Alpha-farnesene synthase aoFS from aliciae fructus and application thereof

Abstract

The application discloses an alpha-farnesene synthase AoFS from Zizania and application thereof, and belongs to the field of terpene biosynthesis. The amino acid sequence of the alpha-farnesene synthase AoFS is shown as SEQ ID NO. 2, which can synthesize a main product alpha-farnesene by catalyzing a farnesyl pyrophosphate (FPP) substrate. It is identified by GC-MS that the fermentation product contains a sesquiterpene compound alpha-farnesene, and the yield shows that the catalytic effect of the alpha-farnesene synthase is better. Therefore, the application can realize the synthesis of alpha-farnesene in microbial cells, effectively reduce the production cost, be favorable to the high-efficiency production of alpha-farnesene, provide technical support for the heterologous preparation of the terpene compound alpha-farnesene by using a synthetic biology method, and is favorable to the selection of the best alpha-farnesene synthase in different chassis strains by researchers so as to produce alpha-farnesene in a high yield.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of terpene biosynthesis, and particularly relates to a alpha-farnesene synthase AoFS from Alpinia oxyphylla and application thereof. BACKGROUND

[0002] Alpinia oxyphylla Miq. is a kind of herbaceous plant of Zingiberaceae Alpinia. The essential oil extracted from different parts of Alpinia oxyphylla fruits is an important ingredient containing various natural compounds and having various medicinal values. So far, the main compounds found in Alpinia oxyphylla include terpenes, phenols and sterols, among which terpenes are important ingredients isolated and identified from the fruits of Alpinia oxyphylla.

[0003] Terpenes are one of the most numerous and most diverse secondary metabolites in plants, and the basic building block is isoprene (C5H8). According to the number of isoprene, terpenes are divided into hemiterpenes (C5), monoterpene (C10), sesquiterpenes (C15), diterpenes (C20), dipterpene (C25), triterpenes (C30), tetraterpenes (C40) and polyterpenes. It has been found through research that terpenes have a wide range of applications in actual production and are developed into natural spices, fuels, food additives, therapeutic agents, metabolic regulators and insecticides. In addition, some terpenes have been clinically proven to have neuroprotective, memory-enhancing, antibacterial and anti-inflammatory, respiratory function-improving, heart-stimulating and sedative and hypnotic effects.

[0004] Farnesene is a non-cyclic sesquiterpene compound, which is initially identified in large quantities in apple skins and is confirmed to be involved in the defense mechanism of plants and responsible for producing characteristic odors. It is later found that farnesene, as an important component of plant essential oils, has properties such as antiseptic, anticancer, neuroprotective and antioxidant. Farnesene is also an important precursor of a variety of molecules and has quite extensive market applications.

[0005] Farnesene synthase is a key enzyme in the farnesene synthesis pathway, which can catalyze the substrate farnesyl pyrophosphate (FPP) to generate farnesene. In order to meet the demand of multiple market applications of farnesene, microbial biosynthesis has significant advantages compared with chemical synthesis and plant extraction methods. However, the microbial biosynthesis method requires the introduction of a highly efficient heterologous farnesene synthase. At present, there is less research on farnesene synthase, and research shows that the catalytic effect of alpha-farnesene synthase from different sources varies with different chassis strains. Therefore, it is beneficial for researchers to select the best alpha-farnesene synthase in different chassis strains to produce alpha-farnesene by excavating more alpha-farnesene synthases. SUMMARY

[0006] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of the present application is to provide a alpha-farnesene synthase AoFS from Alpinia oxyphylla and application thereof.

[0007] The application provides an alpha-farnesene synthase AoFS from a Schisandra source, the AoFS comprises a conservative sequence DDxxD rich in aspartic acid specific to terpene synthase, and the sequence similarity of the AoFS and MdAFS (derived from Malus domestica) and Fsso (derived from Glycine max) is found to be 26.92% and 24.68% respectively through BLAST amino acid sequence alignment (). Figure 3 ), wherein the amino acid sequence of the MdAFS is shown as Q84LB2.2, and the amino acid sequence of the Fsso is shown as NP_001340264.1. The application performs functional characterization on the alpha-farnesene synthase AoFS, and provides application of the AoFS in synthesis of farnesene in Saccharomyces cerevisiae.

[0008] The object of the application is achieved by the following technical scheme:

[0009] An alpha-farnesene synthase AoFS from a Schisandra source, wherein the amino acid sequence is shown as SEQ ID NO. 2.

[0010] A coding gene of the alpha-farnesene synthase AoFS, preferably, the nucleotide sequence is shown as SEQ ID NO. 1.

[0011] The gene can endow the Schisandra pericarp sesquiterpene alpha-farnesene component. The gene sequence comprises a conservative sequence DDxxD rich in aspartic acid specific to terpene synthase, belongs to plant terpene synthase, and has the highest similarity to monoterpenes synthase S-linalool synthase (Q96376.1) derived from the dicotyledon Clarkia breweri in the database, and the amino acid sequence similarity is 41.02%.

[0012] The biological material related to the above alpha-farnesene synthase AoFS is any one or a combination of the following biological materials:

[0013] (a) an expression cassette containing the coding gene;

[0014] (b) a recombinant expression vector containing the coding gene;

[0015] (c) a recombinant expression vector containing the expression cassette in (a);

[0016] (d) a recombinant microorganism containing the coding gene;

[0017] (e) a recombinant microorganism containing the expression cassette in (a);

[0018] (f) Recombinant microorganisms containing the recombinant expression vector described in (b) or (c).

[0019] Furthermore, the expression box described in (a) also includes a promoter and a terminator; the promoter is promoter CCW12 (CCW12 p ), etc., the terminator is terminator CYC1 (CYC1 t )wait.

[0020] Furthermore, the starting vector for the recombinant expression vector described in (b) and (c) is a YEp-type vector, etc.; preferably, it is a YEp352 vector.

[0021] Furthermore, the host microorganisms corresponding to the recombinant microorganisms mentioned in (d), (e), and (f) are selected from prokaryotes or yeasts, etc.; the prokaryotes include bacteria such as Escherichia; the yeasts include yeasts such as Saccharomyces cerevisiae. More specifically, the prokaryote is Escherichia coli (E. coli), specifically Escherichia coli BL21(DE3); the yeast is Saccharomyces cerevisiae CEN.PK2-1Ca strain.

[0022] Preferably, a method for constructing a recombinant Saccharomyces cerevisiae strain includes the following steps: cloning the gene encoding α-farnesene synthase AoFS, whose amino acid sequence is shown in SEQ ID No. 2, into an expression vector to obtain a recombinant expression vector; transforming the recombinant expression vector into host cells of Saccharomyces cerevisiae to construct a recombinant Saccharomyces cerevisiae strain.

[0023] Preferably, the expression vector is the YEp352 vector; and the host cell, *Saccharomyces cerevisiae*, is *Saccharomyces cerevisiae* strain CEN.PK2-1Ca.

[0024] The application of the above-mentioned α-farnesene synthase AoFS, encoding genes, or biomaterials related to α-farnesene synthase AoFS in the preparation of α-farnesene synthase AoFS.

[0025] The application of the above-mentioned α-farnesene synthase AoFS, encoding genes, or biomaterials related to α-farnesene synthase AoFS in the preparation of α-farnesene.

[0026] Furthermore, the application of the aforementioned α-farnesene synthase AoFS, its encoding gene, or biomaterials related to α-farnesene synthase AoFS in the biosynthesis of α-farnesene.

[0027] The application is as follows: using farnesyl pyrophosphate (FPP) as a substrate, α-farnesene is produced by catalysis of α-farnesene synthase AoFS.

[0028] Furthermore, the fermentation medium for producing α-farnesene was SD-ΔUra medium: 20 g / L glucose, 6.7 g / L YNB, 0.62 g / L DO supplement (Δura-Δtrp-Δleu), 0.06 g / L leucine, 0.04 g / L tryptophan, with 20% (v / v) n-dodecane added for biphasic fermentation. The fermentation conditions were: fermentation time 48 h, fermentation temperature 30 °C. The fermentation products were detected by gas chromatography, and unknown fermentation products were further identified using GC-MS.

[0029] The present invention has the following advantages and effects compared with the prior art:

[0030] This invention identifies and functionally characterizes a previously unreported α-farnesene synthase, AoFS, derived from Alpinia oxyphylla. A recombinant expression vector containing this gene was constructed, and heterologous expression was performed in a Saccharomyces cerevisiae strain. The synthesized α-farnesene, the main product, by catalyzing the farnesyl pyrophosphate (FPP) substrate. GC-MS confirmed the presence of the sesquiterpene compound α-farnesene in the fermentation product, and the yield indicated that the α-farnesene synthase exhibited excellent catalytic activity. Therefore, this invention enables the synthesis of α-farnesene in microbial cells, effectively reducing production costs and facilitating the efficient production of α-farnesene. It provides technical support for the heterologous preparation of the terpene compound α-farnesene using synthetic biology methods. Furthermore, the discovery of this novel α-farnesene synthase allows researchers to select the optimal α-farnesene synthase from different substrate strains to achieve high-yield α-farnesene production. Attached Figure Description

[0031] Figure 1 This is an agarose gel electrophoresis image of RNA from the peel of Alpinia oxyphylla.

[0032] Figure 2 This invention illustrates the expression specificity of the AoFS gene in different tissues of Alpinia oxyphylla; S: seed; P: pericarp.

[0033] Figure 3 The amino acid sequence alignment results of AoFS, MdAFS, and Fsso in this invention are shown.

[0034] Figure 4 This is a product containing the AoFS gene obtained by PCR amplification of cDNA from the peel of Alpinia oxyphylla.

[0035] Figure 5 The image shows the recombinant expression plasmid YEp352-AoFS of Saccharomyces cerevisiae in this invention.

[0036] Figure 6The results of GC-MS identification of fermentation products heterologously expressed in Saccharomyces cerevisiae are shown; where A: gas chromatogram of fermentation products of PL00-AoFS; B: mass spectrometry identification results of fermentation products of PL00-AoFS. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0038] Unless otherwise specified, the test methods in the following examples are generally performed under standard experimental conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used are commercially available.

[0039] Example 1: Obtaining the full-length cDNA of the AoFS gene

[0040] S1. Extraction of RNA from Alpinia oxyphylla peel: Fresh Alpinia oxyphylla fruits were harvested and the peel and seeds were separated as soon as possible. After being flash-frozen in liquid nitrogen, the peels were stored at -80℃. Analysis showed that the relative expression level of the AoFS gene in the peel was higher than that in the seeds. Figure 2 Subsequently, the peel of Alpinia oxyphylla was used as the material for RNA extraction. The pipette tips and Eppendorf tubes used for RNA extraction were soaked in 0.1% DEPC overnight at 37°C, then sterilized at 121°C for 25 min. Glassware and mortars were wrapped in aluminum foil and subjected to dry heat treatment at 180°C for 3 h, then cooled before use. Total RNA was extracted from the Alpinia oxyphylla peel using the Novizan RNA extraction kit. RNA integrity was detected by 1% agarose gel electrophoresis. Figure 1 The concentration and purity of the extracted RNA were determined using a multi-functional microplate reader. It was stored at -80℃ for later use. The extracted RNA has three uses: 1. for transcriptome sequencing analysis; 2. for q-PCR experiments; 3. for amplifying the target gene after reverse transcription.

[0041] S2. Using total RNA from the peel of Alpinia oxyphylla as a template, first-strand cDNA was synthesized using the Novizan HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper). The reaction conditions and system are as follows: 1.

[0043] Reagent Amount Total RNA 10 pg - 5 μg RNase-free ddH2O Up to 8 μL

[0044] Heat at 65℃ for 5 minutes, then quickly place on ice to cool rapidly, and let stand on ice for 2 minutes. 2.

[0046] Reagent Amount Mix from previous step 8 μL 5 x gDNA wiper mix 2 μL

[0047] Incubate at 42℃ for 5 minutes. 3.

[0049]

[0050] Using the reverse transcribed cDNA as a PCR template, and based on the annotated gene sequences related to the *Illicium edulis* transcriptome data, primers were designed, and the sequences are as follows:

[0051] Upstream primer F1: 5'-ATGTTCCTTGAAGCACTC-3';

[0052] Downstream primer R1: 5'-CACGGACATTCTTGATAAGGT-3';

[0053] The upstream and downstream primers each contain sequences at their 5' ends identical to those at the ends of the linearized vector. Therefore, the primers used to amplify the target gene during the construction of the recombinant expression vector are as follows (underlined sequences indicate bases homologous to the vector to be ligated):

[0054] Upstream primer F2: 5'- AACACTATATCAATATCTA ATGTTCCTTGAAGCACTC-3';

[0055] Downstream primer R2: 5'- ACTAATTACATGATGCGGC CACGGACATTCTTGATAAGGT-3';

[0056] The synthesized cDNA was then sent to Shanghai Bioengineering Co., Ltd. Using the synthesized cDNA as a template, PCR amplification was performed using the TaKaRa PCR Amplification Kit. The specific reaction conditions and system are as follows:

[0057]

[0058] After the PCR reaction was completed, a preliminary detection of whether the PCR product contained the target fragment band was performed using 1.0% agarose gel electrophoresis. The results are as follows: Figure 4 As shown. After confirming the band size was correct, the PCR product was recovered using the HiPure PCR Pure Mini Kit (Magen), and then its concentration and purity were determined using a multi-functional microplate reader.

[0059] The nucleotide sequence of the AoFS gene is shown in SEQ ID NO.1, from 1 to 1761 bp. The sequence from 1762 to 1798 bp in SEQ ID NO.1 is a partial sequence of the 3'-UTR of the AoFS gene, and the encoded amino acid sequence is shown in SEQ ID NO.2.

[0060] Example 2: Construction of recombinant expression plasmid YEp352-AoFS

[0061] First, the template plasmid YEp352-CCW12 p -CYC1 t (The shuttle plasmid, 2μ, URA, Addgene, USA, contains promoter CCW12 and terminator CYC1) was linearized by PCR amplification to obtain a linearized vector; the PCR primer sequences used are as follows:

[0062] Upstream primer F3: 5'-GCCGCATCATGTAATTAGTT-3';

[0063] Downstream primer R3: 5'-TAGATATTGATATAGTGTTTAAGCG-3';

[0064] The PCR product (AoFS gene) with vector end sequences obtained in Example 1 above was mixed with the linearized vector in a certain proportion and reacted at 37°C for 30 min under the catalysis of recombinase Exnase II.

[0065] The reaction conditions and system are as follows:

[0066] Reagent Amount Exnase II 1 μL 5 x Buffer CE II 2 μL vector (Large fragment length x 0.01) / large fragment concentration segment (Small fragment length x 0.02) / small fragment concentration ddH2O Up to 10 μL

[0067] After cooling 10 μL of the recombinant product on ice, it was directly transformed into *E. coli* DH5α competent cells. The mixture was gently tapped against the tube wall to mix, and incubated on ice for 30 min. After heat shock at 42°C for 90 s, it was immediately cooled on ice for 2-3 min. 800 μL of LB medium (without antibiotics) was added, and the cells were shaken at 37°C for 1 h (220 rpm). The cells were centrifuged at 5,000 rpm for 3 min, and 650 μL of supernatant was discarded. The cells were resuspended in the remaining medium and spread on LB / c / Amp medium using a sterile spreader. + Spread the mixture evenly on a plate, then incubate upside down at 37°C for 12-16 hours. Pick single colonies and plate them on LB / Amp. + Streaking is performed on plates and incubation is carried out for 5–6 hours. Single colonies are then screened for positive results using colony PCR. In a clean bench, 3–5 single colonies are picked and dissolved in 20 μL of ddH₂O. The colonies are then lysed at 95°C for 10 min, centrifuged at 3000 rpm for 2 min, and the resulting supernatant can be used as the template for colony PCR. The reaction system and conditions for colony PCR are as follows:

[0068]

[0069] The primers for colony PCR are shown below:

[0070] Upstream primer F4: 5'-GAACTGTGTGTTAGCTGAAC-3';

[0071] Downstream primer R4: 5'-AgggTTTTCCCAgTCACg-3';

[0072] For colonies that test positive by colony PCR, select the strain corresponding to the band size and inoculate it into LB / Amp. + The culture was carried out overnight at 37°C and 220 rpm in liquid medium. The plasmid was extracted using the HiPure Plasmid Micro Kit (Magen) to obtain the recombinant expression plasmid YEp352-CCW12. p -AoFS-CYC1 t (Abbreviated as YEp352-AoFS) (Plasmid map can be found) Figure 5 Among them, the expression box CCW12 p -AoFS-CYC1 t Inserted between the SacI and HindIII restriction sites in the YEp352 plasmid.

[0073] Simultaneously, a blank vector plasmid YEp352-CCW12 without the AoFS gene was constructed. p -CYC1 t (referred to as YEp352-control) served as a negative control.

[0074] Example 3: Construction of recombinant Saccharomyces cerevisiae strain and identification of fermentation products

[0075] S1. Preparation of competent cells of Saccharomyces cerevisiae PL00

[0076] Activate the PL00 strain preserved at -80℃ using YPD streak plating. Pick a single colony and inoculate it into 5 mL of YPD liquid medium. Incubate at 30℃ and 200 rpm for 12–16 h until OD is reached. 600 Once the OD value reaches between 3.0 and 5.0, take a certain amount of bacterial culture and transfer it to a new 5 mL YPD liquid medium to control the initial OD. 600 Incubate at 0.2-0.4 g / L at 30°C and 220 rpm for approximately 4-6 hours to obtain the final OD value. 600 The concentration should be controlled between 0.8 and 1.0. Prepare competent yeast cells using the kit: Transfer 1 mL of bacterial culture to a 1.5 mL centrifuge tube, centrifuge at 7000 rpm for 3 min, and discard the supernatant. Resuspend the cells in 1 mL of Solution I, centrifuge at 7000 rpm for 3 min, and discard the supernatant. Resuspend the cells in 100 μL of Solution II. The competent yeast cells are now obtained. Aliquot into 25 μL tubes and proceed immediately with plasmid transformation or store at -80℃.

[0077] The construction of strain PL00 involved the following: Using the technology described in patent application number 201910271558.6, the host cell ScCEN.PK2-1Ca underwent genomic modification. Specifically, the restriction factor rox1 in the mevalonate pathway was knocked out, and the expression intensity of erg9, a downstream pathway-related enzyme of the sesquiterpene precursor FPP, was downregulated to increase the supply of the precursor FPP, ultimately obtaining strain ScPK2-M. Based on strain ScPK2-M, the expression intensity of erg12 and tHMG1 was further upregulated using the method described in the literature "Chen Hefeng. Construction and Metabolic Engineering Modification of Valenciaene Cell Factory [D]. South China University of Technology, 2019," ultimately obtaining strain ScPK2-01. Based on the ScPK2-01 strain, the method described in the literature "Liu T,Li W,Chen H,et al.Systematic Optimization of HPO-CPR to Boost(+)-Nootkatone Synthesis in Engineered Saccharomyces cerevisiae[J].Journal ofagricultural and food chemistry,2022,70(49):15548-15559.DOI:10.1021 / acs.jafc.2c07068." was used to overexpress ICE2 and INO2, ultimately obtaining the PL00 strain.

[0078] S2. Recombinant expression plasmids YEp352-AoFS and YEp352-control were transformed into competent cells of Saccharomyces cerevisiae.

[0079] Take a tube of competent Saccharomyces cerevisiae cells (25 μL), add 500 ng of recombinant expression plasmid YEp352-AoFS or YEp352-control to the competent cells, then add 250 μL of Solution III, mix well, vortex for 10 s, and then incubate at 30℃ for 15 min, followed by 1 h. Repeat this process 4 times. Spread the entire bacterial culture onto SD-ΔUra plates (20 g / L glucose, 6.7 g / L YNB, 0.62 g / L DO Supplement (Δura-Δtrp-Δleu), 0.06 g / L leucine, 0.04 g / L tryptophan), and incubate at 30℃ for 2-3 days until single colonies grow. This indicates that the recombinant plasmid has been integrated into the Saccharomyces cerevisiae strain, resulting in recombinant strains PL00-AoFS and PL00-control.

[0080] S3. Detection of α-farnesene by shake-flask fermentation of recombinant strain PL00-AoFS

[0081] A single colony of the recombinant strain PL00-AoFS was picked and inoculated into 5 mL of SD-ΔUra liquid medium. After culturing for 12-16 hours, it was transferred to a shake flask (10 mL of SD-ΔUra liquid medium) and the initial OD was controlled. 600 The concentration was 0.05, and 2 mL of n-dodecane was added to cover the culture. The culture was incubated at 220 rpm and 30°C for 48 hours. After fermentation, the entire upper organic phase was removed, and the OD of the bacterial culture was measured. 600 The organic phase was filtered through a 0.22 μm nylon organic membrane. GC analysis revealed a new peak in the PL00-AoFS fermentation product compared to the PL00-control product around 16 min. Figure 6 A), identified by GC-MS as α-farnesene ( Figure 6 B), with a yield of 304.2 mg / L.

[0082] GC detection conditions: Agilent HP-5 (30m × 0.32mm × 0.25μm) column, nitrogen as carrier gas, carrier gas flow rate 2.3ml / min. Split ratio 15:1, column temperature program as follows:

[0083] Ramp rate Temperature Time 100℃ 10 min 8 °C / min 150℃ 0 min 30 °C / min 250℃ 3 min

[0084] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An α-farnesene synthase AoFS of nootropic origin, characterized in that: The amino acid sequence of the α-farnesene synthase AoFS is shown in SEQ ID NO.

2.

2. The gene encoding the α-farnesene synthase AoFS of the nootropic source as described in claim 1.

3. The gene according to claim 2, characterized in that: The nucleotide sequence of the gene is shown as 1-1761 bp in SEQ ID NO.

1.

4. The biomaterial related to the α-farnesene synthase AoFS of the Alpha-derived source as described in claim 1, characterized in that: It can be any one or more combinations of the following biological materials: (a) An expression cassette containing the gene of claim 2 or 3; (b) A recombinant expression vector containing the gene of claim 2 or 3; (c) A recombinant expression vector containing the expression cassette described in (a); (d) A recombinant microorganism containing the gene described in claim 2 or 3; (e) Recombinant microorganisms containing the expression cassette described in (a); (f) Recombinant microorganisms containing the recombinant expression vector described in (b) or (c).

5. The biomaterial according to claim 4, characterized in that: The expression box described in (a) further includes a promoter and a terminator; the promoter is promoter CCW12 and the terminator is terminator CYC1; The starting vector for the recombinant expression vectors described in (b) and (c) is a YEp type vector; The host microorganisms corresponding to the recombinant microorganisms described in (d), (e), and (f) are selected from prokaryotes or yeast.

6. The biomaterial according to claim 5, characterized in that: The starting vector for the recombinant expression vectors described in (b) and (c) is the YEp352 vector; The host microorganism corresponding to the recombinant microorganisms mentioned in (d), (e), and (f) is the Saccharomyces cerevisiae CEN.PK2-1Ca strain.

7. The use of the gene according to any one of claims 2 to 3 or the biomaterial according to any one of claims 4 to 6 in the preparation of α-farnesene synthase AoFS.

8. The application of the α-farnesene synthase AoFS according to claim 1, the gene according to any one of claims 2 to 3, or the biomaterial according to any one of claims 4 to 6 in the biosynthesis of α-farnesene.

9. The application according to claim 8, characterized in that: α-Farnesene was produced by using farnesyl pyrophosphate (FPP) as a substrate and catalyzing it with α-farnesene synthase (AoFS).

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