A method for producing a recombinant host cell with improved tolerance to terpenes or terpene-containing essential oils and its use

Abstract

The present invention relates to the field of genetic engineering. Specifically, the present invention relates to a recombinant host cell with improved tolerance to terpenes or terpene-containing essential oils or improved terpene production, a production method thereof and uses thereof. More specifically, the recombinant host cell is produced by increasing the expression amount or activity of PDR5 protein in a wild-type host cell, and can be used for terpene production.

Description

[0001] The present application is a divisional application of Chinese patent application No. 201611037629.9 filed on November 23, 2016, entitled “Recombinant host cells with improved tolerance to terpenes or terpene-containing essential oils or improved terpene production, production methods thereof and uses thereof”. Technical Field

[0002] The present invention relates to the field of genetic engineering, and in particular to a recombinant host cell with improved tolerance to terpenes or terpene-containing essential oils or improved terpene production, a production method thereof and a use thereof. Background Art

[0003] Natural active products are generally trace secondary metabolites synthesized by organisms, which mainly play the role of signal transduction, allelopathy, and preventing the invasion of pathogens and insects in organisms. At the same time, due to their important biological activities, they have been widely used in fields such as health care and nutrition. Direct extraction from the original plants is currently the main way to produce such natural products, but this method has many disadvantages, including low content and large differences, difficult product purification, long plant growth cycle, and serious damage to biological resources, especially wild resources.

[0004] At present, the use of synthetic biology principles to design and modify microbial strains to produce natural products has been internationally recognized as one of the most promising methods. For example, the production of artemisinin's precursor artemisinic acid in yeast engineered bacteria reached a maximum of 25g / L (Paddon CJ et al., 2013, Nature, 2013, 496: 528-532). However, many natural products are toxic to microbial hosts, which will seriously affect their production efficiency in cell factories. Therefore, the key to the successful creation of efficient cell factories is to explore functional proteins that can improve the tolerance of microbial hosts to natural products.

[0005] ABC transporters (ATP-binding cassette transporters) that hydrolyze ATP for energy and H-dependent + / Na + Transport proteins such as MATE detoxification proteins (Multidrug and toxic compound extrusion transporter) that transport against the concentration gradient are the main proteins that perform this function. For example, in the study of the transport of the botanical drug berberine, scientists discovered that the MATE efflux protein AtDTX1 can transport berberine out of the cell, and overexpressing the AtDTX1 protein in Saccharomyces cerevisiae can significantly increase the cell's resistance to the toxicity of berberine (Li L et al., J Biol Chem 2002, 277(7): 5360-5368.).

[0006] Terpenoids such as artemisinin, paclitaxel, β-elemene, zedoary oil, and germaene A have been widely used in clinical applications in tumors and infections. However, many important terpenoids are toxic to microbial hosts. For a large number of terpenoids, especially some important terpenoids, it is necessary to explore specific functional proteins that can improve the tolerance of host cells to them, thereby laying the foundation for the efficient biosynthesis of such compounds. SUMMARY OF THE INVENTION

[0007] The present invention provides a host cell with improved tolerance to terpenes or terpene-containing essential oils or improved terpene production and its use. The specific technical scheme is as follows:

[0008] In a first aspect, the present invention provides a recombinant host cell comprising a PDR5 protein with increased expression level or activity compared to a corresponding wild-type host cell.

[0009] In a second aspect, the present invention provides a method for producing a recombinant host cell with improved tolerance to terpenes or terpene-containing essential oils or improved terpene production, which comprises the step of increasing the expression level or activity of PDR5 protein in a wild-type host cell.

[0010] In a specific embodiment of the present invention, the increased expression of the PDR5 protein is achieved by increasing the copy number of the PDR5 gene and / or using a strong promoter.

[0011] In a specific embodiment related to increasing the copy number of a PDR5 gene, the PDR5 gene is a Saccharomyces cerevisiae PDR5 gene.

[0012] In a further specific embodiment related to increasing the copy number of the PDR5 gene, the Saccharomyces cerevisiae PDR5 gene consists of the nucleic acid sequence of SEQ ID NO:1.

[0013] In a third aspect, the present invention provides use of an isolated polynucleotide encoding a PDR5 protein for improving the tolerance of a host cell to terpenes or terpene-containing essential oils or for improving the terpene production of a host cell.

[0014] In a specific embodiment, the polynucleotide encoding the PDR5 protein is the Saccharomyces cerevisiae PDR5 gene.

[0015] In a further embodiment, the Saccharomyces cerevisiae PDR5 gene consists of the nucleic acid sequence of SEQ ID NO:1.

[0016] In a fourth aspect, the present invention provides a use of a PDR5 protein for improving the tolerance of a host cell to terpenes or terpene-containing essential oils or for improving the terpene production of a host cell.

[0017] In a specific embodiment, the PDR5 protein is a Saccharomyces cerevisiae PDR5 protein.

[0018] In a specific embodiment, the Saccharomyces cerevisiae PDR5 protein consists of the amino acid sequence of SEQ ID NO:139.

[0019] In the above aspects of the present invention, the host cell further comprises an exogenous polynucleotide for terpenoid production.

[0020] In the above aspects of the present invention, the host cell is a eukaryotic organism, preferably a Saccharomycetes.

[0021] In the above aspects of the present invention, the host cell is selected from Agaricus, Aspergillus, Bacillus, Candida, Corynebacterium, Escherichia, Fusarium, Gibberella, Kluyveromyce, Laetiporus, Lentinus, Phaffia, Phanerochaete, Pichia, Physcomitrella, Rhodoturula, Saccharomyces, Schizosaccharomyces, Sphaceloma, Xanthophyllomyces and Yarrowiagenera.

[0022] In the above aspects of the present invention, the host cell is selected from the species Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous or Candida albicans, preferably Saccharomyces cerevisiae.

[0023] In the above aspects of the present invention, the terpenes are selected from hemiterpenes, monoterpenes, sesquiterpenes and diterpenes, preferably from monoterpenes and sesquiterpenes, and particularly preferably from sesquiterpenes.

[0024] In the above aspects of the present invention, the terpenes are selected from geranyl esters, geraniol, farnesol, nerolidol, nootkatone, germacene A, elemene, santalol, and clary alcohol, preferably selected from farnesol, nerolidol, germacene A, elemene, and santalol, and the terpene-containing essential oils are selected from zedoary turmeric oil, turmeric oil, sweet orange oil, peppermint oil, perilla oil, and sandalwood oil, preferably selected from zedoary turmeric oil, turmeric oil, and sandalwood oil.

[0025] In a fifth aspect, the present invention provides a method for producing terpenes, comprising culturing the recombinant host cell of the present invention or the recombinant host cell produced by the method of the present invention in a culture medium under conditions that result in the production of terpenes, and recovering the terpenes from the culture medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 . Flowchart for the construction of yeast cell ABC and MATE transporter mutant strains

[0027] Figure 2 Effect of Gemacrene A (GMA) on the growth of mutant strains. When GMA was added to the culture medium, the growth of the strain NK2-ScPDR5 lacking the ABC transporter ScPDR5 (Saccharomyces cerevisiae PDR5) was significantly inhibited compared with the control strain.

[0028] Figure 3.Regulating the expression of ScPDR5 gene changes the tolerance to GMA. Overexpression of ScPDR5 gene in strain NK2 can significantly improve the strain's tolerance to GMA, while the growth of strains NK2, NK2-GFP, and NK2-ScPDR5 is significantly inhibited. The loss of ScPDR5 function leads to a significant decrease in the strain's tolerance to terpenes. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention is not limited to the specific methods, protocols, reagents, etc. described herein, as these may vary. The terms used herein are only used for the purpose of describing specific embodiments rather than for limiting the scope of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art.

[0030] As used herein, the term "host cell" means any cell type that is suitable for carrying out the recombinant manipulations of the invention, e.g., susceptible to transformation, transfection, transduction, etc., with a nucleic acid construct or expression vector comprising a polynucleotide of the invention. The term host cell encompasses any progeny of a parent cell that differs from the parent cell due to mutations that occur during replication.

[0031] Host cells suitable for the present invention include prokaryotes and eukaryotes. Exemplary host cells are eukaryotes, preferably yeasts, such as Agaricus, Aspergillus, Bacillus, Candida, Corynebacterium, Escherichia, Fusarium, Gibberella, Kluyveromyces, Sulfur, Lentinus, Phaffia, Pichia, Moss, Rhodotorula, Saccharomyces, Schizosaccharomyces, Cyclospora, Phaffia and Yarrowia. Preferably selected from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbyella cotton, Candida utilis, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Phaffia or Candida albicans species. Saccharomyces cerevisiae is particularly preferred.

[0032] In a specific embodiment of the present invention, the host cell used is Saccharomyces cerevisiae CEN.PK2-1D (hereinafter referred to as NK2, purchased from the European Collection of Saccharomyces cerevisiae (EUROSCARF)).

[0033] As used herein, the term "recombinant" refers to the change of genetic material by human intervention. Generally, recombinant refers to the manipulation of DNA or RNA in cells or viruses or expression vectors by molecular biology methods.

[0034] As used herein, the term "expression" refers to the process by which an endogenous or exogenous target protein is produced in a cell, which includes but is not limited to transcription, post-transcriptional modification, translation, post-translational modification and secretion.

[0035] The method of the present invention for gene expression by homologous recombination is a conventional method in the art (Shao Z et al., Nucleic Acids Research 2009, 37(2): e16; Dai Z et al., Metabolic Engineering 2013, 20: 146-156).

[0036] As used herein, the term "increased expression" means that the target protein is produced at a higher level in the cell compared to the wild type, that is, overexpressed.

[0037] The increase in the expression of the target protein can be achieved by increasing the copy number of the polynucleotide encoding the target protein, for example, by introducing one or more additional copies of an exogenous polynucleotide into the host cell. As used herein, the term "exogenous polynucleotide" refers to a polynucleotide that is not native to the host cell, including homologous polynucleotides from the same species as the host cell and heterologous polynucleotides from a different species from the host cell.

[0038] Exogenous polynucleotides in expression vectors can be introduced into host cells by many known techniques, including but not limited to heat shock transformation, electroporation, DEAE-dextran transfection, microinjection, liposome-mediated transfection, calcium phosphate precipitation, protoplast fusion, microprojectile bombardment, viral transformation and the like.

[0039] Increasing the expression level of the target protein can also be achieved by using a strong promoter, for example, increasing the strength of the promoter used to drive the expression of the coding sequence or changing the type of the promoter.

[0040] In some embodiments, homologous recombination can be used to replace the promoter of the endogenous gene encoding the target protein with a promoter that causes an increase in the expression of the target protein, such as a strong promoter. The strong promoter is, for example, a constitutive or inducible promoter. In other embodiments, homologous recombination can be used to integrate an inducible or constitutive promoter and a gene encoding the target protein or a transcription factor gene into another locus of the genome.

[0041] The increase in the expression of the target protein can also be achieved by other conventional methods known in the art, such as increasing the strength of the ribosome binding site or Kozak sequence, structurally modifying the coding region, changing the codon usage, increasing the stability of the mRNA transcript, increasing the stability of the protein, and inducing overexpression of the target protein by utilizing the natural mechanism of the host cell (e.g., heat shock, stress, heavy metal or antibiotic exposure). For example, the terpenes of the present invention have been shown to induce overexpression of PDR5.

[0042] Increasing the expression level of the target protein can also be achieved by any combination of the above-mentioned methods, such as using a plasmid containing a promoter that causes overexpression of the target protein in the host cell to introduce an exogenous polynucleotide encoding the target protein into the host cell.

[0043] As used herein, the term "increased activity" refers to an increase in a specific biological function of a target protein compared to a wild-type host cell. In the present invention, the increased activity of a PDR5 protein refers to an increase in the function of a PDR5 protein in a recombinant host cell to transport molecules and ions through a biological membrane compared to a wild-type host cell.

[0044] As used herein, the term "vector" refers to a linear or circular DNA molecule that contains a polynucleotide encoding a target protein and is operably linked to a regulatory sequence for its expression. Vectors used in the present invention include vectors that replicate autonomously in host cells, such as plasmid vectors; and also include vectors that can be integrated into the host cell genome and replicated with the host cell genes. Vectors suitable for the present invention are commercially available.

[0045] As used herein, the term "isolated" polynucleotide refers to a polynucleotide that is separated from at least one component with which it is naturally associated.

[0046] As used herein, the term "transporter" refers to a membrane protein involved in moving molecules and ions through biological membranes. Currently known transporter families include the SMR family, the RND family, the MATE family, and the ABC transporter family.

[0047] ABC transporters utilize the energy from the hydrolysis of adenosine triphosphate (ATP) to participate in the transmembrane transport of a variety of substrates, such as metabolites, lipids and sterols, and drugs. To date, there are 129 and 128 ABC transporters in the Arabidopsis and rice genomes, respectively; there are 31 and 28 ABC transporters in Saccharomyces cerevisiae and Candida albicans, respectively. Taking Saccharomyces cerevisiae as an example, its ABC transporter superfamily members are divided into 6 subfamilies, non-limiting examples include PDR5, PDR10, PDR15, SNQ2, YOR1, YOL075C and PDR18, etc.

[0048] ABC transporters usually have different substrate specificities, and those skilled in the art are not fully aware of the corresponding relationship between different ABC transporters and their substrates. Taking PDR5 as an example, it has been previously reported that deletion of the endogenous PDR5 gene leads to increased tolerance of yeast to butanol. The present invention finds that deletion of the PDR5 gene leads to a decrease in yeast tolerance to terpenes, while overexpression thereof leads to an increase in yeast tolerance to terpenes or terpene-containing essential oils.

[0049] The PDR5 (pleiotropic drug resistance 5) gene encodes a cytoplasmic membrane ABC transporter protein PDR5 involved in the exchange of substances inside and outside the cell. The GenBank number of the Saccharomyces cerevisiae PDR5 gene is Z75061.1.

[0050] As an example, the PDR5 gene used in the present invention is a Saccharomyces cerevisiae PDR5 gene, in particular, a Saccharomyces cerevisiae PDR5 gene consisting of the nucleic acid sequence of SEQ ID NO:1.

[0051] As an example, the PDR5 protein used in the present invention is a Saccharomyces cerevisiae PDR5 protein, in particular, a Saccharomyces cerevisiae PDR5 protein consisting of the amino acid sequence of SEQ ID NO:139.

[0052] Those skilled in the art should understand that the above-mentioned genes / proteins or their sequences are only examples, and any functional analogs of the genes / proteins or equivalent technical solutions of gene / protein sequences obtained by any conventional adjustment of the gene / protein sequences that can also improve the tolerance of host cells to terpenes or terpene-containing essential oils are within the scope of protection of the present invention.

[0053] Exogenous polynucleotides for the production of specific terpenes, such as nerolidol synthase gene, germarene A synthase gene, sclareol production, lysinediol pyrophosphate synthase gene and sclareol synthase gene, etc., can be used in the present invention according to actual production needs.

[0054] As used herein, the term "terpenoid" refers to a large and diverse class of organic molecules derived from five-carbon isoprenoid units assembled and modified in various ways and grouped according to the number of isoprenoid units used in the group members. For example, hemiterpenes have one isoprenoid unit, monoterpenes have two isoprenoid units, sesquiterpenes have three isoprenoid units, diterpenes have four isoprenoid units, triterpenes have six isoprenoid units, and tetraterpenes have eight isoprenoid units. Terpenes have chain-shaped, cyclic, and olefinic bonds with different degrees of saturation. Terpenes can be chemically modified, such as oxidation and carbon chain rearrangement, to form a large number of terpenoids, which also belong to the terpenes of the present invention.

[0055] As used herein, the term "terpene-containing essential oil" refers to volatile aromatic substances containing terpenes extracted from the flowers, leaves, stems, roots or fruits of plants by steam distillation, squeezing, cold soaking or solvent extraction. The recombinant host cell of the present invention also has improved tolerance to terpene-containing essential oils containing terpenes, such as zedoary turmeric oil, turmeric oil, sweet orange oil, peppermint oil, perilla oil and sandalwood oil, preferably zedoary turmeric oil, turmeric oil and sandalwood oil.

[0056] The present invention successfully screened and identified the ABC transporter PDR5 that can improve the tolerance of host cells such as Saccharomyces cerevisiae to terpenes or terpene-containing essential oils, and constructed a host cell with improved tolerance to terpenes or terpene-containing essential oils, laying a foundation for the efficient biosynthesis of terpenes. Example

[0057] To make the technical solutions and advantages of the present invention clearer, the embodiments of the present invention will be further described in detail by examples. It should be understood that the examples should not be construed as limiting, and those skilled in the art can make further adjustments to the embodiments based on the principles of the present invention.

[0058] Example 1: Effects of a series of terpenes on the growth of Saccharomyces cerevisiae CEN.PK2-1D

[0059] Take an appropriate amount of DMSO solvent to prepare the stock solutions of terpenes or terpene-containing essential oils listed in Table 1 (all purchased from Shanghai Yuanye Biotechnology Co., Ltd.) (final concentration is 10 g / L) for use. Activate Saccharomyces cerevisiae CEN.PK2-1D (hereinafter referred to as NK2, purchased from European Saccharomyces cerevisiae Collection Center (EUROSCARF)) in solid selection medium 1, and the solid selection medium 1 is composed of 0.8% yeast selection medium SD-Ura-Trp-Leu-His (purchased from Beijing Fanjinuo Technology Co., Ltd.), 2% glucose, 0.01% Leu. (leucine), 0.005% His. (histidine), 0.01% Ura. (uracil), 0.01% Trp. (tryptophan) and 2% agar powder. Then inoculate in the corresponding liquid selection medium 1, the composition of liquid selection medium 1 is as follows: 0.8% yeast selection medium SD-Ura-Trp-Leu-His, 2% glucose, 0.01% Leu., 0.005% His., 0.01% Ura.0.01% Trp. Cultivate at 30°C, 250rpm for 16 hours to prepare seed solution. Inoculate the seed solution in test tubes containing 4ml liquid selection medium 1, and then add appropriate amount of terpenoid or terpene-containing essential oil stock solution to make the final concentrations as shown in Table 1. Cultivate at 30°C, 250rpm for 8 hours, and measure the sample OD value at 600nm with an enzyme label analyzer. Calculate the inhibition rate with terpenoid or terpene-containing essential oil concentration of 0mg / L as the control reference (inhibition rate = (1-GMA growth OD / control reference OD) × 100%). And the drug concentration in the lowest concentration well with an inhibition rate of more than 50% is calculated as MIC50, unit mg / L.

[0060] Table 1: Inhibition of a series of terpenes or terpene-containing essential oils on Saccharomyces cerevisiae

[0061]

[0062]

[0063] As shown in Table 1, different concentrations of terpenes or terpene-containing essential oils have a significant inhibitory effect on the growth of the common Saccharomyces cerevisiae engineered chassis strain NK2.

[0064] Example 2: Construction of yeast cell ABC and MATE transporter mutant strains

[0065] 1. Construction of pEASY-Trp-URA plasmid

[0066] A: pRS313-TRP-PGK1-MAA45-CYC1t (described in Chinese Patent No. 201610236283.9, which is available to the public from Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences) and B: pHUra-δDNA (Zhubo Dai, Yi Liu, Luqi Huang, Xueli Zhang, Biotechnology and Bioengineering, 2012, 109(11): 2845-2853. The public can obtain it from Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences) as templates, TRP-URA-BamH1 (1253 bp) and BamH1-TRP-URA (1184 bp) were amplified using the primers in Table 2. The amplification system was: 5×Phusion HFBuffer 10 μL, dNTP (10 mM each dNTP) 1 μL, DNA template 20 ng, primers (10 μM) 1 μL each, Phusion High-Fidelity DNA Polymerase (2.5U / μL) 0.5μL, distilled water was added to the total volume of 50μL. Amplification conditions were: 98℃ pre-denaturation for 2 minutes (1 cycle); 98℃ denaturation for 10 seconds, annealing for 10 seconds (annealing temperature 58℃), 72℃ extension for 1 minute (32 cycles); 72℃ extension for 8 minutes (1 cycle).

[0067] Table 2: PCR system information table

[0068]

[0069] BamH1 was used to digest TRP-URA-BamH1 and BamH1-TRP-URA, and the two fragments were purified by gel cutting. 50 ng of each gene fragment was added to the ligation system: 2 μL 10×T4 DNA Ligase Reaction Buffer (NEB), 1 μL T4ligase (NEB, 400,000 cohesive end units / ml), and distilled water was added to 20 μL. The reaction was carried out at room temperature for 2 hours to obtain the ligation product. 1 μL of the ligation product was added to the amplification system: 10 μL 5×Phusion HF Buffer (NEB), 1 μL dNTP (10 mM each dNTP), 20 ng DNA template, 1 μL each of primers TRP-F (10 μM) and URA-R (10 μM), 0.5 μL Phusion High-Fidelity DNA Polymerase (2.5 U / μL) (NEB), and distilled water was added to the total volume of 50 μL. Amplification conditions were as follows: pre-denaturation at 98°C for 2 minutes (1 cycle); denaturation at 98°C for 10 seconds, annealing at 58°C for 10 seconds, and extension at 72°C for 1.5 minutes each (32 cycles); and extension at 72°C for 8 minutes (1 cycle).

[0070] The target size fragment amplification product was cloned into the pEASY-Blunt cloning vector (purchased from Beijing Quanshijin Biotechnology Co., Ltd.). The cloning system is: 1μL PCR amplification product, 1μL pEASY-Blunt cloning vector, gently mixed, reacted at 25℃ for 10 minutes, added 50μL Trans10 competent cells (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), and ice bathed for 30 minutes. Heat shock at 42℃ for 30 seconds, immediately put on ice for 2 minutes. Add 800μL LB medium, incubate at 250rpm, 37℃ for 1 hour, take 200μL of bacterial solution and apply it on the LB plate containing ampicillin. After overnight culture, PCR screen 5 positive single colonies, liquid culture the positive clones, extract the positive clone plasmids for sequencing verification, and the sequencing results show that the target fragment is inserted into the vector pEASY-Blunt to obtain the plasmid: pEASY-TRP-URA.

[0071] 2. Construction of mutant strains

[0072] 2.1 Gene knockout by homologous recombination

[0073] Gene knockout by homologous recombination is the application of the principle of DNA homologous recombination. A homologous DNA fragment containing a nutritional deficiency screening marker is introduced into the recipient cell, so that the homologous fragment replaces the target gene fragment at the original position, and the strain that loses the activity of the gene is screened out by the nutritional deficiency medium. Knockout is mainly divided into two steps. First, the preparation of homologous recombination fragments. The homologous recombination fragments are divided into two parts. The first part is the homologous recombination fragment with a screening marker. The homologous recombination fragment Gene-TU containing a 50bp homologous region and a TRP-URA screening marker is obtained by PCR amplification. The second part is the homologous recombination fragments Gene-Up and Gene-Down containing a 500-600bp homologous region. Second, in vivo homologous recombination is performed. The amplified homologous recombination fragments are integrated into the recipient cells by electroporation to cause homologous recombination. Knockout principle, see Figure 1 .

[0074] 2.2 Preparation of homologous recombination fragments

[0075] C: pEASY-TRP-URA (obtained from step 1 of this example) and D: CEN.PK2-1D genome (genome was extracted using the Kangwei Century Yeast Genome Extraction Kit (Cat. No.: CW0569) according to the manufacturer's instructions) were used as templates and the primers in Table 3 to amplify the knockout modules: M1 (ScPDR5-TU, ScPDR5-UP, ScPDR5-Down), M2 (ScPDR10-TU, ScPDR10-UP, ScPDR10-Down), M3 (ScPDR11-TU, ScPDR11-UP, ScPDR11-Down), M4 (ScPDR12-TU, ScPDR12-UP, ScPDR12-Down), M5 (ScPDR15-TU, ScPDR15-UP, ScPDR15-Down), and M6 (ScPDR16-TU, ScPDR16-UP, ScPDR16-Down). The knockout modules were a mixture of three gene fragments in a molar ratio of 1:1:1. The amplification system was: 5×Phusion HF Buffer 10μl, dNTP (10mM each dNTP) 1μl, DNA template 20ng, primer (10μM) 1μl each, Phusion High-Fidelity DNA Polymerase (2.5U / μl) 0.5μl, and distilled water was added to a total volume of 50μl. The amplification conditions were 98℃ pre-denaturation for 1.5 minutes (1 cycle); 98℃ denaturation for 10 seconds, annealing for 10 seconds (annealing temperature 58℃), 72℃ extension for 2 minutes (32 cycles); 72℃ extension for 8 minutes (1 cycle), and the product was recovered and stored by tapping.

[0076] Table 3: PCR system information table

[0077]

[0078]

[0079]

[0080]

[0081] 2.3 Construction of strains

[0082] The starting yeast Saccharomyces cerevisiae NK2 was cultured overnight in liquid culture medium 1 (liquid culture medium formula reference Example 1), 1 ml (OD about 0.6-1.0) was taken and dispensed into 1.5 ml EP tubes, centrifuged at 4°C and 10000g for 1 min, the supernatant was discarded, the precipitate was washed with sterile water (4°C), centrifuged under the same conditions, and the supernatant was discarded. 1 ml of treatment solution (10 mM LiAc; 10 mM DTT; 0.6 M sorbitol; 10 mM Tris-HCl (pH 7.5, DTT was added only when the treatment solution was used) was added to the bacteria, and placed at 25°C for 20 min. Centrifuged, the supernatant was discarded, 1 ml of 1 M sorbitol (0.22 μM water system membrane sterilization) was added to the bacteria, resuspended, centrifuged, and the supernatant was discarded (resuspended twice with 1 M sorbitol), to a final volume of about 90 μl, and 12 tubes were prepared in the same way. 4.5 μL of knockout modules M1, M2, M3, M4, M5, M6, M7, M8, M9, M10, M11, and M12 (knockout modules can be obtained from step 2.2 of this embodiment) were added respectively, mixed and transferred to an electroporation cup, electroshocked at 2.7 kv for 5.7 ms, 1 ml of 1 M sorbitol was added, resuscitated at 30° C. for 1 h, and coated on the screening solid medium 2, which was composed of 0.8% yeast selection medium SD-Ura-Trp-Leu-His (purchased from Beijing Fanjinuo Technology Co., Ltd.), 0.01% Leu., 0.005% His., 2% glucose and 2% agar powder. The screening culture conditions were: 30° C., culture for more than 36 h. The correct positive clones were identified by PCR verification and sequencing and named as strains NK2-ScPDR5, NK2-ScPDR10, NK2-ScPDR11, NK2-ScPDR12, NK2-ScPDR15, NK2-ScAUS1, NK2-ScYOR1, NK2-ScSNQ2, NK2-ScSTE6, NK2-ScSCE1, NK2-ScSCE2, and NK2-ScSCE3. The strain information is shown in Table 4.

[0083] Table 4: Engineering strain information

[0084]

[0085]

[0086] Example 3: Screening and identification of transporters associated with terpenoid tolerance

[0087] 1. Screening of transporters to improve GMA tolerance

[0088] NK2 was activated in solid selection medium 1 (culture medium formula reference example 1), and NK2-ScPDR5, NK2-ScPDR10, NK2-ScPDR11, NK2-ScPDR12, NK2-ScPDR15, NK2-ScAUS1, NK2-ScYOR1, NK2-ScSNQ2, NK2-ScSTE6, NK2-ScSCE1, NK2-ScSCE2, and NK2-ScSCE3 were activated in solid selection medium 2 (culture medium formula reference example 2), and inoculated into corresponding liquid selection medium 1 (culture medium formula reference example 1) and liquid selection medium 2, respectively. The composition of liquid culture 2 was as follows: 0.8% yeast selection medium SD-Ura-Trp-Leu-His (purchased from Beijing Fanjinuo Technology Co., Ltd.), 0.01% Leu., 0.005% His., and 2% glucose. The seed solution was prepared by culturing at 30° C. and 250 rpm for 16 h. The seed solution was inoculated into 4 ml of the corresponding liquid selection medium containing GMA (final concentration of 5 mg / L) or without GMA in a test tube and cultured at 30°C, 250 rpm for 8 hours, and the sample OD value was measured at 600 nm using an enzyme label analyzer. The inhibition rate was calculated using the drug concentration of 0 mg / L as the control reference. (Inhibition rate = (1-GMA growth OD / control reference OD) × 100%).

[0089] like Figure 2 When GMA was added to the culture medium, the growth of the strain NK2-ScPDR5 lacking the ABC transporter protein ScPDR5 was significantly inhibited compared with the control strain.

[0090] 2. Functional Identification of Transporters that Improve GMA Tolerance

[0091] 2.1 Construction of ScPDR5 overexpressing and control strains

[0092] 2.1.1 Construction of pRS313-TRP-TEF1-MAA45-CYC1t plasmid

[0093] TEF1 (450 bp) was amplified using pM3-ERG9 (described in Chinese patent application 201210453416.X, available from Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences) as template and primers in Primer List 5. The amplification system was: 5×Phusion HF Buffer 10μL, dNTP (10mM each dNTP) 1μL, DNA template 20ng, primers (10μM) 1μL each, Phusion High-Fidelity DNA Polymerase (2.5U / μL) 0.5μL, and distilled water added to a total volume of 50μL. The amplification conditions were 98℃ pre-denaturation for 2 minutes (1 cycle); 98℃ denaturation for 10 seconds, annealing for 10 seconds (annealing temperature 58℃), 72℃ extension for 1 minute (32 cycles); 72℃ extension for 8 minutes (1 cycle).

[0094] Table 5: PCR system information table

[0095]

[0096] The amplified fragment TEF1 and plasmid pRS313-TRP-PGK1-MAA45-CYC1t were double-digested with Pac1 and SexA1, and the fragments TEF1 and pRS313-TRP-MAA45-CYC1t were recovered by gel. 50 ng of each of the obtained fragments were added to the ligation system: 2 μL 10×T4 DNA Ligase Reaction Buffer (NEB), 1 μL T4 ligase (NEB, 400,000 cohesive end units / ml), and distilled water was added to 20 μL. The reaction was carried out at room temperature for 2 hours to obtain the ligation product, which was transferred into Trans10 competent cells and sequenced for verification (the method is the same as that of Example 2: Step 1). The plasmid: pRS313-TRP-TEF1-MAA45-CYC1t was obtained.

[0097] 2.1.2 Construction of control pRS313-TRP-TEF1-GFP-CYC1t plasmid

[0098] GFP (828 bp) was amplified using pYM-N9 (Carsten Janke, Maria M. Magiera and Nicole Rathfelder, et al., Yeast 2004; 21: 947-962. Available from Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences) as template and primers in Primer List 5. The amplification system was: 5×Phusion HF Buffer 10 μL, dNTP (10 mM each dNTP) 1 μL, DNA template 20 ng, primers (10 μM) 1 μL each, Phusion High-Fidelity DNA Polymerase (2.5 U / μL) 0.5 μL, and distilled water added to a total volume of 50 μL. The amplification conditions were pre-denaturation at 98°C for 2 minutes (1 cycle); denaturation at 98°C for 10 seconds, annealing for 10 seconds (annealing temperature 58°C), extension at 72°C for 1 minute (32 cycles); extension at 72°C for 8 minutes (1 cycle).

[0099] The amplified fragment GFP and plasmid pRS313-TRP-TEF1-MAA45-CYC1t (which can be obtained from step 2.1.1 of this example) were double-digested with SexA1 and Asc1, and the fragments GFP and pRS313-TRP-TEF1-…-CYC1t were recovered from the gel. 50 ng of each of the obtained fragments was added to the ligation system: 2 μL 10×T4 DNA Ligase Reaction Buffer (NEB), 1 μL T4 ligase (NEB, 400,000 cohesive end units / ml), and distilled water was added to 20 μL. The reaction was carried out at room temperature for 2 hours to obtain the ligation product, which was transferred into Trans10 competent cells and sequenced for verification (the method is the same as that of step 1 of example 2). The pRS313-TRP-TEF1-GFP-CYC1t plasmid was obtained.

[0100] 2.1.3 Construction of strains NK2+ScPDR5 and NK2+GFP

[0101] Using E: pRS313-TRP-TEF1-MAA45-CYC1t (which can be obtained from step 2.1.1 of this example) and D: CEN.PK2-1D genome (which can be obtained from step 2.2 of Example 2) as templates and primers in List 6, Asse-313-TRP-TEF1-CYC1t (6680 bp), Asse-ScPDR5 (4550 bp), Helper-TEF1 (550 bp), and Helper-CYC1 (550 bp) were amplified. The amplification system was: 10 μL of 5×Phusion HF Buffer, 1 μL of dNTP (10 mM each dNTP), 20 ng of DNA template, 1 μL of each primer (10 μM), 0.5 μL of Phusion High-Fidelity DNA Polymerase (2.5 U / μL), and distilled water was added to make the total volume 50 μL. Amplification conditions were: 98°C pre-denaturation for 2 minutes (1 cycle); 98°C denaturation for 10 seconds, annealing for 10 seconds (annealing temperature 58°C), 72°C extension for 3 minutes (32 cycles); 72°C extension for 8 minutes (1 cycle). Gel was recovered for later use.

[0102] Table 6: PCR system information table

[0103]

[0104]

[0105] The same method as in Example 2: Step 2.3 was used to prepare and transform NK2 competent cells. A total of 6 μL of fragments Asse-313-TRP-TEF1-CYC1t, Asse-ScPDR5, Helper-TEF1, and Helper-CYC1 (molar ratio of 1:1:1:1) and plasmid pRS313-TRP-TEF1-GFP-CYC1t (which can be obtained from this Example: Step 2.2.2) were added, mixed, and transferred to an electric rotating cup respectively. Electric shock was performed at 2.7 kV for 5.7 ms, and 1 ml of 1 M sorbitol was added. The cells were revived at 30°C for 1 h and spread on solid selection medium 3. The solid selection medium 3 was composed of 0.8% yeast selection medium SD-Ura-Trp-Leu-His (purchased from Beijing Fanjinuo Technology Co., Ltd.), 0.01% Leu., 0.005% His. 0.01% Ura., 2% glucose and 2% agar powder. The screening culture conditions were: 30°C, culture for more than 36 hours. The correct positive clones were identified by PCR and named as strains NK2+ScPDR5 and NK2+GFP.

[0106] 2.2 Regulation of ScPDR5 gene expression in response to GMA tolerance

[0107] NK2 was activated in solid selection medium 1 (culture medium formula reference Example 1), and inoculated into corresponding liquid selection medium 1 (culture medium formula reference Example 1) and cultured at 30°C, 250rpm for 16h to prepare seed solution, NK2+GFP, NK2-ScPDR5 (obtained from Example 2) and NK2+ScPDR5 were activated in solid selection medium 3 (culture medium formula reference this example: step 2.1.3), and inoculated into corresponding liquid selection medium 3, the composition of liquid selection medium 3 was as follows: 0.8% yeast selection medium SD-Trp (purchased from Beijing Fanjinuo Technology Co., Ltd.), 2% glucose. Seed solution was prepared by culture at 30°C, 250rpm for 16h. The seed liquid NK2, NK2+GFP, NK2+ScPDR5 and NK2-ScPDR5 were inoculated into 4 ml test tubes of the corresponding liquid selection culture medium containing GMA (final concentration of 5 mg / L) / without GMA, and cultured at 30°C, 250 rpm for 8 hours. The OD value of the samples was measured at 600 nm using an enzyme label analyzer, and the drug concentration of 0 mg / L was used as a control reference.

[0108] like Figure 3 Overexpression of the ScPDR5 gene in strain NK2 can significantly improve the strain's tolerance to GMA, while the growth of strains NK2, NK2-GFP, and NK2-ScPDR5 was significantly inhibited. The loss of ScPDR5 function led to a significant decrease in the strain's tolerance to GMA.

[0109] Example 4: Overexpression of the ScPDR5 gene in Saccharomyces cerevisiae can improve the strain's tolerance to terpenes and terpene-containing essential oils

[0110] Activate NK2 in solid selection medium 1 (culture medium formula reference example 1), and inoculate it in the corresponding liquid selection medium 1 (culture medium formula reference example 1) and culture it at 30°C, 250rpm for 16h to prepare seed solution, activate NK2+GFP, NK2-ScPDR5 and NK2+ScPDR5 in solid selection medium 3 (culture medium formula reference this example: step 2), and inoculate it in the corresponding liquid selection medium 3 (culture medium formula reference example 3) and culture it at 30°C, 250rpm for 16h to prepare seed solution. The seed solution NK2, NK2+GFP, NK2+ScPDR5 and NK2-ScPDR5 were also inoculated in test tubes containing various terpenes (final concentrations as shown in Table 7) / without various terpenes, respectively, and cultured at 30°C, 250rpm for 8 hours, and the sample OD value was measured at 600nm using an enzyme label analyzer. The inhibition rate was calculated using the drug concentration of 0mg / L as the control reference. (Inhibition rate = (1-GMA growth OD / control reference OD) × 100%). Overexpression of the ScPDR5 gene in Saccharomyces cerevisiae can improve the strain's tolerance to terpenes and terpene-containing essential oils. The results are shown in Table 7.

[0111] Table 7: Relationship between regulation of ScPDR5 gene expression and strain tolerance to terpenes and terpene-containing essential oils in Saccharomyces cerevisiae

[0112]

[0113] Table Note: “↑” indicates increased tolerance

[0114] As shown in Table 7, knocking out and overexpressing ScPDR5 in strain NK2 can significantly affect the strain's tolerance to related terpenes or terpene-containing essential oils. In NK2-ScPDR5, the loss of ScPDR5 function significantly reduced the strain's tolerance to related terpenes or terpene-containing essential oils, and the inhibition rate was significantly increased; on the contrary, in NK2+ScPDR5, the overexpression of ScPDR5 significantly increased the strain's tolerance to related terpenes or terpene-containing essential oils, and the inhibition rate was significantly reduced.

[0115] Example 5: Effect of overexpression of ScPDR5 gene on target product yield of terpenoid engineering strains

[0116] 5.1 Acquisition of genetic elements and construction of plasmids used

[0117] 5.1.1 Preparation of genetic elements

[0118] The GES gene (SEQ ID NO: 2), SAAT gene (SEQ ID NO: 3), NES gene (SEQ ID NO: 4), LPS gene (SEQ ID NO: 5), TPS gene (SEQ ID NO: 6) and SaGGPS gene (SEQ ID NO: 7) were fully synthesized at GenScript Biotech Co., Ltd.

[0119] The genomic DNA of the yeast strain NK2-SQ (Establishment of a Yeast Cell Factory for Fermentation Production of Lupeol, Chinese Journal of Traditional Chinese Medicine, Lin Tingting, Wang Dong, Dai Zhubo, Zhang Xueli, Huang Luqi, 2016, 41(6): 1008-1015) was extracted as a template and amplified using the primers in Table 8 to obtain the ERG20 fragment (SEQ ID NO: 8); the synthetic LPS gene and TPS gene were used as templates and amplified using the primers in Table 8 to obtain the cLPS fragment and the cTPS fragment.

[0120] The above amplification system is: TAKARA HS DNA polymerase 5×PS Buffer 10μL, dNTPMix 4μL, primers 1μL each, DNA template 1μL, HS polymerase (2.5U / μL) 0.5μL, add distilled water to a total volume of 50μL. Amplification conditions are: 98℃ pre-denaturation for 3 minutes (1 cycle); 98℃ denaturation for 10 seconds, 55℃ annealing for 15 seconds, 72℃ extension for 2.5 minutes (30 cycles); 72℃ extension for 10 minutes (1 cycle). The obtained PCR product was purified and placed in a -20℃ refrigerator for use.

[0121] Table 8: Primer sequences

[0122]

[0123]

[0124] 5.1.2 Construction of Gene Element Plasmid

[0125] 5.1.2.1 Construction of pM2-cLPS and pM2-SAAT plasmids

[0126] The cLPS amplified in 5.1.1 above, the fully synthesized SAAT and the plasmid pM2-tHMG1 (recorded in Chinese patent ZL201310399947.X) were double-digested with SexA1 and Asc1 to obtain SAAT digestion products, cLPS digestion products and plasmid pM2-tHMG1 digestion backbone; the pM2-tHMG1 digestion backbone was then connected to the digestion products cLPS and SAAT, respectively, to obtain recombinant plasmids pM2-cLPS and pM2-SAAT.

[0127] 5.1.2.2 Construction of pM4-ERG20 and pM4-SaGGPS plasmids

[0128] The ERG20, SaGGPS and plasmid pM11-AtCPR1 (pM11-AtCPR1 is recorded in Chinese patent ZL201310399947.X and is available to the public from Tianjin Institute of Industrial Biotechnology and Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences) obtained in 5.1.1 were double-digested with SexA1 and Asc1, respectively; ERG20 and SaGGPS digestion products and plasmid pM11-AtCPR1 digestion backbone were obtained; and the ERG20 and SaGGPS digestion products were connected to the plasmid pM11-AtCPR1 digestion backbone, respectively, to obtain recombinant plasmids pM4-ERG20 and pM4-SaGGPS.

[0129] 5.1.2.3 Construction of pM3-cTPS and pM3-GES plasmids

[0130] The cTPS amplified in 5.1.1 above, the fully synthesized GES and the plasmid pM3-ERG9 (recorded in Chinese patent application 201210453416.X) were double-digested with SexA1 and Asc1, respectively; the GES digestion product, the cLPS digestion product and the plasmid pM3-ERG9 digestion skeleton were obtained; and the pM3-ERG9 digestion skeleton was connected with the digestion products cTPS and GES, respectively, to obtain recombinant plasmids pM3-cTPS and pM3-GES.

[0131] 5.1.2.4 Construction of pEASY-GAL80-LEU2 plasmid

[0132] GAL80 and LEU2 were amplified using the primers in Primer Table 9, respectively, using NK2-SQ genomic DNA and pRS425 (Sikorski, RS and Hieter, P. 1989, Genetics 122 (1): 19-27, which is publicly available from Tianjin Industrial Biotechnology Institute and Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences) as templates. The amplification system was: 5×Phusion HF Buffer 10 μL, dNTP (10 mM each dNTP) 1 μL, DNA template 20 ng, primers (10 μM) 1.5 μL each, Phusion High-Fidelity DNA Polymerase (2.5 U / μL) 0.5 μL, and distilled water was added to a total volume of 50 μL.

[0133] The amplification conditions were: 98°C pre-denaturation for 3 minutes (1 cycle); 98°C denaturation for 10 seconds, 58°C annealing for 10 seconds, 72°C extension for 1 minute (30 cycles); 72°C extension for 10 minutes (1 cycle). The amplified product GAL80 was cloned into the pEASY-BluntSimple cloning vector for transformation and sequence verification to obtain the plasmid pEASY-GAL80.

[0134] Table 9: Primer sequences

[0135]

[0136]

[0137] pEASY-GAL80 was digested with PmeI, and the target fragment of 5145 bp (30 ng) was purified by gel tapping. 4 μL NEB buffer and 1 μL CIP dephosphorylase (NEB) were added, and distilled water was added to 40 μL. The mixture was treated at 37°C for 1 h, and EDTA with a final concentration of 10 μmol was added. The reaction was terminated at 65°C for 30 min. The target fragment pEASY-GAL80 was recovered by gel tapping and set aside.

[0138] LEU2 (30 ng) was purified by gel tapping, and 4 μL 10×T4 DNA Ligase Reaction Buffer (NEB) and 1 μL T4 Polynucleotide kinase (NEB) were added, and distilled water was added to 40 μL, and phosphorylated at 37°C for 1 h. After gel tapping and recovery, it was ligated with pEASY-GAL80 using T4 DNA ligase (NEB), transformed, and sequenced to obtain plasmid pEASY-GAL80-LEU2.

[0139] 5.1.2.5 pRS313-LEU2-P TEF1 -SynSmFPS-GGGS-NES-T CYC1 Plasmid construction

[0140] SynSmFPS (recorded in Chinese patent 201610961269.5, available to the public from Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences) and NES were used as templates, and the primers in Table 10 were used to amplify SynSmFPS-GGGS and GGGS-SynNES. The amplification system was: 5×Phusion HF Buffer 10μL, dNTP (10mM each dNTP) 1μL, DNA template 20ng, primers (10μM) 1.5μL each, Phusion High-Fidelity DNA Polymerase (2.5U / μL) 0.5μL, and distilled water was added to a total volume of 50μL. The amplification conditions were: 98℃ pre-denaturation for 3 minutes (1 cycle); 98℃ denaturation for 10 seconds, 55℃ annealing for 15 seconds, 72℃ extension for 2 minutes (30 cycles); 72℃ extension for 10 minutes (1 cycle). The obtained PCR products were purified and placed in a -20°C refrigerator for future use.

[0141] Table 10: Primer sequences

[0142]

[0143] SynSmFPS-GGGS and GGGS-NES were used as templates and the primers in Table 10 (SexA1-SynSmFPS and NES-Asc1-R) were used to amplify the SynSmFPS-GGGS-NES fragment.

[0144] The amplification system was: 5×Phusion HF Buffer 10μL, dNTP (10mM each dNTP) 1μL, DNA template SynSmFPS-GGGS and GGGS-NES 20ng each, primer (10μM) 1.5μL each, Phusion High-Fidelity DNA Polymerase (2.5U / μL) 0.5μL, and distilled water was added to a total volume of 50μL. The amplification conditions were: 98℃ pre-denaturation for 3 minutes (1 cycle); 98℃ denaturation for 10 seconds, 58℃ annealing for 10 seconds, 72℃ extension for 2 minutes (30 cycles); 72℃ extension for 10 minutes (1 cycle).

[0145] The amplified product was purified, and then digested with SexA1 and Asc1, and the target fragment SexA1-SynSmFPS-GGGS-NES-Asc1 was recovered by gel cutting and set aside.

[0146] Plasmid pRS313-LEU2-P TEF1 -STpGMAS-T CYC1(Recorded in Chinese Patent No. 201610961269.5, the public can obtain it from Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences) Use SexA1 and Asc1 to cut and cut the rubber to recover the large fragment to obtain the vector pRS313-LEU2-P TEF1 -...-T CYC1 ; The vector pRS313-LEU2-P TEF1 -...-T CYC1 50 ng of each of SexA1-SynSmFPS-GGGS-NES-Asc1 was added to the ligation system: 2 μL 10×T4 DNA Ligase Reaction Buffer (NEB), 1 μL T4 DNA Ligase (NEB, 400,000 cohesive end units / ml), and distilled water was added to 20 μL. The reaction was carried out at room temperature for 2 hours to obtain the ligation product, which was transferred into Trans10 competent cells, and the plasmid was extracted and sequenced to obtain pRS313-LEU2-P TEF1 -SynSmFPS-GGGS-NES-T CYC1 Plasmid.

[0147] Table 11: Plasmid information

[0148]

[0149] 5.2 Monoterpenoid geranyl

[0150] 5.2.1 Preparation of functional modules

[0151] PCR was performed using the PCR templates and primers described in Table 12 to obtain the functional modules: M21 (GAL80-LEU2-up), M22 (P PGK1 -SAAT-T ADH1 ), M23(P TDH3 -ERG20-T TPI1 ), M24(P TEF1 -GES-T CYC1), M25 (GAL80-LEU2-down). The amplification system was: 5×Phusion HF Buffer 10μL, dNTP (10mM each dNTP) 1μL, DNA template 20ng, primers (10μM) 1.5μL each, Phusion High-Fidelity DNA Polymerase (2.5U / μL) 0.5μL, and distilled water was added to the total volume of 50μL. The amplification conditions were: 98℃ pre-denaturation for 3 minutes (1 cycle); 98℃ denaturation for 10 seconds, 58℃ annealing for 10 seconds, and 72℃ extension for 2 minutes each (30 cycles); 72℃ extension for 10 minutes (1 cycle), and the product was recovered and stored by tapping.

[0152] Table 12: PCR system information table

[0153]

[0154]

[0155] 5.2.2 Construction of geranyl ester engineering bacteria

[0156] The same method as in Example 2: Step 3 was used to prepare FPP-001 (recorded in Chinese Patent No. 201610961269.5, which is publicly available from Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences) competent cells. Then, 5 μL of fragments M21, M22, M23, M24, and M25 (molar ratio of 1:1:1:1:1) were added to the competent cells, mixed well, and transferred to an electroporation cup, electroshocked at 2.7 kv for 5.7 ms, added with 1 ml of 1 M sorbitol, resuscitated at 30°C for 1 h, and spread on solid culture medium 4 (the formula of solid culture medium 4 is as follows: 0.8% yeast selection medium SD-Ura-Trp-Leu-His (purchased from Beijing Fanjinuo Technology Co., Ltd.), 0.01% Trp., 2% glucose and 2% agar powder), cultured at 30°C for more than 36 h, and the correct positive clone was identified by PCR and named strain GE-001; on this basis, the GE-001 competent cells were prepared by the same method as in Example 2: Step 3. Then, the control plasmid pRS313-TRP-TEF1-GFP-CYC1t and 4 μL of fragment mixture (Asse-313-TRP-TEF1-CYC1t, Asse-ScPDR5, Helper-TEF1, Helper-CYC1, molar ratio of 1:1:1:1) were added to two tubes of GE-001 competent cells, mixed and transferred to an electroporation cup, electroporated at 2.7 kv for 5.7 ms, added with 1 ml of 1 M sorbitol, resuscitated at 30°C for 1 h, spread on solid culture medium 5 (the formula of solid culture medium 5 is as follows: 0.8% yeast selection medium SD-Ura-Trp-Leu-His (purchased from Beijing Fanjinuo Technology Co., Ltd.), 2% glucose and 2% agar powder), cultured at 30°C for more than 36 h, and the correct positive clones were identified by PCR to obtain the control strain GE-CK and the ScPDR5 overexpression strain GE+ScPDR5.

[0157] 5.2.3 Engineering bacteria culture and product extraction

[0158] Activate GE-CK and GE+ScPDR5 prepared in 5.1.2.2 in solid culture medium 5, prepare seed solution in corresponding liquid selection medium (30°C, 250rpm, 16h), inoculate in 100mL Erlenmeyer flask containing 15mL liquid selection medium at 1% inoculation amount, shake culture at 30°C, 250rpm for 3 days, then add 2.25mL n-hexane, continue shaking culture for 1h. Finally, transfer the liquid in the Erlenmeyer flask to a 50mL centrifuge tube, centrifuge at 5000rpm for 5min, collect the organic phase, pass through an organic nylon membrane (0.22μm), and detect by GC-MS. Detection instrument: Agilent GC-MS Agilent 7890A / 5975C. GC-MS determination conditions: injection port temperature 250℃, injection volume 1μL, no splitting, solvent delay 5min; chromatographic column: HP-5ms (30m*0.25mm); chromatographic conditions: 45℃, 1min, 10℃ / min to 220℃ for 5min; MS conditions: Full Scan: 45-700amu. Reserve. Use the standard of geranyl ester for qualitative and quantitative analysis, which was purchased from Sigma (Cat. No. 45896).

[0159] Results: The geranyl ester production of engineered bacteria GE-CK and GE+ScPDR5 was 0.09 mg / (L·OD) and 0.13 mg / (L·OD) respectively after 3 days of fermentation. The geranyl ester production of the latter was 41% higher than that of the former.

[0160] 5.3-sesquiterpenoid nerolidol

[0161] 5.3.1 Preparation of functional modules

[0162] PCR was performed using the PCR templates and primers described in Table 13 to obtain the functional modules: M26 (GAL80-LEU2-up), M27 (P TEF1 -SynSmFPS-GGGS-NES-T CYC1 ), M28 (GAL80-LEU2-down). The amplification system was: 5×Phusion HF Buffer 10μL, dNTP (10mM each dNTP) 1μL, DNA template 20ng, primers (10μM) 1.5μL each, Phusion High-Fidelity DNA Polymerase (2.5U / μL) 0.5μL, and distilled water was added to the total volume of 50μL. The amplification conditions were: 98℃ pre-denaturation for 3 minutes (1 cycle); 98℃ denaturation for 10 seconds, 58℃ annealing for 10 seconds, and 72℃ extension for 2 minutes each (30 cycles); 72℃ extension for 10 minutes (1 cycle), and the product was recovered and stored by tapping.

[0163] Table 13: PCR system information table

[0164]

[0165]

[0166] 5.3.2 Construction of nerolidol-producing bacteria

[0167] The FPP-001 competent cells were prepared by the same method as in step 3 of Example 2. Then, 3 μL of fragments M26, M27, and M28 (molar ratio of 1:1:1) were added to the competent cells, mixed and transferred to an electroporation cup, electroshocked at 2.7 kv for 5.7 ms, 1 ml of 1 M sorbitol was added, resuscitated at 30°C for 1 h, spread on solid culture medium 4, cultured at 30°C for more than 36 h, and the correct positive clone was identified by PCR and named as strain NE-001; on this basis, the NE-001 competent cells were prepared by the same method as in step 3 of Example 2. Then, the control plasmid pRS313-TRP-TEF1-GFP-CYC1t and 4 μL of fragment mixture (Asse-313-TRP-TEF1-CYC1t, Asse-ScPDR5, Helper-TEF1, Helper-CYC1, molar ratio of 1:1:1:1) were added to two tubes of NE-001 competent cells, mixed and transferred to an electroporation cup, electroporated at 2.7 kv for 5.7 ms, added with 1 ml of 1 M sorbitol, resuscitated at 30 °C for 1 h, spread on solid culture medium 5, and cultured at 30 °C for more than 36 h. The correct positive clones were identified by PCR to obtain the control strain NE-CK and the ScPDR5 overexpression strain NE+ScPDR5, respectively.

[0168] 5.3.3 Engineering bacteria culture and product extraction

[0169] The engineered strains NE-CK and NE+ScPDR5 prepared in 5.3.2 were activated in solid selective medium 5, and seed solution (30°C, 250rpm, 16h) was prepared in the corresponding liquid selective medium, and inoculated into a 100mL Erlenmeyer flask containing 15mL of the corresponding liquid selective medium at a 1% inoculation amount, and cultured at 30°C, 250rpm for 1 day, and then 1.5mL of n-dodecane was added, and the shaking culture was continued for 5 days. Finally, the liquid in the Erlenmeyer flask was transferred to a 50mL centrifuge tube, centrifuged at 5000rpm for 5min, the organic phase was collected, diluted 10 times with n-hexane, passed through an organic nylon membrane (0.22μm), and detected by GC-MS. Detection instrument: Agilent GC-MS Agilent7890A / 5975C. GC-MS determination conditions: injection port temperature 250℃, injection volume 1μL, no splitting, solvent delay 3min; chromatographic column: HP-5ms (30m*0.25mm); chromatographic conditions: 45℃, 1min, 10℃ / min to 300℃ for 5min; MS conditions: Full Scan: 50-750amu. Reserve. The samples were qualitatively and quantitatively analyzed using the standard substance of nerolidol. The standard substance of nerolidol was purchased from Sigma (Cat. No. 81431).

[0170] Results: The nerolidol production of engineered bacteria NE-CK and NE+ScPDR5 reached 13.9 mg / (L·OD) and 15.0 mg / (L·OD) respectively after 6 days of fermentation. The nerolidol production of the latter increased by 8% compared with the former.

[0171] 5.4 Diterpenoid Clareol

[0172] 5.4.1 Preparation of functional modules

[0173] PCR was performed using the PCR templates and primers described in Table 14 to obtain the functional modules: M29 (GAL80-LEU2-up), M30 (P PGK1 -cLPS-T ADH1 ), M31(P TDH3 -SaGGPPS-T TPI1 ), M32(P TEF1 -cTPS-T CYC1), M33 (GAL80-LEU2-down). The amplification system was: 5×Phusion HF Buffer 10μL, dNTP (10mM each dNTP) 1μL, DNA template 20ng, primers (10μM) 1.5μL each, Phusion High-Fidelity DNA Polymerase (2.5U / μL) 0.5μL, and distilled water was added to the total volume of 50μL. The amplification conditions were: 98℃ pre-denaturation for 3 minutes (1 cycle); 98℃ denaturation for 10 seconds, 58℃ annealing for 10 seconds, 72℃ extension for 2 minutes each (30 cycles); 72℃ extension for 10 minutes (1 cycle), and the product was recovered and stored by tapping.

[0174] Table 14: PCR system information table

[0175]

[0176] 5.4.2 Construction of Sclareol Chassis Cells

[0177] The same method as in step 3 of Example 2 was used to prepare FPP-001 competent cells. Then, 5 μL of fragments M29, M30, M31, M32, and M33 (molar ratio of 1:1:1:1:1) were added to the competent cells, mixed well, and transferred to an electroporation cup, electroshocked at 2.7 kv for 5.7 ms, 1 ml of 1 M sorbitol was added, resuscitated at 30° C. for 1 h, applied to solid culture medium 4, cultured at 30° C. for more than 36 h, and the correct positive clone was identified by PCR and named as strain SC-001; on this basis, the same method as in step 3 of Example 2 was used to prepare SC-001 competent cells. Then, pRS313-TRP-TEF1-GFP-CYC1t and 4 μL of fragment mixture (Asse-313-TRP-TEF1-CYC1t, Asse-ScPDR5, Helper-TEF1, Helper-CYC1, molar ratio of 1:1:1:1) were added to two tubes of SC-001 competent cells, mixed and transferred to an electroporation cup, electroporated at 2.7 kv for 5.7 ms, added with 1 ml of 1 M sorbitol, resuscitated at 30 °C for 1 h, spread on solid culture medium 5, and cultured at 30 °C for more than 36 h. The correct positive clones were identified by PCR to obtain the control strain SC-CK and the ScPDR5 overexpression strain SC+ScPDR5.

[0178] 5.4.3 Engineering bacteria culture and product extraction

[0179] Activate the engineered strains SC-CK and SC+ScPDR5 prepared in 5.4.2 in solid selective medium 5, prepare seed solution in the corresponding liquid selective medium (30°C, 250rpm, 16h), inoculate in a 100mL Erlenmeyer flask containing 15mL of the corresponding liquid selective medium at a 1% inoculation amount, and culture at 30°C, 250rpm for 1 day, then add 1.5mL of n-dodecane, and continue to culture for 5 days. Finally, transfer the liquid in the Erlenmeyer flask to a 50mL centrifuge tube, centrifuge at 5000rpm for 5min, collect the organic phase, dilute 10 times with n-hexane, pass through an organic nylon membrane (0.22μm), and detect by GC-MS. Detection instrument: Agilent GC-MS Agilent7890A / 5975C. GC-MS determination conditions: injection port temperature 300℃, injection volume 1μL, no splitting, solvent delay 5min; chromatographic column: HP-5ms (30m*0.25mm); chromatographic conditions: 50℃, 2min, 20℃ / min to 280℃ for 4min, 20℃ / min to 300℃ for 7.5min; MS conditions: Full Scan: 20-600amu. Reserve. Use the standard of sclareol for qualitative and quantitative analysis, which was purchased from Sigma (Cat. No. 49944).

[0180] Results: The production of sclareol by the engineered strains SC-CK and SC+ScPDR5 reached 1.34 mg / (L·OD) and 1.67 mg / (L·OD) respectively after 6 days of fermentation. The production of sclareol by the latter was 25% higher than that by the former.

Claims

1. A method for producing a recombinant host cell with improved tolerance to a terpene-containing essential oil, comprising the step of increasing the expression of a PDR5 protein in a wild-type host cell, wherein the terpene-containing essential oil is selected from the group consisting of zedoary turmeric oil, turmeric oil, sweet orange oil, peppermint oil, perilla oil and sandalwood oil, wherein the host cell is Saccharomyces cerevisiae and the PDR5 protein is Saccharomyces cerevisiae PDR5 protein.

2. The method of claim 1, wherein the increased expression of the PDR5 protein is achieved by increasing the copy number of the PDR5 gene and / or using a strong promoter.

3. The method of claim 1, wherein the amino acid sequence of the Saccharomyces cerevisiae PDR5 protein is as shown in SEQ ID NO: 139, and / or the nucleic acid sequence of the gene encoding the PDR5 protein is as shown in SEQ ID NO:

1.

4. The method of claim 1, wherein the host cell further comprises an exogenous polynucleotide for terpenoid production.

5. The method of claim 1, wherein the terpene-containing essential oil is selected from the group consisting of zedoary turmeric oil, turmeric oil, and sandalwood oil.

6. Use of an isolated polynucleotide encoding a PDR5 protein for improving the tolerance of a host cell to a terpene-containing essential oil, wherein the terpene-containing essential oil is selected from the group consisting of zedoary turmeric oil, turmeric oil, sweet orange oil, peppermint oil, perilla oil and sandalwood oil, wherein the host cell is Saccharomyces cerevisiae and the PDR5 protein is Saccharomyces cerevisiae PDR5 protein.

7. The use according to claim 6, wherein the nucleic acid sequence of the polynucleotide encoding the PDR5 protein is as shown in SEQ ID NO:

1.

8. The use according to claim 6, wherein the amino acid sequence of the PDR5 protein is shown in SEQ ID NO:

139.

9. The use of claim 6, wherein the host cell further comprises an exogenous polynucleotide for terpenoid production.

10. The use of claim 6, wherein the terpene-containing essential oil is selected from the group consisting of zedoary turmeric oil, turmeric oil and sandalwood oil.

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