Saccharomyces cerevisiae engineering strain for producing pinene and application thereof

Abstract

The invention relates to the technical field of bioengineering, in particular to a saccharomyces cerevisiae engineering strain for producing pinene and application of the saccharomyces cerevisiae engineering strain. The invention provides the transporter capable of effectively improving the pinene yield of the strain, and the transporter is over-expressed in the strain, so that the pinene production capacity of the strain is effectively improved, and the pinene yield is remarkably improved. The saccharomyces cerevisiae engineering strain provided by the invention has obviously enhanced pinene production capacity, the pinene yield of the saccharomyces cerevisiae engineering strain is obviously improved compared with that of an original strain, pinene can be efficiently produced, and the saccharomyces cerevisiae engineering strain has a better application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of bioengineering, and particularly relates to an engineered Saccharomyces cerevisiae strain for producing pinene and its application. Background Art

[0002] Monoterpenes are a class of isoprenoid-derived compounds from coniferous plants, with various physiological and pharmacological activities, such as anti-inflammatory, antioxidant, anti-tumor, insecticidal, antibacterial, etc. Currently, they have been used as fragrances, flavors, and insecticides. As a cyclic monoterpene, pinene is a class of isoprenoid-derived compounds with 10 carbons, having various physiological and pharmacological activities. In addition to the above applications, its methyl side chain and cyclic structure endow it with characteristics such as high density and high volume calorific value. In particular, pinene dimer has a density and net combustion heat comparable to that of tactical fuel JP-10. Therefore, as a potential high-density fuel precursor, the green production of pinene has great economic benefits. Currently, pinene is mainly obtained through traditional plant extraction or chemical synthesis, which is not only inefficient but also consumes solvents and damages the environment, not meeting the requirements of the modern energy system of clean, low-carbon, safe, and efficient, and restricting its commercial production and application. Compared with plant extraction, using microorganisms to synthesize monoterpenes such as pinene has the advantages of saving arable land, controllable process, high quality, and green sustainability. Therefore, microbial production is an effective way for the commercial production and application of pinene, with important practical significance.

[0003] Saccharomyces cerevisiae has a clear genetic background and a complete endomembrane system, making it have obvious advantages as a cell factory for terpene compounds. Currently, researchers have used Saccharomyces cerevisiae to achieve the synthesis of various monoterpene compounds, but the yields are generally low and cannot achieve industrial production and application. Summary of the Invention

[0004] The present invention provides an engineered Saccharomyces cerevisiae strain for producing pinene and its application.

[0005] Through the analysis of the existing Saccharomyces cerevisiae strains producing pinene, the present invention finds that although the structural characteristics make pinene relatively easy to be transported out of yeast cells, and most of the pinene produced by yeast cells will be transported into the fermentation broth, its physiological and biochemical characteristics make pinene have certain cytotoxicity to the cytoplasm. The part of pinene remaining in yeast cells will affect the physiological characteristics of yeast cells and reduce the pinene yield. To construct an engineered yeast strain for highly efficient production of pinene, the present invention first enhances the transport ability of yeast cells to pinene, reduces the pinene content in the yeast cytoplasm, reduces the cytoplasmic toxicity of pinene, and increases the pinene yield. The present invention discovers that the overexpression of specific transport proteins in Saccharomyces cerevisiae can significantly increase the yield of pinene.

[0006] Specifically, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides the use of a transporter protein in enhancing the pinene production capacity of a microorganism or constructing a microorganism for producing pinene, wherein the transporter protein is the protein encoded by the STE6 gene of Saccharomyces cerevisiae.

[0008] Preferably, the use includes: expressing the transporter protein in a microorganism, or enhancing the expression and / or activity of the transporter protein in the microorganism.

[0009] The transporter protein STE6 is derived from Saccharomyces cerevisiae, and the amino acid sequence of STE6 is shown in SEQ ID NO.14.

[0010] In the present invention, unless otherwise specified, the amino acid sequence corresponding to a gene name is the amino acid sequence of the protein encoded by that gene.

[0011] For Saccharomyces cerevisiae, the use includes enhancing the expression and / or activity of the above-mentioned transporter protein in Saccharomyces cerevisiae. For microorganisms other than Saccharomyces cerevisiae (such as bacteria like Escherichia coli), the use includes heterologously expressing the above-mentioned transporter protein in the microorganism.

[0012] Preferably, the microorganism is yeast, more preferably Saccharomyces cerevisiae.

[0013] The starting strain for the above-mentioned use is a microorganism capable of synthesizing pinene. Preferably, the pinene yield of the starting strain is higher than 1 mg / L, more preferably higher than 5 mg / L, more preferably higher than 10 mg / L, more preferably higher than 20 mg / L, more preferably higher than 50 mg / L, and more preferably higher than 100 mg / L.

[0014] For a microorganism capable of synthesizing pinene, a currently known pinene-producing microorganism (preferably Saccharomyces cerevisiae) can be selected or constructed by genetic engineering means. Preferably, the starting strain expresses pinene synthase.

[0015] In a second aspect, the present invention provides an engineered Saccharomyces cerevisiae strain, which has enhanced expression and / or activity of a transporter protein compared to the starting strain;

[0016] The transporter protein is the protein encoded by the STE6 gene of Saccharomyces cerevisiae.

[0017] Preferably, the engineered strain has an increased pinene yield compared to the starting strain.

[0018] Preferably, the engineered Saccharomyces cerevisiae strain is an engineered Saccharomyces cerevisiae strain for producing pinene.

[0019] The present invention discovers that the enhanced expression of the above-mentioned specific transporter protein can significantly enhance the pinene production capacity of yeast strains and increase the pinene yield.

[0020] In the present invention, the methods for enhancing the expression and / or activity of the transporter include, but are not limited to: (1) increasing the copy number of the coding gene of the transporter; (2) replacing the transcriptional and / or translational regulatory elements of the coding gene of the transporter with elements having stronger activity, etc. Among them, increasing the copy number can be achieved by integrating the coding gene into the genome or by introducing an expression plasmid carrying the coding gene.

[0021] Preferably, the enhancement of the expression of the transporter is achieved by integrating the expression cassette of the coding gene of the transporter into the genome.

[0022] In some embodiments of the present invention, the expression cassette of the coding gene of the transporter includes a promoter, a transporter coding gene, and a terminator.

[0023] The main limiting factors for the current yeast strains that cannot produce pinene in high yield also include: insufficient precursor supply, metabolic flux limitation (the synthesis pathway of pinene and its competing pathways in Saccharomyces cerevisiae are as Figure 1 and Figure 2 shown). Aiming at the bottleneck problem restricting pinene production, the present invention further strengthens the precursor supply and realizes the efficient production of pinene by Saccharomyces cerevisiae through the combined regulation of the precursor synthase and ERG20 and its downstream genes, as well as the strengthening of the gene combination of the mevalonate (MVA) pathway.

[0024] Furthermore, compared with the starting strain, the engineered strain also has enhanced expression and / or activity of the MVA pathway enzymes.

[0025] The strengthening of the MVA pathway can significantly increase the carbon flux of this pathway, and thus increase the accumulation of GPP. However, the present invention finds that the overall strengthening of the MVA pathway genes will lead to unbalanced metabolic flux and has a poor effect on improving the pinene yield. By specifically combining and strengthening the MVA pathway genes, the pinene yield of the strain is significantly higher than that of the strain with the overall strengthening of the MVA pathway.

[0026] Preferably, the MVA pathway enzymes are the proteins encoded by the IDI1 and tHMG1 genes, and / or the proteins encoded by the ERG10, ERG12, ERG8, and ERG19 genes.

[0027] Preferably, the MVA pathway enzymes are the proteins encoded by the IDI1 and tHMG1 genes and the proteins encoded by the ERG10, ERG12, ERG8, and ERG19 genes. Compared with other combined strengthening methods, using this combined strengthening expression method to strengthen the MVA pathway can be adapted to the efficient synthesis of downstream monoterpenes and more effectively improve the pinene yield.

[0028] In yeast strains, the precursor required for monoterpene synthesis is geranyl pyrophosphate (GPP). GPP is an intermediate compound formed by the condensation of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) catalyzed by farnesyl pyrophosphate synthase (ERG20) in the MVA pathway. At the same time, ERG20 also has high catalytic activity towards GPP. Therefore, it is difficult for GPP to accumulate in yeast strains.

[0029] To reduce the consumption of GPP by ERG20, the above-mentioned engineered strains also express a bifunctional enzyme farnesyl pyrophosphate synthase variant that specifically synthesizes GPP.

[0030] Preferably, the bifunctional enzyme farnesyl pyrophosphate synthase variant that specifically synthesizes GPP is ERG20 selected from the amino acid sequence shown in SEQ ID NO.1 N127W 、ERG20 with the amino acid sequence shown in SEQ ID NO.2 WW 、any one of GPPS2 with the amino acid sequence shown in SEQ ID NO.3.

[0031] To further reduce the consumption of GPP and improve the precursor supply for pinene synthesis, compared with the parental strain, the expression and / or activity of the protein encoded by the endogenous ERG20 and / or ERG9 gene in the above-mentioned Saccharomyces cerevisiae engineered strain is weakened.

[0032] Preferably, the weakening is achieved by replacing the original promoter of the encoding gene of the enzyme with a promoter with weaker activity.

[0033] Preferably, the promoter is selected from P ERG1 、P ERG7 、P BTS1 、P HXT1 、P HXT4 、P HXT6 、P HXT7 、P HXT11 any one of them.

[0034] In the present invention, the nucleotide sequence of promoter P ERG1 is shown in SEQ ID NO.4, the nucleotide sequence of P ERG7 is shown in SEQ ID NO.5, the nucleotide sequence of P BTS1 is shown in SEQ ID NO.6, the nucleotide sequence of P HXT1 is shown in SEQ ID NO.7, the nucleotide sequence of P HXT4 is shown in SEQ ID NO.8, the nucleotide sequence of P HXT6 is shown in SEQ ID NO.9, the nucleotide sequence of P HXT7The nucleotide sequence of P is shown in SEQ ID NO. 10. HXT11 The nucleotide sequence of is shown in SEQ ID NO. 11.

[0035] In some embodiments of the present invention, compared with the parental strain, the expression of the proteins encoded by the endogenous ERG20 and ERG9 genes in the engineered Saccharomyces cerevisiae strain is weakened.

[0036] Preferably, the weakening of the expression of the proteins encoded by the ERG20 and ERG9 genes is achieved by replacing the promoter of the ERG20 gene with P ERG7 or P HXT1 , and replacing the promoter of the ERG9 gene with P ERG7 or P HXT1 to achieve.

[0037] The above-mentioned parental strain is a Saccharomyces cerevisiae strain capable of synthesizing pinene.

[0038] For strains capable of synthesizing pinene, currently known pinene-producing Saccharomyces cerevisiae strains can be selected or constructed by genetic engineering means.

[0039] Preferably, the parental strain expresses pinene synthase Pt30 derived from Pinus taeda.

[0040] In a third aspect, the present invention provides any one of the following applications of the above-mentioned engineered Saccharomyces cerevisiae strain:

[0041] (1) Application in the fermentation production of monoterpenes or their derivatives;

[0042] (2) Application in the breeding of production strains of monoterpenes or their derivatives;

[0043] (3) Application in the construction of engineered strains for the production of monoterpenes or their derivatives.

[0044] Preferably, the monoterpene is pinene.

[0045] The derivatives of pinene include verbenone, verbenol, etc. The derivatives of pinene have the same precursor supply requirements as pinene, and the improvement of pinene production capacity usually also leads to the improvement of the production capacity of its derivatives.

[0046] In the above (2), the breeding methods include but are not limited to mutagenesis breeding, adaptive evolution, etc.

[0047] In the above (3), the construction is carried out by using genetic engineering methods to construct engineered strains.

[0048] Fourthly, the present invention provides a method for fermentatively producing pinene, the method comprising: culturing the engineered Saccharomyces cerevisiae strain described above, and collecting pinene in the culture.

[0049] Preferably, the culturing includes fermentative culturing, and may further include activation culturing and seed culturing before the fermentative culturing.

[0050] Preferably, the temperature of the culturing is 28 - 32 °C.

[0051] The beneficial effects of the present invention at least include: The present invention provides a transporter capable of effectively increasing the pinene yield of the strain. By overexpressing the transporter in the strain, the pinene production capacity of the strain is effectively improved, and the pinene yield is significantly increased.

[0052] The engineered Saccharomyces cerevisiae strain provided by the present invention has an obviously enhanced pinene production capacity. Its pinene yield is significantly improved compared with the starting strain, and it can efficiently produce pinene, having good application prospects. Description of the Drawings

[0053] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0054] Figure 1 It is the synthesis pathway of pinene in Saccharomyces cerevisiae in the invention content of the present invention.

[0055] Figure 2 It is the synthesis pathway of pinene in Saccharomyces cerevisiae and its competing pathway in the invention content of the present invention.

[0056] Figure 3 It is the gas chromatogram of pinene in Example 5 of the present invention.

[0057] Figure 4 It is the mass spectrum of pinene in Example 5 of the present invention.

[0058] Figure 5 It is the fermentation result diagram in a fermenter in Example 6 of the present invention. Detailed Embodiments

[0059] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0060] In a specific embodiment of the present invention, the method for constructing the engineered Saccharomyces cerevisiae strain is as follows:

[0061] 1. Express the pinene synthase gene

[0062] (1) Construction of the pinene expression cassette: The promoter, pinene synthase gene, and terminator are connected to construct an expression cassette;

[0063] (2) Construction of the selection marker expression cassette: The promoter, selection marker, and terminator are connected to construct an expression cassette;

[0064] (3) Construction of the engineered yeast: The obtained expression cassettes are co-transformed into the Saccharomyces cerevisiae strain and integrated into the yeast genome to construct an engineered Saccharomyces cerevisiae strain producing pinene.

[0065] 2. Combined regulation of the precursor enzyme and ERG20

[0066] (1) Construction of the precursor enzyme expression cassette: The promoter, a precursor enzyme gene selected from ERG20 N127W (amino acid sequence as shown in SEQ ID NO.1), ERG20 WW (amino acid sequence as shown in SEQ ID NO.2), GPPS2 (amino acid sequence as shown in SEQ ID NO.3), and the terminator are connected to construct an expression cassette;

[0067] (2) Construction of the combined regulatory expression cassette of ERG20 and its downstream genes: The promoter P ERG1 (nucleotide sequence as shown in SEQ ID NO.4), P ERG7 (nucleotide sequence as shown in SEQ ID NO.5), P BTS1 (nucleotide sequence as shown in SEQ ID NO.6), P HXT1 (nucleotide sequence as shown in SEQ ID NO.7), P HXT4 (nucleotide sequence as shown in SEQ ID NO.8), P HXT6 (nucleotide sequence as shown in SEQ ID NO.9), P HXT7 (nucleotide sequence as shown in SEQ ID NO.10), or P HXT11(The nucleotide sequence is as shown in SEQ ID NO.11) regulates the expression of ERG20 (the amino acid sequence is as shown in SEQ ID NO.12) and its downstream gene ERG9 (the amino acid sequence is as shown in SEQ ID NO.13) respectively.

[0068] (3) Construction of the selection marker expression cassette: The promoter, selection marker, and terminator are ligated to construct the expression cassette.

[0069] (4) Construction of the yeast engineering strain: The obtained expression cassettes are co-transformed into the engineered Saccharomyces cerevisiae strain to integrate them into the yeast genome and construct the yeast engineering strain.

[0070] 3. Enhancement of the mevalonate (MVA) pathway gene combination

[0071] (1) Construction of the MVA pathway gene expression cassette: The promoter, MVA pathway genes (IDI1 and tHMG1, as well as ERG10, ERG12, ERG8, and ERG19), and terminator are ligated to construct the expression cassette;

[0072] (2) Construction of the yeast engineering strain: The obtained expression cassette is transformed into the Saccharomyces cerevisiae strain to integrate it into the yeast genome and construct the yeast engineering strain.

[0073] 4. Overexpression of the endogenous transporter of Saccharomyces cerevisiae to enhance the transport of pinene

[0074] (1) Construction of the transporter gene expression cassette: The promoter, transporter STE6 (the amino acid sequence is as shown in SEQ ID NO.14), and terminator are ligated to construct the expression cassette;

[0075] (2) Construction of the selection marker expression cassette: The promoter, selection marker, and terminator are ligated to construct the expression cassette;

[0076] (3) Construction of the yeast engineering strain: The obtained expression cassettes are co-transformed into the engineered Saccharomyces cerevisiae strain to integrate them into the yeast genome and construct the yeast engineering strain.

[0077] Specifically, for homologous recombination, the above expression cassettes are ligated to the upstream and downstream homologous sequences of the integration site. The homologous sequences can be designed according to the insertion site of the target gene. The selection of the selection marker and the ligation method of the sequences can be carried out according to the conventional technical means in the art. The above gene editing can also be carried out by the CRISPR / Cas9 technology. Specifically, the corresponding guide RNA is obtained in the CRISPR gRNA (guide RNA) Design Tool (http: / / grna.ctegd.uga.edu / ), and the integration plasmid is constructed by ligation.

[0078] The present invention will be further described below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods used in the following embodiments are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0079] Example 1 Preliminary construction of an engineered Saccharomyces cerevisiae strain producing pinene

[0080] The yeast promoter P Gal7 , the pinene synthase gene Pt30 derived from Pinus taeda (the amino acid sequence of the encoded protein is shown in SEQ ID NO. 15), and the yeast terminator T PGK1 were used to construct the expression cassette P Gal7 -Pt30-T PGK1 by ligation and assembly.

[0081] Using the yeast promoter P Gal101 , the GPP precursor enzyme genes (ERG20 N127W , ERG20 WW or GPPS2), and the yeast terminator T TDH1 to construct the precursor enzyme expression cassette P Gal101 -ERG20 N127W / ERG20 WW / GPPS2-T TDH1 by ligation and assembly.

[0082] The precursor enzyme expression cassette P Gal101 -ERG20 N127W / ERG20 WW / GPPS2-T TDH1 , the pinene synthase expression cassette P Gal7 -Pt30-T PGK1 , the expression cassette of the selection marker LEU2, and the upstream and downstream homologous arms of the integration site YPRCtau3 were ligated to the Escherichia coli replicative plasmid PUC19. The successfully constructed plasmid was transferred into Escherichia coli, and the plasmid was extracted for standby after correct sequencing.

[0083] Using the above successfully constructed plasmid as a template, the corresponding linearized fragment was obtained by PCR. The yeast transformation method of multi-fragment co-transformation was carried out by the lithium acetate method. The above linearized fragment was transformed into the Saccharomyces cerevisiae CEN.PK2-1C strain. Using the homologous recombination function of yeast itself, the precursor enzyme expression cassette P Gal101 -ERG20 N127W / ERG20 WW / GPPS2-T TDH1 and the pinene synthase expression cassette P Gal7-Pt30-T PGK1 Integrated into the YPRCtau3 locus of the Saccharomyces cerevisiae genome, screened on a solid plate of defective medium after transformation, and the obtained transformants were identified for positive clones by PCR, stored as glycerol bacteria, and the yeast engineering strains P02 expressing the pinene synthesis gene Pt30 and the GPP precursor enzyme ERG20 were obtained. N127W The yeast engineering strain P01 expressing the pinene synthesis gene Pt30 and the GPP precursor enzyme ERG20 WW And the yeast engineering strain P05 expressing the pinene synthesis gene Pt30 and the GPP precursor enzyme GPPS2.

[0084] Example 2 Combinatorial regulation of ERG20 and its downstream genes

[0085] To reduce the consumption of GPP by ERG20, based on the yeast engineering strain P02 constructed in Example 1 expressing the pinene synthesis gene Pt30 and the precursor enzyme ERG20 N127W Combinatorial regulation of ERG20 and its downstream gene ERG9 was carried out: the promoter P of the yeast endogenous ERG20 gene ERG20 Was replaced with P ERG7 Or P HXT1 , and the pinene yield of the obtained strain reached 15 - 30 mg / L; on the basis of replacing the promoter P of the yeast endogenous ERG20 gene ERG20 With P HXT1 , further replace the promoter P of the yeast endogenous ERG9 gene ERG9 With P ERG1 , P ERG7 , P BTS1 , P HXT1 , P HXT4 , P HXT6 , P HXT7 , P HXT11 With any one of them, and the pinene yield of the obtained strain reached 25 - 50 mg / L, among which the strain with the promoter P of the ERG9 gene ERG9 Replaced with P HXT1 Was named yeast engineering strain P02B.

[0086] Example 3 Gene combination enhancement of the mevalonate (MVA) pathway

[0087] Based on the yeast engineering strain P02B, first, as the key genes of the MVA pathway, IDI1 and tHMG1 were overexpressed simultaneously. Yeast P Gal101 Was used as the promoter, GAL80 was used as the integration point, and the G418 resistance gene was used as the screening marker. The successfully constructed plasmid was transferred into Escherichia coli, and the plasmid was extracted for standby after correct sequencing. Using the successfully constructed plasmid as a template, the corresponding linearized fragment was obtained by PCR, and PGal101 The promoter-driven IDI1 and tHMG1 genes were integrated into the GAL80 locus of the Saccharomyces cerevisiae genome to obtain strains overexpressing the IDI1 and tHMG1 genes.

[0088] Furthermore, the genes of the MVA pathway were combinatorially strengthened. Through comparison, it was found that the yield of the strain with partial strengthening of the MVA pathway genes was higher than that of the strain with overall strengthening. The yeast promoter P Gal101 was ligated with the endogenous ERG10, ERG12, ERG8, and ERG19 genes of Saccharomyces cerevisiae to construct an expression cassette. The P Gal101 -ERG10-ERG12 expression cassette was integrated into the 1309a locus of the genome of the strain overexpressing the IDI1 and tHMG1 genes, and the P Gal101 -ERG8-ERG19 was integrated into the 308a locus to strengthen the genes of the MVA pathway, obtaining the yeast engineering strain P08.

[0089] Example 4 Overexpression of Transporter

[0090] Through screening, it was found that the overexpression of specific endogenous transporters in yeast could significantly increase the yield of pinene. Based on the P08 strain constructed in Example 3, PDR15, PDR10, ESBP6, PDR5, PDR12, YOR1, SNQ2, STE6, PDR11, and PDR18 were overexpressed respectively. It was found that the pinene yield of the strain P08-65 with overexpression of STE6 (the STE6 gene driven by the TEF1p promoter was integrated into the GRE3 locus of the Saccharomyces cerevisiae genome. The specific structure of the expression cassette for integration was: GRE3L-TEF1p-STE6-CPS1t-TRP-GRE3R, where GRE3L and GRE3R were the upstream and downstream homologous arms respectively, CPS1t was the terminator, and TRP was the screening marker) could be significantly increased.

[0091] Example 5 Shake-Flask Fermentation Experiment of Saccharomyces cerevisiae Engineering Strains

[0092] The Saccharomyces cerevisiae engineering strains constructed in the above examples were subjected to shake-flask fermentation experiments. The production capacity of the engineering strains was verified by detecting the change in the pinene yield of the engineering strains. The specific method was as follows:

[0093] 1. Cultivation of seed liquid: Positive clones were picked from the screening plate and inoculated into 1.5 mL of YPD medium, and cultured at 30 °C with shaking at 220 rpm for 18 - 24 hours. Then, inoculated into 5 mL of YPD medium at an inoculation amount of 10% and cultured at 30 °C with shaking at 220 rpm for 24 h.

[0094] 2. Fermentation culture: Inoculate the seed liquid into 20 mL of YPD medium at an inoculation amount of 1%, add 2 mL of isopropyl myristate sterilized at high temperature, and culture with shaking at 30 °C and 250 rpm for 72 h.

[0095] 3. Product extraction and detection: After the fermentation is completed, let the organic phase stand and separate, take 1 mL of the upper organic phase and put it into a 1.5 mL centrifuge tube, centrifuge at 12,000 rpm for 2 min, take 200 μL of the centrifuged organic phase and put it into an injection vial, add 800 μL of n-hexane, shake well and then detect.

[0096] Gas chromatography conditions: Carrier gas: High-purity helium; Volume flow rate: 1 mL / min; Injection volume: 1 μL; Injection port temperature: 230 °C; Split ratio: 30:1; Temperature programming: Hold at 60 °C for 1 min, increase the temperature to 73 °C at a rate of 1 °C / min, then increase the temperature to 165 °C at a rate of 5 °C / min, and finally increase the temperature to 280 °C at a rate of 20 °C / min and hold for 5 min.

[0097] Mass spectrometry conditions: Ionization mode: Electron impact ion source, electron impact energy 70 eV, ion source temperature 230 °C, quadrupole temperature 150 °C, solvent delay: 5 min.

[0098] Among them, the gas chromatography of the pinene standard is as Figure 3 shown, and the mass spectrometry is as Figure 4 shown.

[0099] The detection results of the pinene production of each engineering strain are shown in Table 1.

[0100]

[0101] Fermentation tank fermentation experiment of the Saccharomyces cerevisiae engineering strain in Example 6

[0102] Perform 3 L fermentation tank fermentation on the engineering strain P08-65, and the specific method is as follows:

[0103] 1. Cultivation of the seed liquid: Pick a single colony from the plate and inoculate it into YPD medium, culture with shaking at 30 °C and 220 rpm for 24 hours. Inoculate it into YPD medium at an inoculation amount of 10%, and culture with shaking at 30 °C and 220 rpm for 24 h to obtain the seed liquid.

[0104] 2. Fermentation culture:

[0105] Inoculate the seed solution into a fermenter containing a fermentation medium (formulation as follows: peptone 20 g / L, yeast powder 10 g / L, glucose 30 g / L, corn steep liquor 5 g / L, potassium dihydrogen phosphate 5 g / L, anhydrous magnesium sulfate 3 g / L) at an inoculation amount of 10%. Set the fermentation temperature at 30 °C, the rotation speed at 300 - 800 rpm / min, and the aeration rate at 1 - 3 vvm. Maintain the pH of the fermentation broth at 5.0 by feeding ammonia water solution. After culturing and fermenting for 12 - 24 h, when the sugar concentration in the initial medium of the fermenter is lower than 1 g / L, continuously supplement a 500 g / L glucose mixed solution containing nitrogen sources (peptone 20 g / L, yeast powder 10 g / L) to enable the rapid growth of the strain, and control the sugar concentration in the fermentation broth to be lower than 1 g / L. Stop feeding when the dry weight of the strain reaches the production requirement; add 10% of isopropyl myristate sterilized at high temperature, and start feeding an ethanol solution, controlling the ethanol concentration in the fermentation broth to be lower than 10 g / L and greater than 0 g / L until the production ends.

[0106] The results show that the yield of pinene by the engineered strain P08 - 65 can reach 422.72 ± 15 mg / L( Figure 5 ).

[0107] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a transporter protein in enhancing the pinene production capacity of a microorganism or constructing a microorganism for producing pinene, wherein the transporter protein is the protein encoded by the STE6 gene of Saccharomyces cerevisiae.

2. The use according to claim 1, wherein, the use includes: expressing the transporter protein in a microorganism, or enhancing the expression and / or activity of the transporter protein in the microorganism; preferably, the microorganism is yeast, more preferably Saccharomyces cerevisiae.

3. A Saccharomyces cerevisiae engineering strain, wherein, compared with the starting strain, the engineering strain has enhanced expression and / or activity of a transporter protein; the transporter protein is the protein encoded by the STE6 gene of Saccharomyces cerevisiae; preferably, compared with the starting strain, the engineering strain has increased pinene production.

4. The Saccharomyces cerevisiae engineering strain according to claim 3, wherein, compared with the starting strain, the engineering strain has enhanced expression and / or activity of MVA pathway enzymes; preferably, the MVA pathway enzymes are the proteins encoded by the IDI1 and tHMG1 genes, and / or the proteins encoded by the ERG10, ERG12, ERG8, and ERG19 genes.

5. The Saccharomyces cerevisiae engineering strain according to claim 3 or 4, wherein, the engineering strain expresses a bifunctional farnesyl pyrophosphate synthase variant that specifically synthesizes GPP; Preferably, the bifunctional farnesyl pyrophosphate synthase variant that specifically synthesizes GPP is ERG20 selected from the amino acid sequences shown in SEQ ID NO.1 N127W 、ERG20 with the amino acid sequence shown in SEQ ID NO.2 WW 、any one of GPPS2 with the amino acid sequence shown in SEQ ID NO.

3.

6. The Saccharomyces cerevisiae engineering strain according to any one of claims 3 to 5, wherein, compared with the starting strain, the expression and / or activity of the protein encoded by the endogenous ERG20 and / or ERG9 gene of the Saccharomyces cerevisiae engineering strain is weakened; preferably, the weakening is achieved by replacing the original promoter of the gene with a promoter having weaker activity; More preferably, the promoter is selected from P ERG1 , P ERG7 , P BTS1 , P HXT1 , P HXT4 , P HXT6 , P HXT7 , P HXT11 and any one of them.

7. The Saccharomyces cerevisiae engineering strain according to claim 6, wherein, compared with the starting strain, the expression of the protein encoded by the endogenous ERG20 and ERG9 genes of the Saccharomyces cerevisiae engineering strain is weakened; Preferably, the attenuation is achieved by replacing the promoter of the ERG20 gene with P ERG7 or P HXT1 , and replacing the promoter of the ERG9 gene with P ERG7 or P HXT1 .

8. The Saccharomyces cerevisiae engineering strain according to any one of claims 3 to 7, wherein, the starting strain is a Saccharomyces cerevisiae strain capable of synthesizing pinene; preferably, the starting strain expresses the pinene synthase Pt30 derived from Pinus taeda.

9. Any one of the following applications of the Saccharomyces cerevisiae engineering strain according to any one of claims 3 to 8: (1) Use in the fermentative production of monoterpenes or their derivatives; (2) Use in breeding production strains of monoterpenes or their derivatives; (3) Use in constructing an engineering strain for producing monoterpenes or their derivatives; preferably, the monoterpene is pinene.

10. A method for fermentatively producing pinene, wherein, the method includes: culturing the Saccharomyces cerevisiae engineering strain according to any one of claims 3 to 8, and collecting pinene in the culture.

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