara In-fusion seamless cloning kit. Figure 3 The sequence shown was connected and inserted into the backbone vector Gateway Vector pB4GWnY to obtain plasmid XYL-20190821-Ble. The plasmid structure can be found in Figure 3 (Kan is included in the backbone vector.) Ble is the bleomycin resistance gene from Streptoalloteichus hindustanus (PDB: 1BYL_A).

[0159] The amino acid sequence of the Ble resistance protein is shown in SEQ ID No.6.

[0160] (2) The plasmid XYL-20190821-Ble obtained in step (1) was integrated into the red yeast np11 with the carotenoid synthesis gene knocked out by the ATMT method (for specific operation methods, refer to Lin X, Gao N, Liu S, et al. Characterization of the carotenoid productions and profiles of three Rhodosporidium toruloides mutants from Agrobacterium tumefaciens-mediated transformation. Yeast, 2017, 34(8): 335-342.), and the engineered strain pZPK-20190821-Ntc-ΔCrt-np11-16 capable of high production of limonene was obtained.

[0161] Example 3 Construction of strain pZPK-20190821-KSE-Ntc-ΔCrt-np11-6

[0162] (1) The three genes EfMvaE, EfMvaS and MmMK of the heterologous MVA pathway were connected using the gene sequence of T2A / P2A (specifically MmMK-P2A-EfMvaS-T2A-EfMvaE) to form the gene module KSE. The nucleotide sequence is shown in the sequence listing SEQ ID No. 5, wherein bases 1 to 903 of SEQ ID No. 5 are MmMK, bases 904 to 969 are P2A, bases 970 to 2118 are EfMvaS, bases 2119 to 2181 are T2A, and bases 2182 to 4590 are EfMvaE.

[0163] The nucleotide sequence of SEQ ID No.5, promoter pPGK, Ntc resistance gene, terminator Tnos, promoter pXYL, terminator Thsp were cloned into the Takara In-fusion seamless cloning kit. Figure 4 The sequence shown was connected to insert into the backbone vector Gateway Vector pB4GWnY to obtain plasmid XYL-KSE-Ntc. The plasmid structure is shown in Figure 4 (Kan comes with the skeleton carrier).

[0164] (2) The plasmid XYL-KSE-Ntc obtained in step (1) was integrated into the chromosome of the high-yield limonene engineering strain pZPK-20190821-Ntc-ΔCrt-np11-16 obtained in Example 2 by the ATMT method (for specific operation methods, refer to LinX, Gao N, Liu S, et al. Characterization of the carotenoid productions and profiles of three Rhodosporidium toruloides mutants from Agrobacteriumtumefaciens-mediated transformation. Yeast, 2017, 34(8): 335-342.), obtaining a higher-yield limonene engineering strain pZPK-20190821-KSE-Ntc-ΔCrt-np11-6.

[0165] Example 4 Determination of the yield of limonene produced by the strain

[0166] The strains red yeast np11, red yeast np11 with the carotenoid pathway knocked out (ΔCrt-np11), strain Xyl-LN-Ntc-np11-9 prepared in Example 1, strain pZPK-20190821-Ntc-ΔCrt-np11-16 prepared in Example 2, and strain pZPK-20190821-KSE-Ntc-ΔCrt-np11-6 prepared in Example 3 were fermented using 50 mL centrifuge tubes. Specifically, single colonies of the strains were inoculated into 5 mL of YPD medium (20 g / L glucose, 20 g / L peptone, and 10 g / L yeast extract, diluted to 1 L with water) and cultured at 28 ° C at 200 rpm / min. After fermentation for 8 h, dodecane was added at a volume ratio of 1:5 between dodecane and culture medium.

[0167] After 5 days of two-phase fermentation, 1 mL of n-hexane was added for secondary extraction. The organic layer was separated by centrifugation at 4°C and analyzed for limonene content using a Shimadzu GC-2014C column (60 m × 0.25 mm × 0.25 μm, Kromat, USA). Nitrogen was used as the carrier gas at a flow rate of 1.0 mL / min, and the injection volume was 1 μL. The procedure was as follows: 145°C for 27 min; the injector temperature was maintained at 240°C, and the detector temperature was 260°C; the split ratio was 20:1. A limonene standard curve was established and used to quantify limonene.

[0168] The test results are shown in Figure 5 and Table 1. The results in Table 1 show that the ability of strains pZPK-20190821-Ntc-ΔCrt-np11-16 and pZPK-20190821-KSE-Ntc-ΔCrt-np11-6 to produce limonene was greatly improved.

[0169] Table 1 Limonene production of different strains

[0170]

[0171] Example 5: High-density fermentation of engineered bacterial strains to produce limonene

[0172] The strain pZPK-20190821-KSE-Ntc-ΔCrt-np11-6 prepared in Example 3 was cultured in a 250 mL shake flask for seed liquid. Specifically, a single colony of the pZPK-20190821-KSE-Ntc-ΔCrt-np11-6 strain was inoculated into 50 mL YPD medium (20 g / L glucose, 20 g / L peptone and 10 g / L yeast extract, and water was added to 1 L), and cultured at 200 rpm / min at 28 ° C for 12 h. Then, it was inoculated into a new shake flask at a volume ratio of 10% to prepare secondary seeds. The culture conditions were the same as those for the primary seeds. After 12 hours of secondary seed culture, the mixture was inoculated into a 3L fermentor at a 15% inoculum ratio (i.e., a 15:85 volume ratio of secondary seed liquid to fermentation medium). The pH was controlled at 6.0, ventilation was 2 L / min, and stirring was at 350 rpm. Dodecane was added at a volume ratio of 1:2 to culture medium for in-situ extractive fermentation. Glucose concentration and limonene production were monitored every 12 hours. Limonene was detected using the same method as described in Example 4, with DNS (dinitrosalicylic acid) detection of glucose. When glucose fell below 2 g / L, 20 g / L of glucose, 10 g / L of yeast extract, and 2 g / L of ammonium sulfate were added. After five feedings, limonene production reached 3 g / L.

[0173] Example 6 Application of the technical method and the synthesized gene in other red yeasts

[0174] Rhodotorula glutinis, Rhodotorula acheniorum and Rhodotorula graminis of the genus Rhodotorula were used as starting strains, and limonene-producing Rhodotorula glutinis, Rhodotorula acheniorum and Rhodotorula graminis engineered strains were constructed according to the construction method of the red yeast limonene engineered strain. Rhodotorula glutinis was named Ru-1 (preparation method as described in Example 1), Ru-2 (preparation method as described in Example 2) and Ru-3 (preparation method as described in Example 3); Rhodotorula acheniorum was named Rh-1 (preparation method as described in Example 1), Rh-2 (preparation method as described in Example 2) and Rh-3 (preparation method as described in Example 3); Rhodotorula acheniorum was named Rh-1 (preparation method as described in Example 1), Rh-2 (preparation method as described in Example 2) and Rh-3 (preparation method as described in Example 3); graminis) Ra-1 (prepared as described in Example 1), Ra-2 (prepared as described in Example 2) and Ra-3 (prepared as described in Example 3).

[0175] The obtained engineered strain was cultured in a 50 mL centrifuge tube containing 5 mL of YPD medium (20 g / L glucose, 20 g / L peptone, and 10 g / L yeast extract, diluted to 1 L with water) at 28 °C and 200 rpm / min. After fermentation for 8 h, dodecane was added at a volume ratio of 1:5 between dodecane and culture medium.

[0176] After 5 days of fermentation, 1 mL of n-hexane was added for secondary extraction, and the organic layer was separated by centrifugation at 4° C., and several layers were tested according to the limonene detection method of Example 4.

[0177] The test results are shown in Table 2. The results in Table 2 indicate that other red yeasts also have the ability to produce limonene.

[0178] Table 2 Limonene production of other engineered red yeast limonene

[0179]

[0180] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above disclosure, any person skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto. Sequence Listing <110> Northwest Agriculture and Forestry University <120> A gene, recombinant vector, engineered bacteria and their applications <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1671 <212> DNA <213> Artificial Sequence <400> 1 atggaccgac gctcggctaa ctaccagccc tcgatctggg accacgactt cctccagtcg 60 ctcaactcga actacaccga cgagacctac cgacgccgag ccgaggagct caagggcaag 120 gtcaagatcg ccatcaagga cgtcaccgag ccgctcgacc agctcgagct catcgacaac 180 ctccagcgct tgggcctcgc ttaccgcttc gagaccgaga tccgcaacat cctccacaac 240 atctacaaca acaacaagga ctacgtctgg cgcaaggaga acctctacgc cacctcgctc 300 gagttccgcc tcttgcgcca gcacggctac ccggtctcgc aggaggtctt caacggcttc 360 aaggacgacc agggaggctt catcttcgac gacttcaagg gcatcctctc gctccacgag 420 gcgtcgtact actcgctcga gggcgagtcg atcatggagg aggcgtggca gttcacctcg 480 aagcacctca aggaggtcat gatctcgaag tcgatggagg aggacgtctt cgtcgccgag 540 caggctaagc gcgctctcga gctccctctc cactggaagg tcccgatgct cgaggcacgc 600 tggttcatcc acgtctacga gaagcgcgag gacaagaacc acctcctcct cgagctcgcc 660 aagatggagt tcaacaccct ccaggccatc taccaggagg agctcaagga gatctcgggc 720 tggtggaagg acacgggact cggcgagaag ctctcgttcg ctcgcaaccg cctcgtcgct 780 tcgttcctct ggtcgatggg catcgccttc gagccgcagt tcgcctactg ccgacgcgtc 840 ctcaccatct cgatcgccct catcaccgtc atcgacgaca tctacgacgt ctacggcacc 900 ctcgacgagc tcgagatctt caccgacgca gtcgcacgct gggacatcaa ctacgccctc 960 aagcacctcc cgggctacat gaagatgtgc ttcctcgccc tctacaactt cgtcaacgag 1020 ttcgcctact acgtcctcaa gcagcaggac ttcgacatgc tcctctcgat caagaacgcc 1080 tggctcggcc tcatccaggc gtacctcgtc gaggccaagt ggtatcactc gaagtacacc 1140 ccgaagctcg aggagtacct cgagaacggc ctcgtctcga tcaccggacc actcatcatc 1200 gccatctcgt acctctcagg caccaacccg atcatcaaga aggagctcga gttcctcgag 1260 tcgaacccag acatcgtcca ctggtcgtcg aagatcttcc gcctccagga cgacctcggc 1320 acctcgtcgg acgagatcca gcgaggcgac gtcccgaagt cgatccagtg ctacatgcac 1380 gagaccggcg cttcggagga ggtcgcacgc gagcacatca aggacatgat gcgccagatg 1440 tggaagaagg tcaacgccta caccgccgac aaggactcgc ctctcacccg caccaccacc 1500 gagttcctcc tcaacctcgt ccgcatgtcg cacttcatgt acctccacgg cgacggccac 1560 ggcgtccaga accaggagac catcgacgtc ggcttcacc tcctcttcca gccgatccca 1620 ctcgaggaca aggacatggc cttcaccgcc tcgccaggca ccaagggata g 1671 <210> 2 <211> 783 <212> DNA <213> Artificial Sequence <400> 2 atgtcagcac gtggcctcaa caagatctcc tgctcgctca acctccagac cgagaagctc 60 tgctacgagg acaacgacaa cgacctcgac gaagaactca tgcccaagca catcgccctc 120 atcatggacg gcaaccgacg ctgggcgaag gacaagggac tcgaagtcta cgaaggccat 180 aagcacatca tcccgaagct caaggagatc tgcgacatct cgtcgaagct cggcatccag 240 atcatcaccg ctttcgcttt ctcgaccgag aactggaagc gctcgaagga ggaggtcgac 300 ttcctcctcc agatgttcga ggagatctac gacgagttct cacgctcggg agtccgcgtc 360 tcgatcatcg gctgcaagtc ggacctccca atgaccctcc agaagtgcat cgctctcacc 420 gaaaacca ccaagggcaa caagggactc cacctcgtca tcgccctcaa ctacggaggc 480 tactacgaca tcctccaggc taccagtcg atcgtcaaca aggcgatgaa cggcctcctc 540 600. 600. 600. 600. 600. 600. 600. 600. 600. 600. 600. 600 ccagacctcc tcatccgcac cggaggtgag cagcgcgtct cgaacttcct cctctggcag 660 ctcgcctaca ccgagttcta cttcaccaac accctcttcc ccgacttcgg cgaagaagac ctcaaggagg cgatcatgaa cttccagcag cgccaccgcc gcttcggagg acacacctac tag 783 <210> 3 <211> 2517 <212> DNA <213> Artificial Sequence <400> 3 atggaccgac gctcggctaa ctaccagccc tcgatctggg accacgactt cctccagtcg ctcaactcga actacaccga cgagacctac cgacgccgag ccgaggagct caagggcaag gtcaagatcg ccatcaagga cgtcaccgag ccgctcgacc agctcgagct catcgacaac 240. ctccagcgct tgggcctcgc ttaccgcttc gagaccgaga tccgcaacat cctccacaac atctacaaca acaacaagga ctacgtctgg cgcaaggaga acctctacgc cacctcgctc gagttccgcc tcttgcgcca gcacggctac ccggtctcgc aggaggtctt caacggcttc 360 aaggacgacc agggaggctt catcttcgac gacttcaagg gcatcctctc gctccacgag 420 gcgtcgtact actcgctcga gggcgagtcg atcatggagg aggcgtggca gttcacctcg 480 aagcacctca aggaggtcat gatctcgaag tcgatggagg aggacgtctt cgtcgccgag 540 caggctaagc gcgctctcga gctccctctc cactggaagg tcccgatgct cgaggcacgc 600 tggttcatcc acgtctacga gaagcgcgag gacaagaacc acctcctcct cgagctcgcc 660 aagatggagt tcaacaccct ccaggccatc taccaggagg agctcaagga gatctcgggc 720 tggtggaagg acacgggact cggcgagaag ctctcgttcg ctcgcaaccg cctcgtcgct 780 tcgttcctct ggtcgatggg catcgccttc gagccgcagt tcgcctactg ccgacgcgtc 840 ctcaccatct cgatcgccct catcaccgtc atcgacgaca tctacgacgt ctacggcacc 900 ctcgacgagc tcgagatctt caccgacgca gtcgcacgct gggacatcaa ctacgccctc 960 aagcacctcc cgggctacat gaagatgtgc ttcctcgccc tctacaactt cgtcaacgag 1020 ttcgcctact acgtcctcaa gcagcaggac ttcgacatgc tcctctcgat caagaacgcc 1080 tggctcggcc tcatccaggc gtacctcgtc gaggccaagt ggtatcactc gaagtacacc 1140 ccgaagctcg aggagtacct cgagaacggc ctcgtctcga tcaccggacc actcatcatc 1200 gccatctcgt acctctcagg caccaacccg atcatcaaga aggagctcga gttcctcgag 1260 tcgaacccag acatcgtcca ctggtcgtcg aagatcttcc gcctccagga cgacctcggc 1320 acctcgtcgg acgagatcca gcgaggcgac gtcccgaagt cgatccagtg ctacatgcac 1380 gagaccggcg cttcggagga ggtcgcacgc gagcacatca aggacatgat gcgccagatg 1440 tggaagaagg tcaacgccta caccgccgac aaggactcgc ctctcacccg caccaccacc 1500 gagttcctcc tcaacctcgt ccgcatgtcg cacttcatgt acctccacgg cgacggccac 1560 ggcgtccaga accaggagac catcgacgtc ggcttcaccc tcctcttcca gccgatccca 1620 ctcgaggaca aggacatggc cttcaccgcc tcgccaggca ccaagggagg ctcgggagct 1680 accaacttct cgctcctcaa gcaggcagga gacgtcgagg agaacccagg acctatgtca 1740 gcacgtggcc tcaacaagat ctcctgctcg ctcaacctcc agaccgagaa gctctgctac 1800 gaggacaacg acaacgacct cgacgaagaa ctcatgccca agcacatcgc cctcatcatg 1860 gacggcaacc gacgctgggc gaaggacaag ggactcgaag tctacgaagg ccataagcac 1920 atcatcccga agctcaagga gatctgcgac atctcgtcga agctcggcat ccagatcatc 1980 accgctttcg ctttctcgac cgagaactgg aagcgctcga aggaggaggt cgacttcctc 2040 ctccagatgt tcgaggagat ctacgacgag ttctcacgct cgggagtccg cgtctcgatc 2100 atcggctgca agtcggacct cccaatgacc ctccagaagt gcatcgctct caccgaagaa 2160 accaccaagg gcaacaaggg actccacctc gtcatcgccc tcaactacgg aggctactac 2220 gacatcctcc aggctaccaa gtcgatcgtc aacaaggcga tgaacggcct cctcgacgtc 2280 gaggacatca acaagaacct cttcgaccag gagctcgagt ccaagtgccc gaacccagac 2340 ctcctcatcc gcaccggagg tgagcagcgc gtctcgaact tcctcctctg gcagctcgcc 2400 tacaccgagt tctacttcac caacaccctc ttccccgact tcggcgaaga agacctcaag 2460 gaggcgatca tgaacttcca gcagcgccac cgccgcttcg gaggacacac ctactag <210> 4 <211> 4149 <212> DNA <213> Artificial Sequence <400> 4 atggaccgac gctcggctaa ctaccagccc tcgatctggg accacgactt cctccagtcg ctcaactcga actacaccga cgagacctac cgacgccgag ccgaggagct caagggcaag gtcaagatcg ccatcaagga cgtcaccgag ccgctcgacc agctcgagct catcgacaac 240. ctccagcgct tgggcctcgc ttaccgcttc gagaccgaga tccgcaacat cctccacaac atctacaaca acaacaagga ctacgtctgg cgcaaggaga acctctacgc cacctcgctc gagttccgcc tcttgcgcca gcacggctac ccggtctcgc aggaggtctt caacggcttc 360 aaggacgacc agggaggctt catcttcgac gacttcaagg gcatcctctc gctccacgag 420 gcgtcgtact actcgctcga gggcgagtcg atcatggagg aggcgtggca gttcacctcg 480 aagcacctca aggaggtcat gatctcgaag tcgatggagg aggacgtctt cgtcgccgag 540 caggctaagc gcgctctcga gctccctctc cactggaagg tcccgatgct cgaggcacgc 600 tggttcatcc acgtctacga gaagcgcgag gacaagaacc acctcctcct cgagctcgcc 660 aagatggagt tcaacaccct ccaggccatc taccaggagg agctcaagga gatctcgggc 720 tggtggaagg acacgggact cggcgagaag ctctcgttcg ctcgcaaccg cctcgtcgct 780 tcgttcctct ggtcgatggg catcgccttc gagccgcagt tcgcctactg ccgacgcgtc 840 ctcaccatct cgatcgccct catcaccgtc atcgacgaca tctacgacgt ctacggcacc 900 ctcgacgagc tcgagatctt caccgacgca gtcgcacgct gggacatcaa ctacgccctc 960 aagcacctcc cgggctacat gaagatgtgc ttcctcgccc tctacaactt cgtcaacgag 1020 ttcgcctact acgtcctcaa gcagcaggac ttcgacatgc tcctctcgat caagaacgcc 1080 tggctcggcc tcatccaggc gtacctcgtc gaggccaagt ggtatcactc gaagtacacc 1140 ccgaagctcg aggagtacct cgagaacggc ctcgtctcga tcaccggacc actcatcatc 1200 gccatctcgt acctctcagg caccaacccg atcatcaaga aggagctcga gttcctcgag 1260 tcgaacccag acatcgtcca ctggtcgtcg aagatcttcc gcctccagga cgacctcggc 1320 acctcgtcgg acgagatcca gcgaggcgac gtcccgaagt cgatccagtg ctacatgcac 1380 gagaccggcg cttcggagga ggtcgcacgc gagcacatca aggacatgat gcgccagatg 1440 tggaagaagg tcaacgccta caccgccgac aaggactcgc ctctcacccg caccaccacc 1500 gagttcctcc tcaacctcgt ccgcatgtcg cacttcatgt acctccacgg cgacggccac 1560 ggcgtccaga accaggagac catcgacgtc ggcttcaccc tcctcttcca gccgatccca 1620 ctcgaggaca aggacatggc cttcaccgcc tcgccaggca ccaagggagg ctcgggagct 1680 accaacttct cgctcctcaa gcaggcagga gacgtcgagg agaacccagg acctatgtca 1740 gcacgtggcc tcaacaagat ctcctgctcg ctcaacctcc agaccgagaa gctctgctac 1800 gaggacaacg acaacgacct cgacgaagaa ctcatgccca agcacatcgc cctcatcatg 1860 gacggcaacc gacgctgggc gaaggacaag ggactcgaag tctacgaagg ccataagcac 1920 atcatcccga agctcaagga gatctgcgac atctcgtcga agctcggcat ccagatcatc 1980 accgctttcg ctttctcgac cgagaactgg aagcgctcga aggaggaggt cgacttcctc 2040 ctccagatgt tcgaggagat ctacgacgag ttctcacgct cgggagtccg cgtctcgatc 2100 atcggctgca agtcggacct cccaatgacc ctccagaagt gcatcgctct caccgaagaa 2160 accaccaagg gcaacaaggg actccacctc gtcatcgccc tcaactacgg aggctactac 2220 gacatcctcc aggctaccaa gtcgatcgtc aacaaggcga tgaacggcct cctcgacgtc 2280 gaggacatca acaagaacct cttcgaccag gagctcgagt ccaagtgccc gaacccagac 2340 ctcctcatcc gcaccggagg tgagcagcgc gtctcgaact tcctcctctg gcagctcgcc 2400 tacaccgagt tctacttcac caacaccctc ttccccgact tcggcgaaga agacctcaag 2460 gaggcgatca tgaacttcca gcagcgccac cgccgcttcg gaggacacac ctacggctcg 2520 ggagagggcc gcggctcgct cctcacctgc ggcgacgtcg aggagaaccc aggcccgatc 2580 ctcatccgca ctcgcaaggc tctcaacggc gcaccgtcct cgtcgaccct taccgtccct 2640 tcgaccgacg aggtcaccgc cccgcagctc aagctctcgc cttcgaccgt cgccctcgtc 2700 tcgcagaacg gcattcccga cacccctcgc gacctcgaca cctgcgtcaa gatcttcaac 2760 ggcggtgagg gagcgatgct cctcaacgac gaggagatca tcaccctcgt ccagaagggc 2820 aagctcgccg cctatgcgct cgagaagctt ctcaaggact acgtccgcgc cgtctcgatc 2880 cgccgtgctc tcatctcgcg cgcctcggct cgcaaaaccc tcgaggcgtc cgacctgccg 2940 ttcctccact tcgactactc gcgcgtcatg ggccagtgct gtgagaacgt cgtcggctac 3000 atgcccatcc ccgtcggtat cgcgggaccg ctccgaatcg acggcaacgt cctccccatc 3060 ccgatggcta cgaccgaagg cgcgctcgtc gcctctacct cgcgtggttg caaggccctt 3120 aacgtctcgg gcggcgtcac gaccgtcgtc acgcaggacg cgatgacccg tggcccggct 3180 ctcgacttcc cgagcgtcat catgtgcgcc gccgccaagc gctgggtcga ctcggacgag 3240 ggcagcaaca tcctcaaggc cgcgttcaac tcgacttcga gattcgccag gctcaagagc 3300 ctcaagactg ccatggctgg tcgcacgctc tttgtccgct tcgccaccca gactggcgac 3360 gcgatgggca tgaacatgat ctccaagggc tgcgagcgcg ctctcgatgt catgatgacg 3420 gagcacttcc ccgagatgaa gatcgcgtcg ctctcgggca actactgcac ggacaagaag 3480 ccggccgcga tcaactggat cgagggacga gggaagagtg tcgtcgccga gggcatcatc 3540 cctggcgagg cggtcaagtc gatcctcaag acgaccgtca gcgacctcgt ccgcctcaac 3600 atcaccaaga acctcatcgg ctcggcgatg gccggctccg tcggcggcaa caacgcccac 3660 gcgtccaaca tcctcacggc catctacctc gcgaccggcc aggaccccgc ccagaacgtc 3720 gagtcgagca actgcatgac gctcatggag gccatcaacg acggaaagga cctcttgatc 3780 acctgctcga tgccgtcgat cgaggttggc accgtcggag gcggcaccat cctcctcccg 3840 caggccgcca tgctggacat gctcggcgtc aaaggtccgc acccgacctc gcccggccag 3900 aacgcgcagc agctcgctcg cgtcgtctgc gccgccgtca tggccggcga gctctcgctc 3960 atgtcggccc tcgcggccgg ctcgctcgtt cagagccact tggcgcacaa ccgctcggca 4020 cctgcgacgc ctgccgccca gacaccccag atcggctcgc gcgccgcgac gcctgtcttg 4080 aacggcacgc agcgcctcgc gccgttgacg gtgaccaagg gcaaggacca ccaccaccac 4140 caccactag 4149 <210> 5 <211> 4593 <212> DNA <213> Artificial Sequence <400> 5 atggtctcgt gctcggctcc aggcaagatc tacctcttcg gcgagcacgc cgtcgtctac 60 ggcgagaccg ctatcgcttg cgccgtcgag ctccgcaccc gagtccgagc cgagctcaac 120 gactcgatca ccatccagtc gcagatcggc cgcaccggcc tcgacttcga gaagcacccg 180 tacgtctcgg ccgtcatcga gaagatgcgc aagtcgatcc cgatcaacgg cgtcttcctc 240 accgtcgact cggacatccc ggtcggatcg ggactcggat cgtcggcagc cgtcaccatc 300 gcctcgatcg gtgccctcaa cgagctcttc ggcttcggcc tctcgctcca ggagatcgcc 360 aagctcggcc acgagatcga gatcaaggtc cagggcgccg cttcgcctac cgacacctac 420 gtctcgacct tcggcggcgt cgtcaccatc ccagagcgcc gcaagctcaa gacaccagac 480 tgcggcatcg tcatcggcga cacaggagtc ttctcgtcga ccaaggagct cgtcgccaac 540 gtacgccagc tccgcgagtc gtacccggac ctcatcgagc cgctcatgac ctcgatcggc 600 aagatctcgc gcatcggcga gcagctcgtc ctctcgggag actacgcctc gatcggccgc 660 ctcatgaacg tcaaccaggg cctctcgac gccctcggcg tcaacatcct cgagctctcg 720 cagctcatct actcggctcg cgcagcagga gctttcggcg ctaagatcac cggagcggga 780 ggaggaggct gcatggtcgc tctcaccgca ccagagaagt gcaaccaggt cgccgaggca 840 gtcgcaggag ccggaggaaa ggtcaccatc accaagccga ccgagcaggg cctcaaggtc 900 gacggatcgg gcgccaccaa cttctcgctc ctcaagcagg ccggcgacgt cgaggagaac 960 ccgggaccaa tgaccatcgg catcgacaag atctcgttct tcgtcccgcc gtactacatc 1020 gacatgaccg ccctcgccga ggctcgcaac gtcgacccgg gcaagttcca catcggcatc 1080 ggccaggacc agatggccgt caacccgatc tcgcaggaca tcgtcacctt cgccgccaac 1140 gccgccgagg ctatcctcac caaggagca aaagggcca tcgacatggt catcgtcggc 1200 accgagtcgt cgatcgacga gtcgaaggca gctccagtcg tcctccaccg cctcatggga 1260 atccagccgt tcgcacgctc gttcgagatc aaagggctt gctacggcgc caccgccgga 1320 ctccagctcg ccaagaacca cgtcgccctc cacccggaca agaaggtcct cgtcgtcgcc 1380 gccgacatcg ccaagtacgg cctcaactcg ggcggagagc ctacccaggg agcgggagct 1440 gtcgctatgc tcgtcgcctc ggagcctcgc atcctcgccc tcaaggagga caacgtcatg 1500 ctcacccagg acatctacga cttctggcgc ccgaccggcc acccttaccc tatggtcgac 1560 ggcccgctct cgaacgagac ctacatccag tcgttcgccc aggtctggga cgagcacaag 1620 aagcgcaccg gcctcgactt cgccgactac gacgccctcg ccttccacat cccgtacacc 1680 aagatgggca agaaggccct cctcgccaag atctcggacc agaccgaggc cgagcaggag 1740 cgcatcctcg cccgctacga ggagtcgatc atctactcgc gaagagtcgg caacctctac 1800 accggctcgc tctacctcgg cctcatctcg ctcctcgaga acgctaccac cctcaccgcc 1860 ggcaaccaga tcggcctctt ctcgtacggc tcgggagccg tcgcggagtt cttcaccggc 1920 gagctcgtcg cgggctacca gaaccacctc cagaaggaga cccacctcgc cctcctcgac 1980 aaccgcaccg agctctcgat cgccgagtac gaggccatgt tcgccgagac cctcgacacc 2040 gacatcgacc agaccctcga ggacgagctc aagtactcga tctcggccat caacaacacc 2100 gtccgctcgt accgcaacgg atcgggagag ggacgcggat cgctcctcac ctgcggagac 2160 gtcgaggaga acccaggacc aatgaagacc gtcgtcatca tcgacgccct ccgcaccccg 2220 atcggcaagt acaagggctc gctctcgcag gtctcggccg tcgacctcgg aacccacgtc 2280 accacccagc tcctcaagcg ccactcgacc atctcggagg agatcgacca ggtcatcttc 2340 ggcaacgtcc tccaggccgg caacggacag aacccagcac gtcagatcgc catcaactcg 2400 ggcctctcgc acgagatccc ggccatgacc gtcaacgagg tctgcggctc gggcatgaag 2460 gccgtcatcc tcgccaagca gctcatccag ctcggcgagg ccgaggtcct catcgccgga 2520 ggcatcgaga acatgtcgca ggcaccaaag ctccagcgct tcaactacga gaccgagtcg 2580 tacgacgtcc agttctcgtc gatgatgtac gacggcctca ccgacgcctt ctcgggacag 2640 gctatgggcc tcaccgccga gaacgtcgcc gagaagtacc acgtcacccg cgaggagcag 2700 gaccagttct cggtccactc gcagctcaag gccgctcagg ctcaggccga gggaatcttc 2760 gccgacgaga tcgcaccact cgaggtctcg ggcacgctcg tcgagaagga cgagggcatc 2820 cgcccgaact cgtcggtcga gaagctcggc accctcaaga cggtcttcaa ggaggacggc 2880 accgtcaccg ccggaaacgc ctcgaccatc aacgacggcg cttcggccct catcatcgcc 2940 tcgcaggagt acgccgaggc tcacggactc ccgtacctcg ccatcatccg cgactcggtc 3000 gaggtcggca tcgacccagc ctacatgggc atctcgccga tcaaggccat ccagaagctc 3060 ctcgcacgca accagctcac caccgaggag atcgacctct acgagatcaa cgaggcgttc 3120 gccgccacct cgatcgtcgt ccagcgcgag ctcgccctac cggaggagaa ggtcaacatc 3180 tacggcggag gcatctcgct cggacacgct atcggcgcta ccggagctcg cctcctcacc 3240 tcgctctcgt accagctcaa ccagaaggag aagaagtacg gcgtcgcctc gctctgcatc 3300 ggaggcggcc tcggactcgc tatgctcctc gagcgcccgc agcagaagaa gaactcgcgc 3360 ttctaccaga tgtcgccgga ggagcgtctc gcttcgctcc tcaacgaggg ccagatctcg 3420 gccgacacca agaaggagtt cgagaacacc gccctctcat cgcagatcgc caaccacatg 3480 atcgagaacc agatctcgga gacggaggtc ccgatgggag tcggactcca cctcaccgtc 3540 gacgagaccg actacctcgt cccgatggcc accgaggagc cttcggtcat cgccgctctc 3600 tcgaacggcg ccaagatcgc ccagggcttc aagaccgtca accagcagcg cctcatgcga 3660 ggccagatcg tcttctacga cgtcgccgac ccggagtcgc tcatcgacaa gctccaggtc 3720 cgcgaggccg aggtcttcca gcaggccgag ctctcgtacc cgtcgatcgt caagcgcgga 3780 ggaggactcc gcgacctcca gtaccgcacc ttcgacgagt cgttcgtctc ggtcgacttc 3840 ctcgtcgacg tcaaggacgc gatgggcgcc aacatcgtca acgccatgct cgagggcgtc 3900 gccgagctct tccgcgagtg gttcgccgag cagaagatcc tcttctcgat cctctcgaac 3960 tacgccaccg aatcggtcgt caccatgaag accgctatcc cggtctcgcg cctctcgaag 4020 ggatcgaacg gccgcgagat cgccgagaag atcgtcctcg cctcgcgcta cgcttcgctc 4080 gacccttacc gagcagtcac ccacaacaag ggcatcatga acggcatcga ggccgtcgtc 4140 ctcgctaccg gaaacgacac ccgagcagtc tcggcttcgt gccacgcttt cgccgtcaag 4200 gagggccgct accagggact cacctcgtgg accctcgacg gcgagcagct catcggcgag 4260 atctcggtcc cgctcgctct cgctaccgtc ggaggcgcca ccaaggtcct ccctaagtcg 4320 caggccgccg ccgacctcct cgccgtcacc gacgctaagg agctctcacg cgtcgtcgca 4380 gctgtcggac tcgctcagaa cctcgccgct ctacgagctc tcgtctcgga gggcatccag 4440 aagggccaca tggccctcca ggctcgctcg ctcgctatga ccgtcggcgc taccggcaag 4500 gaggtcgagg ccgtcgctca gcagctcaag cgccagaaga ccatgaacca ggaccgcgcg 4560 atggccatcc tcaacgacct ccgcaagcaa tag 4593 <210> 6 <211> 124 <212> PRT <213> Artificial Sequence <400> 6 Met Ala Lys Leu Thr Ser Ala Val Pro Val Leu Thr Ala Arg Asp Val 1 5 10 15 Ala Gly Ala Val Glu Phe Trp Thr Asp Arg Leu Gly Phe Ser Arg Asp 20 25 30 Phe Val Glu Asp Asp Phe Ala Gly Val Val Arg Asp Asp Val Thr Leu 35 40 45 Phe Ile Ser Ala Val Gln Asp Gln Val Val Pro Asp Asn Thr Leu Ala 50 55 60 Trp Val Trp Val Arg Gly Leu Asp Glu Leu Tyr Ala Glu Trp Ser Glu 65 70 75 80 Val Val Ser Thr Asn Phe Arg Asp Ala Ser Gly Pro Ala Met Thr Glu 85 90 95 Ile Gly Glu Gln Pro Trp Gly Arg Glu Phe Ala Leu Arg Asp Pro Ala 100 105 110 Gly Asn Cys Val His Phe Val Ala Glu Glu Gln Asp 115 120

Claims

1. An engineered bacterium, characterized in that: The engineered bacteria are obtained by transforming the recombinant vector A into a host cell; The recombinant vector A includes a recombinant vector I; The recombinant vector I is an insertion fragment I into the integration backbone vector I; The fragment I contains target gene module I; The nucleotide sequence of the target gene module I is shown in bases 1 to 4128 of SEQ ID No. 4; In the fragment I, upstream of the nucleotide sequence of the target gene module I, there is also a nucleotide sequence of a promoter I; The promoter 1 is XYL; The insertion is to replace the fragment between the ECoRV recognition site and the SpeI recognition site of the integrative backbone vector 1 with the fragment 1, while keeping the other nucleotide sequences of the integrative backbone vector 1 unchanged; The integrative backbone vector I is Gateway Vector pB4GWnY; The host cell is any one of Rhodotorula toruloides, Rhodotorula glutinis, Rhodotorula acheni, and Rhodotorula graminis, in which the carotenoid synthesis pathway is knocked out.

2. The engineered bacteria according to claim 1, characterized in that The recombinant vector A also includes a recombinant vector II; The recombinant vector II is an insertion fragment II into the integration backbone vector II; The fragment II contains the target gene module II; The nucleotide sequence of the target gene module II is shown in SEQ ID No. 5; In the fragment II, the nucleotide sequence of the promoter II is also contained upstream of the nucleotide sequence of the target gene module II; The promoter II is XYL; The insertion is to replace the fragment between the ECoRV recognition site and the SpeI recognition site of the integrative backbone vector II with the fragment II, while keeping the other nucleotide sequences of the integrative backbone vector II unchanged; The integrative backbone vector II is Gateway Vector pB4GWnY.

3. A method for producing limonene, characterized in that: The method comprises the following steps: culturing the engineered bacteria according to claim 1 or 2 to obtain the limonene.

4. The method according to claim 3, characterized in that The method comprises the following steps: The engineered bacteria is fermented in a culture medium I to obtain a fermentation liquid I, dodecane I is added, and the fermentation is continued to obtain the limonene.

5. The method according to claim 4, characterized in that The conditions of the fermentation 1 include: Temperature is 15-35℃; The speed is 100-200 rpm; The time is 8 to 144 hours.

6. The method according to claim 4, characterized in that The conditions for continued fermentation are: Temperature is 15-35℃; The speed is 100-200 rpm; The duration is 4 to 7 days.

7. The method according to claim 4, characterized in that The volume ratio of the dodecane I to the culture medium I is 1:2-5.

8. The method according to claim 4, characterized in that The fermentation liquid obtained by continued fermentation is subjected to secondary extraction with n-hexane to obtain the limonene.

9. The method according to claim 3, characterized in that The method comprises the following steps: The seed liquid and dodecane II are added to the culture medium II, and the culture medium II is fermented to obtain the limonene.

10. The method according to claim 9, characterized in that The volume ratio of the seed solution to the culture medium II is 1-15:85-99; The volume ratio of the dodecane II to the culture medium II is 1:2-5; The conditions of the fermentation II include: pH 3.0-6.5; Ventilation volume is 0.5 to 3 L / min; The stirring speed is 50-350 rpm; Glucose concentration is higher than 2g / L.

11. The method according to claim 10, characterized in that During fermentation II, when the glucose concentration dropped below 2 g / L, the following feeds were performed: Supplement glucose 20-50g / L, yeast extract powder 10-20g / L, and ammonium sulfate 2-10g / L; After 5 to 10 feedings, fermentation II is terminated.

12. The method according to claim 9, characterized in that The seed solution is obtained by the following steps: A single colony is cultured in culture medium A to obtain a first seed liquid, and the first seed liquid is cultured in culture medium B to obtain the seed liquid.

13. The method according to claim 12, characterized in that The volume ratio of the first seed solution to the culture medium B is 1-10:50-100.

14. The method according to claim 12, characterized in that The conditions for culturing A and culturing B independently include: Temperature is 15-35℃; The speed is 100-200 rpm; The time is 12 to 48 hours.

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