Group of mitochondrial signal peptides and application thereof

Abstract

The invention discloses a group of mitochondrial signal peptides and application thereof, and a group of signal peptides with a mitochondrial targeting function is obtained for the first time by screening endogenous mitochondrial key functional proteins of rhodotorula. The invention further realizes expression of a target gene through mitochondrial signal peptide mediation, realizes expression of a target product synthetic pathway gene in rhodotorula mitochondria, and successfully constructs synthetic pathways of terpene such as limonene, linalool, patchouli alcohol and kaurene, branched chain alcohol such as isobutanol and organic acid such as 3-hydracrylic acid. Further, carbon metabolic flow of pyruvic acid flowing to the target product is dredged, and synthesis of the mitochondrial region indoor target product is finally realized. The characterization of a group of rhodotorula mitochondrial signal peptides disclosed by the invention can realize the synthesis of mitochondrial regional chamber target products, and lays a foundation for the research on the realization of metabolic engineering transformation in subcellular organelles.

Description

Technical Field

[0001] The invention relates to a group of mitochondrial signal peptides and applications thereof, and belongs to the field of genetic engineering. Background Art

[0002] With the rapid development of synthetic biology and metabolic engineering, the successful construction of green, environmentally friendly and sustainable microbial cell engineering has attracted widespread attention. Although most engineered metabolic pathways are located in the yeast cytoplasm, compartmentalization engineering that redirects the entire metabolic pathway to subcellular organelles also represents an attractive metabolic engineering strategy. Mitochondria, as the power source of cells, are generally considered to be the most promising subcellular organelle compartmentalization engineering. Recombinant mitochondrial entire metabolic engineering has important advantages in utilizing a rich precursor pool (such as pyruvate, acetyl-CoA, ATP, NADH and fatty acyl-CoA, etc.), a stronger redox microenvironment, bypassing competitive pathways, and minimizing transport restrictions. In order to achieve mitochondrial compartmentalization, an N-terminal mitochondrial targeting sequence is required to relocate the targeted enzyme to the mitochondria. It has been reported that the repeated use of the same targeting sequence to target the entire metabolic pathway gene to the mitochondria can easily lead to pathway gene instability or competition problems. Therefore, in order to expand the application of mitochondrial compartmentalization engineering in multi-gene biosynthetic pathways, it is of great significance to explore and screen more efficient targeting sequences. Currently, mitochondrial compartmentalization has been shown in Saccharomyces cerevisiae to improve the production of a series of high value-added compounds, such as valencene, isobutanol, isoprene, etc. However, no signal peptide sequence with mitochondrial targeting function has been characterized in Rhodotorulatoruloides, which limits the application of compartmentalization engineering in the mitochondrial multigene biosynthetic pathway of Rhodotorula.

[0003] At present, the production of terpenoids such as limonene, 1,8-cineole, kaurene and carotenoids, as well as oil derivatives such as fatty alcohols and fatty acids, has been achieved in the cytoplasm of Rhodosporidium toruloides. At the same time, as a non-traditional yeast, R. toruloides np11 also has the following advantages: (1) as a unicellular microorganism, it is easy to grow rapidly using simple culture media; (2) as a haploid, it is easy to be genetically engineered and screened; (3) it has the ability to efficiently synthesize acetyl-CoA; (4) it has a wealth of omics analysis technologies, etc., providing a strain platform for the microbial production of target products. However, the genetic characteristics of R. toruloides itself cannot effectively synthesize limonene, linalool, patchouli alcohol, kaurene, isobutanol and 3-hydroxypropionic acid, etc., so metabolic engineering strategies are needed. By simultaneously introducing the target product synthesis pathway gene fused with the mitochondrial signal peptide sequence into the mitochondria of R. toruloides cells, the metabolic flow of the pyruvate node is promoted to the synthesis of the target product, so as to achieve the synthesis of the target product in the mitochondria of R. toruloides. However, the functional characterization and identification of mitochondrial signal peptide sequences in R. toruloides and the study of compartmentalized production of related products have not been reported. Summary of the invention

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

[0005] The first technical problem to be solved by the present invention is to characterize a group of signal peptides with mitochondrial targeting function.

[0006] To solve the above problems, the technical solution of the present invention is:

[0007] 1) Screened a group of signal peptide genes from Rhodosporidium toruloides with potential mitochondrial targeting function;

[0008] 2) predicting the effective sequences of the group of potential mitochondrial signal peptides;

[0009] 3) Cloning the EGFP gene fused with the effective sequence of mitochondrial signal peptide;

[0010] 4) Connecting the amplified MTS-EGFP gene to the expression vector to obtain a recombinant expression vector;

[0011] 5) introducing the recombinant expression vector obtained in step 4) into the Rhodosporidium toruloides recipient bacteria;

[0012] 6) Verify the targeting function of the group of mitochondrial signal peptides, such as MTS2, MTS3, MTS4 and MTS10.

[0013] A group of mitochondrial signal peptides (MTSs for short), wherein the mitochondrial signal peptides are selected from the following 11 mitochondrial signal peptide sequences: the NCBI accession numbers corresponding to the 11 mitochondrial signal peptides are: EMS19018.1 (MTS1), EMS19424.1 (MTS2), EMS19541.1 (MTS3), EMS20697.1 (MTS4), EMS20271.1 (MTS5), EMS21738.1 (MTS6), EMS25036.1 (MTS7), EMS25048.1 (MTS8), EMS26107.1 (MTS9), RHTO_07719 (MTS10), and XP_016275910.1 (MTS11).

[0014] The amino acid sequence of the mitochondrial signal peptide is shown in SEQ ID NO.1-11.

[0015] A gene of the mitochondrial signal peptide, wherein the GC content of the gene nucleotide sequence of the mitochondrial signal peptide is 50-75%.

[0016] A recombinant expression vector comprising the gene nucleotide sequence of the mitochondrial signal peptide.

[0017] Preferably, the recombinant expression vector contains at least one mitochondrial signal peptide gene nucleotide sequence.

[0018] Preferably, the recombinant expression vector is a recombinant plasmid.

[0019] An engineered bacterium for over-expressing a target product synthesis pathway gene mediated by a mitochondrial signal peptide is constructed, wherein the engineered bacterium comprises a recombinant expression vector of the gene nucleotide sequence of the mitochondrial signal peptide.

[0020] Preferably, the engineered bacteria contain at least one recombinant expression vector.

[0021] An application of mitochondrial signal peptides, using the mitochondrial signal peptides alone or in combination to construct engineered bacteria containing but not limited to limonene, linalool, patchouli alcohol, kaurene, isobutanol and 3-hydroxypropionic acid, to achieve the synthesis of mitochondrial compartmentalized target products.

[0022] Furthermore, the polycistronic nucleotide sequence of the target product synthesis pathway gene is connected through a 2A connecting peptide gene, and the 2A connecting peptide amino acid sequence is as shown in SEQ ID NO.13-14.

[0023] The engineered bacteria is red yeast, which is a species in the genus Rhodotorula, including but not limited to Rhodotorula toruloides, Rhodotorula glutinis, Rhodotorula mucilaginosa, Rhodotorula graminis, Rhodotorula rubra, Rhodotorula lactosa, Rhodotorula sphaerocarpum, Rhodotorula marina, Rhodotorula acheniorum and Rhodotorula bogoriensis.

[0024] The second technical problem to be solved by the present invention is to provide the application of the group of mitochondrial signal peptides (taking limonene products as an example).

[0025] To solve the above problems, the technical solution of the present invention is:

[0026] 1) Cloning a 3-methyl 3-hydroxyglutaryl-CoA synthase mutant EfMvaS from Enterococcus faecalis that is fused with the gene nucleotide sequence of the mitochondrial signal peptide MTS2 and has been codon-optimized A110G The nucleotide sequence of the gene;

[0027] 2) The codon-optimized enhanced green fluorescent protein (EGFP) gene from jellyfish was cloned;

[0028] 3) cloning the gene nucleotide sequence fused with the mitochondrial signal peptide MTS10 and after codon optimization, including but not limited to the gene nucleotide sequence of acetyl-CoA acetyltransferase / 3-hydroxy-3-methylglutaryl-CoA reductase EfMvaE from E. faecalis;

[0029] 4) The codon-optimized nucleotide sequence of the red fluorescent protein (RFP) gene from the mushroom coral was cloned;

[0030] 5) cloning the gene nucleotide sequence of mevalonate kinase MmMK from Methanosarcina mazei, which is fused with the gene nucleotide sequence of mitochondrial signal peptide MTS3 and after codon optimization;

[0031] 6) cloning the gene nucleotide sequence fused with the mitochondrial signal peptide MTS4 and the gene nucleotide sequence including but not limited to the phosphomevalonate kinase PMK from red yeast;

[0032] 7) cloning the gene nucleotide sequence fused with the mitochondrial signal peptide MTS2 and the gene nucleotide sequence including but not limited to the diphosphomevalonate decarboxylase DPDC from red yeast;

[0033] 8) cloning the gene nucleotide sequence of the mitochondrial signal peptide MTS3 fused with the gene nucleotide sequence and codon-optimized, including but not limited to the gene nucleotide sequence of the nerol diphosphate synthase NPPS from Solanum lycopersicum and the gene nucleotide sequence of the fusion protein NPPS-GGGS-LS of the limonene synthase CltLS from Citrus limon;

[0034] 9) cloning the gene nucleotide sequence fused with the mitochondrial signal peptide MTS10 and the gene nucleotide sequence including but not limited to the isopentenyl pyrophosphate isomerase IDI from red yeast;

[0035] 10) The PCR products obtained in step 1), step 2) and step 3) are connected to the expression vector PZPK to obtain the vector PZPK-MTS2-EfMvaS-EGFP-MTS10-EfMvaE;

[0036] 11) ligating the PCR products obtained in step 4), step 5) and step 6) into the expression vector PZPK to obtain the expression vector PZPK-RFP-MTS3-MmMK-MTS4-PMK;

[0037] 12) The PCR products obtained in step 7), step 8) and step 9) are ligated into the expression vector PZPK to obtain the expression vector PZPK-MTS2-DPDC-MTS3-NPPS-GGGS-LS-MTS10-IDI;

[0038] 13) The expression vectors obtained in step 11), step 12) and step 13) are sequentially transferred to yeast by Agrobacterium pyogenes-mediated transformation method ATMT;

[0039] 14) Verifying the engineered bacteria.

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

[0041] In one embodiment of the present invention, the limonene engineering bacteria is Rhodosporium toruloides CGMCC 2.1389, and the accession number of the China General Microbiological Culture Collection Center is CGMCC 2.1389.

[0042] According to the application of the present invention, in one embodiment of the present invention, the step 10) includes the following steps:

[0043] S10.1 The nucleotide sequences of the 3-methyl 3-hydroxyglutaryl-CoA synthase EfMvaS gene fused with the mitochondrial signal peptide MTS2, the green fluorescent protein EGFP gene, and the acetyl-CoA acetyltransferase / 3-hydroxy-3-methylglutaryl-CoA reductase EfMvaE gene fused with the mitochondrial signal peptide MTS10 are connected through a 2A connecting peptide gene, and the upstream and downstream contain promoter and terminator sequences.

[0044] S10.2 Insert the fragment between the EcoRV recognition site and the SpeI recognition site in the integration vector to construct an Agrobacterium-mediated binary expression vector recombinant plasmid.

[0045] S10.3 Red yeast is used as a host strain to infect and transform the recombinant plasmid to obtain a genetically engineered strain containing 3-methyl 3-hydroxyglutaryl-CoA synthase, green fluorescent protein and acetyl-CoA acetyltransferase / 3-hydroxy-3-methylglutaryl-CoA reductase.

[0046] According to the application of the present invention, in one embodiment of the present invention, the step 11) includes the following steps:

[0047] S11.1 The nucleotide sequences of the red fluorescent protein RFP gene, the mevalonate kinase MmMK gene fused with MTS3 and the phosphomevalonate kinase PMK gene fused with MTS4 are connected through a 2A connecting peptide gene, and the upstream and downstream contain promoter and terminator sequences.

[0048] S11.2 Insert the fragment between the EcoRV recognition site and the SpeI recognition site in the integration vector to construct a binary expression vector recombinant plasmid mediated by Agrobacterium oxytoca.

[0049] S11.3 The genetically engineered strain obtained in step S10.3 is used as a host strain to infect and transform the recombinant plasmid to obtain a genetically engineered strain of red fluorescent protein, mevalonate kinase and phosphomevalonate kinase.

[0050] According to the application of the present invention, in one embodiment of the present invention, the step 12) includes the following steps:

[0051] S12.1 The diphosphomevalonate decarboxylase DPDC gene fused with the mitochondrial signal peptide MTS2, the fusion protein NPPS-GGGS-LS gene of the nerol diphosphate synthase NPPS from tomato fused with the mitochondrial signal peptide MTS3 and the limonene synthase CltLS from lemon, and the isopentenyl pyrophosphate isomerase IDI gene fused with the mitochondrial signal peptide MTS10 are connected through a 2A connecting peptide gene, and the upstream and downstream contain promoter and terminator sequences.

[0052] S12.2 Insert the fragment between the EcoRV recognition site and the SpeI recognition site in the integration vector to construct a binary expression vector recombinant plasmid mediated by soil Pseudomonas aeruginosa.

[0053] S12.3 The genetically engineered strain obtained in step S11.3 is used as a host strain to infect and transform the recombinant plasmid to obtain a genetically engineered strain of mevalonate diphosphate decarboxylase, nerol diphosphate synthase, a fusion protein of lemon-derived limonene synthase, and isopentenyl pyrophosphate isomerase.

[0054] According to the group of mitochondrial signal peptides and their applications described in the present invention, MTSs are used alone or in combination to construct engineered bacteria including but not limited to limonene, linalool, patchouli alcohol, kaurene, isobutanol and 3-hydroxypropionic acid, so as to achieve the synthesis of mitochondrial compartmentalized target products.

[0055] The beneficial effects of this application include:

[0056] The present invention screened and characterized a group of mitochondrial signal peptides in Rhodosporidium toruloides for the first time; and enhanced the synthesis efficiency of terpenes, branched-chain alcohols and organic acids, etc., heterologously expressed in Rhodosporidium toruloides by constructing target product synthesis pathway genes mediated by mitochondrial signal peptides, and achieved the following results:

[0057] The screening of a group of mitochondria provided by the present invention was characterized and verified for the first time in Rhodosporidium toruloides, expanding the enabling tools for Rhodosporidium toruloides to achieve mitochondrial compartmentalization of subcellular structures.

[0058] The application of a group of mitochondria provided by the present invention is the first to use a group of mitochondrial signal peptide-mediated free expression method in Rhodosporidium toruloides to obtain engineered strains producing limonene, linalool, patchouli alcohol, kaurene, isobutanol and 3-hydroxypropionic acid, thereby realizing the heterologous expression of mitochondrial compartmentalization of the target product. The genetic operations required are few, simple and efficient, and the strain transformation cycle is short.

[0059] The engineered bacteria of limonene, linalool, patchouli alcohol, kaurene, isobutanol and 3-hydroxypropionic acid provided by the present invention have stable heterologous gene expression, no need to add antibiotics and inducers during fermentation, thus reducing production costs, and the fermentation process is simple and controllable with high economy.

[0060] The series of engineered bacteria provided by the present invention (taking limonene as an example) have the following process for fermenting limonene: the engineered bacteria are cultured in a shake flask at 28°C and 180rpm until mid-logarithmic phase, and inoculated into a 3L fermenter with 10% inoculum for fermentation; the fermentation parameters are pH 6.0, 28°C, and 180rpm. The limonene engineered bacteria provided by the present invention use glucose as a substrate, and the 3L fermenter is batch-fed for 192h, with a yield of 4g / L, and has good industrial application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 It is the validation of the targeting function of the mitochondrial signal peptide by adding proline;

[0062] Figure 2 This is a schematic diagram of the construction of the limonene engineering bacterial expression vector;

[0063] Figure 3 This is a GCMS comparison chart of limonene synthesized by limonene engineering bacteria and its standard product;

[0064] Figure 4 It is the yield of limonene fermented by limonene engineered bacteria. DETAILED DESCRIPTION

[0065] The present invention is further explained below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and implement it. However, the following embodiments are for illustrative purposes rather than for limiting the scope of the present invention.

[0066] Unless otherwise specified, the experimental materials and reagents in the following examples can be purchased from commercial sources.

[0067] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0068] The starting strain red yeast (Rhodosporidium toruloides) in the example was obtained from Dalian Institute of Chemical Physics, which is a conventional red yeast np11 in the art 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.).

[0069] Primers were synthesized by Yangling Tianrun Aoke Biotechnology Co., Ltd. PCR products were sequenced by Yangling Tianrun Aoke Biotechnology Co., Ltd.

[0070] Detection method of limonene:

[0071] In the embodiment, a gas chromatograph-mass spectrometer (GC-MS) is used to perform qualitative analysis on the sample. A liquid injection method, a flame ionization detector (FID) detector, a chromatographic column is an HP-INNOWax capillary column (60mx 250μm x 0.25μm), helium (He) is used as a carrier gas, and the flow rate is 1.5mL / min; the injection volume is 1μL, and the split ratio is 20:1; the GC heating program is as follows: 40°C for 3min, heated to 160°C at 4°C / min, heated to 220°C at 7°C / min, and maintained for 8min. The MSD transmission line and ion source temperatures are 280°C and 230°C, respectively; the electron ion source (EI), the energy is 70eV, and the scanning range is 29-350u.

[0072] In the embodiment, the sample is quantitatively analyzed by gas chromatograph (GC). The limonene in the sample is quantified by gas chromatography (GC). The quantitative analysis is 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 is used as the carrier gas, the flow rate is 1.0mL / min, the injection volume is 1μL, and the split ratio is 20:1; the GC column box program is 145°C for 27min; the injector and detector temperatures are 240°C and 260°C, respectively; a limonene standard curve is established to quantitatively analyze the limonene content in the sample.

[0073] Example 1: Screening and characterization of a panel of mitochondria

[0074] E1.1 Based on the multi-omics data analysis of Rhodosporidium toroculans, 11 key functional proteins in the inner membrane and matrix of the mitochondria of Rhodotorula were selected, and MTSs with potential mitochondrial targeting function were predicted by combining Mitoprot and TargetP online tools. The amino acid sequences of the MTSs gene fragments are shown in the sequence table SEQ ID NO.1-11.

[0075] The NCBI accession numbers and valid sequences corresponding to the above MTSs are shown in the following table:

[0076] Table 1 Characteristics of mitochondrial targeting sequences (MTSs) clones

[0077]

[0078] Table 1

[0079]

[0080] E1.2 used overlap extension PCR to amplify the gene fragment in E1.1 and extended the desired gene fragment with the N-terminus of EGFP. The PCR conditions were 98℃ pre-denaturation for 5min; 98℃ denaturation for 10sec, 57℃ annealing for 5sec, 72℃ extension for 5min, for a total of 30 cycles. The last overlap extension obtained the MTS-EGFP gene fragment with restriction sites at both ends.

[0081] The primers used in the above experimental process are shown in the following table:

[0082] Table 2 Primer sequence list

[0083]

[0084] Table 2

[0085]

[0086]

[0087] E1.3 Prepare Escherichia coli DH5α competent cells, and select plasmid pZPK with nourseothricin resistance as its expression vector. The above-mentioned gene fragment MTS-EGFP was cloned between the restriction enzyme cutting sites EcoRV and SpeI by enzyme cutting and ligation, and transferred into Escherichia coli DH5α competent cells by heat shock at 42°C. After a single colony grows on the LB screening solid plate containing the corresponding resistance, the upstream primer MTS-F of the target gene and the downstream primer tHSP-R of the terminator are used for colony PCR verification. The transformants with correct bands are inoculated into 5 mL of LB culture medium with the corresponding resistance and cultured for 12-16 hours. The corresponding plasmid is extracted and sequenced for verification to obtain the pZPK-Ntc-pXYL-EcoRV-MTS-EGFP-SpeI-tHSP recombinant plasmid.

[0088] Among them, Ntc is the nourseothricin resistance gene, and the amino acid sequence of its encoded protein is GenBank: AAS47018.1.

[0089] E1.4 Prepare soil Bacillus competent cells, and electrophore the pZPK-Ntc-pXYL-MTS-EGFP-tHSP recombinant plasmid constructed in step E1.3 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 MTS-F of the target gene and the downstream primer tHSP-R of the terminator to perform colony PCR verification. The verified single colony of soil Bacillus is inoculated into 5 mL of LB medium with 5 μL of kanamycin and cultured for 12-16 hours. The corresponding plasmid is extracted and digested again with restriction endonucleases EcoRV and SpeI for verification.

[0090] E1.5 Inoculate the single colony of Pseudomonas aeruginosa verified correctly in step E1.4 into 5 mL LB liquid culture medium containing the corresponding antibiotics and culture it for about 15 hours until OD600 = 0.4-0.6. At the same time, inoculate the Rhodosporidium toruloides np11 into YPD liquid culture medium and culture it for 15 hours until OD600 = 0.4-0.6. Wash the cells twice with sterile water, resuspend them, take 100 μL of the bacterial solution from each plate, mix them, and drop them on the sterile filter paper of the IM induction plate. After culturing at 25°C for 48 hours, transfer them to the YPD plate containing nourseothricin resistance and cephalosporin, and continue culturing at 30°C for 48 hours.

[0091] E1.6 Pick as many single colonies as possible from step E1.5, and verify them by multiple subcultures on the screening plate. Finally, the correct single colony is verified to be the correct genetically engineered bacterium, and is named Z1 (genotype is np11-pXYL-MTS-EGFP-tHSP).

[0092] E1.7 The transformants selected in step E1.6 are first preliminarily screened using an automated fluorescence microscope.

[0093] E1.8 Add the transformants selected in step E1.7 to fresh YPD medium and culture for 16-17 hours, then use MitoTracker probe to stain the mitochondria of the cells. The specific operation steps are consistent with the reagent instructions.

[0094] E1.9 The transformants stained with mitochondria in step E1.8 were characterized and identified by bio-laser confocal microscopy. Figure 2 shown.

[0095] Example 2: Verification of the targeting function of mitochondrial signal peptide by adding proline

[0096] E2.1 According to the transcriptional coding mechanism of polycistronic mediated by 2A connecting peptide and the results of Example 1, the expression plasmid obtained in Example 1E1.3 was added with methionine and proline at the N-terminus of MTSs to obtain the pZPK-Ntc-pXYL-EcoRV-MP-MTS-EGFP-SpeI-tHSP recombinant plasmid. The amino acid sequence of the 2A connecting peptide sequence gene is shown in the sequence table SEQ ID NO.13-14.

[0097] E2.2 is similar to Example 1E1.3. The pZPK-Ntc-pXYL-MP-MTS-EGFP-tHSP recombinant plasmid constructed in step E2.1 was heat-shocked into Escherichia coli DH5α competent cells and colony PCR verification was performed using primers for MP-MTS-F and tHSP-R. The transformants containing the correct bands were inoculated into 5 mL of LB medium with 5 μL of kanamycin and cultured for 12-16 h. The plasmid was extracted and sequenced to obtain the pZPK-Ntc-pXYL-EcoRV-MP-MTS-EGFP-SpeI-tHSP recombinant plasmid.

[0098] The primers used in the above experimental process are shown in the following table:

[0099] Table 3 Primer sequence list

[0100]

[0101] Table 3

[0102]

[0103] E2.3 is similar to Example 1E1.4-6, except that the soil Bacillus carrying the recombinant plasmid is used to infect the strain np11 to obtain strain Z2 (genotype: np11-NTC-pXYL-EcoRV-MP-MTS-EGFP-SpeI-tHSP).

[0104] E2.4 is similar to Example 1E1.7-9, the transformants with mitochondrial staining were characterized and identified by biological laser confocal microscopy, such as Figure 2 As shown, the MTS2, MTS3, MTS4, MTS5, MTS6 and MTS10 are not affected by proline, while the MTS1, MTS7, MTS8, MTS9 and MTS11 can be used to direct the first gene in the expression cassette.

[0105] Example 3: Construction of MTSs-mediated limonene engineering strain

[0106] E3.1 The MTSs with good targeting function obtained in Example 2 were synthesized by Suzhou Hongxun Biotechnology Co., Ltd., and two vectors, pUC57-MTS2-EcoRV-P2A-MTS10-Stu1 and pUC57-EcoRV-P2A-MTS3-EcoR1-T2A-MTS4-Spe1, were obtained.

[0107] E3.2 Based on the codon preference of Rhodosporidium toruloides, a mutant of 3-methyl 3-hydroxyglutaryl-CoA synthase EfMvaS from Enterococcus faecalis was cloned. A110GThe amino acid sequence of the gene. The 3-methyl 3-hydroxyglutaryl coenzyme A synthase mutant EfMvaS A110G The amino acid sequence of the gene is shown in the sequence listing SEQ ID NO.12.

[0108] E3.3 Based on the gene fragment EfMvaS A110G The primers MTS2-fu-EfMvaS were designed and synthesized based on the upstream and downstream sequences of the fragment inserted into the vector. A110G -F and EfMvaS A110G -fu-P2A-R, PCR amplification of the gene fragment EfMvaS in E3.3 A110G , and the correct gene fragment was recovered by agarose gel electrophoresis.

[0109] E3.4 The recombinant plasmid pUC57-MTS2-EcoRV-P2A-MTS10-Stu1 obtained in Example 3E3.1 was digested with EcoRV and purified to obtain a linearized vector fragment.

[0110] E3.5 using TAKARA HD Cloning Kit connects the linearized vector to the target gene fragment with the homology arms of the vector insertion site. The specific operation steps are consistent with the kit instructions.

[0111] E3.6 Similar to Example 1E1.3, the recombinant plasmid was heat-shocked into E. coli DH5α competent cells and colony PCR was performed with primers for PUC57-F and PUC57-R. Transformants containing the correct bands were inoculated into 5 mL of LB medium containing 5 μL of kanamycin and cultured for 12-16 h. The plasmid was extracted and sequenced to obtain PUC57-MTS2-EfMvaS A110G -P2A-MTS10-Stu1 recombinant plasmid.

[0112] E3.7 is similar to Example 3E3.2. According to the codon preference of Rhodosporidium toruloides, the amino acid sequence of the acetyl-CoA acetyltransferase / 3-hydroxy-3-methylglutaryl-CoA reductase EfMvaE gene from Enterococcus faecalis was cloned. The NCBI accession number of the acetyl-CoA acetyltransferase / 3-hydroxy-3-methylglutaryl-CoA reductase EfMvaE is WP_002382276.1.

[0113] E3.8 is similar to Example 3E3.3. Primers MTS10-fu-EfMvaE-F and EfMvaE-fu-PUC57-R were designed and synthesized based on the sequence of the gene fragment EfMvaE and the upstream and downstream sequences of its inserted vector fragment. The gene fragment EfMvaE in E3.7 was amplified by PCR, and the correct gene fragment was recovered by agarose gel electrophoresis.

[0114] E3.9 The recombinant plasmid PUC57-MTS2-EfMvaS obtained in Example 3E3.6 was A110G -P2A-MTS10-Stu1 was digested with Stu1 and purified to obtain a linearized vector fragment.

[0115] E3.10 is similar to Example 3E3.5-6, using TAKARA HD Cloning Kit was used to connect the linearized vector with the target gene fragment with the homology arm of the vector insertion site. The recombinant plasmid was heat-shocked into E. coli DH5α competent cells and the primers PUC57-F and PUC57-R were used for colony PCR verification. The transformants containing the correct bands were inoculated into 5 mL of LB medium with 5 μL of kanamycin and cultured for 12-16 hours. The plasmid was extracted and sequenced to obtain PUC57-MTS2-EfMvaS A110G -P2A-MTS10-EfMvaE recombinant plasmid.

[0116] E3.11 Amplify the gene fragment MTS2-EfMvaS in E3.10 using primer pair XYL-fu-MTS2-F and P2A-fu-EGFP-R A110G -P2A, the correct gene fragment was recovered by agarose gel electrophoresis.

[0117] E3.12 The expression vector pZPK-Ble-pXYL-EcoRV-EGFP-P2A-SnaB1-tHSP was digested with EcoRV and purified to obtain a linearized vector fragment.

[0118] E3.13 is similar to Example 3E3.5-6, using TAKARA HD Cloning kit, connect the linearized vector and the target gene fragment with the homology arm of the vector insertion site. Heat shock the recombinant plasmid into E. coli DH5α competent cells and perform colony PCR verification with primers XYL-F and tHSP-R. Transformants containing the correct bands were inoculated into 5mL LB medium with 5μL kanamycin and cultured for 12-16h. The plasmid was extracted and sequenced to obtain PZPK-Ble-pXYL-MTS2-EfMvaS A110G -P2A-EGFP-P2A-SnaB1-tHSP recombinant plasmid.

[0119] E3.14 used primer pair P2A-fu-MTS10-F and EfMvaE-fu-tHSP-R to amplify the gene fragment MTS10-EfMvaE in E3.10, and the correct gene fragment was recovered by agarose gel electrophoresis.

[0120] E3.15 The recombinant vector PZPK-Ble-pXYL-MTS2-EfMvaS obtained in Example 3E3.13 A110G -P2A-EGFP-P2A-SnaB1-tHSP was digested with SnaB1 and purified to obtain a linearized vector fragment.

[0121] E3.16 is similar to Example 3E3.5-6, using TAKARA HD Cloning kit was used to connect the linearized vector with the target gene fragment with the homology arm of the vector insertion site. The recombinant plasmid was heat-shocked into E. coli DH5α competent cells and colony PCR was performed with primers for EGFP-F and tHSP-R. The transformants containing the correct bands were inoculated into 5 mL of LB medium with 5 μL of kanamycin and cultured for 12-16 hours. The plasmid was extracted and sequenced to obtain PZPK-Ble-pXYL-MTS2-EfMvaS A110G -P2A-EGFP-P2A-MTS10-EfMvaE-tHSP recombinant plasmid.

[0122] E3.17 is similar to Example 1E1.4-6. The soil Bacillus infection strain np11 with the recombinant plasmid was continuously subcultured on a YPD plate containing Bleococcus resistance and cephalosporin, and finally a high-expression green fluorescence strain Z3-1 (genotype np11-Ble-pXYL-MTS2-EfMvaS A110G -P2A-EGFP-P2A-MTS10-EfMvaE-tHSP).

[0123] E3.18 Based on the codon preference of Rhodosporidium toruloides, the amino acid sequence of the mevalonate kinase MmMK gene from Methanosarcina mazei was cloned. The NCBI accession number of the mevalonate kinase MmMK is AGF97182.1.

[0124] E3.19 designed and synthesized primers P2A-MTS3-fu-MmMK-F and MmMK-fu-T2A-R based on the sequence of the gene fragment MmMK and the upstream and downstream sequences of its inserted vector fragment, PCR amplified the gene fragment MmMK in E3.18, and recovered the correct gene fragment by agarose gel electrophoresis.

[0125] E3.20 The recombinant plasmid pUC57-EcoRV-P2A-MTS3-EcoR1-T2A-MTS4-Spe1 obtained in Example 3E3.1 was digested with EcoR1 and purified to obtain a linearized vector fragment.

[0126] E3.21 is similar to Example 3E3.5-6, using TAKARA HD Cloning Kit was used to connect the linearized vector with the target gene fragment with the homology arm of the vector insertion site. The recombinant plasmid was heat-shocked into E. coli DH5α competent cells and the primers PUC57-F and PUC57-R were used for colony PCR verification. The transformants containing the correct bands were inoculated into 5mL LB medium with 5μL kanamycin and cultured for 12-16h. The plasmid was extracted and sequenced to obtain the pUC5-EcoRV-P2A-MTS3-MmMK-T2A-MTS4-Spe1 recombinant plasmid.

[0127] E3.22 cloned the amino acid sequence of the endogenous phosphomevalonate kinase PMK gene of Rhodosporidium toruloides. The NCBI accession number of the phosphomevalonate kinase PMK is XP_016272321.1.

[0128] E3.23 designed and synthesized primers MTS4-fu-PMK-F and PMK-fu-PUC57-R based on the sequence of the gene fragment PMK and the upstream and downstream sequences of its inserted vector fragment, PCR amplified the gene fragment PMK in E3.22, and recovered the correct gene fragment by agarose gel electrophoresis.

[0129] E3.24 The recombinant plasmid pUC57-EcoRV-P2A-MTS3-MmMK-T2A-MTS4-Spe1 obtained in Example 3E3.21 was digested with Spe1 and purified to obtain a linearized vector fragment.

[0130] E3.25 is similar to Example 3E3.5-6, using TAKARA HD Cloning Kit was used to connect the linearized vector with the target gene fragment with the homology arm of the vector insertion site. The recombinant plasmid was heat-shocked into E. coli DH5α competent cells and colony PCR was performed with primers for MTS4-F and PUC57-R. The transformants containing the correct bands were inoculated into 5 mL of LB medium with 5 μL of kanamycin and cultured for 12-16 hours. The plasmid was extracted and sequenced to obtain the pUC57-EcoRV-P2A-MTS3-MmMK-T2A-MTS4-PMK recombinant plasmid.

[0131] E3.26 used primer pair RFP-fu-P2A-MTS3-F and PMK-fu-tHSP-R to amplify the gene fragment P2A-MTS3-MmMK-T2A-MTS4-PMK in E3.25, and the correct gene fragment was recovered by agarose gel electrophoresis.

[0132] E3.27 The expression vector pZPK-Ntc-pXYL-RFP-EcoRV-tHSP was digested with EcoRV and purified to obtain a linearized vector fragment.

[0133] E3.28 is similar to Example 3E3.5-6, using TAKARA HD Cloning kit was used to connect the linearized vector with the target gene fragment with the homology arm of the vector insertion site. The recombinant plasmid was heat-shocked into E. coli DH5α competent cells and colony PCR was performed with primers XYL-F and tHSP-R. The transformants containing the correct bands were inoculated into 5mL LB medium with 5μL kanamycin and cultured for 12-16h. The plasmid was extracted and sequenced to obtain the PZPK-Ntc-pXYL-RFP-P2A-MTS3-MmMK-T2A-MTS4-PMK-tHSP recombinant plasmid.

[0134] E3.29 is similar to Example 1E1.4-6. The soil Pseudomonas aeruginosa carrying the recombinant plasmid is used to infect the strain Z3-1 obtained in Example 3E3.17, and the strain is continuously subcultured on a YPD plate containing nourseothricin resistance and cephalosporin, ultimately obtaining the green fluorescence and red fluorescence highly expressing strain Z3-2 (genotype: Z3-1-Ntc-pXYL-RFP-P2A-MTS3-MmMK-T2A-MTS4-PMK-tHSP).

[0135] E3.30 cloned the amino acid sequence of the endogenous diphosphomevalonate decarboxylase DPDC gene of Rhodosporidium toruloides. The NCBI accession number of the diphosphomevalonate decarboxylase DPDC is XP_016275120.1.

[0136] E3.31 designed and synthesized primers MTS2-fu-DPDC-F and DPDC-fu-P2A-R based on the sequence of the gene fragment DPDC and the upstream and downstream sequences of its inserted vector fragment, PCR amplified the gene fragment DPDC in E3.30, and recovered the correct gene fragment by agarose gel electrophoresis.

[0137] E3.32 is similar to Example 3E3.5-6, using TAKARA HD Cloning kit was used to connect the linearized vector fragment obtained in Example 3E3.4 with the target gene fragment with the homology arm of the vector insertion site. The recombinant plasmid was heat-shocked into E. coli DH5α competent cells and the primers PUC57-F and PUC57-R were used for colony PCR verification. The transformants containing the correct bands were inoculated into 5 mL of LB medium with 5 μL of kanamycin and cultured for 12-16 hours. The plasmid was extracted and sequenced to obtain the PUC57-MTS2-DPDC-P2A-MTS10-Stu1 recombinant plasmid.

[0138] E3.33 cloned the amino acid sequence of the endogenous isopentenyl pyrophosphate isomerase IDI gene of Rhodosporidium toruloides. The NCBI accession number of the isopentenyl pyrophosphate isomerase IDI is XP_016270310.1.

[0139] E3.34 designed and synthesized primers MTS4-fu-IDI-F and IDI-fu-PUC57-R based on the sequence of gene fragment IDI and the upstream and downstream sequences of its inserted vector fragment, PCR amplified the gene fragment IDI in E3.33, and recovered the correct gene fragment by agarose gel electrophoresis.

[0140] E3.35 The vector pUC57-EcoRV-P2A-MTS3-EcoR1-T2A-MTS4-Spe1 obtained in Example 3E3.1 was digested with Spe1 and purified to obtain a linearized vector fragment.

[0141] E3.36 is similar to Example 3E3.5-6, using TAKARA HD Cloning kit was used to connect the linearized vector fragment obtained in Example 3E3.35 with the target gene fragment with the homology arm of the vector insertion site. The recombinant plasmid was heat-shocked into E. coli DH5α competent cells and the primers PUC57-F and PUC57-R were used for colony PCR verification. The transformants containing the correct bands were inoculated into 5 mL of LB medium with 5 μL of kanamycin and cultured for 12-16 hours. The plasmid was extracted and sequenced to obtain the pUC57-EcoRV-P2A-MTS3-EcoR1-T2A-MTS4-IDI recombinant plasmid.

[0142] The E3.37 clone fuses the amino acid sequence of the fusion protein NPPS-GGGS-LS gene of the codon-optimized nerol diphosphate synthase NPPS from tomato and the limonene synthase CltLS from lemon. The NCBI accession number corresponding to the nerol diphosphate synthase NPPS is NP_001234633.1; the NCBI accession number corresponding to the limonene synthase CltLS is Q8L5K3.1.

[0143] E3.38 designed and synthesized primers MTS3-fu-NPPS-GGGS-LS-F and NPPS-GGGS-LS-fu-P2A-R based on the sequence of the gene fragment NPPS-GGGS-LS and the upstream and downstream sequences of its inserted vector fragment. PCR was used to amplify the gene fragment NPPS-GGGS-LS in E3.37, and the correct gene fragment was recovered by agarose gel electrophoresis.

[0144] E3.39 is similar to Example 3E3.5-6, using TAKARA HD Cloning kit, connect the linearized vector fragment obtained in Example 3E3.35 with the target gene fragment with the homology arm of the vector insertion site. Heat shock the recombinant plasmid into E. coli DH5α competent cells and use primers to verify PUC57-F and PUC57-R by colony PCR. Transformants containing correct bands are inoculated into 5mL LB medium with 5μL kanamycin and cultured for 12-16h. After plasmid extraction and sequencing verification, pUC57-EcoRV-P2A-MTS3-NPPS-GGGS-LS-T2A-MTS4-IDI recombinant plasmid is obtained.

[0145] E3.40 used primer pair XYL-fu-MTS2-F and P2A-fu-tHSP-R to amplify the gene fragment MTS2-DPDC-P2A in E3.36, and the correct gene fragment was recovered by agarose gel electrophoresis.

[0146] E3.42 The expression vector pZPK-G418-pXYL-EcoRV-tHSP was digested with EcoRV and purified to obtain a linearized vector fragment.

[0147] E3.43 is similar to Example 3E3.5-6, using TAKARA HD Cloning kit, connect the linearized vector fragment obtained in Example 3E3.42 with the target gene fragment with the homology arm of the vector insertion site. Heat shock the recombinant plasmid into E. coli DH5α competent cells and perform colony PCR verification with primers XYL-F and tHSP-R. Transformants containing the correct bands are inoculated into 5 mL LB medium with 5 μL kanamycin and cultured for 12-16 hours. The plasmid is extracted and sequenced to obtain the pZPK-G418-pXYL-MTS2-DPDC-P2A-EcoRV-tHSP recombinant plasmid.

[0148] E3.44 used primer pair DPDC-fu-P2A-F and T2A-fu-tHSP-R to amplify the gene fragment MTS3-NPPS-GGGS-LS-T2A in E3.39, and the correct gene fragment was recovered by agarose gel electrophoresis.

[0149] E3.45 The recombinant plasmid pZPK-G418-pXYL-MTS2-DPDC-P2A-EcoRV-tHSP obtained in Example 3E3.43 was digested with EcoRV and purified to obtain a linearized vector fragment.

[0150] E3.46 is similar to Example 3E3.5-6, using TAKARA HD Cloning kit, connect the linearized vector fragment obtained in Example 3E3.45 with the target gene fragment with the homology arm of the vector insertion site. Heat shock the recombinant plasmid into E. coli DH5α competent cells and perform colony PCR verification with primers for DPDC-F and tHSP-R. Transformants containing correct bands are inoculated into 5mL LB medium with 5μL kanamycin and cultured for 12-16h. After plasmid extraction and sequencing verification, pZPK-G418-pXYL-MTS2-DPDC-P2A-MTS3-NPPS-GGGS-LS-T2A-EcoRV-tHSP recombinant plasmid is obtained.

[0151] E3.47 used primer pair T2A-fu-MTS4-F and IDI-fu-tHSP-R to amplify the gene fragment MTS4-IDI in E3.36, and the correct gene fragment was recovered by agarose gel electrophoresis.

[0152] E3.48 The recombinant plasmid pZPK-G418-pXYL-MTS2-DPDC-P2A-MTS3-NPPS-GGGS-LS-T2A-EcoRV-tHSP obtained in Example 3E3.46 was digested with EcoRV and purified to obtain a linearized vector fragment.

[0153] E3.49 is similar to Example 3E3.5-6, using TAKARA HD Cloning kit, connect the linearized vector fragment obtained in Example 3E3.48 with the target gene fragment with the homology arm of the vector insertion site. Heat shock the recombinant plasmid into Escherichia coli DH5α competent cells and perform colony PCR verification with primers T2A-fu-MTS4-F and tHSP-R. The transformant containing the correct band is inoculated into 5mL LB medium with 5μL kanamycin and cultured for 12-16h. The plasmid is extracted and sequenced to obtain the pZPK-G418-pXYL-MTS2-DPDC-P2A-MTS3-NPPS-GGGS-LS-T2A-MTS4-IDI-tHSP recombinant plasmid.

[0154] E3.50 is similar to Example 1E1.4-6, the soil Pseudomonas aeruginosa carrying the recombinant plasmid is used to infect the strain Z3-2 obtained in Example 3E3.29, and the strain is continuously subcultured on a YPD plate containing geneticin resistance and cephalosporin, ultimately obtaining a high-yield limonene engineered strain Z3-3 (genotype: Z3-2-G418-pXYL-MTS2-DPDC-P2A-MTS3-NPPS-GGGS-LS-T2A-MTS10-IDI-tHSP).

[0155] The above experimental process is as follows Figure 3 The primers used are shown in the following table:

[0156] Table 4 Primer sequence list

[0157]

[0158] Table 4

[0159]

[0160]

[0161] Example 4: Fermentation of limonene using genetically engineered bacteria

[0162] The strain red yeast np11 and the strain Z3-3 prepared in Example 3 were fermented in a 250 mL conical flask.

[0163] Specifically, a single colony of the strain was inoculated into 5 mL YPD medium (20 g / L glucose, 20 g / L peptone and 10 g / L yeast extract, and water was added to 1 L) in a 50 mL centrifuge tube and cultured at 28 ° C at 180 rpm / min. After fermentation for 48 hours, 50 mL YPD medium was added to a 250 mL conical flask and seed solution was inoculated with an initial OD600 of 0.5. At the same time, 20% n-dodecane extractant was added for in-situ extraction, and cultured at 28 ° C, 180 rpm for 72 to 120 hours. At the end of the fermentation, 9000 g was centrifuged for 1 min, and the upper organic layer was taken for GC detection of limonene production. Specifically, the test results are shown in Table 5.

[0164] Table 5 Limonene production by fermentation of limonene by limonene engineering bacteria

[0165] As shown below:

[0166]

[0167] Specifically, sample Z3-3, limonene standard, and original starting strain Rhodosporidium toruloides np11 were qualitatively tested by GCMS, and the test results were as follows: Figure 4As shown, the mass spectrum shows that there are obvious characteristic peaks of limonene at 68 and 93, which are highly consistent with 68 and 93 in the standard, while the original starting strain Rhodosporidium toruloides np11 has no chromatographic peaks, indicating that the genetically engineered strain Z3-3 does have the ability to synthesize limonene from scratch.

[0168] Example 5: High-density fermentation of genetically engineered bacteria to produce limonene

[0169] The strain Z3-3 single colony prepared in Example 3 was inoculated in 50mL YPD medium (20g / L glucose, 20g / L peptone and 10g / L yeast extract) 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 in a total volume of 180mL YPD medium at a volume ratio of 10% to prepare a second-level seed solution, which was cultured at 28°C and 180rpm for 12-16h. The second-level seed solution was washed with sterile water and inoculated in a 3L bioreactor with a working volume of 1.8L according to a volume ratio of 10%, with a pH of 6.0 (regulated by 2M sodium hydroxide), aeration of 2L / min, and a stirring speed of 350rpm. The glucose concentration and limonene production were monitored every 12h. The limonene detection method was the same as described above, and glucose was detected by DNS method (3,5-dinitrosalicylic acid method). MM medium containing 20 g / L glucose was used as the initial medium. When the glucose concentration in the fermentation broth was lower than 5 g / L, feeding was started. The feeding was linked to the dissolved oxygen concentration in the fermentation broth to maintain the dissolved oxygen at 10%.

[0170] Specifically, the composition of MM medium containing 20 g / L glucose is: 20 g / L glucose, 5 g (NH4) 2 SO 4 3gK 2 PO 4 ,0.5 g MgSO 4 ·7H2O, 2mL trace metal solution and 1mL vitamin solution were diluted to 1L with distilled water to prepare MM medium containing different carbon sources. Among them, the trace metal solution ( / 100mL) contained 0.45g CaCl 2 2H 2 O,0.45g ZnSO 4 7H 2 O, 0.3 g FeSO 4 7H 2 O,0.1g H 3 BO 3 ,0.1g MnCl 2 4H 2 O,0.04g Na 2 MoO 4 2H 2O,0.03g CoCl 2 6H 2 O,0.01g CuSO 4 5H 2 O, 0.01g KI, 1.5g EDTA. Vitamin solution ( / 100mL) contains 5mg biotin, 20mg p-aminobenzoic acid, 1g niacin, 1g calcium pantothenate, 1g pyridoxine hydrochloride, 0.1g thiamine hydrochloride (vitamin B1), and 2.5g inositol.

[0171] Finally, glucose was used as the substrate and the fermentation was carried out in a 3L fermenter in batch mode for 192 hours, with a yield of 4 g / L.

[0172] Example 6: Construction and fermentation of MTSs-mediated linalool engineering strains

[0173] E6.1 artificially synthesizes the amino acid sequence of the fusion protein GPPS::McLis gene of codon-optimized geranyl diphosphate synthase GPPS from North American fir and linalool synthase McLis from lemon mint. The fusion protein is connected by a GGGS flexible linker. The NCBI accession numbers of the fusion protein GPPS and McLis are AAN01134.1 and Q8H2B4.1, respectively.

[0174] E6.2 The vector pUC57-EcoRV-P2A-MTS3-EcoR1-T2A-MTS4-IDI vector obtained in Example 3E3.36 was digested with EcoRV and purified to obtain a linearized vector fragment.

[0175] E6.3 Primers MTS2-F and DPDC-fu-P2A-R were designed and synthesized based on the sequence of the gene fragment DPDC and the upstream and downstream sequences of its inserted vector fragment, and the gene fragment MTS2-P2A-DPDC in Example 3E3.32 was amplified by PCR. The correct gene fragment was recovered by agarose gel electrophoresis.

[0176] E6.4 is similar to Example 3E3.5-6, using TAKARA HD Cloning kit was used to connect the linearized vector fragment obtained in Example 7E7.3 with the target gene fragment with the homology arm of the vector insertion site. The recombinant plasmid was heat-shocked into E. coli DH5α competent cells and the primers PUC57-F and PUC57-R were used for colony PCR verification. The transformants containing the correct bands were inoculated into 5 mL of LB medium with 5 μL of kanamycin and cultured for 12-16 hours. The plasmid was extracted and sequenced to obtain pUC57-MTS2-DPDC-P2A-MTS3-EcoR1-T2A-MTS4-IDI.

[0177] E6.5 used primer pair XYL-fu-MTS2-F and IDI-fu-tHSP-R to amplify the gene fragment MTS2-DPDC-P2A-MTS3-EcoR1-T2A-MTS4-IDI in E6.4, and the correct gene fragment was recovered by agarose gel electrophoresis.

[0178] E6.6 is similar to Example 3E3.5-6, using TAKARA HD Cloning kit, connect the linearized vector fragment obtained in Example 3E3.42 with the target gene fragment with the homology arm of the vector insertion site. Heat shock the recombinant plasmid into E. coli DH5α competent cells and perform colony PCR verification with primers XYL-F and tHSP-R. Transformants containing the correct bands are inoculated into 5 mL LB medium with 5 μL kanamycin and cultured for 12-16 hours. The plasmid is extracted and sequenced to obtain the pZPK-G418-pXYL-MTS2-DPDC-P2A-MTS3-EcoR1-T2A-MTS4-IDI-tHSP recombinant plasmid.

[0179] E6.7 The expression vector pZPK-G418-pXYL-MTS2-DPDC-P2A-MTS3-EcoR1-T2A-MTS4-IDI-tHSP was digested with EcoR1 and purified to obtain a linearized vector fragment.

[0180] E6.8 used primer pair MTS3-fu-GPPS-F and McLis-fu-T2A-R to amplify the gene fragment GPPS::McLis in E6.1, and the correct gene fragment was recovered by agarose gel electrophoresis.

[0181] E6.9 is similar to Example 3E3.5-6, using TAKARA HD Cloning kit, connect the linearized vector fragment obtained in Example 7E7.7 with the target gene fragment with the homology arm of the vector insertion site. Heat shock the recombinant plasmid into E. coli DH5α competent cells and perform colony PCR verification with primers P2A-F and MTS4-R. Transformants containing the correct bands are inoculated into 5 mL LB medium with 5 μL kanamycin and cultured for 12-16 hours. The plasmid is extracted and sequenced to obtain the pZPK-G418-pXYL-MTS2-DPDC-P2A-MTS3-GPPS::McLis-T2A-MTS4-IDI-tHSP recombinant plasmid.

[0182] E6.10 is similar to Example 1E1.4-6, the soil Pseudomonas aeruginosa carrying the recombinant plasmid is used to infect the strain Z3-2 obtained in Example 3E3.29, and the strain is continuously passaged on a YPD plate containing geneticin resistance and cephalosporin, ultimately obtaining a high-yield linalool engineered strain Z3-4 (genotype: Z3-2-G418-pXYL-MTS2-DPDC-P2A-MTS3-NPPS-GGGS-LS-T2A-MTS10-IDI-tHSP).

[0183] The strain red yeast np11 and the strain Z3-4 prepared in Example 6E6.10 were fermented in a 250 mL conical flask.

[0184] Specifically, a single colony of the strain was inoculated into 5 mL YPD medium (20 g / L glucose, 20 g / L peptone and 10 g / L yeast extract, and water was added to 1 L) in a 50 mL centrifuge tube and cultured at 28 ° C and 180 rpm / min. After fermentation for 48 hours, 50 mL YPD medium was added to a 250 mL conical flask and seed solution was inoculated with an initial OD600 = 0.5, and 20% myristic acid isopropyl ester extractant was added for in situ extraction, and cultured at 28 ° C, 180 rpm for 72 to 120 hours. At the end of the fermentation, centrifugation was performed at 9000 g for 1 min, and the upper organic layer was taken for GC detection of linalool production. The obtained strains can produce a certain amount of linalool.

[0185] The primers used in the above experimental process are shown in the following table:

[0186] Table 6 Primer sequence list

[0187]

[0188] Example 7: Construction and fermentation of MTSs-mediated patchouli alcohol engineering strain

[0189] The artificial synthesis of E7.1 includes but is not limited to the amino acid sequence of the fusion protein FPPS::PTS gene of the endogenous farnesyl pyrophosphate synthase FPPS of Rhodosporidium toruloides and the codon-optimized patchouli alcohol PTS from patchouli. The amino acid sequence of the fusion protein is connected by a GSG flexible linker. The NCBI accession numbers corresponding to the FPPS and PTS are XP_016272719.1 and Q49SP3.1, respectively.

[0190] E7.2 used primer pair MTS3-fu-FPPS-F and PTS-fu-T2A-R to amplify the gene fragment FPPS::PTS in E7.1, and the correct gene fragment was recovered by agarose gel electrophoresis.

[0191] E7.3 is similar to Example 3E3.5-6, using TAKARA HD Cloning kit, connect the linearized vector fragment obtained in Example 7E7.7 with the target gene fragment with the homology arm of the vector insertion site. Heat shock the recombinant plasmid into E. coli DH5α competent cells and perform colony PCR verification with primers P2A-F and MTS4-R. Transformants containing the correct bands are inoculated into 5 mL LB medium with 5 μL kanamycin and cultured for 12-16 hours. The plasmid is extracted and sequenced to obtain the pZPK-G418-pXYL-MTS2-DPDC-P2A-MTS3-FPPS::PTS-T2A-MTS4-IDI-tHSP recombinant plasmid.

[0192] E7.4 is similar to Example 1E1.4-6, the soil Pseudomonas aeruginosa carrying the recombinant plasmid is used to infect the strain Z3-2 obtained in Example 3E3.29, and the strain is continuously subcultured on YPD plates containing geneticin resistance and cephalosporin, and finally a high-yield patchouli alcohol engineered strain Z3-5 (genotype is Z3-2-G418-pXYL-MTS2-DPDC-P2A-MTS3-FPPS::PTS-T2A-MTS10-IDI-tHSP) is obtained.

[0193] The strain red yeast np11 and the strain Z3-5 prepared in Example 7E7.4 were fermented in a 250 mL conical flask.

[0194] Specifically, a single colony of the strain was inoculated into 5 mL YPD medium (20 g / L glucose, 20 g / L peptone and 10 g / L yeast extract, and water was added to make the volume 1 L) in a 50 mL centrifuge tube and cultured at 28° C. and 180 rpm / min. After fermentation for 48 hours, 50 mL YPD medium was added to a 250 mL conical flask and seed solution was inoculated at an initial OD600 of 0.5. At the same time, 20% n-dodecane extractant was added for in-situ extraction, and cultured at 28° C. and 180 rpm for 72 to 120 hours. At the end of the fermentation, centrifugation was performed at 9000 g for 1 minute, and the upper organic layer was taken for GC detection of patchouli alcohol production. The obtained strains were able to produce a certain amount of patchouli alcohol.

[0195] The primers used in the above experimental process are shown in the following table:

[0196] Table 7 Primer sequence list

[0197]

[0198] Example 8: Construction and fermentation of MTSs-mediated kaurene engineering strains

[0199] The artificial synthesis of E8.1 includes but is not limited to the amino acid sequence of the fusion protein FPPS::GfKS gene of the endogenous farnesyl farnesyl pyrophosphate synthase GGPPS of Rhodosporidium toruloides and the codon-optimized kaurene synthase GfKS from Gibberella fujikura. The amino acid sequence of the fusion protein is connected by a GSG flexible linker. The NCBI accession numbers of the GGPPS and GfKS are XP_016271675.1 and XP_023431478.1, respectively.

[0200] In E8.2, the gene fragment FPPS::GfKS in E8.1 was amplified using the primer pair MTS3-fu-GGPPS-F and GfKS-fu-T2A-R, and the correct gene fragment was recovered by agarose gel electrophoresis.

[0201] E8.3 is similar to Example 3E3.5-6, using TAKARA HD Cloning kit, connect the linearized vector fragment obtained in Example 7E7.7 with the target gene fragment with the homology arm of the vector insertion site. Heat shock the recombinant plasmid into E. coli DH5α competent cells and perform colony PCR verification with primers P2A-F and MTS4-R. Transformants containing the correct bands are inoculated into 5 mL LB medium with 5 μL kanamycin and cultured for 12-16 hours. The plasmid is extracted and sequenced to obtain the pZPK-G418-pXYL-MTS2-DPDC-P2A-MTS3-GGPPS::GfKS-T2A-MTS4-IDI-tHSP recombinant plasmid.

[0202] E8.4 is similar to Example 1E1.4-6, the soil Pseudomonas aeruginosa carrying the recombinant plasmid is used to infect the strain Z3-2 obtained in Example 3E3.29, and the strain is continuously subcultured on YPD plates containing geneticin resistance and cephalosporin, and finally a high-yield kaurene engineered strain Z3-6 (genotype is Z3-2-G418-pXYL-MTS2-DPDC-P2A-MTS3-GGPPS::GfKS-T2A-MTS10-IDI-tHSP) is obtained.

[0203] The strain red yeast np11 and the strain Z3-6 prepared in Example 8E8.3 were fermented in a 250 mL conical flask.

[0204] Specifically, similar to Example 7, at the end of fermentation, centrifugation was performed at 9000 g for 1 min, and the upper organic layer was taken for GC detection of kaurene production. All the obtained strains were able to produce a certain amount of kaurene.

[0205] The primers used in the above experimental process are shown in the following table:

[0206] Table 8 Primer sequence list

[0207]

[0208] Example 9: Construction and fermentation of MTSs-mediated isobutanol engineering strains

[0209] The artificial synthesis of E9.1 includes but is not limited to codon-optimized α-ketoacid decarboxylase (α-KDC) and alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae. The NCBI accession numbers of the α-ketoacid decarboxylase α-KDC and alcohol dehydrogenase ADH are NP_116635.1 and AAA34411.1, respectively.

[0210] E9.2 used primer pairs MTS2-fu-KDC-F and KDC-fu-P2A-R, MTS10-fu-ADH-F and ADH-fu-Stu1-R to amplify the gene fragments α-KDC and ADH in E9.1, respectively, and the correct gene fragments were recovered by agarose gel electrophoresis.

[0211] E9.3 is similar to Example 3E3.4-13. The recombinant plasmid was heat-shocked into E. coli DH5α competent cells and colony PCR was performed with primers XYL-F and tHSP-R. The transformants containing the correct bands were inoculated into 5 mL of LB medium with 5 μL of kanamycin and cultured for 12-16 h. The plasmid was extracted and sequenced to obtain the pZPK-Ble-pXYL-MTS2-KDC-P2A-MTS10-ADH-tHSP recombinant plasmid.

[0212] E9.4 is similar to Example 1E1.4-6, and the soil Pseudomonas aeruginosa strain NP11 carrying the recombinant plasmid is continuously subcultured on YPD plates containing bleomycin resistance and cephalosporin, and finally a high-yield isobutanol engineered strain Z3-7 (genotype: Ble-pXYL-MTS2-KDC-P2A-MTS10-ADH-tHSP) is obtained.

[0213] The strain red yeast np11 and the strain Z3-7 prepared in Example 9E9.4 were fermented in a 250 mL conical flask.

[0214] Specifically, similar to Example 7, at the end of fermentation, centrifugation was performed at 9000 g for 1 min, and the upper organic layer was taken for GC detection of isobutanol production. The obtained strains were able to produce a certain amount of isobutanol.

[0215] The primers used in the above experimental process are shown in the following table:

[0216] Table 9 Primer sequence list

[0217]

[0218] Table 9

[0219]

[0220] Example 10: Construction and fermentation of MTSs-mediated 3-hydroxypropionic acid engineering strains

[0221] E10.1 artificial synthesis 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.

[0222] The gene fragment MCR in E10.1 was amplified in E10.2 using primer pair MTS2-fu-MCR-F and MCR-fu-P2A-R, and the correct gene fragment was recovered by agarose gel electrophoresis.

[0223] E10.3 is similar to Example 3E3.4-13. The recombinant plasmid was heat-shocked into E. coli DH5α competent cells and colony PCR was performed with primers XYL-F and tHSP-R. The transformants containing the correct bands were inoculated into 5 mL of LB medium with 5 μL of kanamycin and cultured for 12-16 h. The plasmid was extracted and sequenced to obtain the pZPK-Ble-pXYL-MTS2-MCR-tHSP recombinant plasmid.

[0224] E10.4 is similar to Example 1E1.4-6, the soil Pseudomonas aeruginosa strain NP11 carrying the recombinant plasmid was continuously subcultured on YPD plates containing bleomycin resistance and cephalosporin, and finally a high-yield 3-hydroxypropionic acid engineered strain Z3-8 (genotype: Ble-pXYL-MTS2-MCR-tHSP) was obtained.

[0225] The strain red yeast np11 and the strain Z3-8 prepared in Example 10E10.4 were fermented in a 250 mL conical flask.

[0226] Specifically, similar to Example 7, at the end of the fermentation, HPLC (Shimadzu LC-20AT, Japan) equipped with a RID detector and a PDA detector and an Aminex HPX-87H column (Bio-Rad) were used for analysis. The culture sample was centrifuged, and the supernatant was filtered through a 0.22 μm filter membrane and 0.5 mM H 2 SO 4 The eluent, with a flow rate of 0.5 mL / min and a column temperature of 65 °C, was used to measure 3-hydroxypropionic acid, and the obtained strains were able to produce a certain amount of 3-hydroxypropionic acid.

[0227] The primers used in the above experimental process are shown in the following table:

[0228] Table 10 Primer sequence list

[0229]

[0230] Example 11: Application of a group of mitochondrial signal peptides in other red yeasts

[0231] R. glutinis, R. mucilaginosa, R. graminis, R. rubra, R. lactosa, R. sphaerocarpum, R. marina, R. acheniorum and R. bogoriensis were used as host strains to express the recombinant plasmid PZPK-Ble-pXYL-MTS2-EfMvaS in Example 3. A110G -P2A-EGFP-P2A-MTS10-EfMvaE-tHSP,PZPK-Ntc-pXYL-RFP-P2A-MTS3-MmMK-T2A-MTS4- PMK-tHSP and pZPK-G418-pXYL-MTS2-DPDC-P2A-MTS3-NPPS-GGGS-LS-T2A-MTS4-IDI-tHSP.

[0232] The method for constructing the genetically engineered bacteria is as shown in Example 3, and limonene is extracted and detected as shown in Example 4. The obtained strains can produce a certain amount of limonene.

[0233] 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. Equivalent substitutions or changes made by technicians in this technical field on the basis of the present invention are all 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 slight changes or modifications are equivalent to equivalent implementation cases, and are all within the scope of the technical solution.

Claims

1. A group of mitochondrial signal peptides, characterized in that The mitochondrial signal peptide is selected from the following 11 mitochondrial signal peptide sequences: the NCBI accession numbers corresponding to the 11 mitochondrial signal peptides are: EMS19018.1, EMS19424.1, EMS19541.1, EMS20697.1, EMS20271.1, EMS21738.1, EMS25036.1, EMS25048.1, EMS26107.1, RHTO_07719, XP_016275910.

1.

2. A set of mitochondrial signal peptides according to claim 1, characterized in that: The amino acid sequence of the mitochondrial signal peptide is shown in SEQ ID NO.1-11.

3. A mitochondrial signal peptide gene as claimed in claim 1, characterized in that: The GC content of the nucleotide sequence of the gene of the mitochondrial signal peptide is 50-75%.

4. A recombinant expression vector comprising the gene of the mitochondrial signal peptide according to claim 3, characterized in that: The recombinant expression vector contains at least one gene of the mitochondrial signal peptide.

5. The recombinant expression vector of the mitochondrial signal peptide gene according to claim 4, characterized in that: The recombinant expression vector is a recombinant plasmid.

6. An engineered bacterium for overexpressing a target product synthesis pathway gene mediated by a mitochondrial signal peptide, characterized in that: The engineered bacteria comprises the recombinant expression vector of the gene of the mitochondrial signal peptide as claimed in claim 3.

7. The engineered bacteria for overexpressing the target product synthesis pathway gene mediated by the mitochondrial signal peptide according to claim 6, characterized in that: The engineered bacteria contain at least one recombinant expression vector.

8. The engineered bacteria for overexpressing the target product synthesis pathway gene mediated by the mitochondrial signal peptide according to claim 7, characterized in that: The recombinant expression vector is used alone or in combination with the mitochondrial signal peptide gene to construct engineered bacteria including but not limited to limonene, linalool, patchouli alcohol, kaurene, isobutanol and 3-hydroxypropionic acid, so as to achieve the synthesis of mitochondrial compartmentalized target products.

9. The engineered bacteria for overexpressing the target product synthesis pathway gene mediated by the mitochondrial signal peptide according to claim 8, characterized in that: During the construction of the engineered bacteria, the polycistronic nucleotide sequences of the target product synthesis pathway genes are connected via a 2A connecting peptide gene, and the amino acid sequence of the 2A connecting peptide is as shown in SEQ ID NO.13-14.

10. The engineered bacteria for overexpressing a target product synthesis pathway gene mediated by a mitochondrial signal peptide according to any one of claims 6 to 9, characterized in that: The engineered bacteria is red yeast, which is a species in the genus Rhodotorula, including but not limited to Rhodotorula toruloides, Rhodotorula glutinis, Rhodotorula mucilaginosa, Rhodotorula graminis, Rhodotorula rubra, Rhodotorula lactosa, Rhodotorula sphaerocarpum, Rhodotorula marina, Rhodotorula acheniorum and Rhodotorula bogoriensis.

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