Saccharomyces cerevisiae engineering bacteria for producing nootkatone as well as construction method and application thereof

Abstract

The invention provides a saccharomyces cerevisiae engineering bacterium for producing nootkatone, the saccharomyces cerevisiae engineering bacterium takes a saccharomyces cerevisiae strain as a chassis strain and is modified by at least one of the following a)-d): a) transferring exogenous genes EgVS, ATR1, HPO or ADH3; b) overexpressing an endogenous gene PDR1; c) knocking out the endogenous gene OPI1; d) truncating the endogenous gene HMG1; in the chassis strain, an endogenous gene ERG20 is over-expressed, an endogenous gene ERG9 is down-regulated, and an endogenous gene ROX1 is knocked out; the promoter of the endogenous gene ERG20 is TEF1; the promoter of the endogenous gene ERG9 is COX9. The invention also provides a corresponding construction method and application in production of nootkatone. The saccharomyces cerevisiae engineering bacteria constructed by the invention can be used for production of nootkatone, and through series optimization, the yield of nootkatone can reach 258.467 mg / L.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to an engineered yeast strain for producing nootkatone, a construction method thereof and an application thereof. Background Art

[0002] Nootkatone (5,6-dimethyl-8-isopropenyl-bicyclo-(4,4,0)-dec-1-en-3-one), also known as cinnamone and cinnamone, is an unsaturated enone belonging to the yarrow family of bicyclic sesquiterpenes. It was first discovered and its structure identified in the 1960s from Alaskan oak bark oil and grapefruit peel oil. Nootkatone has a thorny aroma and is a key flavor component in grapefruit peel and juice.

[0003] Nootkatone is the most important and expensive aromatic compound in grapefruit, considered one of the primary chemical components of its odor and flavor. It can be used to formulate various citrus flavors, such as grapefruit and orange, providing unique aromas and flavors to foods, beverages, and cosmetics. Nootkatone also exhibits anti-proliferative and pro-apoptotic properties, suggesting potential applications in medicine. Studies have shown that nootkatone can reduce body fat percentage, making it a highly sought-after chemical in the cosmetics and food industries. Furthermore, nootkatone has been shown to be an effective repellent or insecticide against pests such as deer ticks, lone star ticks, and mosquitoes. It is an environmentally friendly insecticide, as it is a volatile essential oil that is not persistent in the environment. Compared to plant-based essential oil repellents such as citronella, peppermint, and lemongrass oils, nootkatone's mosquito repellency can last for several hours and is non-toxic to humans. Furthermore, nootkatone has applications in tobacco and feed additives. In tobacco, nootkatone can enhance the aroma of cigarettes, enrich their flavor, and reduce their irritation. Nootkatone has been widely used in various fields, but its synthesis method needs to be further upgraded to meet the industry's demand for nootkatone.

[0004] Currently, there are three production methods for nootkatone, including plant extraction, chemical synthesis and biosynthesis.

[0005] Plant extraction is a method for obtaining natural-grade nootkadone. Essential oils are rich in secondary metabolites such as terpenes, flavonoids, and alkaloids. These volatile and aromatic secondary metabolites are key components of natural flavors and fragrances. The key to plant extraction is to preserve the active ingredients in the essential oil and improve extraction efficiency. Common extraction methods include distillation, supercritical CO2 extraction, and pressing. However, the nootkadone content in grapefruit peel essential oil is relatively low. According to Oxford Biotrans, 400,000 kilograms of grapefruit are required to produce 1 kilogram of nootkadone. Furthermore, natural nootkadone exists in multiple stereoisomers, which reduces the purity of the target product. Separating different stereoisomers is not feasible in industrial production, increasing extraction costs and reducing efficiency. Furthermore, grapefruit peel quality is difficult to control, as weather changes, fluctuating growing conditions, and seasonal variations can significantly vary the raw material. Due to many factors, the efficiency of nootkatone extraction through plants is low and the market price is high, which limits the development and application of nootkatone.

[0006] Given the numerous limitations of plant-based extraction methods for producing nootkatone, which have struggled to meet market demand, researchers have turned to chemical synthesis to fill this gap. Based on the molecular structure of nootkatone, various chemical synthesis methods have been established, including the Robinson cyclization of cyclohexanone derivatives, the Diels–Alder reaction of cyclohexa-1,4-diene derivatives and dienophiles, the cyclization and ring expansion of cyclopentanone, the intramolecular Sakurai reaction, and the Diels–Alder / Aldol cascade reaction. Chemical synthesis methods have drawbacks such as numerous reaction steps, cumbersome procedures, the requirement for stringent reaction conditions, and difficulty in product isolation. Furthermore, the addition of large amounts of toxic chemical reagents during the reaction creates byproducts that cannot be recycled, leading to environmental pollution. Due to the safety risks associated with chemical synthesis, nootkatone obtained through chemical methods cannot be used as a natural flavoring in food and pharmaceutical applications, significantly limiting its application.

[0007] Because both plant extraction and chemical synthesis methods have limitations in producing nootkatone, researchers have begun utilizing synthetic biology techniques to engineer microbial metabolic pathways to produce nootkatone. According to the "National Food Safety Standard: General Rules for Food Flavorings," spices produced using enzymatic or microbial processes also fall under the category of natural flavors. Therefore, constructing a cell factory in microorganisms that can efficiently synthesize nootkatone is an ideal production method. Summary of the Invention

[0008] In order to solve the problems existing in the prior art, the present invention provides an engineered yeast strain for producing nootkatone, a construction method thereof, and an application thereof.

[0009] The first object of the present invention is to provide an engineered Saccharomyces cerevisiae strain for producing nootkatone, wherein the engineered Saccharomyces cerevisiae strain is based on a Saccharomyces cerevisiae strain and undergoes at least one of the following modifications a) to d):

[0010] a) Introducing exogenous genes EgVS, ATR1, HPO or ADH3;

[0011] b) overexpression of the endogenous gene PDR1;

[0012] c) Knockout of the endogenous gene OPI1;

[0013] d) truncation of the endogenous gene HMG1;

[0014] In the chassis strain, the endogenous gene ERG20 is overexpressed, the endogenous gene ERG9 is downregulated, and the endogenous gene ROX1 is knocked out; the promoter of the endogenous gene ERG20 is TEF1; and the promoter of the endogenous gene ERG9 is COX9.

[0015] In the present invention, the chassis strain uses a Saccharomyces cerevisiae strain as a starting strain, overexpresses the endogenous gene ERG20, downregulates the endogenous gene ERG9, and knocks out the endogenous gene ROX1; the promoter of the endogenous gene ERG20 is TEF1; the promoter of the endogenous gene ERG9 is COX9. The chassis strain can be any Saccharomyces cerevisiae strain, preferably a CEN.PK series strain, which is a typical strain for terpenoid synthesis, and undergoes the above-mentioned modifications.

[0016] Preferably, the starting strain of the chassis strain is the Saccharomyces cerevisiae strain CEN.PK113-11C.

[0017] That is, the chassis strain is a Saccharomyces cerevisiae strain CEN.PK113-11C that overexpresses ERG20, downregulates ERG9, knocks out ROX1, and limits the promoter of ERG20 to TEF1 and the promoter of ERG9 to COX9. This chassis strain is named strain JL2 in this article.

[0018] As a preference, the chassis strain is transformed as follows: exogenous genes EgVS, ATR1, HPO, and ADH3 are introduced;

[0019] Preferably, the promoter of the exogenous gene ATR1 is adjusted to QCR10p or COX9; preferably, the promoter of the exogenous gene ATR1 is adjusted to QCR10p.

[0020] Preferably, the chassis strain is modified as follows: the endogenous gene HMG1 is truncated; the gene sequence after truncating HMG1 is SEQ ID NO.8 in the sequence listing;

[0021] Preferably, the promoter of the exogenous gene ATR1 is adjusted to COX9.

[0022] Preferably, the chassis strain is modified as follows: overexpressing the endogenous gene PDR1, and adjusting the promoter of the endogenous gene PDR1 to TDH3p;

[0023] Preferably, the endogenous gene OPI1 is knocked out;

[0024] As a further preference, the promoter of the endogenous gene ERG9 is adjusted to CTR3.

[0025] Preferably, among the exogenous genes, EgVS is derived from Endocellionglaciale, ATR1 is derived from Arabidopsisthaliana, HPO is derived from Hyoscyamusmuticus, and ADH3 is derived from Pichiapastoris.

[0026] Preferably, the 5' end of each of the endogenous gene and the exogenous gene is connected to a promoter, and the 3' end is connected to a terminator.

[0027] The second object of the present invention is to provide a method for constructing an engineered strain of Saccharomyces cerevisiae that produces nootkatone, constructing an expression module of a promoter-exogenous gene / endogenous gene-terminator, and transforming the expression module and a linearized backbone vector into a chassis strain.

[0028] The third object of the present invention is to provide the use of the above-mentioned engineered yeast Saccharomyces cerevisiae in the preparation of nootkatone.

[0029] The fourth object of the present invention is to provide a method for preparing nootkatone, which comprises fermenting the above-mentioned engineered yeast Saccharomyces cerevisiae.

[0030] Preferably, the fermentation medium used in the fermentation is: SC / Delft liquid medium containing dodecane and copper sulfate; the volume ratio of dodecane to SC / Delft liquid medium is 1:5; the concentration of copper sulfate is 50 μM-400 μM;

[0031] Preferably, the fermentation medium used in the fermentation is: a Delft liquid medium containing dodecane and copper sulfate; the volume ratio of the dodecane to the Delft liquid medium is 1:5; the concentration of the copper sulfate is 50 μM;

[0032] Preferably, the fermentation conditions are: initial OD 0.1-0.5, culture at 30° C., 200 rpm-220 rpm for 96 h-108 h;

[0033] Preferably, the fermentation conditions are: initial OD 0.5, culture at 30° C., 220 rpm for 108 h.

[0034] The brewer's yeast engineered bacteria constructed by the present invention can be used for the production of nootkatone. Through a series of optimizations, the yield of nootkatone can reach a maximum of 258.467 mg / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0036] Figure 1 The results of adjusting the exogenous genes for nootkatone synthesis and key enzymes in the mevalonate pathway are presented, and the results of the yield improvement by two-phase fermentation are also demonstrated.

[0037] Figure 2 The relative fold difference of expression intensity of different promoters and the corresponding yield changes.

[0038] Figure 3 The optimization of culture conditions led to an increase in yield and changes in the intermediate product nocardol.

[0039] Figure 4 The effect of ATR1 expressed by promoters of different strengths on yield changes after optimization of culture conditions.

[0040] Figure 5 The combined upregulation of PDR1 and OPI1 knockout promoted the synthesis of nootkatone.

[0041] Figure 6 The nootkatone production in Saccharomyces cerevisiae was obtained by adjusting the ERG9 promoter to CTR3. DETAILED DESCRIPTION

[0042] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent companies. The quantitative tests in the following examples were performed in triplicate, and the results were averaged.

[0043] Example 1 Method for constructing recombinant Saccharomyces cerevisiae and recombinant plasmids

[0044] 1. Method for constructing recombinant Saccharomyces cerevisiae in the present invention

[0045] Unless otherwise specified, the present invention uses the Crispr-Cas9 gene editing method to construct recombinant Saccharomyces cerevisiae (the specific reference article is doi.org / 10.1038 / s41467-019-09005-3). The method is as follows:

[0046] 1.1 Guide plasmids were constructed using the GOLDENGATE method. The PCR amplification conditions for the fragments are shown in Table 1.

[0047] Table 1 PCR amplification of DNA fragments

[0048] Composition system volume 2 × PhantaFlashMasterMix 25 μL F-primer (10 μM) 2μL R-primer (10 μM) 2μL Template (10-500ng) xμL <![CDATA[ddH2O]]> up to 50 μL

[0049] 2×Phanta Flash Master Mixt was purchased from Nanjing Novozymes Biotechnology Co., Ltd. PCR amplification was performed according to the instructions, and the obtained PCR products were recovered using the company's gel recovery kit.

[0050] 1.2 Perform the GOLDEN GATE reaction. The reaction system is shown in Table 2, and the reaction procedure is shown in Table 3.

[0051] Table 2 GOLDEN GATE reaction system

[0052] Composition system volume <![CDATA[T4NDA ligase]]> 0.4μL BsaI enzyme 1.6μL <![CDATA[T4Buffer]]> 2μL PCR fragment Determined by the concentration of the fragment Vector (pCas-lacz) Determined by the concentration of the fragment <![CDATA[ddH2O]]> up to 20 μL

[0053] Determine the mass of vector plasmid to add based on its size. For fragments 0-3 kb, add 50 ng; for fragments 3-6 kb, add 100 ng; and for fragments 6-9 kb, add 150 ng. Maintain a 1:1 molar ratio of vector plasmid to DNA fragment in the reaction system.

[0054] Table 3 GOLDEN GATE reaction procedure

[0055]

[0056] 1.3 After the completion of GOLDEN GATE, colon competent cells were used for transformation and plasmid extraction. The preparation and transformation of colon competent cells were performed using methods known in the art.

[0057] 1.4 All other fragments were prepared according to the PCR amplification method in Table 4.

[0058] Table 4 PCR amplification conditions of gene fragments

[0059] Composition system volume F-primer (100 μM) 20 μL R-primer (100 μM) 20 μL 1×KODase 1 μL 2mMDNTPs 43μL <![CDATA[25mMMgSO4]]> 6μL 10×KODBuffer 10 μL

[0060] The obtained PCR products were recovered using the gel recovery kit from Nanjing Novozymes Biotechnology Co., Ltd.

[0061] 1.5 Construction of recombinant Saccharomyces cerevisiae

[0062] First, prepare Saccharomyces cerevisiae competent cells according to conventional methods, and then transfer the exogenous gene into Saccharomyces cerevisiae. All transformation plasmids should not be less than 500ng, and the donor should not be less than 1500ng. After the transformation is completed, apply SC-URA solid medium, pick single clones for PCR verification, apply 5-FoA solid medium with correct bands to remove the plasmid, and then streak the correct clones with the plasmid removed on YPD plates to separate single clones. When performing production verification, prepare Saccharomyces cerevisiae competent cells according to conventional methods. Then, because the genes that can synthesize nootkatone are expressed on the plasmid, the plasmid is transferred into Saccharomyces cerevisiae, and all transformation plasmids should also not be less than 500ng. After the transformation is completed, apply SC-URA-HIS to the plate, and after the clones grow, pick three relatively large and similarly sized clones for fermentation production verification.

[0063] 1.6 Fermentation culture of recombinant Saccharomyces cerevisiae

[0064] The fermentation medium of recombinant Saccharomyces cerevisiae was SC liquid medium (complete amino acid medium, reference article is doi.org / 10.1038 / s41467-019-09005-3), with a culture volume of 20 ml, an initial OD of 0.1, and culture at 30°C and 220 rpm for 108 h.

[0065] 2. Method for constructing the recombinant plasmid in the present invention

[0066] 2.1 PCR amplification conditions of gene fragments are shown in Table 4.

[0067] 2.2 Plasmid Construction Homologous recombination was used, employing the Gibson assembly method. 2× Clon Express Mix was purchased from Nanjing Novozyme Technology Co., Ltd. The reaction conditions were: reaction at 50°C for 15 minutes. The reaction system is shown in Table 5.

[0068] Table 5 Construction of plasmids

[0069] Composition system volume carrier 20ng / kb Clip 1 20-40ng / kb Clip 2 and other clips 20-40ng / kb 2×ClonExpressMix The sum of the volume of the vector and all fragments

[0070] Example 2 Nootkatone synthesis gene selection, mevalonate (MVA) pathway key enzyme modification and culture condition optimization

[0071] Key enzymes for nootkatone synthesis were selected, and the expression of key enzymes in the mevalonate pathway was adjusted to increase the flux of the nootkatone synthesis pathway with farnesyl pyrophosphate (FPP) as a precursor.

[0072] 1. Strains, plasmids, and primers

[0073] Escherichia coli (E. coli DH5α) was used for plasmid amplification. The plasmids constructed in this example are shown in Table 6. The yeast strains constructed in this example are shown in Table 7. The starting strain of Saccharomyces cerevisiae was the CEN.PK113-11C (MATa SUC2 MAL2-8c his3 Δ1 ura3-52) strain, which was used for gene expression and pathway construction. The primers used in this example are shown in Table 8.

[0074] Table 6 Plasmids used in this example

[0075]

[0076] The gene sequence of plasmid PstgURA3 is as follows (SEQ ID NO.1):

[0077]

[0078] The gene sequence of plasmid PUGG1 is as follows (SEQ ID NO.2):

[0079]

[0080] Table 7 Strains used in this example

[0081]

[0082] Table 8 Primers used in this example

[0083]

[0084]

[0085]

[0086] 2 Construction of Saccharomyces cerevisiae strain JL2

[0087] The present invention detected trace amounts of nootkatone in strain CEN.PK113-11C and confirmed it as the target product through mass spectrometry (MS) analysis. Due to the extremely low yield, it is suspected that the key production pathways were not modified. Therefore, the key enzymes in the mevalonate (MVA) pathway were optimized to construct strain JL2.

[0088] Specific measures include upregulating the expression of ERG20 (farnesylpyrophosphate synthetase) (replacing the original promoter with the TEF1 promoter, reference article: doi.org / 10.1038 / s41467-024-45557-9), downregulating the expression of ERG9 (squalene synthase) (replacing the original promoter with the COX9 promoter, reference article: doi.org / 10.1038 / s41467-024-45557-9), and knocking out the inhibitor of the mevalonate (MVA) pathway, ROX1 (heme-dependent inhibitor, Regulation by Oxygen) (whole gene knockout), thereby increasing the production of nootkatone to 0.6 mg / L.

[0089] 2.1 Upregulate the expression of ERG20 and replace the original promoter of ERG20 with the TEF1 promoter

[0090] PCR amplification was performed using the CEN.PK113-11C genome as a template using TEF1-ERG20-F and TEF1-ERG20-R primers. The fragment recovered after gel extraction served as the DNA donor. A plasmid for co-expressing the gRNA sequence and Cas9 protein gene was amplified using the PstgURA3 plasmid as a template using pcas-ERG20-pgf and Leu2.1URA3R primers. The gel-extracted fragment was combined with pcas-lacz to construct a guide plasmid using Golden Gate. The guide plasmid and DNA donor were transformed into the Saccharomyces cerevisiae CEN.PK113-11C strain by electroporation, and the strain was obtained after plasmid loss.

[0091] 2.2 Downregulate the expression of ERG9 and replace the original promoter of ERG9 with the COX9 promoter

[0092] PCR amplification was performed using the CEN.PK113-11C genome as a template using primers Cox9-ERG9-F and Cox9-ERG9-R. The fragment recovered after gel extraction served as the DNA donor. A plasmid for co-expressing the gRNA sequence and Cas9 protein gene was amplified using the PstgURA3 plasmid as a template using primers pcas-ERG9-pgf and Leu2.1URA3R. The gel-recovered fragment was combined with pcas-lacz to construct a guide plasmid using Golden Gate. The guide plasmid and DNA donor were transformed into the Saccharomyces cerevisiae CEN.PK113-11C strain by electroporation, and the strain was obtained after plasmid loss.

[0093] 2.3 Knockout of ROX1, an inhibitor of the MVA pathway (full gene knockout)

[0094] PCR amplification was performed using ROX1-orf-donor-F and ROX1-orf-donor-R primers, with each other serving as templates. The fragment obtained after alcohol precipitation served as the DNA donor. A plasmid for co-expressing the gRNA sequence and Cas9 protein gene was amplified using the PstgURA3 plasmid as a template and primers pcas-ROX1-gf and Leu2.1URA3R. The gel-recovered fragment was combined with pcas-lacz to construct a guide plasmid using Golden Gate. The guide plasmid and DNA donor were transformed into the Saccharomyces cerevisiae CEN.PK113-11C by electroporation, and the Saccharomyces cerevisiae strain JL2 was obtained after plasmid loss.

[0095] In the present invention, the detection methods for nootkatone production and its precursor valencene are described in the reference article doi / full / 10.1021 / acs.jafc.1c07035. Nootkatone standards were purchased from Sigma-Aldrich, Catalog No. 74437. Valencene standards were purchased from Sigma-Aldrich, Catalog No. 75056.

[0096] 3 Construction of pJLP1 and pJLP2 plasmids

[0097] In this example, the synthetic genes selected were EgVS (valencene synthase, Eryngium glaciale valencene synthase), ATR1 (cytochrome P450 reductase, cytochrome P450 reductase), HPO (monooxygenase, premna-spirodiene oxygenase), and ADH3 (alcohol dehydrogenase). To facilitate genomic gene editing, the expression cassettes of the EgVS and ADH3 genes were expressed on a URA-tagged high-copy plasmid to construct plasmid pJLP1. ATR1 and HPO were expressed on a HIS-tagged high-copy plasmid to construct plasmid pJLP2. The reference article is doi.org / 10.1038 / s41467-024-45557-9. The constructed plasmids are shown in Table 6.

[0098] Among them, the source of EgVS is Endocellion glaciale, and the sequence source article used is doi.org / 10.1016 / j.ymben.2022.03.005.

[0099] ATR1 is from Arabidopsis thaliana, HPO is from Hyoscyamus muticus, and ADH3 is from Pichia pastoris. The sequence sources of these three genes are as follows: doi / full / 10.1021 / acs.jafc.1c07035.

[0100] The EgVS gene sequence is as follows (SEQ ID NO. 3):

[0101]

[0102]

[0103] The ATR1 gene sequence is as follows (SEQ ID NO.4):

[0104]

[0105] The HPO gene sequence is as follows (SEQ ID NO.5):

[0106]

[0107]

[0108] The ADH3 gene sequence is as follows (SEQ ID NO.6):

[0109]

[0110] 3.1 Construction of plasmid pJLP1

[0111] Using plasmid PUGG1 as a template, PCR amplification was performed with primers PUGG1-MCR-F and EgVS-R1, and gel recovery yielded a linear vector fragment. Using the synthetic gene EgVS as a template, PCR amplification was performed with primers EgVS-F1 and EgVS-R, and gel recovery yielded fragment 1. Using the CEN.PK113-11C genome as a template, PCR amplification was performed with primers CCW12-F1 and CCW12-ADH3PPop-R, and with primers ADH1t-ADH3PPop-F and ADH1t-R1, and gel recovery yielded fragments 2 and 3. Using the synthetic gene ADH3 as a template, PCR amplification was performed with primers ADH3PPop-F and ADH3PPop-R, and gel recovery yielded fragment 4. The linear vector, fragment 1, fragment 2, fragment 3 and fragment 4 were homologously ligated using the method of step 2.2 of Example 1, and E. coli was transformed and the plasmid was extracted to obtain plasmid pJLP1.

[0112] 3.2 Construction of plasmid pJLP2

[0113] Using plasmid PUGG1 as a template, PCR amplification was performed with primers His-F and FBA1t-R, and gel recovery was performed to obtain a linear vector fragment. Using the CEN.PK113-11C genome as a template, PCR amplification was performed with primers CDC19-AtCPR1-F and CDC19-R, with primers FBA1t-ATR1-F and ATR1-R, with primers HPO-His-F and PUC57-HPO-R, and with primers PUC57-HPO-F and HPO-His-R, and gel recovery was performed to obtain fragments 1, 2, 3, and 4. Using the synthetic gene ATR1 as a template, PCR amplification was performed with primers ATR1-F and CDC19-ATR1-R, to obtain fragment 5. Using the synthetic gene ADH3 as a template, PCR amplification was performed with primers HPO-TEF1-F and HPO-TEF1-R, to obtain fragment 6. The linear vector, fragment 1, fragment 2, fragment 3, fragment 4, fragment 5 and fragment 6 were homologously ligated using the method of step 2.2 of Example 1, and E. coli was transformed and the plasmid was extracted to obtain plasmid pJLP2.

[0114] The method used in constructing pJLP1 and pJLP2 plasmids is shown in step 2 of Example 1.

[0115] 4 Construction of Saccharomyces cerevisiae strain JL3

[0116] Plasmids pJLP1 and pJLP2 were transformed into Saccharomyces cerevisiae strain JL2 according to the method in step 1.5 of Example 1 to obtain Saccharomyces cerevisiae strain JL3. Fermentation was carried out according to the method in step 1.6 of Example 1, and the nootkatone yield was determined to be 0.6 mg / L.

[0117] 5. Fermentation of Saccharomyces cerevisiae strain JL3

[0118] 4 ml of dodecane was added to 20 ml of SC liquid medium (two-phase fermentation) to a final volume of 24 ml. Saccharomyces cerevisiae strain JL3 was fermented according to the fermentation culture conditions of step 1.6 of Example 1, and the yield of nootkatone was finally increased to 1.8 mg / L.

[0119] Figure 1 The results of adjusting the exogenous gene for nootkatone synthesis and the key enzymes of the mevalonate pathway are presented, and the results of the two-phase fermentation on the yield improvement are also presented (both groups used 20 ml SC liquid culture medium with 4 ml dodecane added).

[0120] Example 3 Effect of ATR1 enzyme on yield under different promoter strengths

[0121] Different expression intensities of ATR1 affect the final yield. High expression of the HPO enzyme facilitates the reaction, so different intensities of endogenous Saccharomyces cerevisiae promoters were selected to express the ATR1 gene, based on the high expression of HPO. Ultimately, ATR1 was expressed using promoters between CDC19 and POX1, based on the promoter strength coefficient. Because ATR1 and HPO are expressed on the plasmid pJLP2, this plasmid was used as the initial template for constructing all plasmids in this example.

[0122] The yeast strains of this example are shown in Table 9, the plasmids are shown in Table 10, and the primers are shown in Table 11.

[0123] Table 9 Strains used in this example

[0124]

[0125]

[0126] Table 10 Plasmids used in this example

[0127]

[0128]

[0129]

[0130] 2 Construction of plasmid pJLP7

[0131] Using plasmid pJLP2 as a template, PCR amplification with primers QCR10-ATR1-F and His-R1 was performed, followed by gel recovery to obtain a linear vector fragment. Using plasmid pJLP2 as a template, PCR amplification with primers HPO-His-F and QCR10-ATR1-R was performed, followed by gel recovery to obtain fragment 1. Using the CEN.PK113-11C genome as a template, PCR amplification with primers ATR1-QCR10-F and ATR1-QCR10-R was performed, followed by gel recovery to obtain fragment 2. The vector fragment was then homologously ligated with fragments 1 and 2, E. coli was transformed, and the plasmid was extracted to obtain plasmid pJLP7.

[0132] The promoter sequence of the QCR10 (ubiquinol-cytochrome C oxidoreductase) gene is as follows (SEQ ID NO. 7 in the sequence listing):

[0133]

[0134] The construction methods of the remaining plasmids are similar to those of plasmid pJLP7 and will not be repeated here.

[0135] 3 Construction method of Saccharomyces cerevisiae strain JL8

[0136] Plasmids pJLP1 and pJLP7 were transformed into strain JL2 according to the method of step 1.5 of Example 1 to obtain strain JL8.

[0137] The construction methods of the remaining strains are the same as those of the Saccharomyces cerevisiae strain JL8 and will not be repeated here.

[0138] 4. Fermentation of Saccharomyces cerevisiae strains JL4-JL11 was carried out according to the method in step 5 of Example 2, and the production of nootkatone was detected. The results are as follows: Figure 2 shown.

[0139] Depend on Figure 2 The results show that: 1. The yield varies with the strength of the ATR1 promoter and shows a certain trend. 2. The yield is highest when the weak promoter QCR10 is used, reaching 14.7653333 mg / L, which is strain JL8.

[0140] Figure 2 The relative fold difference of expression intensity of different promoters and the corresponding yield changes.

[0141] Example 4: Further optimization of culture conditions to increase yield

[0142] To further improve the flux of the MVA pathway, we truncated the key gene HMG1 (HMG-CoA reductase, HMG-CoAreductase GeneBankID NO: KM821404) in the genome, constructed the Saccharomyces cerevisiae strain JL12, and then transformed the plasmids pJLP1 and pJLP7 to construct the Saccharomyces cerevisiae strain JL13.

[0143] The yeast strains constructed in this example are shown in Table 12, and the primers are shown in Table 13.

[0144] Table 12 Bacteria used in the experiment

[0145] Strain name Strain information Background bacteria source JL12 XII5::(PGK1p-tHMG1-CYC1t) JL2 This chapter builds JL13 pJLP1,pJLP7 JL12 This chapter builds

[0146] Table 13 Primers used in the experiment

[0147]

[0148] 2 Construction method of Saccharomyces cerevisiae strain JL12

[0149] PCR amplification was performed using laboratory-stored HMG1 as a template using primers XII-5-tHMG1-F and XII-5-tHMG1-R. The fragment recovered after gel extraction served as the DNA donor. A plasmid for co-expressing the gRNA sequence and Cas9 protein gene was amplified using the PstgURA3 plasmid as a template using primers pcas-XII-5-gf and Leu2.1URA3R. The recovered fragment was combined with pcas-lacz via Golden Gate to construct a guide plasmid. The guide plasmid and DNA donor were transformed into Saccharomyces cerevisiae JL2 by electroporation, resulting in strain JL12 after plasmid loss.

[0150] The truncated tHMG1 gene sequence is as follows (SEQ ID NO.8 in the sequence listing):

[0151]

[0152]

[0153] 3 Construction method of Saccharomyces cerevisiae strain JL13

[0154] Plasmids pJLP1 and pJLP7 were transformed into Saccharomyces cerevisiae strain JL12 according to the method of step 1.5 of Example 1 to obtain Saccharomyces cerevisiae strain JL13.

[0155] 4. Fermentation method of Saccharomyces cerevisiae strain JL13

[0156] 4.1 Experimental Group: Saccharomyces cerevisiae strain JL13 was fermented in 4 ml dodecane + 20 ml Delft, with an initial OD of 0.5, cultured at 30°C and 220 rpm for 108 h. The production of nootkatone was determined to be 54 mg / L.

[0157] 4.2 Control: Saccharomyces cerevisiae strain JL13 was fermented in a medium consisting of 4 ml dodecane and 20 ml SC, with an initial OD of 0.5, cultured at 30°C and 220 rpm for 108 h. The nootkatone production was determined to be 11.3697 mg / L.

[0158] The experimental results are shown in Figure 3 .Depend on Figure 3 b It can be seen that nootkatol, the precursor substance of nootkatone synthesis, fully participated in the reaction after the culture medium was changed, indicating that the optimization of the culture medium is more important for production.

[0159] Figure 3 The optimization of culture conditions led to an increase in yield and changes in the intermediate product nocardol.

[0160] Example 5 Effects of different promoters on nootkadone production under Delft medium conditions

[0161] After switching to Delft culture medium, we confirmed that the previously measured yield-promoter strength curve persisted. We then retested several strains and found a consistent trend. The weaker COX9 (Subunit VIIa of cytochrome c oxidase) promoter produced the highest yield, reaching 80.986 mg / L. This is consistent with strain JL17. Table 14 shows the corresponding strain information.

[0162] Table 14 Strains used in the experiment

[0163] Strain name Strain information Background bacteria source JL14 pJLP1,pJLP2 JL12 This chapter builds JL15 pJLP1,pJLP6 JL12 This chapter builds JL16 pJLP1,pJLP7 JL12 This chapter builds JL17 pJLP1,pJLP9 JL12 This chapter builds

[0164] 1 Construction of plasmid pJLP9

[0165] Using plasmid pJLP2 as a template, PCR amplification with primers Cox9-ATR1-F and His-R1 was performed, followed by gel recovery to obtain a linear vector fragment. Using plasmid pJLP2 as a template, PCR amplification with primers HPO-His-F and Cox9-ATR1-R was performed, followed by gel recovery to obtain fragment 1. Using the CEN.PK113-11C genome as a template, PCR amplification with primers ATR1-Cox9-F and ATR1-Cox9-R was performed, followed by gel recovery to obtain fragment 2. The vector fragment was then homologously ligated with fragments 1 and 2, E. coli was transformed, and the plasmid was extracted to obtain plasmid pJLP9.

[0166] The Cox9 gene promoter sequence is as follows (SEQ ID NO.9 in the sequence listing):

[0167]

[0168] 2 Construction method of Saccharomyces cerevisiae strain JL17

[0169] Plasmids pJLP1 and pJLP9 were transformed into strain JL12 according to the method of step 1.5 of Example 1 to obtain strain JL17.

[0170] Fermentation was performed according to the method in step 4.1 of Example 4, and the nootkatone production of strain JL17 was determined to be 80.986 mg / L.

[0171] Figure 4 The effect of ATR1 expressed by promoters of different strengths on yield changes after optimization of culture conditions.

[0172] Example 6: Upregulation of PDR1 increases nootkatone production, and superposition of PDR1 with OPI1 further increases production.

[0173] Certain ABC transporters may affect overall terpenoid production. Since the transcription factor PDR1 (pleiotropic drug resistance protein 1) regulates ABC transporter expression, we upregulated PDR1 expression and used CRISPR editing to replace its promoter with TDH3p, the gene for glyceraldehyde-3-phosphate dehydrogenase. Results showed that upregulating PDR1 enhanced flux in the nootkatone biosynthesis pathway, increasing the yield to 154 mg / L in strain JL19 and also increasing valencene production. Next, we knocked out the OPI1 (overproducer of inositol synthase 1) gene, which increases the endoplasmic reticulum membrane area in Saccharomyces cerevisiae. Results showed increased nootkatone production, while valencene production decreased slightly. Combining these two targets in strain JL21 boosted the yield to 202.536 mg / L.

[0174] The yeast strains constructed in this example are shown in Table 15, and the primers are shown in Table 16.

[0175] Table 15 Strains used in the experiment

[0176] Strain name Strain information Background bacteria source JL18 PDR1p::TDH3p JL12 This chapter builds JL19 pJLP1,pJLP9 JL18 This chapter builds JL20 opi1△ JL18 This chapter builds JL21 pJLP1,pJLP9 JL20 This chapter builds

[0177] Table 16 Primers used in the experiment

[0178]

[0179] 2 Construction method of Saccharomyces cerevisiae strain JL18

[0180] PCR amplification was performed using the CEN.PK113-11C genome as a template using primers TDH3-PDR1-F and TDH3-PDR1-R. The fragment recovered after gel extraction served as the DNA donor. A plasmid for co-expressing the gRNA sequence and Cas9 protein gene was amplified using the PstgURA3 plasmid as a template using primers pcas-PDR1-pgf and Leu2.1URA3R. The recovered fragment was combined with pcas-lacz via Golden Gate to construct a guide plasmid. The guide plasmid and DNA donor were transformed into Saccharomyces cerevisiae JL12 by electroporation, and strain JL18 was obtained after plasmid loss.

[0181] 3 Construction method of Saccharomyces cerevisiae strain JL19

[0182] Plasmids pJLP1 and pJLP9 were transformed into strain JL18 according to the method of step 1.5 of Example 1 to obtain strain JL19.

[0183] The TDH3p gene promoter sequence is as follows (SEQ ID NO.10 in the sequence listing):

[0184]

[0185] 4 Construction method of Saccharomyces cerevisiae strain JL20

[0186] PCR amplification was performed using primers OPI1-orf-donor-F and OPI1-orf-donor-R, each serving as a template. The fragment obtained after alcohol precipitation served as the DNA donor. A plasmid co-expressing the gRNA sequence and Cas9 protein gene was amplified by PCR using the PstgURA3 plasmid as a template and primers pcas-OPI1-gf and Leu2.1URA3R. The gel-recovered fragment was combined with pcas-lacz to construct a guide plasmid using Golden Gate. The guide plasmid and DNA donor were transformed into Saccharomyces cerevisiae JL18 by electroporation, and strain JL20 was obtained after plasmid loss.

[0187] 5. Construction method of Saccharomyces cerevisiae strain JL21

[0188] Plasmids pJLP1 and pJLP9 were transformed into strain JL20 according to the method of step 1.5 of Example 1 to obtain strain JL21.

[0189] 6. Fermentation was carried out according to the fermentation method of step 4.1 in Example 4, and the yield of nootkatone was determined to be 202.536 mg / L.

[0190] Figure 5 The combined upregulation of PDR1 and OPI1 knockout promoted the synthesis of nootkatone.

[0191] Example 6 Further regulating key enzymes in the mevalonate pathway to increase nootkadone production

[0192] Comparing the growth rate of the modified strain with that of the wild-type strain revealed that switching the ERG9 promoter to COX9 had little effect on growth. Therefore, we believed that downregulation of the ERG9 gene could be further enhanced. Through literature research, we used CRISPR knockout technology to modify its promoter to the copper-regulated CTR3 promoter. The CTR3 gene is known as high-affinity copper transporter 3 (Cu transporter 3), ultimately constructing strain JL23. During fermentation, we added copper sulfate at varying concentrations, from 0 μM to 400 μM. Results showed that at 50 μM, strain JL23 achieved a final yield of 258.467 mg / L.

[0193] The yeast strains constructed in this example are shown in Table 17, and the primers used in this example are shown in Table 18.

[0194] Table 17 Bacteria used in the experiment

[0195] Strain name Strain information Background bacteria source JL22 ERG9p::CTR3p JL20 This chapter builds JL23 pJLP1,pJLP9 JL22 This chapter builds

[0196] Table 18 Bacteria used in the experiment

[0197]

[0198] 2 Construction method of Saccharomyces cerevisiae strain JL22

[0199] PCR amplification was performed using the CEN.PK113-11C genome as a template with primers CTR3-ERG9-F and CTR3-ERG9-R. The fragment recovered after gel extraction served as the DNA donor. A plasmid for co-expressing the gRNA sequence and Cas9 protein gene was amplified by PCR using the PstgURA3 plasmid as a template with primers pcas-ERG9-pgf and Leu2.1URA3R. The recovered fragment was combined with pcas-lacz via Golden Gate to construct a guide plasmid. The guide plasmid and DNA donor were transformed into Saccharomyces cerevisiae JL20 by electroporation, and strain JL22 was obtained after plasmid loss.

[0200] The CTR3 gene promoter sequence is as follows (SEQ ID NO.11 in the sequence listing):

[0201]

[0202] 3 Construction method of Saccharomyces cerevisiae strain JL23

[0203] Plasmids pJLP1 and pJLP9 were transformed into strain JL22 according to the method of step 1.5 of Example 1 to obtain strain JL23.

[0204] 4. Fermentation of Saccharomyces cerevisiae strain JL23

[0205] The fermentation method of step 4.1 in Example 4 was followed, and a certain concentration of copper sulfate was added to the culture medium for fermentation. The yield of nootkatone was measured. When the copper sulfate concentration ranged from 50 μM to 400 μM, the yield was above 230 mg / L. When the copper sulfate concentration was 50 μM, the highest yield was 258.467 mg / L.

[0206] Figure 6 The nootkatone production in Saccharomyces cerevisiae was obtained by adjusting the ERG9 promoter to CTR3.

[0207] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. An engineered strain of Saccharomyces cerevisiae for producing nootkatone, characterized in that: The engineered yeast strain of Saccharomyces cerevisiae is based on the Saccharomyces cerevisiae strain and undergoes at least one of the following modifications a) to d): a) Introducing exogenous genes EgVS, ATR1, HPO or ADH3; b) overexpression of the endogenous gene PDR1; c) Knockout of the endogenous gene OPI1; d) truncation of the endogenous gene HMG1; In the chassis strain, the endogenous gene ERG20 is overexpressed, the endogenous gene ERG9 is downregulated, and the endogenous gene ROX1 is knocked out; the promoter of the endogenous gene ERG20 is TEF1; and the promoter of the endogenous gene ERG9 is COX9.

2. The engineered yeast strain for producing nootkatone according to claim 1, wherein: The starting strain of the chassis strain is the cerevisiae strain CEN.PK113-11C.

3. The engineered yeast of Saccharomyces cerevisiae for producing nootkatone according to claim 1, wherein: The chassis strain was transformed as follows: exogenous genes EgVS, ATR1, HPO, and ADH3 were introduced; Preferably, the promoter of the exogenous gene ATR1 is adjusted to QCR10p or COX9; preferably, the promoter of the exogenous gene ATR1 is adjusted to QCR10p.

4. The engineered yeast of Saccharomyces cerevisiae for producing nootkatone according to claim 3, wherein: The chassis strain was modified as follows: the endogenous gene HMG1 was truncated; the gene sequence after truncating HMG1 is SEQ ID NO.8 in the sequence listing; Preferably, the promoter of the exogenous gene ATR1 is adjusted to COX9.

5. The engineered yeast strain for producing nootkatone according to claim 4, wherein: The chassis strain was modified as follows: the endogenous gene PDR1 was overexpressed, and the promoter of the endogenous gene PDR1 was adjusted to TDH3p; Preferably, the endogenous gene OPI1 is knocked out; As a further preference, the promoter of the endogenous gene ERG9 is adjusted to CTR3.

6. The engineered yeast strain for producing nootkatone according to any one of claims 1 to 5, characterized in that: Among the exogenous genes, EgVS is derived from Endocellionglaciale, ATR1 is derived from Arabidopsis thaliana, HPO is derived from Hyoscyamus muticus, and ADH3 is derived from Pichiapastoris.

7. The engineered yeast strain for producing nootkatone according to any one of claims 1 to 6, characterized in that: The 5' end of each of the endogenous gene and the exogenous gene is connected to a promoter, and the 3' end is connected to a terminator.

8. A method for constructing an engineered strain of Saccharomyces cerevisiae for producing nootkatone, characterized in that: An expression module of promoter-exogenous gene / endogenous gene-terminator is constructed, and the expression module and the linearized backbone vector are transformed into a chassis strain.

9. Use of the engineered yeast Saccharomyces cerevisiae according to any one of claims 1 to 7 in the preparation of nootkatone.

10. A method for preparing nootkatone, characterized in that: Fermentation using the engineered yeast Saccharomyces cerevisiae according to any one of claims 1 to 7; Preferably, the fermentation medium used in the fermentation is: SC / Delft liquid medium containing dodecane and copper sulfate; the volume ratio of dodecane to SC / Delft liquid medium is 1:5; the concentration of copper sulfate is 50 μM-400 μM; Preferably, the fermentation medium used in the fermentation is: a Delft liquid medium containing dodecane and copper sulfate; the volume ratio of the dodecane to the Delft liquid medium is 1:5; the concentration of the copper sulfate is 50 μM; Preferably, the fermentation conditions are: initial OD 0.1-0.5, culture at 30° C., 200 rpm-220 rpm for 96 h-108 h; Preferably, the fermentation conditions are: initial OD 0.5, culture at 30° C., 220 rpm for 108 h.

Images