Saccharomyces cerevisiae engineering strain for producing caryophyllene and application thereof

Abstract

The invention relates to the technical field of bioengineering, in particular to a saccharomyces cerevisiae engineering strain for producing caryophyllene and application of the saccharomyces cerevisiae engineering strain. The invention provides a saccharomyces cerevisiae engineering strain which has enhanced expression and / or activity of mevalonic acid pathway enzyme and one or more modifications selected from the following (1)-(2): (1) weakened expression and / or activity of sterol synthesis pathway enzyme; (2) attenuated expression and / or activity of fatty acid synthetic pathway enzymes; compared with an original strain, the saccharomyces cerevisiae engineering strain has the advantage that the sesquiterpene yield is increased. Compared with an original strain, the saccharomyces cerevisiae engineering strain has the advantages that the production capacity of the sesquiterpenes such as the caryophyllene is obviously improved, the sesquiterpenes such as the caryophyllene can be efficiently synthesized, and the saccharomyces cerevisiae engineering strain has better application value.

Description

Technical Field

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

[0002] Sesquiterpenes are a type of terpene compound with various physiological and pharmacological activities. Caryophyllene is a bicyclic sesquiterpene compound that not only has an inhibitory effect on colon cancer cells but also has significant antifungal activity. In addition, its methyl side chain and cyclic structure endow it with characteristics such as high density and high volume calorific value, and its density and net combustion heat are close to those of terpene dimer fuels. Therefore, caryophyllene is also a potential high-density fuel precursor with good application value. Currently, caryophyllene is mainly obtained through traditional plant extraction or chemical synthesis methods, which are not only inefficient but also consume solvents and pollute the environment, not meeting the requirements of the modern energy system of clean, low-carbon, safe, and efficient, thus restricting its commercial production and application. Compared with plant extraction, using microorganisms to synthesize caryophyllene has the advantages of saving arable land, controllable process, high quality, and green and sustainable production methods. Therefore, microbial production is an effective way for the commercial production and application of caryophyllene, with important practical significance.

[0003] Saccharomyces cerevisiae has a clear genetic background and a complete endomembrane system. Its endogenous MVA pathway can synthesize FPP, the common precursor of sesquiterpenes, and is suitable as a cell factory for producing caryophyllene. Currently, researchers have achieved the synthesis of caryophyllene using Saccharomyces cerevisiae, but its yield is still low and cannot meet the requirements of production practice. Summary of the Invention

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

[0005] Through the analysis of the existing engineered Saccharomyces cerevisiae strains for producing caryophyllene, the present invention finds that the main limiting factors for the low yield of caryophyllene in Saccharomyces cerevisiae are insufficient precursor supply and metabolic flux limitation. In addition, the serious homogenization of metabolic regulation strategies is another factor restricting high yield. To address the above bottleneck problems, by trying various metabolic engineering regulation strategies, the present invention strengthens the mevalonate (MVA) pathway and combines strategies such as weakening the competitive pathways of the target product precursors to strengthen precursor supply and balance the metabolic flux, ultimately achieving the efficient production of caryophyllene by Saccharomyces cerevisiae. The synthesis pathway of caryophyllene and its competitive pathways in Saccharomyces cerevisiae are as Figure 1 and Figure 2 shown.

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

[0007] In a first aspect, the present invention provides an engineered Saccharomyces cerevisiae strain which, compared with the parental strain, has enhanced expression and / or activity of mevalonate pathway enzymes, and one or more modifications selected from the following (1)-(2):

[0008] (1) Weakened expression and / or activity of sterol synthesis pathway enzymes;

[0009] (2) Weakened expression and / or activity of fatty acid synthesis pathway enzymes;

[0010] Compared with the parental strain, the engineered Saccharomyces cerevisiae strain has an increased sesquiterpene yield.

[0011] The present invention discovers that the combination of enhanced expression and / or activity of mevalonate pathway enzymes and weakened expression and / or activity of sterol synthesis pathway enzymes and / or fatty acid synthesis pathway enzymes can significantly promote the production of sesquiterpenes by the strain and enhance the sesquiterpene production ability of the strain.

[0012] Preferably, compared with the parental strain, the engineered Saccharomyces cerevisiae strain has enhanced expression and / or activity of mevalonate pathway enzymes, weakened expression and / or activity of sterol synthesis pathway enzymes, and weakened expression and / or activity of fatty acid synthesis pathway enzymes.

[0013] Preferably, compared with the parental strain, the engineered Saccharomyces cerevisiae strain has enhanced expression of mevalonate pathway enzymes, weakened expression of sterol synthesis pathway enzymes, and weakened expression of fatty acid synthesis pathway enzymes.

[0014] The above-mentioned sterol synthesis pathway enzymes are one or more selected from the proteins encoded by the ERG9, ERG2, ERG3, ERG4, ERG5, ERG6, ERG24, ERG25, ERG26, ERG27 genes.

[0015] The above-mentioned fatty acid synthesis pathway enzymes are one or more selected from the proteins encoded by the ACC1, FAS1, FAS2 genes.

[0016] The above-mentioned mevalonate pathway enzymes are the proteins encoded by the IDI1 and / or tHMG1 genes.

[0017] Among the above-mentioned pathway enzymes, the present invention has determined the key regulatory nodes and their combinations through screening.

[0018] Preferably, the sterol synthesis pathway enzyme is the protein encoded by the ERG9 gene, and / or the proteins encoded by the ERG4, ERG5, ERG6, ERG24, and ERG27 genes. Weakening the above-mentioned enzymes in the sterol synthesis pathway can more effectively regulate the metabolic flux of the sterol synthesis pathway and provide more precursor substances for the synthesis of sesquiterpenes such as caryophyllene.

[0019] Preferably, the fatty acid synthesis pathway enzyme is a protein encoded by the ACC1, FAS1 or FAS2 gene. Weakening the above enzymes in the fatty acid synthesis pathway can more effectively regulate the metabolic flux of the fatty acid synthesis pathway, enabling more metabolic flux to flow from storage carbon sources to the target product, providing more carbon flux for the mevalonate pathway, and thus importing more carbon flux into the synthesis of sesquiterpenes such as caryophyllene.

[0020] Preferably, the mevalonate pathway enzyme is a protein encoded by the IDI1 and tHMG1 genes. The present invention has found that IDI1 and tHMG1, as key enzymes in the mevalonate pathway, simultaneously regulating these two enzymes can significantly enhance the metabolic flux of the mevalonate pathway, providing more precursor substances for the synthesis of sesquiterpenes such as caryophyllene.

[0021] In the present invention, the weakening is preferably achieved by replacing the original promoter of the encoding gene of the enzyme with a promoter having weaker activity.

[0022] The promoter is selected from P ERG1 (the nucleotide sequence is shown in SEQ ID NO.1), P ERG7 (the nucleotide sequence is shown in SEQ ID NO.2), P BTS1 (the nucleotide sequence is shown in SEQ ID NO.3), P HXT1 (the nucleotide sequence is shown in SEQ ID NO.4), P HXT4 (the nucleotide sequence is shown in SEQ ID NO.5), P HXT6 (the nucleotide sequence is shown in SEQ ID NO.6), P HXT7 (the nucleotide sequence is shown in SEQ ID NO.7), P HXT11 (the nucleotide sequence is shown in SEQ ID NO.8), any one of them.

[0023] Among them, P BTS1 is a diterpene synthesis-related promoter, P ERG1 , P ERG7 are sterol synthesis-related promoters, P HXT1 , P HXT4 , P HXT6 , P HXT7 , P HXT11 are glucose-induced promoters.

[0024] Preferably, the expression of the sterol synthesis pathway enzyme is weakened by replacing the promoter of the ERG9 gene with P ERG1 , P HXT1 or P ERG7 , and replacing the promoter of the ERG4 gene with P BTS1 , and replacing the promoter of the ERG5 gene with PHXT1 1. Replace the promoter of the ERG6 gene with P BTS1 2. Replace the promoter of the ERG24 gene with P ERG7 and replace the promoter of the ERG27 gene with P ERG7 to achieve.

[0025] Using the above promoters to regulate the expression of enzymes in the sterol synthesis pathway is more conducive to balancing the synthesis of precursor substances of the target product and the metabolic flux of the terminal pathway of the target product, and promoting the accumulation of the target product.

[0026] Preferably, the expression of the enzyme weakening the fatty acid synthesis pathway is achieved by replacing the promoter of the ACC1 gene with P ERG7 , or by replacing the promoter of the FAS1 or FAS2 gene with P HXT1 or P ERG7 to achieve.

[0027] Using the above promoters to regulate the expression of enzymes in the fatty acid synthesis pathway is more conducive to balancing the synthesis of precursor substances of the target product and the metabolic flux of the terminal pathway of the target product, and promoting the accumulation of the target product.

[0028] In the present invention, the enhanced expression of the mevalonate pathway enzyme is preferably achieved by increasing the copy number of the IDI1 and tHMG1 genes.

[0029] Preferably, the enhanced expression of the mevalonate pathway enzyme is achieved by integrating the expression cassettes of the IDI1 and tHMG1 genes on the genome.

[0030] The expression cassettes of the IDI1 and tHMG1 genes include: yeast P Gal101 promoter and the IDI1 and tHMG1 genes.

[0031] The IDI1 and tHMG1 genes are the endogenous IDI1 and tHMG1 genes of Saccharomyces cerevisiae.

[0032] In some embodiments of the present invention, the engineered Saccharomyces cerevisiae strain is obtained by integrating the expression cassettes of the IDI1 and tHMG1 genes on the genome based on the starting strain, and performing promoter replacement in any one of the following (1)-(2) and promoter replacement in any one of the following (3)-(5):

[0033] (1) Replace the promoter of the ERG9 gene with P ERG1 , P HXT1 or P ERG7 ;

[0034] (2) Replace the promoter of the ERG9 gene with P ERG1 , P HXT1 or PERG7 and replacing the promoter of the ERG4 gene with P BTS1 replacing the promoter of the ERG5 gene with P HXT1 replacing the promoter of the ERG6 gene with P BTS1 replacing the promoter of the ERG24 gene with P ERG7 and replacing the promoter of the ERG27 gene with P ERG7 ;

[0035] (3) replacing the promoter of the ACC1 gene with P ERG7 ;

[0036] (4) replacing the promoter of the FAS1 gene with P ERG7 ;

[0037] (5) replacing the promoter of the FAS2 gene with P HXT1 or P ERG7 .

[0038] The above-mentioned yeast engineering strain contains an enhanced mevalonate pathway, a metabolic flux distribution pattern adapted to the efficient synthesis of sesquiterpenes, a balanced metabolic flux, and an enhanced carbon flux of the mevalonate pathway, and can efficiently synthesize sesquiterpenes such as caryophyllene.

[0039] In the present invention, the starting strain is a Saccharomyces cerevisiae strain capable of synthesizing sesquiterpenes.

[0040] In the present invention, the sesquiterpene is preferably caryophyllene.

[0041] Preferably, the starting strain is a Saccharomyces cerevisiae strain capable of synthesizing caryophyllene.

[0042] Preferably, the starting strain expresses the caryophyllene synthase gene QHS1 derived from Artemisia annua. The present invention discovers that the caryophyllene synthase gene from the above source can better match the regulation of the above mevalonate pathway, sterol synthesis pathway, and fatty acid synthesis pathway, enabling better balance between the precursor synthesis metabolic flux and the caryophyllene synthesis metabolic flux, and significantly promoting the production and yield improvement of caryophyllene.

[0043] In some embodiments of the present invention, the starting strain is a strain obtained by expressing the caryophyllene synthase gene QHS1 derived from Artemisia annua in the Saccharomyces cerevisiae strains BY4742, BY4741, CEN.PK2-1C or CEN.PK2-1D.

[0044] The above-mentioned Saccharomyces cerevisiae engineering strain can be constructed by known genetic engineering techniques, such as homologous recombination, CRISPR / Cas9 technology, etc.

[0045] Second aspect, the present invention provides any one of the following applications of the above-mentioned engineered Saccharomyces cerevisiae strain:

[0046] (1) Application in the fermentative production of sesquiterpenes or their derivatives;

[0047] (2) Application in the selection of production strains of sesquiterpenes or their derivatives;

[0048] (3) Application in the construction of engineered strains for the production of sesquiterpenes or their derivatives.

[0049] Preferably, the sesquiterpene in the above applications is caryophyllene.

[0050] The derivatives of caryophyllene include but are not limited to caryophyllene oxide, caryophyllene alcohol, etc. The derivatives of caryophyllene have the same precursor supply requirements as caryophyllene, and generally, the improvement of caryophyllene production capacity will also lead to the improvement of the production capacity of its derivatives.

[0051] In the above (2), the selection methods include but are not limited to mutagenesis breeding, adaptive evolution, etc.

[0052] In the above (3), the construction is carried out by using genetic engineering methods to construct engineered strains.

[0053] Third aspect, the present invention provides a method for fermentatively producing caryophyllene, the method comprising: culturing the above-mentioned engineered Saccharomyces cerevisiae strain and collecting caryophyllene in the culture.

[0054] Preferably, the culturing includes fermentative culturing, and before the fermentative culturing, it may also include activation culturing and seed culturing.

[0055] Preferably, the temperature of the culturing is 28 - 32 °C.

[0056] The beneficial effects of the present invention at least include: The engineered Saccharomyces cerevisiae strain provided by the present invention has significantly improved production capacity of sesquiterpenes such as caryophyllene compared with the starting strain, can efficiently synthesize sesquiterpenes such as caryophyllene, and the yield of sesquiterpenes such as caryophyllene is significantly increased, having good application value. Brief Description of the Drawings

[0057] 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.

[0058] Figure 1 It is a synthetic pathway diagram of caryophyllene in Saccharomyces cerevisiae in the invention content of the present invention.

[0059] Figure 2 This is the synthetic pathway of caryophyllene and its competing pathway diagram in Saccharomyces cerevisiae in the summary of the invention of the present invention.

[0060] Figure 3 This is the gas chromatogram of caryophyllene in Example 5 of the present invention.

[0061] Figure 4 This is the mass spectrum of caryophyllene in Example 5 of the present invention. Detailed implementation manners

[0062] 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 with reference to the accompanying drawings in the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without creative efforts shall fall within the protection scope of the present invention.

[0063] In the detailed implementation manners of the present invention, the construction method of the Saccharomyces cerevisiae engineering strain is as follows:

[0064] 1. Express caryophyllene synthesis genes

[0065] (1) Construction of the caryophyllene expression cassette: A promoter, a caryophyllene synthesis gene, and a terminator are connected to construct an expression cassette;

[0066] (2) Construction of the selection marker expression cassette: A promoter and a selection marker are connected to construct an expression cassette;

[0067] (3) Construction of the yeast engineering strain: The obtained expression cassettes are co-transformed into the starting strain, integrated into the yeast genome, and a Saccharomyces cerevisiae engineering strain producing caryophyllene is constructed.

[0068] 2. Strengthen the mevalonate pathway genes

[0069] (1) Construction of the MVA expression cassette: A promoter and MVA pathway genes (IDI1 and tHMG1) are connected to construct an expression cassette;

[0070] (2) Construction of the selection marker expression cassette: A promoter and a selection marker are connected to construct an expression cassette;

[0071] (3) Construction of the yeast engineering strain: The obtained expression cassettes are co-transformed into the Saccharomyces cerevisiae engineering strain producing caryophyllene constructed in 1 above, integrated into the yeast genome, and a Saccharomyces cerevisiae engineering strain with enhanced mevalonate pathway genes is constructed.

[0072] 3. Weaken the sterol synthesis pathway genes

[0073] (1) Replace the original promoter of the ERG9 gene (the amino acid sequence of the encoded protein is as shown in SEQ ID NO.9) with a promoter of a different regulatory type (selected from one of P ERG1 、P ERG7 、P BTS 、P HXT1 、P HXT4 、P HXT6 、P HXT7 、P HXT11 ).

[0074] (2) Replace the original promoter of the sterol synthesis pathway genes: ERG2 (the amino acid sequence of the encoded protein is as shown in SEQ ID NO.12), ERG3 (the amino acid sequence of the encoded protein is as shown in SEQ ID NO.13), ERG4 (the amino acid sequence of the encoded protein is as shown in SEQ ID NO.14), ERG5 (the amino acid sequence of the encoded protein is as shown in SEQ ID NO.15), ERG6 (the amino acid sequence of the encoded protein is as shown in SEQ ID NO.16), ERG24 (the amino acid sequence of the encoded protein is as shown in SEQ ID NO.18), ERG25 (the amino acid sequence of the encoded protein is as shown in SEQ ID NO.19), ERG26 (the amino acid sequence of the encoded protein is as shown in SEQ ID NO.20) and / or ERG27 (the amino acid sequence of the encoded protein is as shown in SEQ ID NO.21) with a promoter of a different regulatory type (selected from one of P ERG1 、P ERG7 、P BTS 、P HXT1 、P HXT4 、P HXT6 、P HXT7 、P HXT11 ).

[0075] The above promoter replacement can be carried out using the CRISPR / Cas9 technology, and the corresponding guide RNA can be obtained in the CRISPR gRNA (guide RNA) Design Tool (http: / / grna.ctegd.uga.edu / ).

[0076] 4. Enhance the carbon flux of the mevalonate pathway

[0077] Use promoters of different regulatory types (P ERG1 、P ERG7 、P BTS1 、P HXT1 、P HXT4 、P HXT6 、P HXT7 、P HXT11Replace the original promoter of the fatty acid synthesis pathway gene: ACC1 (the amino acid sequence of the encoded protein is as shown in SEQ ID NO.22), FAS1 (the amino acid sequence of the encoded protein is as shown in SEQ ID NO.23), and / or FAS2 (the amino acid sequence of the encoded protein is as shown in SEQ ID NO.24) with one of the following.

[0078] The above promoter replacement can be carried out using the CRISPR / Cas9 technology, and the corresponding guide RNA is obtained in the CRISPR gRNA (guide RNA) Design Tool (http: / / grna.ctegd.uga.edu / ).

[0079] The homologous sequences described in the present invention can be designed according to the insertion site of the target gene.

[0080] 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.

[0081] Example 1 Construction of an engineered Saccharomyces cerevisiae strain expressing the caryophyllene synthesis gene

[0082] The yeast promoter P Gal101 , the caryophyllene synthesis gene QHS1 derived from Artemisia annua (the amino acid sequence of the encoded protein is as shown in SEQ ID NO.25), and the yeast terminator T PGK1 are used to construct the expression cassette P Gal101 -QHS1-T PGK1 through ligation and assembly.

[0083] Using the Gibson assembly method, the expression cassette P Gal101 -QHS1-T PGK1 , the expression cassette of the antibiotic selection marker NrsR (P TEF1 -NrsR-T TEF1 ), and the upstream and downstream homologous arms of the integration site YPRCtau3 (YPRCtau3L, YPRCtau3R) are ligated with the Escherichia coli replicative plasmid PUC19. The successfully ligated plasmid is transferred into Escherichia coli, and the plasmid is extracted for standby after correct sequencing.

[0084] Using the above constructed plasmid as a template, the corresponding linearized fragment YPRCtau3L-P Gal101 -QHS1-T PGK1 -P TEF1-NrsR-T TEF1 -YPRCtau3R, a yeast transformation method for multi-fragment co-transformation using the lithium acetate method. The linearized fragment is transformed into the Saccharomyces cerevisiae strain CEN.PK2-1C. Utilizing the homologous recombination function of yeast itself, the expression cassettes P Gal101 -QHS1-T PGK1 and P TEF1 -NrsR-T TEF1 are integrated into the YPRCtau3 locus of the Saccharomyces cerevisiae genome to obtain a Saccharomyces cerevisiae engineering strain expressing the caryophyllene synthase gene. After transformation, screening is carried out on a solid plate of the medium containing nourseothricin. The obtained transformants are identified for positive clones by PCR and stored in glycerol.

[0085] Example 2 Enhancement of genes in the MVA pathway

[0086] The key genes IDI1 and tHMG1 (sequences can be obtained from the database) in the MVA pathway of Saccharomyces cerevisiae are overexpressed simultaneously. The yeast promoter P Gal101 is selected as the promoter, GAL80 as the integration site, and the G418 resistance gene as the screening marker. The plasmid connected with the P Gal101 promoter, the Saccharomyces cerevisiae IDI1 and tHMG1 genes, the antibiotic screening marker G418 expression cassette, and the homologous arms upstream and downstream of the integration site is transferred into Escherichia coli. After correct sequencing, the plasmid is extracted for standby. Using the successfully constructed plasmid as a template, the corresponding linearized fragment is obtained by PCR. The linearized fragment is transformed into the Saccharomyces cerevisiae engineering strain expressing the caryophyllene synthase gene constructed in Example 1. The IDI1 and tHMG1 genes regulated by the P Gal101 promoter and the antibiotic screening marker G418 expression cassette are integrated into the GAL80 locus of the Saccharomyces cerevisiae genome to obtain the Saccharomyces cerevisiae engineering strain C01.

[0087] Example 3 Regulation of genes in the sterol synthesis pathway

[0088] The precursor required for caryophyllene synthesis is farnesyl pyrophosphate (FPP), the starting compound of the sterol synthesis pathway. To achieve high production of caryophyllene, the distribution of metabolic flux is first optimized by regulating the gene ERG9 in the sterol synthesis pathway to increase the supply of the caryophyllene synthesis precursor. Different types of regulatory promoters are selected for combined regulation. These promoters include: promoters related to diterpene synthesis (P BTS1 ), promoters related to sterol synthesis (P ERG1 , P ERG7 ), glucose-induced promoters (P HXT1 , P HXT4 , P HXT6 , P HXT7 , P HXT11 ).

[0089] In strain C01, the promoter of the endogenous P gene of Saccharomyces cerevisiae was replaced with P ERG9 or P ERG1 or P ERG7 or P BTS1 or P HXT1 or P HXT4 or P HXT6 or P HXT7 or P HXT11 , and the caryophyllene yield could reach 150 - 500 mg / L. Specifically, in strain C01, the promoter P ERG9 of the endogenous ERG9 gene of yeast was replaced with P ERG7 to obtain strain C06. The promoter P ERG9 of the endogenous ERG9 gene of yeast was replaced with P HXT1 to obtain strain C07. The promoter P ERG9 of the endogenous ERG9 gene of yeast was replaced with P ERG1 to obtain strain C08.

[0090] The genes in the sterol synthesis pathway other than ERG9 also include ERG1 (encoding the protein sequence shown in SEQ ID NO.10), ERG2, ERG3, ERG4, ERG5, ERG6, ERG7 (encoding the protein sequence shown in SEQ ID NO.11), ERG11 (encoding the protein sequence shown in SEQ ID NO.17), ERG24, ERG25, ERG26, ERG27. Therefore, the downstream genes ERG2, ERG3, ERG4, ERG5, ERG6, ERG24, ERG25, ERG26, ERG27 of the sterol synthesis pathway were further regulated to balance the metabolic flux and further increase the caryophyllene yield.

[0091] In strain C07, promoters with different regulation types (P ERG1 , P ERG7 , P BTS1 , P HXT1 , P HXT4 , P HXT6 , P HXT7 , P HXT11 ) were used to further regulate the remaining genes (ERG2, ERG3, ERG4, ERG5, ERG6, ERG24, ERG25, ERG26, ERG27) in the sterol synthesis pathway. The endogenous P ERG2 and / or P ERG3 and / or P ERG4 and / or P ERG5 and / or P ERG6 and / or P ERG24 and / or P ERG25 and / or P ERG26 and / or PERG27 The gene promoter is replaced with P ERG1 or P ERG7 or P BTS1 or P HXT1 or P HXT4 or P HXT6 or P HXT7 or P HXT11 , the caryophyllene yield can reach 700 - 900 mg / L. Replace the endogenous P of yeast ERG2 or P ERG3 or P ERG27 gene promoter with P ERG1 or P ERG7 or P BTS1 or P HXT4 , the caryophyllene yield can reach 750 - 900 mg / L. Replace the endogenous P of yeast ERG4 or P ERG6 or P ERG25 gene promoter with P BTS1 or P HXT1 or P HXT6 or P HXT7 , the caryophyllene yield can reach 700 - 850 mg / L. Replace the endogenous P of yeast ERG5 or P ERG24 or P ERG26 gene promoter with P ERG1 or P HXT6 or P HXT4 or P HXT11 , the caryophyllene yield can reach 700 - 850 mg / L.

[0092] Specifically, in strain C07, replace the promoter P of the ERG4 gene ERG4 with P BTS1 , replace the promoter P of the ERG5 gene ERG5 with P HXT1 , replace the promoter P of the ERG6 gene ERG6 with P BTS1 , replace the promoter P of the ERG24 gene ERG24 with P ERG7 and replace the promoter P of the ERG27 gene ERG27 with P ERG7 , to obtain strain C11.

[0093] Example 4 Strengthen the carbon flux of the MVA pathway

[0094] The fatty acid synthesis pathway and the MVA pathway require the same starting substrate acetyl coenzyme A. Therefore, to enhance the carbon flux of the MVA pathway, the fatty acid synthesis starting genes ACC1, FAS1, and FAS2 are regulated, so that more metabolic flux flows from the storage carbon source to the target product, improving the caryophyllene production ability of the strain.

[0095] Using promoters (P ERG1 , P ERG7 , P BTS1 , P HXT1 , P HXT4 , P HXT6 , P HXT7 , P HXT11 ) of different regulatory types to further regulate the fatty acid synthesis initiation genes ACC1, FAS1, and FAS2. In strain C11, replacing the promoters of yeast endogenous P ACC1 and / or P FAS1 and / or P FAS2 genes with P ERG1 or P ERG7 or P BTS1 or P HXT1 or P HXT4 or P HXT6 or P HXT7 or P HXT11 , the caryophyllene yield can reach 800 - 1100 mg / L; replacing the promoter of yeast endogenous P ACC1 gene with P ERG1 or P ERG7 or P BTS1 or P HXT4 or P HXT7 , the caryophyllene yield can reach 800 - 900 mg / L; replacing the promoter of yeast endogenous P FAS1 gene with P ERG7 or P HXT1 or P HXT6 or P HXT7 , the caryophyllene yield can reach 850 - 950 mg / L; replacing the promoter of yeast endogenous P FAS2 gene with P ERG7 or P BTS1 or P HXT1 or P HXT4 or P HXT6 or P HXT11 , the caryophyllene yield can reach 900 - 1100 mg / L.

[0096] Specifically, in strain C11, replacing the promoter P ACC1 of the yeast endogenous ACC1 gene with P ERG7 to obtain strain C17. On the basis of strain C11, replacing the promoter P FAS1 gene of the yeast endogenous FAS1 gene with P ERG7 to obtain strain C20. On the basis of strain C11, replacing the promoter P FAS2 gene of the yeast endogenous FAS2 gene with P HXT1, C21 strain was obtained. Based on strain C11, the promoter P of the yeast endogenous FAS2 gene was FAS2 gene replaced with P ERG7 , and C22 strain was obtained.

[0097] Example 5 Fermentation Experiment of Engineered Saccharomyces cerevisiae Strains

[0098] Flask fermentation experiments were carried out on the engineered Saccharomyces cerevisiae strains constructed in the above examples. The production capacity of the engineered strains was verified by detecting the change in caryophyllene production of the engineered strains. The specific method is as follows:

[0099] 1. Cultivation of seed liquid: Pick positive clones from the screening plate and inoculate them into 1.5 mL of YPD medium. Incubate at 30 °C with shaking at 220 rpm for 18 - 24 hours. Inoculate according to a 10% inoculum size into 5 mL of YPD medium and incubate at 30 °C with shaking at 220 rpm for 24 h.

[0100] 2. Fermentation culture: Inoculate the seed liquid into 20 mL of YPD medium at a 1% inoculum size, add 2 mL of isopropyl myristate sterilized by high temperature, and incubate at 30 °C with shaking at 250 rpm for 120 h.

[0101] 3. After fermentation, the caryophyllene production was determined by GC or GC-MS. The specific method is as follows: After the organic phase was allowed to stand and separate, 1 mL of the upper organic phase was taken into a 1.5 mL centrifuge tube and centrifuged at 12000 rpm for 2 min. 200 μL of the centrifuged organic phase was taken into an injection vial, 800 μL of n-hexane was added, and after shaking well, it was detected.

[0102] 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.

[0103] 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.

[0104] Among them, the gas chromatography of the caryophyllene standard product is as Figure 3 shown, and the mass spectrometry is as Figure 4 shown.

[0105] The detection results of caryophyllene production of each engineered strain are shown in Table 1.

[0106] Table 1

[0107]

[0108]

[0109] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; 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 on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An engineered Saccharomyces cerevisiae strain, characterized in that, compared with the parental strain, the engineered strain has enhanced expression and / or activity of mevalonate pathway enzymes, and one or more modifications selected from the following (1)-(2): (1) Weakened expression and / or activity of sterol synthesis pathway enzymes; (2) Weakened expression and / or activity of fatty acid synthesis pathway enzymes; compared with the parental strain, the engineered Saccharomyces cerevisiae strain has increased sesquiterpene production.

2. The engineered Saccharomyces cerevisiae strain according to claim 1, characterized in that, the sterol synthesis pathway enzymes are one or more selected from the proteins encoded by the ERG9, ERG2, ERG3, ERG4, ERG5, ERG6, ERG24, ERG25, ERG26, ERG27 genes; and / or, the fatty acid synthesis pathway enzymes are one or more selected from the proteins encoded by the ACC1, FAS1, FAS2 genes; and / or, the mevalonate pathway enzymes are the proteins encoded by the IDI1 and / or tHMG1 genes.

3. The engineered Saccharomyces cerevisiae strain according to claim 1 or 2, characterized in that, the sterol synthesis pathway enzyme is the protein encoded by the ERG9 gene, and / or, the proteins encoded by the ERG4, ERG5, ERG6, ERG24 and ERG27 genes; the fatty acid synthesis pathway enzyme is the protein encoded by the ACC1, FAS1 or FAS2 gene; the mevalonate pathway enzyme is the protein encoded by the IDI1 and tHMG1 genes.

4. The engineered Saccharomyces cerevisiae strain according to any one of claims 1 to 3, characterized in that, the weakening is achieved by replacing the original promoter of the enzyme-encoding gene with a promoter having weaker activity; 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.

5. The engineered Saccharomyces cerevisiae strain according to claim 4, characterized in that, The expression of the attenuated sterol synthesis pathway enzyme is achieved by replacing the promoter of the ERG9 gene with P ERG1 , P HXT1 or P ERG7 , and replacing the promoter of the ERG4 gene with P BTS1 , replacing the promoter of the ERG5 gene with P HXT1 , replacing the promoter of the ERG6 gene with P BTS1 , replacing the promoter of the ERG24 gene with P ERG7 and replacing the promoter of the ERG27 gene with P ERG7 ; The expression of the attenuated fatty acid synthesis pathway enzyme is achieved by replacing the promoter of the ACC1 gene with P ERG7 , or by replacing the promoter of the FAS1 or FAS2 gene with P HXT1 or P ERG7 .

6. The engineered Saccharomyces cerevisiae strain according to any one of claims 1 to 5, characterized in that, the enhanced expression of the mevalonate pathway enzyme is achieved by increasing the copy number of the IDI1 and tHMG1 genes; preferably, the enhanced expression of the mevalonate pathway enzyme is achieved by integrating the expression cassettes of the IDI1 and tHMG1 genes on the genome; The expression cassettes of the IDI1 and tHMG1 genes comprise: yeast P Gal101 promoter and the IDI1 and tHMG1 genes.

7. The engineered Saccharomyces cerevisiae strain according to any one of claims 1 to 6, characterized in that, the parental strain is a Saccharomyces cerevisiae strain capable of synthesizing sesquiterpenes.

8. The engineered Saccharomyces cerevisiae strain according to any one of claims 1 to 7, characterized in that, the sesquiterpene is caryophyllene; preferably, the parental strain expresses the caryophyllene synthase gene QHS1 derived from Artemisia annua.

9. Any one of the following applications of the engineered Saccharomyces cerevisiae strain according to any one of claims 1 to 8: (1) Application in the fermentative production of sesquiterpenes or their derivatives; (2) Application in the breeding of production strains of sesquiterpenes or their derivatives; (3) Application in the construction of engineered strains for the production of sesquiterpenes or their derivatives; preferably, the sesquiterpene is caryophyllene.

10. A method for the fermentative production of caryophyllene, characterized in that, The method includes: culturing the engineered Saccharomyces cerevisiae strain according to any one of claims 1 to 8, and collecting caryophyllene in the culture.

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