Peroxisome signal peptide and application thereof

Abstract

The invention discloses a peroxisome positioning signal peptide and application thereof. The invention relates to an application of an N-terminal II-type peroxisome positioning signal peptide alone or in combination with a C-terminal I-type signal peptide in the biosynthesis of a compartment high-value compound. The invention comprises the construction of an auxiliary carrier of two peroxisome signal peptides, and covers the application of the auxiliary carrier in the biosynthesis of terpene substances such as limonene, linalool and patchouli alcohol, and flavonoid substances such as daidzein, genistein, 3-hydracrylic acid and coenzyme Q10. The peroxisome signal peptide disclosed by the invention can provide a new element for metabolic engineering modification of subcellular organelles of rhodotorula expansa.

Description

Technical Field

[0001] The invention relates to a type II peroxisome signal peptide and application thereof, and belongs to the field of genetic engineering. Background Art

[0002] A sustainable alternative to traditional production of valuable compounds is to construct cell factories through microbial engineering, which solves the production of compounds worth billions of dollars. Cell factories are used to produce important industrial products, including pharmaceuticals, proteins, commodity chemicals and biofuels, by synthetic biology and genetic engineering of hosts such as bacteria, fungi, mammals and plant cells (Li M, Hou F, Wu T, et al. Recent advances of metabolic engineering strategies in natural isoprenoid production using cell factories [J]. Natural product reports, 2020, 37 (1): 80-99.). As cell engineering becomes more and more complex, the introduction of longer and more complex heterologous metabolic pathways increases the possibility of crosstalk with natural cell functions in host cells, while increasing the risk of cytotoxicity and the impact on host cell growth. Subcellular compartmentalization is an effective strategy for dealing with non-productive or harmful crosstalk. In synthetic biology, compartmentalization has been proven to be an effective strategy to separate toxic heterologous components from the host cytoplasm, thereby limiting cytotoxicity, improving growth and increasing product yield. Currently, compartmentalization strategies have been used to effectively synthesize various high-value products, including terpenes, monoterpenoid indole alkaloids, and fatty alcohols, in Saccharomyces cerevisiae, Yarrowia lipolytica, and Pichia pastoris (Ren Y, Liu S, Jin G, et al. Microbial production of limonene and its derivatives: achievements and perspectives. Biotechnology Advances, 2020, 44: 107628.). Among subcellular organelles, peroxisomes, with their monolayer membranes, high permeability, dynamic changes in number with growth, and no effect on cell growth due to modification, only require simple C-terminal type I and N-terminal type II signal peptides for localization, enabling efficient synthesis of high-value products through compartmentalization.Compounds such as monoterpenes and fatty alcohols have been synthesized through peroxisomal compartmentalization, but this has not been reported in Rhodotorula toruloides (Dusséaux S, Wajn WT, Liu Y, et al. Transforming yeast peroxisomes into microfactories for the efficient production of high-value isoprenoids. Proceedings of the National Academy of Sciences, 2020, 117(50): 31789-31799.).

[0003] In order to achieve compartmentalized synthesis of products in the peroxisomes of R. toruloides, peroxide signal peptides are needed to locate the expression position of genes. It is difficult to construct a multi-gene expression pathway in Rhodotorula yeast by relying solely on C-terminal type I peroxisomal localization peptides, and the localization activity of C-terminal type I signal peptides in Rhodotorula yeast has not been fully verified (Kim J, Coradetti ST, Kim YM, et al. Multi-omics driven metabolic network reconstruction and analysis of lignocellulosic carbon utilization in Rhodosporidium toruloides. Frontiers in Bioengineering and Biotechnology, 2021, 8: 612832.). It is urgent to find a type II signal peptide that can be efficiently positioned in red yeast and verify the role of type I signal peptides to provide the necessary gene elements for the peroxisomal compartmentalization of multi-gene pathways. Summary of the Invention

[0004] To overcome the deficiencies in the prior art, the present invention aims to provide peroxisomal signal peptides and their applications, in order to construct a complete target product synthesis pathway in the peroxisome of Rhodosporidium toruloides, achieve the production of the target product, and provide potential guidance for the development of the synthesis of high-value compounds using red yeast.

[0005] The first technical problem to be solved by the present invention is to characterize a signal peptide with peroxisome targeting function.

[0006] In order to solve the above problems, the technical solution of the present invention is:

[0007] 1) Screening for type II signal peptides from R. toruloides that have peroxisome targeting function;

[0008] 2) Verify the type I signal peptide with peroxisome targeting function in R. toruloides;

[0009] 3) verifying the effective sequence of the peroxisome type II signal peptide gene;

[0010] 4) Clone the EGFP gene with an effective peroxisomal signal peptide sequence;

[0011] 4) Ligating the amplified PTS-EGFP gene with the expression vector to obtain a recombinant expression vector;

[0012] 5) Introducing the recombinant expression vector obtained in step 4) into the R. toruloides platform bacteria;

[0013] 6) Verify the targeting function of the type II peroxisome signaling peptide.

[0014] A type II peroxisome signal peptide (PTS2) is selected from endogenous 3-ketoacyl-CoA thiolase from R. toruloides. The NCBI accession number for the peroxisome signal peptide gene is XM_016420776. The amino acid sequence of the type II peroxisome signal peptide is shown in SEQ ID NO. 1.

[0015] The GC content of the nucleotide sequence of the type II peroxisome signal peptide gene can be adjusted between 55-75%.

[0016] Preferably, the nucleotide sequence of the peroxisome signal peptide gene is truncated to a gene sequence as shown in SEQ ID NO. 2-7, which can all function as a signal peptide.

[0017] An auxiliary vector comprising the above-mentioned type II peroxisome signal peptide or a combination thereof with a type I peroxisome signal peptide to mediate the expression of a target biosynthetic pathway gene is a recombinant vector.

[0018] Preferably, the auxiliary vector contains at least one type II peroxisome signal peptide sequence.

[0019] Preferably, the amino acid and corresponding nucleotide sequences of the type I peroxisome signal peptide are SEQ ID NOs. 8-1, 8-2, 8-3, and 8-4, and the specific sequences are as follows:

[0020] SEQ ID NO.8-1 SKL TCGAAGCTC SEQ ID NO.8-2 AKL GCGAAGCTG SEQ ID NO.8-3 AKM GCGAAGATG SEQ ID NO.8-4 ARL GCGCGGCTG

[0021] An engineered bacterium for peroxisome signal peptide-mediated target product synthesis pathway genes, characterized in that the constructed engineered bacterium comprises an auxiliary vector constructed with the above-mentioned peroxisome signal peptide sequence.

[0022] Furthermore, the auxiliary vector can quickly construct the peroxisome compartmentalized expression of the target product synthesis pathway gene, relying on the rapid assembly of the polycistronic nucleotide sequence with 2A peptide connection and fixed enzyme cleavage sites. The 2A peptide amino acid sequence is SEQ ID NO.9, and the auxiliary vector sequence is SEQ ID NO.10.

[0023] The invention discloses an application of a peroxisome signal peptide. The peroxisome signal peptide auxiliary vector can be used alone or in combination to rapidly construct engineered bacteria containing, but not limited to, terpenes such as limonene, linalool, and patchouliol, flavonoids such as daidzein, genistein, 3-hydroxypropionic acid, and coenzyme Q10, thereby realizing the peroxisome compartmentalization and synthesis of high-value compounds.

[0024] The recombinant bacteria is a red yeast, which is a species of the genus Rhodotorula, including but not limited to Rhodotorula toruloides, Rhodotorula glutinis, Rhodotorula spherical, Rhodotorula acheniorum, Rhodotorula graminis, Rhodotorula marina, Rhodotorula mucilaginosa, Rhodotorula rubra, Rhodotorula lactosa, R. sphaerocarpum and R. bogoriensis.

[0025] The second technical problem to be solved by the present invention is to provide an application of the peroxisome signal peptide.

[0026] In order to solve the above problems, the technical solution of the present invention is:

[0027] 1) Cloning the codon-optimized gene sequence of the peroxisome signal peptide PTS2, including but not limited to the gene sequence of neryl diphosphate synthase NPPS from Solanum lycopersicum, and the gene sequence of the fusion protein NPPS-GGGS-LS of the limonene synthase CltLS from Citrus limon;

[0028] 2) cloning a gene nucleotide sequence fused with the peroxisome signal peptide PTS2 and a gene nucleotide sequence including but not limited to a truncated hydroxymethylglutaryl-CoA reductase HMGR from red yeast;

[0029] 3) cloning a gene nucleotide sequence fused with the peroxisome signal peptide PTS1 and a gene nucleotide sequence including but not limited to the isopentenyl pyrophosphate isomerase PrIDI from Phaffia rhodozyma;

[0030] 4) cloning the gene nucleotide sequence of mevalonate kinase MmMK from Methanosarcina mazei, which is fused with the peroxisome signal peptide PTS2 and has been codon-optimized;

[0031] 5) cloning the gene nucleotide sequence fused with the peroxisome signal peptide PTS2 and the gene nucleotide sequence including but not limited to the phosphomevalonate kinase PMK from red yeast;

[0032] 6) Cloning the gene nucleotide sequence of the peroxisome signal peptide PTS1 fused thereto and codon-optimized, including but not limited to, the gene nucleotide sequence of diphosphomevalonate decarboxylase PDC from Rhodotorula;

[0033] 7) Cloning a codon-optimized gene sequence containing the peroxisome signal peptide PTS1, including but not limited to a gene sequence of acetyl-CoA acetyltransferase / 3-hydroxy-3-methylglutaryl-CoA reductase EfMvaE from Enterococcus faecalis;

[0034] 8) Cloning of the gene nucleotide sequence fused to the peroxisome signal peptide PTS2 and after codon optimization, including but not limited to the hydroxymethylglutaryl-CoA synthase mutant EfMvaS from E. faecalis A110G The nucleotide sequence of the gene;

[0035] 9) The polymerase chain reaction (PCR) products obtained in step 1), step 2) and step 3) were ligated into the binary expression vector PZPK by enzyme digestion and ligation to obtain the vector PZPK-PTS2-HMGR-PTS2-NPPS::LS-IDI-SKL

[0036] 10) The PCR products obtained in steps 4), 5), and 6) were ligated into the binary expression vector PZPK by enzyme digestion and ligation to obtain the expression vector PZPK-PTS2-MK-PTS2-PMK-PDC-SKL;

[0037] 11) The PCR products obtained in step 7) and step 8) were ligated into the binary expression vector PZPK by enzyme digestion to obtain the expression vector PZPK-PTS2-S A110G-E-SKL;

[0038] 12) The expression vectors obtained in step 9), step 10) and step 11) are sequentially transformed into yeast via Agrobacterium pyogenes-mediated transformation (ATMT);

[0039] 13) Select transformants containing all the above genes for fermentation testing.

[0040] According to the present invention, the red yeast is a species of the genus Rhodotorula, including but not limited to R. toruloides, R. glutinis, R. acheniorum, R. graminis, R. marina, R. mucilaginosa, R. rubra, R. lactosa, R. sphaerocarpum and R. bogoriensis.

[0041] In one embodiment of the present invention, the limonene engineering bacteria is Rhodosporidium toruloides CGMCC 2.1389, and the China General Microbial Culture Collection Number is CGMCC 2.1389.

[0042] According to the application of the present invention, in one embodiment of the present invention, step 9) includes the following steps:

[0043] S9.1 cloning of the gene nucleotide sequence fused with the peroxisome signal peptide PTS2 requires the helper vector pUC-PTS: pUC-PTS2-EcoRV-P2A-PTS2-Stu1-P2A-PTS2-Sca1-P2A-SpeI-PTS1.

[0044] S9.2 The NPPS-GGGS-LS fusion protein gene, HMGR gene and PrIDI gene sequences were introduced into the restriction sites and homologous arms of the corresponding site PTS2 upstream and downstream of the genes through primers, and were inserted into the auxiliary vector pUC-PTS by homologous recombination to obtain the corresponding protein gene sequences with PTS.

[0045] S9.3 introduces restriction sites or homologous arms of promoter and terminator upstream and downstream of the gene, which contains NPPS-GGGS-LS fusion protein, HMGR and PrIDI gene nucleotide sequences fused with peroxisome signal peptide PTS, and connects them into the binary expression vector PZPK through restriction ligation and homologous recombination.

[0046] S9.4 The recombinant plasmid containing NPPS-GGGS-L, HMGR and PrIDI genes was transformed into Bacillus aeruginosa by electroporation to construct a binary expression vector recombinant plasmid mediated by Bacillus aeruginosa.

[0047] According to the application of the present invention, in one embodiment of the present invention, the step 10) includes the following steps:

[0048] S10.1 is similar to S9.2. The MmMK gene, PMK gene and PDC gene sequences fused with the gene nucleotide sequence of the peroxisome signal peptide PTS are obtained through an auxiliary vector. Restriction sites or homologous arms of the promoter and terminator are introduced upstream and downstream of the gene through primers, and they are respectively connected to the binary expression vector PZPK through restriction enzyme ligation and homologous recombination.

[0049] S10.2 The recombinant plasmid containing MmMK, PMK and PDC genes is transformed into Bacillus thuringiensis by electroporation to construct a binary expression vector recombinant plasmid mediated by Bacillus thuringiensis.

[0050] According to the application of the present invention, in one embodiment of the present invention, the step 11) includes the following steps:

[0051] Similar to S11.1 S9.2, the EfMvaE and EfMvaS fused with the peroxisome signal peptide PTS gene nucleotide sequence are obtained through the auxiliary vector. A110G The gene nucleotide sequence is introduced into restriction sites or homologous arms of promoter and terminator at the upstream and downstream of the gene through primers, and is respectively connected to the binary expression vector PZPK through restriction ligation and homologous recombination.

[0052] S11.2 will include EfMvaE and EfMvaS A110G The recombinant plasmid of the gene is transferred into Bacillus aeruginosa by electroporation to construct a binary expression vector recombinant plasmid mediated by Bacillus aeruginosa.

[0053] According to the peroxisome signal peptide and its application described in the present invention, the use of the peroxisome signal peptide PTSs auxiliary expression vector alone or in combination can quickly construct engineered bacteria containing but not limited to terpenes such as limonene, linalool, patchouli alcohol, flavonoids such as daidzein, genistein, 3-hydroxypropionic acid and coenzyme Q10, thereby realizing the peroxisome compartmentalization synthesis of high-value compounds.

[0054] The beneficial effects of this application include:

[0055] This invention screened and characterized a type II peroxisomal signal peptide and its combination with a type I signal peptide in R. toruloides for the first time. Furthermore, by constructing genes for the target product synthesis pathway mediated by the peroxisomal signal peptide, the synthesis efficiency of terpenes, flavonoids, 3-hydroxypropionic acid, and coenzyme Q10, etc., heterologously expressed in R. toruloides, was enhanced, achieving the following results:

[0056] The present invention provides a type II peroxisome signal peptide, which is characterized and verified in R. toruloides for the first time, expanding the enabling tools of R. toruloides to achieve subcellular peroxisome compartmentalization.

[0057] The present invention provides an application of a peroxisome signal peptide, which for the first time utilizes a peroxisome signal peptide-mediated gene expression method in R. toruloides to obtain an engineered bacterium that produces terpenes such as limonene, linalool, and patchouliol, and flavonoids such as daidzein, genistein, 3-hydroxypropionic acid, and coenzyme Q10, thereby achieving heterologous expression of the target product in the peroxisome compartment. The genetic operations required are few, simple, and efficient, and the strain transformation cycle is short.

[0058] The engineered bacteria for producing limonene, linalool, patchouli alcohol, 3-hydroxypropionic acid, daidzein, genistein and coenzyme Q10 provided by the present invention have stable heterologous gene expression, do not require the addition of antibiotics and inducers during fermentation, thereby reducing production costs, and have a simple and controllable fermentation process with high economy.

[0059] The present invention provides a series of engineered bacteria (using limonene as an example) for fermentation production of limonene. The process involves culturing the engineered bacteria in shake flasks at 28°C and 180 rpm until mid-logarithmic phase, then inoculating the bacteria in a 3-L fermentor at a 10% inoculum size. Fermentation parameters are pH 6.0, 22°C, and 500 rpm. Using glucose as the substrate, the 3-L fermentor achieved a yield of 4 g / L in fed-batch fermentation for 240 hours, demonstrating excellent potential for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is a schematic diagram of the peroxisome signal peptide targeting function and auxiliary vector construction;

[0061] Figure 2 This is a graph showing the yield of limonene, a product synthesized by limonene-engineered bacteria;

[0062] Figure 3 This is the qualitative diagram of limonene, a product synthesized by limonene engineering bacteria, and its stability verification; DETAILED DESCRIPTION

[0063] The present invention will be further described below with reference to the accompanying drawings and specific examples so that those skilled in the art can better understand the present invention and implement it. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0064] Unless otherwise specified, the experimental materials and reagents in the following examples can be purchased from commercial sources.

[0065] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0066] The starting strain Rhodosporidium toruloides in the examples was obtained from the Dalian Institute of Chemical Physics and is a conventional Rhodosporidium toruloides np11 in the art (Zhu Z, Zhang S, Liu H, et al. A multi-omic map of the lipid-producing yeast Rhodosporidium toruloides. Nature communications, 2012, 3(1): 1112.) that has been published.

[0067] Primers were synthesized by Yangling Tianrun Aoke Biotechnology Co., Ltd. PCR products were sequenced by Yangling Tianrun Aoke Biotechnology Co., Ltd.

[0068] Detection method of limonene:

[0069] In the embodiment, a gas chromatography-mass spectrometer (GC-MS) was used to perform qualitative analysis on the sample. Liquid injection was used, a flame ionization detector (FID) detector was used, the chromatographic column was an HP-INNOWax capillary column (60mx 250μmx 0.25μm), helium (He) was used as the carrier gas, the flow rate was 1.5mL / min; the injection volume was 1μL, the split ratio was 20:1; the GC heating program was as follows: 40°C for 3min, heated to 160°C at 4°C / min, heated to 220°C at 7°C / min, and held for 8min. The MSD transmission line and ion source temperatures were 280°C and 230°C, respectively; the electron ion source (EI) had an energy of 70eV and a scan range of 29 to 350u.

[0070] In the embodiment, the sample was quantitatively analyzed using a gas chromatograph (GC). The limonene in the sample was quantified using a gas chromatograph (GC). The quantitative analysis was performed on a Shimadzu GC-2014C (Shimadzu, Japan) equipped with a KB-1 chromatographic column (60m×0.25mm×0.25μm, Kromat, USA), using an FID detector; nitrogen was used as the carrier gas, the flow rate was 1.0mL / min, the injection volume was 1μL, and the split ratio was 20:1; the GC column oven program was 145°C for 27min; the injector and detector temperatures were 240°C and 260°C, respectively; a limonene standard curve was established to quantitatively analyze the limonene content in the sample.

[0071] Example 1: Characterization of a set of peroxisome type II targeting peptides (PTS2)

[0072] The peroxisomal 3-ketoacyl-CoA thiolase (3-KT) from E1.1 R. toruloides contains a nonapeptide motif typical of PTS2, and PTS2 requires the core nonapeptide and adjacent motifs for function. The first 40 amino acid residues of endogenous 3-KT (PTS2-40AA) were cloned and expressed in fusion with a green fluorescent protein (EGFP) for fluorescence detection. The NCBI accession number for the endogenous 3-KT gene is XM_016420776. To characterize peroxisomal localization, endogenous peroxisome biogenesis factor 11 (RtPex11), a peroxidase membrane protein, was fused to red fluorescent protein to generate RtPex11-RFP. Colocalization with PTS2-40AA-EGFP confirmed peroxisomal localization.

[0073] Overlap extension PCR was used to amplify the PTS2-40AA gene fragment from E1.1 at the N-terminus of EGFP using primers PTS2-1 with SpeI-EGFP-R, PTS2-2 with SpeI-EGFP-R, and PTS2-3 with SpeI-EGFP-R, sequentially. PCR conditions included 98°C pre-denaturation for 5 minutes, followed by 30 cycles of denaturation at 98°C for 10 seconds, annealing at 57°C for 5 seconds, and extension at 72°C for 5 minutes. The resulting PTS2-40AA-EGFP gene fragment contained restriction sites at both ends.

[0074] The primers used in the above experimental process are shown in the table below:

[0075] Table 1 Primer sequence list

[0076]

[0077]

[0078] E1.3 Prepare Escherichia coli DH5α competent cells, and select the plasmid pZPK with nourseothricin resistance as its expression vector. The above-mentioned gene fragment PTS2-40AA-EGFP was cloned between the restriction enzyme sites EcoRV and SpeI by enzyme digestion and ligation, and then heat-shocked into Escherichia coli DH5α competent cells at 42°C. After a single colony grew on an LB screening plate containing the corresponding resistance, colony PCR verification was performed using the upstream primer XYL-F and the downstream primer tHSP-R of the terminator. The correct transformant was inoculated into 5 mL of LB medium with the corresponding resistance and cultured for 12 hours. The corresponding plasmid was extracted and sequenced for verification to obtain the pZPK-Ntc-pXYL-EcoRV-PTS2-40AA-EGFP-SpeI-tHSP recombinant plasmid.

[0079] Among them, Ntc is the nourseothricin resistance gene, and the amino acid sequence of its encoded protein is GenBank: AAS47018.1.

[0080] E1.4 Prepare soil bacillus competent cells, and electroporate the pZPK-Ntc-pXYL-EcoRV-PTS2-40AA-EGFP-SpeI-tHSP recombinant plasmid constructed in step E1.3 into the soil bacillus competent cells. After a single colony grows on an LB screening plate containing the corresponding resistance, colony PCR verification is performed using the upstream primer XYL-F of the target gene and the downstream primer tHSP-R of the terminator. The verified single colony of soil bacillus is inoculated into 5 mL of LB medium with 0.1% kanamycin and cultured for 16-18 hours. The corresponding plasmid is extracted and digested again with restriction endonucleases EcoRV and SpeI for verification.

[0081] E1.5 Inoculate the single colony of Bacillus subtilis verified correctly in step E1.4 into 5 mL LB liquid culture medium containing 0.1% corresponding antibiotics and culture for about 16 hours to OD600 = 0.4-0.6. At the same time, inoculate the R. toruloides pigment production-deficient bacteria ΔCar-np11 into YPD liquid culture medium and culture for 18 hours to OD600 = 0.4-0.6. Wash the bacteria twice with sterile water, resuspend them, take 100 μL of the bacterial solution from each, mix them, and drop them onto sterile filter paper on the IM induction plate. After culturing at 25°C for 48 hours, transfer them to YPD plates containing nourseothricin resistance and cephalosporin, and continue culturing at 30°C for 48 hours.

[0082] E1.6 Pick as many single colonies as possible from step E1.5 and subculture them multiple times on screening plates for verification. Finally, the correct single colony is the correct genetically engineered bacterium and is named PTS2-40AA (genotype is np11-pXYL-PTS2-40AA-EGFP-tHSP).

[0083] E1.7 Perform preliminary screening of the transformants selected in step E1.6 using an automated fluorescence microscope, and select transformants with a dot-like fluorescence distribution.

[0084] E1.8 is similar to steps E1.2-4. Primers XYL-fu-Pex11-F and Thsp-fu-Pex11-R, and Pex11-fu-RFP-F and Thsp-fu-RFP-R are used to clone the RtPex11 and RFP fragments, respectively. They are then cloned into the restriction enzyme sites EcoRV and SpeI by homologous recombination to construct the pZPK-Ntc-pXYL-EcoRV-RtPex11-RFP-SpeI-tHSP recombinant plasmid, which is then transformed into soil abscesses.

[0085] E1.9 The soil bacillus obtained in step E1.8 and the transformants in step E1.7 were infected and passaged, and characterized by biological laser confocal microscopy. Figure 1 As shown, PTS2-40AA is able to target EGFP to peroxisomes.

[0086] E1.10: Similar to Example 1E1.2, overlapping extension PCR was performed using the primers in Table 1 to clone progressively truncated fragments of PTS2-40AA at the N-terminus and C-terminus of EGFP to obtain the shortest PTS2 sequence. The resulting fragments were N0C10-GFP, N0C12-GFP, N0C14-GFP, N2C10-GFP, N3C10-GFP, and N4C11-GFP. The nucleotide sequences of the progressively truncated fragments of PTS2-40AA at the N-terminus and C-terminus are shown in Table 2 below.

[0087] E1.11 was similar to Example 1E1.2-7. The fragment obtained in E1.10 was cloned by homologous recombination between the EcoRV and SpeI restriction enzyme sites to construct the pZPK-Ntc-pXYL-EcoRV-PTS2-EGFP-SpeI-tHSP recombinant plasmid, which was then transformed into Agrobacterium spp. By infecting ΔCar-np11 and gradually passaged, strains N0C10, N0C12, N0C14, N2C10, N3C10, and N4C11 were obtained.

[0088] Table 2 Nucleotide sequences of different truncations of PTS2-40AA

[0089]

[0090] E1.12 The soil bacillus obtained in step E1.8 and the transformants in step E1.11 were infected and passaged, and characterized by biological laser confocal microscopy. Figure 1As shown, the N-terminus of PTS2-40AA can truncate up to 4 amino acids, and the C-terminus can truncate up to 11 amino acids, namely N4C11, which can still localize EGFP to the peroxisome.

[0091] Example 2: Characterization of type I peroxisome targeting peptide (PTS1)

[0092] E2.1 selected four combinations based on the general sequence of peroxisome type I targeting peptides [S / A / C]-[K / R / H]-[L / M], and sequentially expressed them in fusion with green fluorescent proteins. Fluorescence detection was performed using R. toruloides as the host. The four green fluorescent proteins characterized by PTS1 fusion were: EGFP-SKL, EGFP-AKL, EGFP-AKM, and EGFP-ARL.

[0093] In E2.2, using SKL as an example, primers EcoRV-EGFP-F and EcoRV-EGFP-F were used to amplify the EGFP gene fragment from E2.1 by PCR. The desired tripeptide gene fragment was then extended to its C-terminus. PCR conditions were 98°C pre-denaturation for 5 minutes, followed by 30 cycles of denaturation at 98°C for 10 seconds, annealing at 57°C for 5 seconds, and extension at 72°C for 5 minutes. This yielded an EGFP-SKL gene fragment with restriction sites at both ends.

[0094] E2.3 Prepare Escherichia coli DH5α competent cells, and select the plasmid pZPK with nourseothricin resistance as its expression vector. The above-mentioned gene fragment EGFP-SKL was cloned between the restriction enzyme sites EcoRV and SpeI by enzyme digestion and ligation, and then heat-shocked into Escherichia coli DH5α competent cells at 42°C. After a single colony grew on the LB screening solid plate containing the corresponding resistance, colony PCR verification was performed using the upstream primer EGFP-F of the target gene and the downstream primer tHSP-R of the terminator. The transformants with correct bands were inoculated into 5 mL of LB medium with the corresponding resistance and cultured for 12 hours. The corresponding plasmid was extracted and sequenced for verification to obtain the pZPK-Ntc-pXYL-EcoRV-EGFP-SKL-SpeI-tHSP recombinant plasmid.

[0095] Among them, Ntc is the nourseothricin resistance gene, and the amino acid sequence of its encoded protein is GenBank: AAS47018.1.

[0096] E2.4 Prepare soil Bacillus competent cells, and electroporate the pZPK-Ntc-pXYL-EcoRV-EGFP-SKL-SpeI-tHSP recombinant plasmid constructed in step E2.3 into the soil Bacillus competent cells. After a single colony grows on the LB screening solid plate containing the corresponding resistance, use the upstream primer EGFP-F of the target gene and the downstream primer tHSP-R of the terminator to perform colony PCR verification. The verified correct soil Bacillus single colony is inoculated into 5 mL of LB medium with 0.1% kanamycin and cultured for 16-18 hours. The corresponding plasmid is extracted and enzyme digested again with restriction endonucleases EcoRV and SpeI for verification.

[0097] The primers used in the above experimental process are shown in Table 3 below:

[0098] Table 3 Primer sequence list

[0099]

[0100] E2.5 Inoculate the single colony of Bacillus subtilis verified correctly in step E2.4 into 5 mL LB liquid culture medium containing 0.1% corresponding antibiotics and culture for about 16 hours to OD600 = 0.4-0.6. At the same time, inoculate the R. toruloides pigment production-deficient bacteria ΔCar-np11 into YPD liquid culture medium and culture for 18 hours to OD600 = 0.4-0.6. Wash the bacteria twice with sterile water and resuspend them. Take 100 μL of the bacterial solution from each and mix them. Then drop them onto sterile filter paper on the IM induction plate. After culturing at 25°C for 48 hours, transfer them to YPD plates containing nourseothricin resistance and cephalosporin and continue culturing at 30°C for 48 hours.

[0101] E2.6 Pick as many single colonies as possible from step E2.5 and subculture them multiple times on screening plates for verification. Finally, the correct single colony is the correct genetically engineered bacterium and is named EGFP-SKL (genotype is np11-pXYL-EGFP-SKL-tHSP).

[0102] E2.7 Perform preliminary screening of the transformants selected in step E2.6 using an automated fluorescence microscope, and select transformants with a dot-like fluorescence distribution.

[0103] E2.8 is similar to Example 1E1.8-9. The soil abscessus obtained in step E1.8 and the transformant in E2.7 were infected and passaged, and characterized and identified by biological laser confocal microscopy.

[0104] E2.8 is similar to Example 2E2.2-8. The EGFP-AKL, EGFP-AKM and EGFP-ARL engineered bacteria were constructed using the primers in Table 3. After infection and passage, Figure 1As shown, SKL, AKL, ARL and AKM are all able to target EGFP to peroxisomes. The nucleotide sequences of SKL, AKL, ARL and AKM are shown in Table 4 below:

[0105] Table 4 Sequence list of type I peroxisome targeting peptides

[0106] SEQ ID NO.8-1 SKL TCGAAGCTC SEQ ID NO.8-2 AKL GCGAAGCTG SEQ ID NO.8-3 AKM GCGAAGATG SEQ ID NO.8-4 ARL GCGCGGCTG

[0107] Example 3: Verification of the effect of proline on PTS2 targeting function

[0108] E3.1 Based on the polycistronic transcriptional coding mechanism mediated by the 2A linker peptide and the results of Example 1, the expression plasmid pZPK-N4C11 obtained in Example 1E1.10 was used as a template and the primers in Table 4 were used to clone the Pro-N4C11-EGFP fragment with proline added to the N-terminus. The amino acid sequence of the 2A linker peptide sequence is shown in SEQ ID NO. 9.

[0109] E3.2: Similar to Example 1E1.2-7, the fragment obtained in E3.1 was cloned by homologous recombination between the restriction enzyme sites EcoRV and SpeI to construct the recombinant plasmid pZPK-Ntc-pXYL-EcoRV-Pro-N4C11-EGFP-SpeI-tHSP. This recombinant plasmid was then transformed into Agrobacterium spp. Pro-N4C11 was obtained by infection with ΔCar-np11 and subsequent passage.

[0110] E3.3 The soil bacillus obtained in step E1.8 and the transformants in E3.2 were infected and passaged, and characterized by biological laser confocal microscopy. Figure 1 As shown, the resulting localization of PTS2(N4C11) was not affected by proline.

[0111] The primers used in the above experimental process are shown in the table below:

[0112] Table 5 Primer sequence list

[0113]

[0114] Example 4: Construction of PTSs-mediated limonene engineering strain

[0115] E4.1 The two types of PTS2 and PTS1 with good targeting functions obtained according to Examples 1-3 can be introduced into peroxisomal expression through a multi-gene pathway mediated by a 2A connecting peptide.

[0116] In order to facilitate the construction of vectors for multi-gene localization to peroxisomes, E4.2 was synthesized by Suzhou Hongxun Biotechnology Co., Ltd. to obtain the auxiliary expression vector pUC-PTS2-EcoRV-P2A-PTS2-Stu1-P2A-SpeI-PTS1, which can quickly assemble multi-gene pathways localized to peroxisomes.

[0117] E4.3 optimized the exogenous gene based on the codon preference of R. toruloides. The amino acid sequence of the fusion protein NPPS-GGGS-LS gene, a protein derived from S. lycopersicum's nerol diphosphate synthase (NPPS) and C. limon's limonene synthase (CltLS), was cloned using primers PTS2-fu2-fu-NPPS-F and P2A-fu2-LS-R to generate the PTS2-NPPS-GGGS-LS fragment. The NCBI accession number for NPPS is NP_001234633.1; the NCBI accession number for CltLS is Q8L5K3.1.

[0118] E4.4 The recombinant plasmid pUC-PTS2-EcoRV-P2A-PTS2-Stu1-P2A-SpeI-PTS1 obtained in Example 4E4.2 was digested with Stu1 and then purified to obtain a linearized vector fragment.

[0119] E4.5 using TAKARA HD Cloning Kit: Ligate the linearized vector to the target gene fragment with homology arms to the vector insertion site. The specific operation steps are consistent with the kit instructions.

[0120] E4.6 is similar to Example 1E1.3. The recombinant plasmid was transformed into Escherichia coli DH5α competent cells and colony PCR verification was performed using primers pUC-F and pUC-R. Transformants containing the correct bands were inoculated into 5 mL of LB medium with 0.1% kanamycin and cultured for 12 h. The plasmid was extracted and sequenced to obtain the pUC-PTS2-EcoRV-P2A-PTS2-NPPS-GGGS-LS-P2A-SpeI-PTS1 recombinant plasmid.

[0121] E4.7 is similar to Example 4E4.3-6. The gene PTS2-tHMGR fragment was cloned using primers PTS2-full-tHMGR-F and P2A-full-HMGR-R. The recombinant plasmid obtained in E4.6 was digested with EcoRV and purified to obtain the vector. The linearized vector fragment was ligated using the HD Cloning Kit with the target gene fragment PTS2-tHMGR, which contained homology arms to the vector insertion site. The recombinant plasmid was transformed into Escherichia coli DH5α. After colony PCR and sequencing verification, the recombinant plasmid pUC-PTS2-tHMGR-P2A-PTS2-NPPS-GGGS-LS-P2A-SpeI-PTS1 was obtained.

[0122] E4.8 is similar to Example 4E4.3-6. The heterologous isopentenyl pyrophosphate isomerase PrIDI gene was cloned using primers P2A-fu3-PrIDI-F and Thsp-fu-skl-PrIDI-R to obtain PrIDI-SKL. The recombinant plasmid obtained in E4.7 was digested with SpeI and purified to obtain the vector. The linearized vector fragment was ligated using the HD Cloning Kit with the target gene fragment PrIDI-SKL, which contained homology arms to the vector insertion site. The recombinant plasmid was transformed into Escherichia coli DH5α and verified by colony PCR and sequencing to obtain the recombinant plasmid pUC-PTS2-tHMGR-P2A-PTS2-NPPS-GGGS-LS-P2A-PrIDI-PTS1.

[0123] E4.9 is similar to Example 1E1.2-6. Using the recombinant plasmid in E4.8 as a template, the fragment PTS2-tHMGR-P2A-PTS2-NPPS-GGGS-LS-P2A-PrIDI-PTS1 was cloned. The resulting fragment was cloned between the restriction enzyme sites EcoRV and SpeI of pZPK by homologous recombination to construct the pZPK-Ble-pXYL-PTS2-tHMGR-P2A-PTS2-NPPS-GGGS-LS-P2A-PrIDI-PTS1-tHSP recombinant plasmid, which was then transformed into soil abscesses. The R. toruloides pigment-producing deficient strain ΔCar-np11 was infected with the soil bacillus carrying the recombinant plasmid and serially passaged on YPD plates containing bleomycin resistance and cephalosporin, eventually obtaining the limonene-producing strain QD2 (genotype: ΔCar-np11-Ble-pXYL-PTS2-tHMGR-P2A-PTS2-NPPS-GGGS-LS-P2A-PrIDI-PTS1-tHSP).

[0124] E4.10 was similar to Example 4E4.3, using primers PTS2-fu2-SF and P2A-fu2-SR to clone the hydroxymethylglutaryl-CoA synthase mutant EfMvaS from E. faecalis. A110GThe nucleotide sequence of the gene containing acetyl-CoA acetyltransferase / 3-hydroxy-3-methylglutaryl-CoA reductase EfMvaE from E. faecalis was cloned using primers P2A-fu3-EF and SKL-fu3-ER.

[0125] E4.11 The expression vector pUC-PTS2-EcoRV-P2A-PTS2-Stu1-P2A-PTS2-Sca1-P2A-SpeI-PTS1 was digested with Sca1 and then purified to obtain a linearized vector fragment.

[0126] E4.12 is similar to Example 4E4.5-6, using TAKARA The HD Cloning Kit was used to ligate the linearized vector fragment obtained in Example 4E4.12 with the target gene fragment containing homology arms to the vector insertion site. The recombinant plasmid was transformed into E. coli DH5α cells and verified by colony PCR using primers for PUC-F and PUC-R. Transformants containing the correct band were inoculated into 5 mL of LB medium supplemented with 0.1% kanamycin and cultured for 12 hours. The plasmid was extracted and sequenced to obtain pUC-PTS2-EcoRV-P2A-PTS2-Stu1-P2A-PTS2-EfMvaS. A110G -P2A-SpeI-PTS1 recombinant plasmid.

[0127] E4.13 The expression vector pUC-PTS2-EcoRV-P2A-PTS2-Stu1-P2A-PTS2-EfMvaS A110G -P2A-SpeI-PTS1 was digested with SpeI and then purified to obtain a linearized vector fragment.

[0128] E4.14 is similar to Example 4E4.5-6, using TAKARA The HD Cloning Kit was used to ligate the linearized vector fragment obtained in Example 4E4.14 with the target gene fragment containing homology arms to the vector insertion site. The recombinant plasmid was transformed into E. coli DH5α competent cells and verified by colony PCR using primers for PUC-F and PUC-R. Transformants containing the correct band were inoculated into 5 mL of LB medium supplemented with 0.1% kanamycin and cultured for 12 hours. The plasmid was extracted and sequenced to obtain pUC-PTS2-EcoRV-P2A-PTS2-Stu1-P2A-PTS2-EfMvaS. A110G -P2A-EfMvaE-PTS1 recombinant plasmid.

[0129] E4.15 is similar to Example 4E4.3, using the recombinant plasmid pUC-PTS2-EcoRV-P2A-PTS2-Stu1-P2A-PTS2-EfMvaS A110G -P2A-EfMvaE-PTS1 was used as a template and primers xyl-fu-PTS2-SF and Thsp-fu-SKL-ER were used to obtain the fragment PTS2-EfMvaS A110G -E-PTS1.

[0130] E4.16 The expression vector pZPK-NTC-pXYL-EcoRV-SpeI-tHSP was double-digested with EcoRV and SpeI and then purified to obtain a linearized vector fragment.

[0131] E4.17 is similar to Example 4E4.5-6, using TAKARA The HD Cloning Kit was used to ligate the linearized vector fragment obtained in Example 4E4.11 with the target gene fragment containing homology arms to the vector insertion site. The recombinant plasmid was transformed into E. coli DH5α competent cells and verified by colony PCR using primers for XYL-F and tHSP-R. Transformants containing the correct band were inoculated into 5 mL of LB medium supplemented with 0.1% kanamycin and cultured for 12 hours. The plasmid was extracted and sequenced to obtain pZPK-NTC-pXYL-PTS2-EfMvaS. A110G -E-SKL-tHSP recombinant plasmid.

[0132] E4.18 was similar to Example 1E1.4-6. The strain QD2 obtained by infecting E4.9 with the soil bacillus carrying the E4.18 recombinant plasmid was serially passaged on YPD plates containing nourseothricin resistance and cephalosporin, and finally the high-yield limonene engineered strain QD3 (genotype QD2-NTC-pXYL-PTS2-EfMvaS A110G -E-SKL-tHSP).

[0133] E4.19 was similar to Example 4E4.3-9. The fragment PTS2-MmMK was cloned using primers PTS2-ful-MK-F and P2A-ful-MK-R, the fragment PTS2-PMK was cloned using primers PTS2-fu2-PMK-F and P2A-fu2-PMK-R, and the fragment PDC-SKL was cloned using primers P2A-fu3-PDC-F and Thsp-fu-sk1-PDC-R. The vector pUC-PTS2-MK-P2A-PTS2-PMK-P2A-PDC-PTS1 was constructed by homologous recombination and ligation. This vector was used as a template to clone the fragment PTS2-MK-P2A-PTS2-PMK-P2A-PDC-PTS1. The pZPK-G418-pXYL-PTS2-MK-P2A-PTS2-PMK-P2A-PDC-PTS1-tHSP recombinant plasmid was constructed by homologous recombination and transformed into Bacillus pyogenes to infect the QD2 strain. The strain was continuously passaged on YPD plates containing geneticin resistance and cephalosporin, and finally the limonene-producing strain QD4 (genotype: QD2-G418-pXYL-PTS2-MK-P2A-PTS2-PMK-P2A-PDC-PTS1-tHSP) was obtained. The soil Bacillus pyogenes carrying the E4.18 recombinant plasmid was used to infect the strain QD4 obtained by E4.19. The strain was continuously passaged on YPD plates containing nourseothricin resistance and cephalosporin, and finally the high-yield limonene engineered strain QD5 (genotype: QD4-NTC-pXYL-PTS2-EfMvaS A110G -E-SKL-tHSP).

[0134] E4.20: Similar to Example 4E4.3, the acetoacetyl-CoA synthase gene fragment T7 from Streptomyces qinzhouensis was cloned using primers PTS2-fu2-T7-F and P2A-fu2-T7-R. The NPPS-LS gene fragment was cloned using primers PTS2-fu1-NPPS-F and P2A-fu1-LS-R. The endogenous ATP-dependent citrate lyase gene fragment RtACL was cloned using primers PTS2-fu-RtACL-F and P2A-fu-RtACL-R.

[0135] E4.21 is similar to Example 4E4.11-12. The expression vector pUC-PTS2-EcoRV-P2A-PTS2-Stu1-P2A-PTS2-Sca1-P2A-SpeI-PTS1 is digested with the corresponding restriction sites and then purified to obtain a linearized vector fragment. The linearized vector was ligated with the target gene fragment with homology arms of the vector insertion site by homologous recombination to obtain the recombinant plasmids pUC-PTS2-EcoRV-P2A-PTS2-T7-P2A-PTS2-ScaI-P2A-SpeI-PTS1, pUC-PTS2-NPPS-LS-P2A-PTS2-Stu1-P2A-PTS2-ScaI-P2A-SpeI-PTS1, and pUC-PTS2-EcoRV-P2A-PTS2-RtACL-P2A-PTS2-ScaI-P2A-SpeI-PTS1.

[0136] E4.22 was similar to Example 4E4.15-18. The recombinant plasmids obtained in E4.21 were used as templates to clone the PTS2-T7, PTS2-NPPS-LS, and PTS2-RtACL fragments. A110G -E-SKL-tHSP recombinant plasmid was digested with EcoRV and ligated to PTS2-T7, PTS2-NPPS-LS, and PTS2-RtACL fragments to construct the recombinant plasmid pZPK-NTC-pXYL-PTS2-T7-PTS2-EfMvaS A110G -E-SKL-tHSP, pZPK-NTC-pXYL-PTS2-NPPS-LS-PTS2-EfMvaS A110G -E-SKL-tHSP, pZPK-NTC-pXYL-PTS2-RtACL-PTS2-EfMvaS A110G -E-SKL-tHSP.

[0137] E4.23 was similar to Example 1E1.4-6. The strain QD4 obtained by infecting E4.19 with the soil bacillus carrying the recombinant plasmid E4.22 was serially passaged on YPD plates containing nourseothricin resistance and cephalosporin, and finally the limonene-producing engineered strain QD6 (genotype QD4-NTC-pXYL-PTS2-RtACL-PTS2-EfMvaS A110G -E-SKL-tHSP), QD7 (genotype is QD4-NTC-pXYL-PTS2-T7-PTS2-EfMvaS A110G-E-SKL-tHSP), QD8 (genotype is QD4-NTC-pXYL-PTS2-NPPS-LS-PTS2-EfMvaS A110G -E-SKL-tHSP).

[0138] E4.24 is similar to Example 4E4.20-23, cloning fragments ACL-ACCT, PTS2-T7, and ligating them into the recombinant plasmid pZPK-NTC-pXYL-PTS2-NPPS-LS-PTS2-EfMvaS in E4.22. A110G -E-SKL-tHSP, and the recombinant plasmid pZPK-pXYL-ACL-ACCT-tHSP-pPGK-PTS2-T7-NTC-tNOS-pXYL-PTS2-NPPS-LS-PTS2-EfMvaS was obtained. A110G -E-SKL-tHSP. The strain QD4 obtained by infection at E4.19 was strain QD9 (genotype: QD4-pXYL-ACL-ACCT-tHSP-pPGK-PTS2-T7-NTC-tNOS-pXYL-PTS2-NPPS-LS-PTS2-EfMvaS A110G -E-SKL-tHSP).

[0139] The primers used in the above experimental process are shown in the table below:

[0140] Table 6 Primer sequence list

[0141]

[0142]

[0143] Table 6

[0144]

[0145]

[0146] Example 5: Fermentation of limonene using genetically engineered bacteria

[0147] The R. toruloides pigment production-deficient strain ΔCar-np11 and the engineered strain obtained in Example 4 were fermented in a 250 mL conical flask.

[0148] Specifically, a single colony of the strain was inoculated into 5 mL of MM medium (2% glucose, 0.5% (NH4)2SO4, 3% KH2PO4, 0.05% MgSO4·7H2O, 1 mL / L vitamin solution and 1 mL / L trace element solution, and water was added to 1 L). The seed solution was cultured in a 50 mL centrifuge tube at 28°C and 180 rpm / min. After fermentation for 24 hours, the seed solution was inoculated into 50 mL of MM medium in a 250 mL conical flask at an initial OD600 of 0.5. At the same time, 20% n-dodecane extractant was added for in situ extraction, and the mixture was cultured at 22°C and 180 rpm for 96 hours. After fermentation, the mixture was centrifuged at 9000 g for 1 minute, and the upper organic layer was taken for GC detection of limonene production. Specifically, the test results are as follows: Figure 2 As shown in Table 6, the limonene production engineered bacteria have strong stability.

[0149] Specifically, the sample QD5, the limonene standard, and the starting strain ΔCar-np11 were qualitatively tested by GCMS, and the test results were as follows: Figure 3 As shown in the chromatogram, the sample and standard peak at the same time, while the starting strain has no peak at the same time. The mass spectra of both the sample and standard exhibit distinct limonene-specific peaks at specific ions 68 and 93, while the starting strain ΔCar-np11 does not. This demonstrates that the genetically engineered strain QD5 is capable of heterologous de novo synthesis of limonene in peroxisomes.

[0150] Table 7 Limonene production by fermentation of limonene by engineered bacteria

[0151]

[0152] Example 6: High-density fermentation of genetically engineered bacteria to produce limonene

[0153] A single colony of strain QD9 prepared in Example 4 was inoculated into 50mL YPD (20% peptone, 20% glucose, 10% yeast extract powder, dissolved in 1L water) culture medium to prepare a first-level seed solution, which was cultured at 28°C and 180rpm for 12-16h. The first-level seed solution was inoculated into a YPD culture medium with a total volume of 200mL at a 5% inoculum size to prepare a second-level seed solution, which was cultured at 28°C and 180rpm for 12-16h. The second-level seed solution was inoculated into a 3L bioreactor with a working volume of 2L at a 10% volume ratio inoculation size, with a pH of 6.0 (regulated by 2M sodium hydroxide and 2M sulfuric acid), an aeration volume of 2L / min, a stirring speed of 400rpm, and a defoaming agent of 0.1%. The strain biomass, glucose concentration, and limonene production were monitored every 12h. The limonene detection method was the same as described above, and glucose was detected using the DNS method (3,5-dinitrosalicylic acid method). A semi-synthetic medium containing 50 g / L glucose was used as the initial medium. Feeding was started when the glucose concentration in the fermentation broth was lower than 5 g / L. The feed was linked to the dissolved oxygen concentration in the fermentation broth to maintain the dissolved oxygen at 20%.

[0154] Specifically, the composition of the semi-synthetic culture medium containing 50 g / L glucose is as follows: 50 g / L glucose, 10 g / L yeast extract, 5 g / L (NH₄)₂SO₄, 5 g / L MgSO₄·7H₂O, 2 g / L KH₂PO₄, 2 g / L Na₂HPO₄, 0.1% trace metal solution, and 0.1% vitamin solution, brought to 2 L with distilled water. The trace metal solution (per 100 mL) contains 0.45 g CaCl₂₂H₂O, 0.45 g ZnSO₄ₐH₂O, 0.3 g FeSO₄ₐH₂O, 0.1 g H₃BO₃, 0.1 g MnCl₂₄H₂O, 0.04 g Na₂MoO₄₂H₂O, 0.03 g CoCl₂₶H₂O, 0.01 g CuSO₄ₐH₂O, 0.01 g KI, and 1.5 g EDTA. The vitamin solution ( / 100 mL) contains 5 mg biotin, 20 mg p-aminobenzoic acid, 1 g niacin, 1 g calcium pantothenate, 1 g pyridoxine hydrochloride, 0.1 g thiamine hydrochloride (vitamin B1), and 2.5 g inositol.

[0155] Finally, glucose was used as the substrate and the fermentation was carried out in a 2L fermenter in a fed-batch manner for 168 h, with a yield of 4 g / L.

[0156] Example 7: Construction and fermentation of PTSs-mediated linalool engineering strains

[0157] E7.1 artificially synthesized the amino acid sequence of the codon-optimized fusion protein GPPS-GGGS-McLis gene, including, but not limited to, the geranyl diphosphate synthase GPPS from Abies grandis and the linalool synthase McLis from lemon mint. The fusion protein is connected by a GGGS flexible linker. The NCBI accession numbers for the GPPS and McLis fusion proteins are AAN01134.1 and Q8H2B4.1, respectively.

[0158] E7.2 The auxiliary vector pUC-PTS2-EcoRV-P2A-PTS2-Stu1-P2A-SpeI-PTS1 obtained in Example 4E4.2 was digested with EcoRV and purified to obtain a linearized vector fragment. The GPPS-GGGS-McLis gene fragment with homology arms was then ligated by homologous recombination to obtain pUC-PTS2-EcoRV-P2A-PTS2-GPPS-GGGS-McLis-P2A-SpeI-PTS1.

[0159] E7.3 is similar to Example 4E4.3-6, using TAKARA Using an HD Cloning Kit, ligate the linearized vector fragment obtained in Example 7E7.2 with the target gene fragment GPPS-GGGS-McLis, containing homology arms to the vector insertion site, in Example 7E7.1. Transform the recombinant plasmid into competent E. coli DH5α cells and verify by colony PCR using primers PUC-F and PUC-R. Transformants containing the correct band were inoculated into 5 mL of LB medium supplemented with 0.1% kanamycin and cultured for 12 hours. The plasmid was extracted and verified by sequencing to obtain the recombinant plasmid pUC-PTS2-EcoRV-P2A-PTS2-GPPS-GGGS-McLis-P2A-SpeI-PTS1.

[0160] E7.4 was similar to Example 4E4.7-8, and the fragments PTS2-tHMGR and PrIDI-SKL were respectively inserted into the E7.3 recombinant plasmid pUC-PTS2-EcoRV-P2A-PTS2-GPPS-GGGS-McLis-P2A-SpeI-PTS1 to obtain the pUC-PTS2-tHMGR-P2A-PTS2-GPPS-GGGS-McLis-P2A-PrIDI-SKL recombinant plasmid.

[0161] E7.5 was similar to Example 4E4.9. Using the recombinant plasmid in E7.4 as a template, the fragment PTS2-tHMGR-P2A-PTS2-GPPS-GGGS-McLis-P2A-PrIDI-SKL was cloned. The resulting fragment was cloned between the restriction enzyme sites EcoRV and SpeI of pZPK by homologous recombination to construct the pZPK-Ble-pXYL-PTS2-tHMGR-P2A-PTS2-GPPS-GGGS-McLis-P2A-PrIDI-SKL-tHSP recombinant plasmid, which was then transformed into Agrobacterium spp. The R. toruloides pigment-production-deficient strain ΔCar-np11 was infected with the recombinant plasmid-carrying Bacillus subtilis. The strain was serially passaged on YPD plates containing bleomycin resistance and cephalosporin, ultimately obtaining the high-linalool-producing engineered strain FZ2 (genotype: ΔCar-np11-Ble-pXYL-PTS2-tHMGR-P2A-PTS2-GPPS-GGGS-McLis-P2A-PrIDI-SKL-tHSP).

[0162] E7.6 is similar to Example 4E4.9. The pZPK-NTC-pXYL-PTS2-EfMvaS A110G -E-SKL-tHSP and soil abscesses carrying the pZPK-G418-pXYL-PTS2-MK-P2A-PTS2-PMK-P2A-PDC-PTS1-tHSP recombinant plasmids in E4.19 successively infected the FZ2 strain, and the engineered strain FZ3 was obtained after stable subculture.

[0163] The strain ΔCar-np11 and the strain FZ3 prepared in Example 7E7.6 were fermented in 250 mL conical flasks.

[0164] Specifically, a single colony of the strain was inoculated into 5 mL of MM medium and cultured at 28°C, 180 rpm / min. After 24 hours of fermentation, the strain was transferred to 50 mL of MM medium at an initial OD600 of 0.5. In situ extraction was performed with the addition of 20% isopropyl myristate. The strain was then cultured at 28°C, 180 rpm, for 72–120 hours. At the end of fermentation, the strain was centrifuged at 9000 g for 1 minute, and the upper organic phase was collected and analyzed for linalool production by GC.

[0165] Similar to Example 6, glucose was used as the substrate and the fermentation was carried out in a 2 L fermentor in a fed-batch manner for 168 h, with a yield of 5 g / L.

[0166] Example 8: Construction and fermentation of PTSs-mediated patchouli alcohol engineering strain

[0167] The synthetic E8.1 protein includes, but is not limited to, the amino acid sequence of the FPPS-GGGS-PT gene, a fusion protein of the endogenous farnesyl pyrophosphate synthase (FPPS) from R. toruloides and the codon-optimized patchouli alcohol (PTS) from Patchouli. The amino acid sequences of the fusion protein are connected by a GSG flexible linker. The NCBI accession numbers for FPPS and PT are XP_016272719.1 and Q49SP3.1, respectively.

[0168] E8.2 is similar to Example 7E7.2-4, and the recombinant plasmid pUC-PTS2-tHMGR-P2A-PTS2-FPPS-GGGS-PT-P2A-PrIDI-SKL is constructed using an auxiliary vector.

[0169] E8.3 is similar to Example 7E7.5. Using the recombinant plasmid in E8.2 as a template, the fragment PTS2-tHMGR-P2A-PTS2-FPPS-GGGS-PT-P2A-PrIDI-SKL was cloned. The resulting fragment was cloned between the restriction enzyme sites EcoRV and SpeI of pZPK by homologous recombination to construct the pZPK-Ble-pXYL-PTS2-tHMGR-P2A-PTS2-FPPS-GGGS-PT-P2A-PrIDI-SKL-tHSP recombinant plasmid, which was then transformed into soil abscesses. The R. toruloides pigment-production-deficient strain ΔCar-np11 was infected with the soil abscess carrying the recombinant plasmid and serially passaged on YPD plates containing bleomycin resistance and cephalosporin, eventually obtaining the high-yield patchouli alcohol engineered strain GH2 (genotype: ΔCar-np11-pZPK-Ble-pXYL-PTS2-tHMGR-P2A-PTS2-FPPS-GGGS-PT-P2A-PrIDI-SKL-tHSP).

[0170] E8.4 is similar to Example 7E7.6. The pZPK-NTC-pXYL-PTS2-EfMvaS A110G -E-SKL-tHSP and soil abscesses carrying the pZPK-G418-pXYL-PTS2-MK-P2A-PTS2-PMK-P2A-PDC-PTS1-tHSP recombinant plasmids in E4.19 successively infected the GH2 strain, and the engineered bacteria GH3 were obtained after stable subculture.

[0171] The strain ΔCar-np11 and the strain GH3 prepared in Example 8E8.4 were fermented in 250 mL conical flasks.

[0172] Specifically, a single colony of the strain was inoculated into 5 mL of MM medium and cultured at 28°C, 180 rpm / min. After 24 hours of fermentation, the strain was transferred to 50 mL of MM medium at an initial OD600 of 0.5. In situ extraction was performed with the addition of 20% n-dodecane. The strain was then cultured at 28°C, 180 rpm, for 72-120 hours. At the end of fermentation, the strain was centrifuged at 9000 g for 1 minute, and the supernatant was collected for GC analysis of patchouli alcohol production.

[0173] Similar to Example 6, glucose was used as the substrate and the fermentation was carried out in a 2 L fermentor in a fed-batch manner for 168 h, with a yield of 4 g / L.

[0174] Example 9: Construction and fermentation of PTSs-mediated kaurene engineering strains

[0175] The E9.1 synthetic protein includes, but is not limited to, the amino acid sequence of the fusion protein FPPS-GGGS-GfKS gene, which is a fusion protein of the endogenous farnesyl-farnesyl pyrophosphate synthase (GGPPS) from R. toruloides and a codon-optimized kaurene synthase (GfKS) from Gibberella fujikura. The amino acid sequences of the fusion protein are connected by a GSG flexible linker. The corresponding NCBI accession numbers for GGPPS and GfKS are XP_016271675.1 and XP_023431478.1, respectively.

[0176] E9.2 is similar to Example 7E7.2-4, and the recombinant plasmid pUC-PTS2-tHMGR-P2A-PTS2-FPPS-GGGS-GfKS-P2A-PrIDI-SKL is constructed using an auxiliary vector.

[0177] E9.3 is similar to Example 7E7.5. Using the recombinant plasmid in E9.2 as a template, the fragment PTS2-tHMGR-P2A-PTS2-FPPS-GGGS-GfKS-P2A-PrIDI-SKL was cloned, and the resulting fragment was cloned between the restriction enzyme sites EcoRV and SpeI of pZPK by homologous recombination to construct the pZPK-Ble-pXYL-PTS2-tHMGR-P2A-PTS2-FPPS-GGGS-GfKS-P2A-PrIDI-SKL-tHSP recombinant plasmid, which was then transformed into soil abscesses. The R. toruloides pigment-production-deficient strain ΔCar-np11 was infected with soil abscesses carrying the recombinant plasmid and serially passaged on YPD plates containing bleomycin resistance and cephalosporin, ultimately obtaining the high-kaurene-producing engineered strain BK2 (genotype: ΔCar-np11-pZPK-Ble-pXYL-PTS2-tHMGR-P2A-PTS2-FPPS-GGGS-GfKS-P2A-PrIDI-SKL-tHSP).

[0178] E9.4 is similar to Example 7E7.6. The pZPK-NTC-pXYL-PTS2-EfMvaS A110G -E-SKL-tHSP and soil abscesses carrying the recombinant plasmids pZPK-G418-pXYL-PTS2-MK-P2A-PTS2-PMK-P2A-PDC-PTS1-tHSP in E4.19 successively infected the BK2 strain, and the engineered bacteria BK3 were obtained after stable subculture.

[0179] The strain ΔCar-np11 and the strain BK3 prepared in Example 8E9.4 were fermented in 250 mL conical flasks.

[0180] Specifically, a single colony of the strain was inoculated into 5 mL of MM medium and cultured at 28°C, 180 rpm / min. After 24 hours of fermentation, the strain was transferred to 50 mL of MM medium at an initial OD600 of 0.5. In situ extraction was performed with the addition of 20% n-dodecane. The culture was then incubated at 28°C, 180 rpm, for 72-120 hours. At the end of fermentation, the mixture was centrifuged at 9000 g for 1 minute, and the supernatant organic layer was collected for GC analysis of kaurene production.

[0181] Similar to Example 6, glucose was used as the substrate and the fermentation was carried out in a 2 L fermentor in a fed-batch manner for 168 h, with a yield of 2 g / L.

[0182] Example 10: Construction and fermentation of PTSs-mediated 3-hydroxypropionic acid engineering strains

[0183] The artificial synthesis of E10.1 includes but is not limited to the codon-optimized malonyl-CoA reductase (MCR) from Chloroflexus aurantiacus. The NCBI accession number of the malonyl-CoA reductase MCR is WP_012258473.1.

[0184] The gene fragment MCR in E10.1 was amplified using primer pair MTS1-fu-MCR-F and MCR-fu-P2A-R at E10.2, and the correct gene fragment was recovered by agarose gel electrophoresis.

[0185] E10.3 was similar to Example 4E4.3-6. The recombinant plasmid was heat-shocked into Escherichia coli DH5α competent cells and colony PCR verification was performed using primers XYL-F and tHSP-R. Transformants containing the correct bands were inoculated into 5 mL of LB medium supplemented with 5 μL of kanamycin and cultured for 12 h. The plasmid was extracted and sequenced to obtain the pZPK-Ble-pXYL-PTS2-MCR-tHSP recombinant plasmid.

[0186] E10.4 is similar to Example 4E4.9. The soil bacillus infection strain NP11 carrying the recombinant plasmid was serially passaged on YPD plates containing bleomycin resistance and cephalosporin, and finally a high-yield 3-hydroxypropionic acid engineered strain QJ1 (genotype: Ble-pXYL-PTS2-MCR-tHSP) was obtained.

[0187] The strain Rhodotorula np11 and the strain QJ1 prepared in Example 10E10.4 were fermented in 250 mL conical flasks.

[0188] Specifically, at the end of fermentation, analysis was performed using an HPLC (Shimadzu LC-20AT, Japan) equipped with a RID detector and a PDA detector and an Aminex HPX-87H column (Bio-Rad). Culture samples were centrifuged, and the supernatant was filtered through a 0.22 μm filter membrane using 0.5 mM H2SO4 as the eluent at a flow rate of 0.5 mL / min and a column temperature of 65°C for the measurement of 3-hydroxypropionic acid.

[0189] Similar to Example 6, glucose was used as the substrate and the fermentation was carried out in a 2 L fermentor in a fed-batch manner for 168 h, with a yield of 10 g / L.

[0190] Example 11: Construction and fermentation of PTSs-mediated daidzein engineering strain

[0191] The E11.1 synthetic enzymes include, but are not limited to, codon-optimized p-coumaryl-CoA ligase (At4CL) from Arabidopsis thaliana, chalcone synthase (VvS393) from grapevine, chalcone isomerase (MsCHI) from alfalfa, chalcone reductase (GmCHR) from soybean, 2-hydroxyisoflavone synthase (GeHIS) from Glycyrrhiza uralensis, and 2-hydroxyisoflavone dehydratase (GmHID) from soybean. The NCBI accession numbers for these key enzymes are: NP_175579.1, NP_001267879.1, XP_003592761.1, Q9SXS3.1, NP_001353935.1, and NP_001237228.1.

[0192] E11.2 is similar to Example 4E4.11-15, and the auxiliary vector is used to construct the pUC-PTS2-At4CL-P2A-PTS2-VvS393-P2A-PTS2-MsCHI and pUC-PTS2-GmCHR-P2A-PTS2-GeHIS-P2A-PTS2-GmHID recombinant plasmids.

[0193] E11.3 is similar to Example 4E4.16-18. The recombinant plasmids pUC-PTS2-At4CL-P2A-PTS2-VvS393-P2A-PTS2-MsCHI and pUC-PTS2-GmCHR-P2A-PTS2-GeHIS-P2A-PTS2-GmHID are used as templates to clone the fragments PTS2-At4CL-P2A-PTS2-VvS393-P2A-PTS2-MsCHI and PTS2-GmCHR-P2A-PTS2-GeHIS-P2A-PTS2-GmHID. The binary expression vectors pZPK-NTC-pXYL-PTS2-At4CL-P2A-PTS2-VvS393-P2A-PTS2-MsCHI-tHSP and pZPK-BLE-pXYL-PTS2-GmCHR-P2A-PTS2-GeHIS-P2A-PTS2-GmHID-tHSP were constructed by enzyme digestion and ligation.

[0194] E11.4 is similar to Example 4E4.19. The abscessus carrying the expression vector obtained in E11.3 was sequentially infected into the wild R. toruloides strain np11. After continuous subculture on the corresponding resistance plate, the engineered strain DE1 producing daidzein was finally obtained (genotype: np11-NTC-BLE-pXYL-PTS2-At4CL-P2A-PTS2-VvS393-P2A-PTS2-MsCHI-tHSP-pXYL-PTS2-GmCHR-P2A-PTS2-GeHIS-P2A-PTS2-GmHID-tHSP).

[0195] Similar to Example 6, glucose was used as the substrate and the fermentation was carried out in 2LYPD medium in a fed-batch manner for 240 h. The yield reached 30 mg / L, which was twice the yield of cytoplasmic synthesis.

[0196] Example 12: Construction and fermentation of PTSs-mediated genistein-producing strains

[0197] The E11.1 synthetic enzymes include, but are not limited to, codon-optimized p-coumaryl-CoA ligase (At4CL) from Arabidopsis thaliana, chalcone synthase (VvS393) from grapevine, chalcone isomerase (MsCHI) from alfalfa, 2-hydroxyisoflavone synthase (GeHIS) from Glycyrrhiza uralensis (Glycyrrhiza uralensis) and 2-hydroxyisoflavone dehydratase (GmHID) from soybean. The NCBI accession numbers for these key enzymes are: NP_175579.1, NP_001267879.1, XP_003592761.1, NP_001353935.1, and NP_001237228.1.

[0198] E11.2 is similar to Example 4E4.11-15, using auxiliary vectors to construct pUC-PTS2-At4CL-P2A-PTS2-VvS393-P2A-PTS2-MsCHI and pUC-PTS2-GeHIS-P2A-PTS2-GmHID recombinant plasmids.

[0199] E11.3 was similar to Example 4E4.16-18. Using the recombinant plasmids pUC-PTS2-At4CL-P2A-PTS2-VvS393-P2A-PTS2-MsCHI and pUC-PTS2-GeHIS-P2A-PTS2-GmHID as templates, the fragments PTS2-At4CL-P2A-PTS2-VvS393-P2A-PTS2-MsCHI and PTS2-GeHIS-P2A-PTS2-GmHID were cloned. The binary expression vectors pZPK-NTC-pXYL-PTS2-At4CL-P2A-PTS2-VvS393-P2A-PTS2-MsCHI-tHSP and pZPK-BLE-pXYL-PTS2-GeHIS-P2A-PTS2-GmHID-tHSP were constructed by enzyme digestion and ligation.

[0200] E11.4 is similar to Example 4E4.19. The abscessus carrying the expression vector obtained in E11.3 was sequentially infected into the wild R. toruloides strain np11. After continuous subculture on the corresponding resistance plate, the genistein-producing engineered strain GE1 (genotype: np11-NTC-BLE-pXYL-PTS2-At4CL-P2A-PTS2-VvS393-P2A-PTS2-MsCHI-tHSP-pXYL-PTS2-GeHIS-P2A-PTS2-GmHID-tHSP) was finally obtained.

[0201] Similar to Example 6, glucose was used as the substrate and the fermentation was carried out in 2LYPD medium in a fed-batch manner for 240 h. The yield reached 90 mg / L, which was twice the yield of cytoplasmic synthesis.

[0202] Example 13: Construction and fermentation of PTSs-mediated coenzyme Q10 engineered strains

[0203] The synthetic E13.1 enzymes include, but are not limited to, codon-optimized decaprenyl diphosphate synthase (ddsA) and p-hydroxybenzoate octenyltransferase (ubiA) from Escherichia coli and derived from Rhodococcus sp. The NCBI accession number for decaprenyl diphosphate synthase is API81680.1, and the NCBI accession number for p-hydroxybenzoate octenyltransferase is EHS5572144.1. The codon-optimized sequences are SEQ ID NOs. 11-12, respectively.

[0204] The NCBI accession numbers of the endogenous ubiquinone biosynthetic monooxygenase COQ4-10 in E13.2 R. toruloides are: RHTO_06687, RHTO_06918, RHTO_05146, RHTO_01107, RHTO_03730, RHTO_02142 and RHTO_07367, respectively.

[0205] E13.3 is similar to Example 4E4.11-15, except that the auxiliary vector is used to construct the pUC-PTS2-ddsA-P2A-PTS2-ubiA recombinant plasmid.

[0206] E13.4. Similar to Example 4E4.16-18, using the recombinant plasmid from E12.4 as a template, clone the fragment PTS2-ddsA-P2A-PTS2-ubiA. Then, construct the binary expression vector pZPK-NTC-pXYL-PTS2-ddsA-P2A-PTS2-ubiA-tHSP by enzyme digestion and ligation.

[0207] E13.5 is similar to Example 4E4.19. The soil bacillus carrying the expression vector obtained in E13.4 was sequentially infected into the wild R. toruloides strain np11. After continuous subculture on the corresponding resistance plates, the coenzyme Q10-producing engineered strain Q10-1 (genotype: np11-NTC-pXYL-PTS2-ddsA-P2A-PTS2-ubiA-tHSP) was finally obtained.

[0208] E13.6 is similar to Example 13E13.3-5, except that the endogenous fragment in E13.2 is introduced into the peroxisome of the Q10-1 strain for expression, resulting in the engineered bacterium Q10-2 capable of producing coenzyme Q10.

[0209] Similar to Example 6, glucose was used as the substrate and the fermentation was carried out in 2LYPD medium in a fed-batch manner for 240 h, with a yield of 500 mg / L.

[0210] Example 14: Application of a peroxisomal signal peptide in other red yeasts

[0211] R. glutinis, R. acheniorum, R. graminis, R. marina, R. mucilaginosa, R. rubra, R. lactosa, R. sphaerocarpum and R. bogoriensis were used as host strains to express the recombinant plasmid pZPK-LDP-HpaI-tHMGR-SKL-KpnI-SV40-pPGK-Ble-Tnos-pGPD-Eco RV-NPPS-GGGS-LS-SKL-t35-pACC-StuI-PrIDI-SKL-SpeI-tHSP, pZPK-LDP-HpaI-MmMK-SKL-KpnI-SV40-pPGK-G418-Tnos-pGPD-EcoRV-P MK-SKL-t35-pACC-StuI-PDC-SKL-SpeI-Thsp, pZPK-pXYL-ACL-ACCT-tHSP-pPGK-PTS2-T7-NTC-tNOS-pXYL-PTS2-NPPS-LS-PTS2-EfMvaS A110G -E-SKL-tHSP.

[0212] The method for constructing the genetically engineered bacteria is as shown in Example 4, and limonene is extracted and detected as shown in Example 5. The obtained strains can all produce a certain amount of limonene.

[0213] The embodiments described above are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims. The above are only a few embodiments of the present application, and are not intended to limit the present application in any form. Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the present application. Any technician familiar with the profession, without departing from the scope of the technical solution of the present application, using the above disclosed technical content to make some changes or modifications are equivalent to equivalent implementation cases and are within the scope of the technical solution.

Claims

1. A peroxisome signal peptide, characterized in that The peroxisomal signal peptide is derived from endogenous 3-ketoacyl-CoA thiolase of Rhodosporidium toruloides. The NCBI accession number of the 3-ketoacyl-CoA thiolase gene is XM_016420776.

2. The peroxisome signal peptide according to claim 1, characterized in that The amino acid sequence of the peroxisome signal peptide is shown in SEQ ID NO.

1.

3. A peroxisome signal peptide gene according to claim 1, characterized in that: The N-terminal type II peroxisome signal peptide gene has a GC content of 55-75%, and can play a signal peptide positioning function even when truncated to a gene sequence shown in SEQ ID NO. 2-7.

4. An auxiliary carrier, characterized in that The auxiliary vector contains at least the gene of the type II peroxisome signal peptide, and the amino acid and corresponding nucleotide sequences of the type I peroxisome signal peptide are SEQ ID NO.8-1, SEQ ID NO.8-2, SEQ ID NO.8-3, and SEQ ID NO.8-4.

5. The auxiliary carrier according to claim 4, characterized in that The auxiliary vector is a recombinant plasmid.

6. The auxiliary carrier according to claim 4, characterized in that The auxiliary vectors for type II and type I peroxisomal signal peptide-mediated expression of target biosynthetic pathway genes can be used to rapidly construct peroxisomal-targeted biosynthetic pathway expression cassettes, and the expression cassettes are based on 2A peptides to achieve rapid multicistronic assembly; the amino acid sequence of the 2A peptide is SEQ ID NO.9, and the auxiliary vector sequence is SEQ ID NO.

10.

7. An engineered bacterium, characterized in that: Comprising the auxiliary vector according to any one of claims 4 to 6.

8. The engineered bacteria according to claim 7, characterized in that The type II peroxisome signal peptide is used alone or in combination with a type I signal peptide to assist in the construction of an engineered bacterium containing, but not limited to, terpene substances; the terpene substance includes one of limonene, linalool, and patchouliol; the flavonoid substance is one of daidzein and genistein; the organic acid is 3-hydroxypropionic acid; and the coenzyme is coenzyme Q10.

9. The engineered bacteria according to claim 8, characterized in that The engineered bacteria is red yeast, which is a species in the genus Rhodotorula, including but not limited to toruloides red yeast R. toruloides, glutinis red yeast R. glutinis, spherical red yeast R. acheniorum, graminis red yeast R. marina, mucilaginosa red yeast R. mucilaginosa, deep red yeast R. rubra, lactose red yeast R. lactosa, R. sphaerocarpum, and Bogor red yeast R. bogoriensis.

10. Use of the engineered bacteria according to any one of claims 7 to 9 in fermentation production.

Images