Enzyme composition, strain and application thereof in preparation of santalol

Abstract

The invention relates to the field of bioengineering, in particular to an enzyme composition, a strain and application of the strain in preparation of santalol. The present invention provides an enzyme composition comprising: a santalene synthase, a cytochrome P450 monooxygenase, and a cytochrome P450 reductase 2. The invention provides a saccharomyces cerevisiae strain for producing santalol and application of the saccharomyces cerevisiae strain for producing santalol, which is characterized in that a key enzyme santalene synthase variant and cytochrome P450 in a santalol biosynthesis path are integrated onto a saccharomyces cerevisiae genome of a high-yield substrate farnesyl pyrophosphate (FPP), and a path for producing santalol by heterologous metabolism is constructed in saccharomyces cerevisiae; the constructed recombinant saccharomyces cerevisiae strain for producing santalol has an important application prospect in synthesis of santalol.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and more particularly to enzyme compositions, strains, and their application in the preparation of santalol. Background Technology

[0002] Santal alcohol is a naturally occurring sesquiterpene alcohol compound with the molecular formula C. 15 H 24 O, mainly existing in α- and β-isomers, is the main component of sandalwood essential oil. Sandalwood essential oil possesses pharmacological activities such as antibacterial, anti-inflammatory, and nerve-sedative effects, and has unique value in the fields of fragrance and fine chemicals. Traditional methods of extracting sandalwood essential oil from sandalwood heartwood are limited in production due to the slow growth of trees, low oil yield, and endangered trees, and cannot meet the growing market demand.

[0003] With the rapid development of synthetic biology technology, the sustainable production of terpenoids using microbial cell factories has become an industry consensus. The biosynthetic pathway of santalol has now been elucidated. The biosynthesis of santalol begins with isoprene units generated via the mevalonate (MVA) or methyl erythrose phosphate (MEP) pathway. These units are then catalyzed by farnesyl pyrophosphate synthases (FPPs) to form the C15 precursor FPP. The linear FPP is then cyclized into the tricyclic sesquiterpene skeleton santalene by santalene synthase. Subsequently, hydroxylation modification of C12 via cytochrome P450 yields santalol.

[0004] Saccharomyces cerevisiae, with its complete and efficient MVA pathway, mature gene editing platform, high flexibility in metabolic pathway optimization, and genetic stability, has become a commonly used production platform for terpenoid products. The industrialization of producing chemicals such as artemisinic acid and squalene using Saccharomyces cerevisiae has been achieved. This provides a reference and precedent for the heterologous metabolism production of santalol using Saccharomyces cerevisiae. Summary of the Invention

[0005] In view of this, the present invention provides an enzyme composition, a strain, and its application in the preparation of santalol. The present invention provides a *Saccharomyces cerevisiae* strain for producing santalol and its application, by integrating a variant of santalene synthase, a key enzyme in the santalol biosynthetic pathway, and cytochrome P450 into the genome of a *Saccharomyces cerevisiae* strain that highly produces the substrate farnesyl pyrophosphate (FPP), thereby constructing a heterologous metabolic pathway for santalol production in *Saccharomyces cerevisiae*, and constructing a recombinant *Saccharomyces cerevisiae* strain for santalol production, which has significant application prospects in the synthesis of santalol.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] The present invention provides an enzyme composition comprising: santalene synthase, cytochrome P450 monooxygenase and cytochrome P450 reductase 2.

[0008] In some embodiments of the present invention, the santalene synthase in the above-described enzyme composition is derived from Clausena lansium; and / or

[0009] The cytochrome P450 monooxygenase was derived from Santalum album; and / or

[0010] The cytochrome P450 reductase 2 was derived from Arabidopsis thaliana.

[0011] In some embodiments of the present invention, the santalene synthase in the above-described enzyme composition has the following characteristics:

[0012] (1) An amino acid sequence as shown in SEQ ID NO:1; or

[0013] (2) An amino acid sequence obtained by substituting, deleting, or adding one or more amino groups to the amino acid sequence shown in (1), and which has the same or similar function as the amino acid sequence shown in (1); or

[0014] (3) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (1) or (2); and / or

[0015] The cytochrome P450 monooxygenase has the following characteristics:

[0016] (4) An amino acid sequence as shown in SEQ ID NO:2; or

[0017] (5) An amino acid sequence obtained by substituting, deleting, or adding one or more amino groups to the amino acid sequence shown in (4), and which has the same or similar function as the amino acid sequence shown in (4); or

[0018] (6) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (4) or (5); and / or

[0019] The cytochrome P450 reductase 2 has the following characteristics:

[0020] (7) An amino acid sequence as shown in SEQ ID NO:3; or

[0021] (8) An amino acid sequence obtained by substituting, deleting, or adding one or more amino groups to the amino acid sequence shown in (7), and which has the same or similar function as the amino acid sequence shown in (7); or

[0022] (9) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (7) or (8).

[0023] In some embodiments of the present invention, in the above enzyme composition, the sequence of SEQ ID NO: 1 is: MSTQQVSSENIVRNAANFHPNIWGNHFLTCPSQTIDSWTQQHHKELKEEVRKMMVSDANKPAQRLRLIDTVQRLGVAYHFEKEIDDALEKIGHDPFDDKDDLYIVSLCFRLLRQHGIKISCDVFEKFKDDDGKFKASLMNDVQGMLSLYEAAHLAIHGEDILDEAIVFTTTHLKSTVSNSPVNSTFAEQIRHSLRVPLRKAVPRLESRYFLDIYSRDDLHDKTLLNFAKLDFNILQAMHQKEASEMTRWWRDFDFLKKLPYIRDRVVELYFWILVGVSYQPKFSTGRIFLSKIICLETLVDDTFDAYGTFDELAIFTEAVTRWDLGHRDALPEYMKFIFKTLIDVYSEAEQELAKEGRSYSIHYAIRSFQELVMKYFCEAKWLNKGYVPSLDDYKSVSLRSIGFLPIAVASFVFMGDIATKEVFEWEMNNPKIIIAAETIVRFLDDIAGHRFEQKREHSPSAIECYKNQHGVSEEEAVKALSLEVANSWKDINEELLLNPMAIPLPLLQVILDLSRSADFMYGNAQDRFTHSTMMKDQVDLVLKDPVKLDD。

[0024] In some embodiments of the present invention, in the above enzyme composition, the sequence of SEQ ID NO:2 is: MSPATAVILTLLVALGLSILLRRRQKRNNLPPGPPALPIIGNIHILGTLPHQSLYNLAKKYGPIMSMRLGLVPAVVISSPEAAELVLKTHDIVFASRPRLQVADYFHYGTKGVILTEYGTYWRNMRRLCTVKLLNTVKIDSFAGTRKKEVASFVQSLKEASVAHKMVNLSARVANVIENMVCLMVIGRSSDERFKLKEVIQEAAQLAGAFNIGDYVPFLMPLDLQGLTRRIKSGSKAFDDILEVIIDEHVQDIKDHDDEQHGDFIDVLLAMMNKPMDSREGLSIIDRTNIKAILVDMIGAAMDTSTSGVEWAISELIKHPRVMKKLQDEVKTVIGMNRMVEEADLPKLPYLDMVVKETMRLHPPGPLLVPRESMEDITINGYYIPKKSRIIVNAWAIGRDTNAWSNNAHEFFPERFMSSNVDLQGQDFQLIPFGSGRRGCPGMRLGLTTVRLVLAQLIHCFDLELPKGTVATDLDMSEKFGLAMPRAQHLLAFPTYRLES。

[0025] In some embodiments of the present invention, the sequence of SEQ ID NO:3 in the above enzyme composition is: *.

[0026] The present invention also provides a nucleic acid molecule encoding the above-described enzyme composition.

[0027] In some embodiments of the present invention, the nucleic acid molecule encoding the santalene synthase in the above-mentioned nucleic acid molecule has:

[0028] (10) The nucleotide sequence shown in SEQ ID NO:4;

[0029] (11) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (10);

[0030] (12) A sequence that has at least 80% homology with the nucleotide sequence shown in (10);

[0031] (13) A complementary sequence to the sequence shown in (10), (11) or (12); and / or

[0032] The nucleic acid molecule encoding the cytochrome P450 monooxygenase has:

[0033] (14) The nucleotide sequence shown in SEQ ID NO:5;

[0034] (15) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (14);

[0035] (16) A sequence that has at least 80% homology with the nucleotide sequence shown in (14);

[0036] (17) A complementary sequence to the sequence shown in (14), (15) or (16); and / or

[0037] The nucleic acid molecule encoding the cytochrome P450 reductase 2 has:

[0038] (18) The nucleotide sequence shown in SEQ ID NO:6;

[0039] (19) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (18);

[0040] (20) A sequence that has at least 80% homology with the nucleotide sequence shown in (18);

[0041] (21) Complementary sequences to sequences shown in (18), (19) or (20).

[0042] The present invention also provides an expression cassette comprising: the above-described nucleic acid molecule and an acceptable expression element.

[0043] In some embodiments of the present invention, the above-mentioned expression box includes: expression box 1 and / or expression box 2;

[0044] The expression cassette 1 includes: a nucleic acid molecule encoding the santalene synthase;

[0045] The expression cassette 2 includes: a nucleic acid molecule encoding the cytochrome P450 monooxygenase, a nucleic acid molecule encoding the cytochrome P450 reductase 2, and a linker peptide (GGGGS) 4.

[0046] In some embodiments of the present invention, the acceptable expression element in the above-described expression box includes: a promoter and a terminator; the promoter includes: P GAP and / or P ADH1The terminator includes: T CYC1 and / or T ADH1 .

[0047] In some embodiments of the present invention, the acceptable expression element in the above-described expression box includes: URA screening label.

[0048] The present invention also provides an expression vector comprising: the above-described nucleic acid molecule and / or the above-described expression cassette.

[0049] The present invention also provides strains for transformation and / or transfection of the above expression vectors.

[0050] In some embodiments of the present invention, the chassis strains of the above-mentioned strains, including the transformed and / or transfected strains, include: Saccharomyces cerevisiae.

[0051] In some embodiments of the present invention, the above-mentioned strain of Saccharomyces cerevisiae includes: SyBE_Sc0125XJ01.

[0052] In some embodiments of the present invention, in the above-mentioned strains, the insertion site of the nucleic acid molecule and / or the expression cassette is the HO site of the Saccharomyces cerevisiae.

[0053] The present invention also provides the use of the above-described enzyme composition, the above-described nucleic acid molecule, the above-described expression cassette, the above-described expression vector, and / or the strains described above in the preparation of santalol.

[0054] The present invention also provides a method for preparing santalol, wherein the santalol is obtained by inoculating and culturing the above-mentioned strains and then fermenting them.

[0055] In some embodiments of the present invention, the fermentation culture in the above preparation method includes: two-phase fermentation; the two-phase fermentation includes the step of adding 10% of the extractant dodecane.

[0056] This invention is based on a laboratory-produced high-FPP Saccharomyces cerevisiae strain and introduces exogenous santalene synthase Sansyn. F441V The gene was expressed using a flexible linker peptide (GGGGS)4 fusion to express cytochrome P450 monooxygenase CYP736A167 and cytochrome P450 reductase 2 (46tATR2) with a 46-amino acid truncated N-terminus. Both expression cassettes were integrated into the HO site of the Saccharomyces cerevisiae genome, resulting in a Saccharomyces cerevisiae strain capable of producing santalol, which provides a strong foundation for subsequent optimization to increase santalol production. Attached Figure Description

[0057] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0058] Figure 1 This illustrates the santalol metabolic pathway based on the MVA pathway.

[0059] Figure 2 A schematic diagram showing the structure of a gene fragment integrated into the HO site of the Saccharomyces cerevisiae genome;

[0060] Figure 3 GC-MS chromatogram showing the two-phase fermentation products of recombinant Saccharomyces cerevisiae;

[0061] Figure 4 This indicates the yield of santalol produced by the fermentation of recombinant brewing yeast. Detailed Implementation

[0062] This invention discloses an enzyme composition, a bacterial strain, and its application in the preparation of santalol.

[0063] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0064] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0065] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0066] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0067] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately." Here, "approximately" generally means an actual value within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0068] This invention provides a method for constructing a brewing yeast strain for producing santalol, comprising the following steps:

[0069] (1) A high-FPP-producing Saccharomyces cerevisiae strain from the laboratory was used as the starting strain.

[0070] (2) Synthesis of santanene synthase with codon optimization from Saccharomyces cerevisiae F441V The gene (Sansyn) synthesizes the Saccharomyces cerevisiae gene, which uses the (GGGGS)4 protein linker peptide to link the cytochrome P450 monooxygenase CYP736A167 and the cytochrome P450 reductase 2 (46tATR2) truncated at the N-terminus of 46 amino acids, namely CYP736A167-(GGGGS)4-46tATR2 (abbreviated as CYP-L-tATR).

[0071] (3) An expression cassette fragment was constructed based on overlap extension PCR technology. Fragment 1: P GAP +Sansyn+T CYC1 Fragment 2: P ADH1 +CYP-L-tATR+T ADH1 ;

[0072] (4) Based on homologous recombination, two expression cassette fragments and URA screening tags were integrated into the HO site of Saccharomyces cerevisiae to construct a recombinant yeast strain that produces santalol.

[0073] (5) Shake flask fermentation: After culturing in YPD medium for 24 h, 10% dodecane extractant was added. After two-phase fermentation for 72 h, the upper extract phase was collected and the product santalol was detected by GC-MS.

[0074] The santanene synthase (Sansyn) used in this invention, optimized with codons from *Saccharomyces cerevisiae*. F441V The genes (GGGGS)4 flexible linker peptide-linked cytochrome P450 monooxygenase CYP736A167 and the N-terminal 46-amino acid truncated cytochrome P450 reductase 2 (46tATR2) genes were both synthesized by Jiutian Gene Technology (Tianjin) Co., Ltd.

[0075] All primers used in this invention were synthesized by Suzhou Genewiz Biotechnology Co., Ltd.

[0076] The high-fidelity DNA polymerase KOD one used in the PCR of this invention was purchased from Toyobo (Shanghai) Biotechnology Co., Ltd.

[0077] In this invention, the substrate strain of the recombinant strain is SyBE_Sc0125XJ01, and its genotype is shown in Table 1.

[0078] The nucleic acid sequence of Sansyn described in this invention is SEQ ID NO.4; the nucleic acid sequence of CYP736A167 is SEQ ID NO.5; the nucleic acid sequence of 46tATR2 is SEQ ID NO.6; and the nucleic acid sequence of linker (GGGGS)4 is SEQ ID NO.7.

[0079] Table 1. Strains used in this study

[0080]

[0081] Table 2 Gene sequences involved in this invention

[0082]

[0083]

[0084]

[0085]

[0086] In Examples 1 to 5 and Comparative Examples 1 to 3 of the present invention, the raw materials and reagents used can all be purchased from the market.

[0087] The present invention will be further illustrated below with reference to the embodiments:

[0088] Example 1: Construction of the santalene synthase expression cassette

[0089] Using the genome of Saccharomyces cerevisiae BY4742 as a template, primers P-GAP-F / P-GAP-R and T-CYC1-F / T-CYC1-R were designed to amplify the promoter P from the genome. GAP and Termination T CYC1 Using sansyn as a template, primers Sansyn-GAP-F / Sansyn-CYC1-R were designed to amplify the 5' end containing the promoter P. GAP A 35bp homologous arm with homology at the 3' end, and the 3' end has a terminator T. CYC1 The 31bp homologous arm fragment at the 5' end was purified to obtain individual fragments. Based on overlap extension PCR, the three fragments were mixed and used as a template for amplification using primers P-GAP-F / T-CYC1-ADH1-R to obtain the santalene synthase expression cassette P. GAP +Sansyn+T CYC1 The fragment, its 3' segment has a connection with promoter P ADH1 The 20bp homologous arm uses the following primer sequences:

[0090] P-GAP-F:TCATTATCAATACTGCCATTTCAAA (as shown in SEQ ID NO:8);

[0091] P-GAP-R:TTTGTTTGTTTATGTGTGTTTATTCGAAACTAAGT (as shown in SEQ ID NO:9);

[0092] T-CYC1-F:TCATGTAATTAGTTATGTCACGCTTACATTCAC (as shown in SEQ ID NO:10);

[0093] T-CYC1-R:GCAAATTAAAGCCTTCGAGCGT (as shown in SEQ ID NO:11);

[0094] Sansyn-GAP-F:ACTTAGTTTCGAATAAACACACATAAACAAACAAAATGTCCACTCAACAAGTTTCTTCTG (as shown in SEQ ID NO: 12);

[0095] Sansyn-CYC1-R:GAATGTAAGCGTGACATAACTAATTACATGACTAATCATCCAATTTAACAGGGTCCTTCA (as shown in SEQ ID NO: 13);

[0096] T-CYC1-ADH1-R:CCCGGAAACAACAAAAAGGATGGCAAATTAAAGCTTCGAG

[0097] (as shown in SEQ ID NO:14).

[0098] Example 2: Construction of the Cytochrome P450 Expression Kit

[0099] Using the genome of Saccharomyces cerevisiae BY4742 as a template, primers P-ADH1-F / P-ADH1-R and T-ADH1-F / T-ADH1-R were designed to amplify the promoter P from the genome. ADH1 and Termination T ADH1 Using the synthesized CYP-L-tATR gene as a template, primers CYP-ADH1-F / tATR-ADH1-R were designed to amplify the 5' end of the gene containing the promoter P. ADH1 A 41bp homologous arm with homology at the 3' end, the 3' end of which carries a terminator T ADH1The 31bp homologous arm fragment at the 5' end was purified to obtain individual fragments. The three fragments were spliced ​​into one fragment by overlap extension PCR, and amplified using primers P-ADH1-F / T-ADH1-R to obtain the cytochrome P450 expression cassette P... ADH1 +CYP-L-tATR+

[0100] T ADH1 Excerpt.

[0101] The primer sequences used are as follows:

[0102] P-ADH1-F:ATCCTTTTGTTGTTTCCGGG (as shown in SEQ ID NO:15);

[0103] P-ADH1-R:AGTTGATTGTATGCTTGGTATAGCTTGAAA (as shown in SEQ ID NO:16);

[0104] T-ADH1-F:GCGAATTTCTTATGATTTATGATTTTT (as shown in SEQ ID NO:17);

[0105] T-ADH1-R:CCGGTAGAGGTGTGGTCAATAAG (as shown in SEQ ID NO:18);

[0106] CYP-ADH1-F:TCTGCACAATATTTCAAGCTATACCAAGCATACAATCAACTATGTCCCCAGCTACTGCTG (as shown in SEQ ID NO:19);

[0107] tATR-ADH1-R: TAATAAAAATCATAAATCATAAGAAATTCGCTCACCAAACATCTCTCAAGTATCTACCGG (as shown in SEQ ID NO: 20).

[0108] Example 3 Construction of the Santal Synthesis Gene Expression Kit

[0109] Using the santalene synthase expression cassette and cytochrome P450 expression cassette fragments constructed in Examples 1 and 2 as templates, the two fragments were spliced ​​together using overlap extension PCR, and amplified using primers P-GAP-F / T-ADH1-R to obtain expression cassette P. GAP +Sansyn+T CYC1 +P ADH1 +CYP-L-tATR+T ADH1Fragment (hereinafter referred to as BOX1 fragment). Example 4: Integration of the HO site into the Saccharomyces cerevisiae genome and verification of recombinant strains.

[0110] Using the genome of high-FPP Saccharomyces cerevisiae SyBE_Sc0125XJ01 as a template, a 500bp upstream homologous region fragment (HOR fragment) was amplified using primers HOR-F / HOR-R; a 500bp downstream homologous region fragment (HOL fragment) was amplified using primers HOL-TADH1-F / HOL-R, which has a 20bp homologous arm at its 5' end that is homologous to the 3' end of the BOX1 fragment.

[0111] Using pRS426 plasmid as a template, the URA selection tag fragment P was amplified using primers P-URA3-F / URA3-R. URA3 +URA3 has a 25bp homologous arm at its 5' end that is homologous to the 3' end of the HOR fragment.

[0112] The purified HOR fragment and the URA selection tag fragment were spliced ​​together using overlap extension PCR to form a single DNA fragment. This fragment was then amplified using primers HOR-F / URA3-GAP-R to obtain the HOR-URA3 fragment, which has a 32bp homologous arm at its 3' end, identical to the 5' end of the BOX1 fragment. The purified BOX1 fragment was then mixed with the HOL fragment as a template and amplified using primers P-GAP-F / HOR-R to obtain the BOX1-HOL fragment.

[0113] The HOR-URA3 and BOX1-HOL fragments were transformed into *Saccharomyces cerevisiae* XJ01 substrate culture using the lithium acetate method. After transformation, the transformants were screened using SC-URA solid plates (synthetic yeast nitrogen source YNB 6.7 g / L, glucose 20 g / L, a mixed amino acid powder lacking tryptophan, leucine, histidine, and uracil 2 g / L, 2% agar powder; tryptophan, leucine, and histidine were replenished in 100% stock solution). The selected transformants were verified by PCR using JP-CYP-F / R primers, and the correctly amplified fragment length was 517 bp. Single colonies with correct verification were isolated and purified, and the genome was extracted and sent to the company for sequencing of the BOX1 fragment. The successfully sequenced recombinant strain (Sy-txc-1) was stored for later use, with the product santalol to be detected during fermentation.

[0114] HOR-F:GCTGTAAAAAGTATGGCTTGGATG (as shown in SEQ ID NO:21);

[0115] HOR-R:GATCTCTTGGTATGTCAGCTACTGT (as shown in SEQ ID NO:22);

[0116] HOL-TADH1-F:ATTGACCACACCTCTACCGGAGGAAATCATTGAAGGAAG (as shown in SEQ ID NO:23);

[0117] HOL-R:CGTAATGCCCAATTTTTCGCC (as shown in SEQ ID NO:24);

[0118] P-URA3-F:GATCTCTTGGTATGTCAGCTACTGTAGAGTGCACCATACCACAGCT T (as shown in SEQ ID NO:25);

[0119] URA3-R: TTAGTTTTGCTGGCCGCATC (as shown in SEQ ID NO:26);

[0120] URA3-GAP-R:GATGCGGCCAGCAAAACTAATCATTATCAATACTGCCATTTCA AAGAATACG (as shown in SEQ ID NO:27);

[0121] JP-CYP-F:ATTTCTTCTCCAGAAGCTGCTG (as shown in SEQ ID NO:28);

[0122] JP-CYP-R:GTTCGTCGATGATGACTTCC (as shown in SEQ ID NO:29).

[0123] Example 5: Detection of Santal Fermentation by Recombinant Saccharomyces cerevisiae Strain

[0124] Experimental methods:

[0125] Seed culture medium: SC-URA liquid medium (synthetic yeast nitrogen source YNB 6.7g / L, glucose 20g / L, mixed amino acid powder lacking tryptophan, leucine, histidine and uracil 2g / L, tryptophan, leucine and histidine were replenished in the form of 100* stock solution);

[0126] Fermentation medium: YPD liquid medium (yeast extract 10g / L, peptone 20g / L, glucose 20g / L).

[0127] The strain obtained in Example 4 was inoculated into 4 mL of seed culture medium and cultured at 30°C and 220 rpm for 14-16 h, with the initial cell concentration OD... 600=0.1 was inoculated into 100 mL of fermentation medium and cultured at 30 °C and 220 rpm for 12 h. Then, 5 mL of 200 g / L galactose (inducer) and 10 mL of dodecane (extraction agent) were added. After two-phase fermentation for 72 h, samples were taken to detect the OD of the culture medium. 600 Data was collected, including the cell concentration at the end of fermentation, and the upper organic phase was used to detect the products.

[0128] Quantitative detection method for santalol: Take 100 mL of fermentation broth at the end of fermentation, centrifuge at 5000 rpm to separate the cells, collect the upper organic phase, and filter 1 mL through a 0.22 μm organic filter membrane into a GC-MS vial. Detection method: HP-5MS capillary column (non-polar, 30 m × 0.25 mm × 0.25 μm); injection port temperature: 250 ℃; carrier gas: high-purity helium (purity > 99.999%), flow rate: 0.8 mL / min; injection method: split injection, injection volume: 1, split ratio: 30:1; temperature program: initial temperature 40 ℃, hold for 3 min, then increase to 130 ℃ at 10 ℃ / min, then increase to 180 ℃ at 2 ℃ / min, and finally increase to 300 ℃ at 50 ℃ / min and hold for 10 min; mass spectrometry conditions: EI. + Ionization; Ionization voltage: 70 eV; Scan range: 40-400 amu; SIM: m / z 93, 94, 105, 107, 119, 122, 202 [dwell time 50 msec]; Ion source temperature: 200 °C; Transfer line temperature: 300 °C; Spectral search: searched using WILEY and NIST08 libraries.

[0129] Comparative Example 1 constructed a strain with the GAL1 promoter replacing the GAP promoter, Sy-txc-2.

[0130] 1. Construction of the santalene synthase expression cassette

[0131] Using the genome of Saccharomyces cerevisiae BY4742 as a template, primers P-GAL1-F / P-GAL1-R and T-CYC1-F / T-CYC1-R were designed to amplify the promoter P from the genome. GAL1 and Termination T CYC1 Using sansyn as a template, primers Sansyn-GAL1-F / Sansyn-CYC1-R were designed to amplify the 5' end containing the promoter P. GAL1 A 25bp homologous arm with homology at the 3' end, the 3' end bearing a terminator T CYC1 The 31bp homologous arm fragment at the 5' end was purified to obtain individual fragments. Based on overlap extension PCR, the three fragments were mixed and used as a template for amplification using primers P-GAL1-F / T-CYC1-ADH1-R to obtain the santalene synthase expression cassette P. GAL1+Sansyn+T CYC1 The fragment, its 3' segment has a connection with promoter P ADH1 20 bp homologous arm, the primer sequences used are as follows:

[0132] P-GAL1-F: TGGAACTTTCAGTAATACGCTTAAC (as shown in SEQ ID NO:30);

[0133] P-GAL1-R: ATAGTTTTTTCTCCTTGACGTTAAAG (as shown in SEQ ID NO:31);

[0134] T-CYC1-F:TCATGTAATTAGTTATGTCACGCTTACATTCAC (as shown in SEQ ID NO:10);

[0135] T-CYC1-R:GCAAATTAAAGCCTTCGAGCGT (as shown in SEQ ID NO:11);

[0136] SANSYN-GAL1-F:TTTAACGTCAAGGAGAAAAAAACTATATGTCCACTCAACAAGTTTCTTC (as shown in SEQ ID NO: 32);

[0137] Sansyn-CYC1-R:GAATGTAAGCGTGACATAACTAATTACATGACTAATCATCCAATTTAACAGGGTCCTTCA (as shown in SEQ ID NO: 13);

[0138] T-CYC1-ADH1-R:CCCGGAAACAACAAAAGGATGGCAAATTAAAGCTTCGAG (as shown in SEQ ID NO:14).

[0139] 2. The construction of the cytochrome P450 expression cassette is the same as in Example 2.

[0140] 3. Construction of the santalol synthesis gene expression cassette

[0141] Using the santalene synthase expression cassette and cytochrome P450 expression cassette fragments constructed in steps 1 and 2 as templates, the two fragments were spliced ​​together using overlap extension PCR, and amplified using primers P-GAL1-F / T-ADH1-R to obtain the expression cassette P. GAL1 +Sansyn+T CYC1 +P ADH1 +CYP-L-tATR+T ADH1 The clip (hereinafter referred to as the BOX2 clip).

[0142] 4. Integration of HO sites into the Saccharomyces cerevisiae genome

[0143] The integration method is the same as in Example 4, except that: the purified HOR fragment and the URA selection tag fragment are spliced ​​together using overlap extension PCR to form a single DNA fragment, which is then amplified using primers HOR-F / URA3-GAL1-R to obtain the HOR-URA3 fragment, which has a 25bp homologous arm at its 3' end that is homologous to the 5' end of the BOX2 fragment. The purified BOX2 fragment and the HOL fragment are mixed as a template and amplified using primers P-GAL1-F / HOR-R to obtain the BOX2-HOL fragment.

[0144] URA3-GAL1-R:GTTAACGGTATTACTGAAAGTTCCATTAGTTTTGCTGGCCGC

[0145] (as shown in SEQ ID NO:33).

[0146] Comparative Example 2 constructed a strain with the GAL1 promoter replacing both the GAP and ADH1 promoters, Sy-txc-3.

[0147] 1. Construction of the santalene synthase expression cassette

[0148] 1) Using the genome of Saccharomyces cerevisiae BY4742 as a template, primers P-GAL1-F / P-GAL1-R and T-CYC1-F / T-CYC1-R were designed to amplify the promoter P from the genome. GAL1 and Termination T CYC1 Using sansyn as a template, primers Sansyn-GAL1-F / Sansyn-CYC1-R were designed to amplify the 5' end containing the promoter P. GAL1 A 25bp homologous arm with homology at the 3' end, the 3' end bearing a terminator T CYC1 The 31bp homologous arm fragment at the 5' end was purified to obtain individual fragments. Based on overlap extension PCR, the three fragments were mixed and used as a template for amplification using primers P-GAL1-F / T-CYC1-GAL1-R to obtain the santalene synthase expression cassette P. GAL1 +Sansyn+T CYC1 The fragment, its 3' segment has a connection with promoter P GAL1 24 bp homologous arm, the primer sequences used are as follows:

[0149] P-GAL1-F: TGGAACTTTCAGTAATACGCTTAAC (as shown in SEQ ID NO:30);

[0150] P-GAL1-R: ATAGTTTTTTCTCCTTGACGTTAAAG (as shown in SEQ ID NO:31);

[0151] T-CYC1-F:TCATGTAATTAGTTATGTCACGCTTACATTCAC (as shown in SEQ ID NO:10);

[0152] T-CYC1-R:GCAAATTAAAGCCTTCGAGCGT (as shown in SEQ ID NO:11);

[0153] SANSYN-GAL1-F:TTTAACGTCAAGGAGAAAAAAACTATATGTCCACTCAACAAGTTTCTTC (as shown in SEQ ID NO: 32);

[0154] Sansyn-CYC1-R:GAATGTAAGCGTGACATAACTAATTACATGACTAATCATCCAATTTAACAGGGTCCTTCA (as shown in SEQ ID NO: 13);

[0155] T-CYC1-GAL1-R:TTAAGCGTATTACTGAAAGTTCCAGCAAATTAAAGCTTCGA GCG (as shown in SEQ ID NO: 34).

[0156] 2) Using the genome of Saccharomyces cerevisiae BY4742 as a template, primers P-GAL1-F / P-GAL1-R and T-ADH1-F / T-ADH1-R were designed to amplify the promoter P from the genome. GAL1 and Termination T ADH1 Using the synthesized CYP-L-tATR gene as a template, primers CYP-GAL1-F / tATR-ADH1-R were designed to amplify the 5' end of the gene containing the promoter P. GAL1 A 25bp homologous arm with homology at the 3' end, the 3' end bearing a terminator T ADH1 The 31bp homologous arm fragment at the 5' end was purified to obtain individual fragments. The three fragments were spliced ​​into one fragment by overlap extension PCR, and amplified using primers P-GAL1-F / T-ADH1-R to obtain the cytochrome P450 expression cassette P. GAL1 +CYP-L-tATR+T ADH1 Excerpt.

[0157] The primer sequences used are as follows:

[0158] P-GAL1-F: TGGAACTTTCAGTAATACGCTTAAC (as shown in SEQ ID NO:30);

[0159] P-GAL1-R: ATAGTTTTTTCTCCTTGACGTTAAAG (as shown in SEQ ID NO:31); T-ADH1-F: GCGAATTTCTTATGATTTATGATTTTT (as shown in SEQ ID NO:17);

[0160] T-ADH1-R:CCGGTAGAGGTGTGGTCAATAAG (as shown in SEQ ID NO:18);

[0161] CYP-GAL1-F:TTTAACGTCAAGGAGAAAAAACTATATGTCCCCAGCTACTGC (as shown in SEQ ID NO:35);

[0162] tATR-ADH1-R: TAATAAAAATCATAAATCATAAGAAATTCGCTCACCAAACATCTCTCAAGTATCTACCGG (as shown in SEQ ID NO: 20).

[0163] 2. The construction of the cytochrome P450 expression cassette is the same as in Example 2.

[0164] 3. Construction of the santalol synthesis gene expression cassette

[0165] Using the santalene synthase expression cassette and cytochrome P450 expression cassette fragments constructed in steps 1 and 2 as templates, the two fragments were spliced ​​together using overlap extension PCR, and amplified using primers P-GAL1-F / T-ADH1-R to obtain the expression cassette P. GAL1 +Sansyn+T CYC1 +P GAL1 +CYP-L-tATR+T ADH1 The clip (hereinafter referred to as the BOX3 clip).

[0166] 4. Integration of HO sites into the Saccharomyces cerevisiae genome

[0167] The integration method is the same as in Example 4, except that: the purified HOR fragment and the URA selection tag fragment are spliced ​​together using overlap extension PCR to form a single DNA fragment, which is then amplified using primers HOR-F / URA3-GAL1-R to obtain the HOR-URA3 fragment, which has a 25bp homologous arm at its 3' end that is homologous to the 5' end of the BOX3 fragment. The purified BOX3 fragment is then mixed with the HOL fragment as a template and amplified using primers P-GAL1-F / HOR-R to obtain the BOX3-HOL fragment.

[0168] URA3-GAL1-R:GTTAAGCGTATTACTGAAAGTTCCATTAGTTTTGCTGGCCGC (as shown in SEQ ID NO:36).

[0169] The effect of adding an inducing agent on the experiment in Comparative Example 3

[0170] In Example 5, the difference lies in whether or not the inducer galactose is added. The rest of the procedures are the same.

[0171] The results are as follows:

[0172] Sy-txc-1:

[0173] The three parallel groups with added galactose had final fermentation OD values ​​of 31.10, 31.14, and 30.64, corresponding to α-santalol concentrations of 35.36, 40.95, and 37.65, respectively.

[0174] The three parallel groups without galactose had final fermentation OD values ​​of 20.86, 21.26, and 20.95, corresponding to α-santalol concentrations of 10.12, 11.32, and 9.98, respectively.

[0175] Sy-txc-2:

[0176] The three parallel groups with added galactose had final fermentation OD values ​​of 31.52, 31.62, and 30.41, corresponding to α-santalol concentrations of 37.05, 36.70, and 35.67, respectively.

[0177] The three replicates in the group without galactose showed final fermentation OD values ​​of 24.82, 24.74, and 24.94, corresponding to α-santalol concentrations of 0.952, 1.044, and 0.897, respectively.

[0178] Sy-txc-3:

[0179] The three parallel groups with added galactose had final fermentation OD values ​​of 34.81, 34.75, and 34.62, corresponding to α-santalol concentrations of 33.64, 32.45, and 32.31, respectively.

[0180] In the three parallel groups without galactose, the final OD values ​​of fermentation were 26.95, 27.51, and 26.35, corresponding to α-santalol concentrations of 0.78, 1.03, and 0.76, respectively.

[0181] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An enzyme composition, characterized in that, include: Santalene synthase, cytochrome P450 monooxygenase, and cytochrome P450 reductase 2.

2. The enzyme composition according to claim 1, characterized in that, The santalene synthase has the following characteristics: (1) An amino acid sequence as shown in SEQ ID NO:1; or (2) An amino acid sequence obtained by substituting, deleting, or adding one or more amino groups to the amino acid sequence shown in (1), and which has the same or similar function as the amino acid sequence shown in (1); or (3) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (1) or (2); and / or The cytochrome P450 monooxygenase has the following characteristics: (4) An amino acid sequence as shown in SEQ ID NO:2; or (5) An amino acid sequence obtained by substituting, deleting, or adding one or more amino groups to the amino acid sequence shown in (4), and which has the same or similar function as the amino acid sequence shown in (4); or (6) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (4) or (5); and / or The cytochrome P450 reductase 2 has the following characteristics: (7) An amino acid sequence as shown in SEQ ID NO:3; or (8) An amino acid sequence obtained by substituting, deleting, or adding one or more amino groups to the amino acid sequence shown in (7), and which has the same or similar function as the amino acid sequence shown in (7); or (9) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (7) or (8).

3. A nucleic acid molecule encoding the enzyme composition as described in claim 1 or 2.

4. The nucleic acid molecule as described in claim 3, characterized in that, The nucleic acid molecule encoding the santalene synthase has: (10) The nucleotide sequence shown in SEQ ID NO:4; (11) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (10); (12) A sequence that has at least 80% homology with the nucleotide sequence shown in (10); (13) A complementary sequence to the sequence shown in (10), (11) or (12); and / or The nucleic acid molecule encoding the cytochrome P450 monooxygenase has: (14) The nucleotide sequence shown in SEQ ID NO:5; (15) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (14); (16) A sequence that has at least 80% homology with the nucleotide sequence shown in (14); (17) A complementary sequence to the sequence shown in (14), (15) or (16); and / or The nucleic acid molecule encoding the cytochrome P450 reductase 2 has: (18) The nucleotide sequence shown in SEQ ID NO:6; (19) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (18); (20) A sequence that has at least 80% homology with the nucleotide sequence shown in (18); (21) Complementary sequences to sequences shown in (18), (19) or (20).

5. An expression box, characterized in that, include: The nucleic acid molecule as described in claim 3 or 4, and the acceptable expression element.

6. An expression carrier, characterized in that, include: The nucleic acid molecule as described in claim 3 or 4 and / or the expression cassette as described in claim 5.

7. A bacterial strain, characterized in that, Transformation and / or transfection of the expression vector as described in claim 6.

8. The strain according to claim 7, characterized in that, The chassis strains used for transformation and / or transfection include: Saccharomyces cerevisiae.

9. The use of the enzyme composition of claim 1 or 2, the nucleic acid molecule of claim 3 or 4, the expression cassette of claim 5, the expression vector of claim 6, and / or the strain of claim 7 or 8 in the preparation of santalol.

10. A method for preparing santalol, characterized in that, The santalol is obtained by inoculating and fermenting the strain as described in claim 7 or 8.

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