S3G4685 gene and application thereof in synthesis of triterpenoid active products

Abstract

The invention discloses an S3G4685 gene and an application of the S3G4685 gene in synthesis of triterpenoid active products. The nucleotide sequence of the S3G4685 gene is as shown in SEQ ID NO. 1, and the amino acid sequence of the protein of the S3G4685 gene is as shown in SEQ ID NO. 2. The S3G4685 gene can significantly enhance the key metabolic flux of a terpene synthesis pathway in a host strain and improve the yield of squalene and downstream terpenoids thereof. The gene, the recombinant strain and the fermentation method provided by the invention provide a new biological catalysis strategy for efficient and sustainable production of terpenoids, and have important industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and metabolic engineering, specifically involving the S3G4685 gene and its application in the synthesis of triterpenoid bioactive products. Background Technology

[0002] Terpenes are a diverse class of natural products with broad bioactivity, widely used in medicine, cosmetics, fragrances, and biofuels. Based on the number of carbon atoms in their skeleton, terpenes can be classified into monoterpenes, sesquiterpenes, diterpenes, and triterpenes. Among them, triterpenes have attracted considerable attention due to their significant pharmacological activities. For example, ganoderic acids, the main active triterpenoid component in Ganoderma lucidum, have been proven to possess various bioactivities, including antitumor, immunomodulatory, hepatoprotective, and antiviral effects, and have significant development value in the pharmaceutical and functional food fields.

[0003] Squalene, as a key precursor to triterpenoids, plays a crucial role in cholesterol synthesis, steroid hormone metabolism, and the biosynthesis of high-valent terpenoids such as ganoderic acid. Currently, the production of terpenoids mainly relies on plant extraction or chemical synthesis. For example, the production of ganoderic acid still primarily depends on the cultivation and extraction of Ganoderma lucidum fruiting bodies, which suffers from problems such as long growth cycles, low extraction efficiency, and unstable product content; while chemical synthesis involves cumbersome steps, low yields, and significant environmental pollution.

[0004] Microbial cell factories offer a viable pathway for the green and sustainable production of terpenoids. Saccharomyces cerevisiae and filamentous fungi, due to their mature genetic manipulation, rapid growth, and ease of large-scale cultivation, have become common hosts for constructing terpenoid biosynthetic pathways. However, the flux of terpenoid biosynthetic pathways in natural strains is limited, and metabolic regulation is complex. In particular, the metabolic flux upstream of squalene synthase (SQS) often becomes a bottleneck, resulting in low yields of target products such as ganoderic acid, thus limiting their industrial application.

[0005] In recent years, enhancing the expression of key genes in the terpene synthesis pathway through genetic engineering has become an effective strategy for increasing yield. However, current technologies still lack efficient, universal, and stable endogenous or exogenous enhancing factors, especially functional genes in filamentous fungi that efficiently promote squalene accumulation and thus enhance triterpene product synthesis.

[0006] Therefore, discovering new genes with broad-spectrum terpene synthesis capabilities and constructing efficient and stable recombinant strains are of great significance for promoting the industrialization of microbial production of high-value terpene compounds such as ganoderic acid. Summary of the Invention

[0007] The purpose of this invention is to provide a new gene and its encoded protein that can significantly enhance the ability of microorganisms to synthesize terpenoids, thereby solving the technical problems of low terpenoid yield and limited metabolic flux in existing microbial cell factories, and providing a new key component for the efficient and sustainable production of high-value triterpenoids such as squalene and ganoderic acid.

[0008] The S3G4685 gene, the nucleotide sequence of which is shown in SEQ ID NO. 1.

[0009] S3G4685 protein, the amino acid sequence of which is shown in SEQ ID NO.2; or the amino acid sequence thereof having one or more amino acid residues substituted and / or deleted and / or added while still having squalene-producing efficacy.

[0010] Specifically, substitution and / or deletion and / or addition of one or more amino acid residues refers to substitution and / or deletion and / or addition of no more than 10, 5, or 3 amino acid residues. In a more specific embodiment, a tag sequence is attached to the amino terminus or carboxyl terminus, preferably Poly-Arg, Poly-His, FLAG, Strep-tag II, c-myc, and more specifically RRRRR, HHHHHH, DYKDDDDK, WSHPQFEK, EQKLISEEDL. To facilitate the purification of the protein in (a), tags as shown in Table 1 can be attached to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID NO. 1.

[0011] Table 1. Sequence of Labels

[0012] A recombinant vector containing the S3G4685 gene.

[0013] The recombinant strain containing the recombinant vector is either *Saccharomyces cerevisiae* or a filamentous fungus.

[0014] Application of the S3G4685 gene in the synthesis of triterpenoid active products.

[0015] The triterpenoid active product is squalene.

[0016] The molecular structure of squalene is as follows: .

[0017] A method for increasing the yield of terpenoids in Saccharomyces cerevisiae or filamentous fungi, wherein the S3G4685 gene is expressed in the strain.

[0018] Since terpene synthesis is a highly energy-intensive metabolic process that may generate reactive oxygen species (ROS), the S3G4685 protein identified in this invention may participate in maintaining cellular redox homeostasis, indirectly ensuring the efficient operation of the synthesis pathway. It may also act as a partner protein providing electrons to cytochrome P450 oxidases (CYPs), directly participating in the functionalization of the triterpene skeleton. This gene can effectively enhance the metabolic flux of the terpene synthesis pathway in host cells and is a key component in constructing efficient microbial cell factories for terpene compounds, possessing significant industrial application prospects and market value.

[0019] Beneficial effects of the present invention: The present invention, through its effect on fungi Emericellopsis Transcriptome sequencing and differential expression analysis were performed on sp. XJ1056 under four culture conditions showing significant differences in triterpenoid production, identifying eight candidate genes related to redox coenzyme metabolic cycles. These genes may indirectly or directly regulate the highly energy-intensive metabolic pathway of terpenoid synthesis by influencing intracellular reducing power or energy supply. Gene overexpression validation in the original strain revealed that one gene (named S3G4685) significantly increased triterpenoid production in the host strain. Furthermore, this gene was obtained through cDNA amplification, and a recombinant plasmid for expression in *Saccharomyces cerevisiae* was constructed. Heterologous expression and fermentation tests showed that the squalene production of the recombinant *Saccharomyces cerevisiae* strain expressing S3G4685 increased from 57.75 mg / L to 73.98 mg / L, an increase of 28.09%. These results indicate that this gene has a broad-spectrum function of enhancing terpenoid synthesis in both filamentous fungi and yeast. Attached Figure Description

[0020] Figure 1 Fungi under four culture conditions Emericellopsis sp Triterpenoid yield of XJ1056.

[0021] Figure 2 This is a heatmap of the expression levels of the 30 genes most associated with triterpenoid production in transcriptome analysis.

[0022] Figure 3 The standard curve of squalene was determined using GC-QQQ chromatography-mass spectrometry. Detailed Implementation

[0023] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0024] The experimental materials used in the following experiments and their sources are as follows: Strains and vectors: filamentous fungi Emericellopsissp. XJ1056 and yeast S. cerevisiae BJ5464-NpgA and the yeast expression vector PRS425m were both derived from the Institute of Biotechnology, Chinese Academy of Agricultural Sciences.

[0025] Enzymes and kits: RNA reverse transcription kit was purchased from TAKARA; ClonExpress MultiS One Step Cloning Kit-C113 and DNA polymerase were purchased from Novizan; DNA marker was purchased from Kangwei Century; plasmid mini-preparation kit was purchased from Tiangen; universal DNA purification gel recovery kit was purchased from Thermo Fisher Scientific; E. coli DH5α competent cells were purchased from Kangwei Century; other reagents were domestically produced analytical grade products.

[0026] Culture medium: LB broth (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0) was used for *E. coli*. SC- - Leu-deficient medium (1% glucose, 6.7% Difco) TM Yeast Nitrogen Base with AminoAcids and Leu / Trp DO Supplement. YPD medium (1% yeast extract, 2% peptone, 2% glucose). YPD low-glucose medium (1% yeast extract, 2% peptone, 1% glucose). If preparing a solid medium, add 2% agar powder.

[0027] For other experimental methods not specified in the examples, they were performed according to conventional methods. Molecular cloning was performed according to the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989) or according to the conditions recommended by the manufacturer.

[0028] Example 1: Predicting functional genes related to triterpenoid production by analyzing transcriptome Through fungi Emericellopsis Transcriptome sequencing and differential expression analysis were performed on sp. XJ1056 under four culture conditions showing significant differences in triterpenoid production, identifying eight candidate genes related to redox coenzyme metabolic cycles. The experimental methods are as follows: 1) Experimental materials and data sources filamentous fungi EmericellopsisSp. XJ1056 was used as the research subject. Fermentation was conducted under four culture conditions (labeled A, B, C, and D) with significantly different triterpenoid yields, and cell samples were collected. Four biological replicates were set up for each condition. Total RNA was extracted from each sample using the TRIzol method. After quality control, strand-specific transcriptome sequencing (Illumina NovaSeq platform, PE150) was performed by Shanghai Ling'en Biotechnology Co., Ltd.

[0029] The total yield of triterpenoids was tested under four conditions. It was found that the highest yield (61.32 mg / L) was observed under condition A, while the yield decreased significantly under conditions B, C, and D. Figure 1 ).

[0030] 2) Transcriptome data analysis and differential expression screening The obtained raw sequencing data underwent quality filtering (removing low-quality reads and adapter sequences), and high-quality reads were aligned to the target sequence using HISAT2 software. Emericellopsis The XJ1056 reference genome was used. Transcript assembly and gene expression quantification were performed using StringTie, with FPKM values ​​used as an indicator of gene expression levels.

[0031] Pairwise differential expression analysis was performed using the DESeq2 package in R for four conditions (A vs B, A vs C, A vs D). The criteria for screening differentially expressed genes were: |log2(Fold Change)| > 4 and a corrected p-value (padj) < 0.05. A total of 235 differentially expressed genes were obtained from all comparison groups for subsequent analysis.

[0032] 3) Weighted Gene Co-expression Network Analysis (WGCNA) To systematically identify gene modules that exhibit highly synergistic changes with triterpenoid yield traits, the WGCNA method was used for analysis. The specific steps are as follows: a. Data preprocessing: Genes with FPKM > 1 in at least one sample were selected to construct an expression matrix, totaling 8980 genes.

[0033] b. Construct a co-expression network: Using the WGCNA R package, select an appropriate soft thresholding power to convert expression similarity into an adjacency matrix, and further convert it into a topological overlap matrix (TOM).

[0034] c. Gene Module Identification: Dynamic Tree Cut was used to perform hierarchical clustering of genes. Based on the dissimilarity of the TOM matrix, genes with highly correlated expression patterns were clustered into different modules. A total of 12 modules were identified, each marked with a different color.

[0035] d. Module-trait association analysis: The correlation between the eigengene (ME) of each gene module and triterpenoid yield was calculated. Three modules (Module 1, Module 6, and Module 11) were found to be significantly positively correlated with high yield conditions (correlation coefficient r > 0.7, p < 0.001), and two modules (Module 7 and Module 12) were significantly negatively correlated (Table 2).

[0036] Table 2 Module Information and its Correlation with Output

[0037] 4) Candidate gene screening and functional enrichment analysis Functional annotation and enrichment analysis: Interproscan analysis was performed on the core genes selected, and genes related to "redox", "coenzyme binding", and "terpene skeleton biosynthesis" were screened out.

[0038] 5) Candidate gene identification: Candidate genes were further screened based on the following criteria: (i) Expression was significantly upregulated under high-yield conditions; (ii) High phenotypic correlation with triterpenoid production ( Figure 2 ); (iii) The annotation function is related to the metabolism or binding of redox coenzymes (NAD / NADP / FAD, etc.); Ultimately, eight candidate genes that met the above criteria were selected, including the target gene that was subsequently named S3G4685.

[0039] Through the above-mentioned multi-condition transcriptome sequencing, differential expression analysis, co-expression network construction and functional annotation, it was successfully obtained from... Emericellopsis Eight candidate genes highly correlated with triterpenoid production and whose function is related to redox coenzyme metabolism were screened from sp. XJ1056, laying a solid foundation for subsequent verification of their function through genetic manipulation.

[0040] Example 2: Heterologous expression of expression-related candidate functional genes in yeast By overexpressing candidate genes in MC yeast using the TDH3 promoter, genes that can help increase squalene production were screened, and finally, a gene associated with high terpene production was identified. The specific experimental method is as follows: 1) Obtaining candidate gene cDNA In heterologous expression in yeast, the cDNA gene needs to be integrated into the yeast genome to express the fungal gene. Emericellopsis sp. XJ1056 was inoculated on YES solid medium and cultured for 7 days. RNA was extracted from it. The specific steps are as follows: a. Scrape the bacterial cells with a pipette tip, absorb the moisture with absorbent paper, and then place the cells in a mortar. Pour liquid nitrogen into the mortar and grind the bacterial cells vigorously until they become bacterial powder.

[0041] b. Load the bacterial powder into a 1.5 mL EP tube, quickly add 1 mL of Trizol, incubate at room temperature for 10-15 min, centrifuge at 12000 rpm for 10 min at 4℃, and then transfer the supernatant to a new 1.5 mL EP tube.

[0042] c. Add 200 μL of chloroform to the tube, mix well and invert, let stand at room temperature for 15 min, centrifuge at 12000 rpm for 10 min at 4℃, and transfer the supernatant to a new 1.5 mL EP tube.

[0043] d. Add 500 μL of isopropanol to the tube, let it stand at room temperature for 10 min, centrifuge at 12000 rpm for 10 min at 4℃, discard the supernatant, and let the RNA settle at the bottom of the tube.

[0044] e. Add 1 mL of 75% ethanol (prepared with DEPC water) to wash the precipitate, centrifuge at 7000 rpm for 5 min at 4℃, and discard the supernatant. Let it stand at room temperature for 10 min to evaporate the ethanol.

[0045] f. Dissolve the RNA in 50 μL of DEPC water, measure the concentration using NanoDrop, and confirm whether the RNA has been degraded using 1% agarose gel electrophoresis.

[0046] After RNA extraction, the Takara reverse transcription kit (PrimeScript) was used. TMThe RT reagent kit with gDNA Eraser was used to remove gDNA from RNA and reverse transcribe it into cDNA. The reaction mixture for gDNA removal was: 2 μL 5× gDNA Eraser buffer, 1 μL gDNA Eraser, 1 μg RNA, and RNase-free dH2O, adjusted to 10 μL. The reaction program was: 2℃ for 2 min, then stored at 4℃. The reaction mixture for reverse transcription was: 10 μL of the previous reaction mixture, 4 μL RNase-free dH2O, 4 μL 5× PrimeScript Buffer, 1 μL PrimeScript RT Enzyme Mix, and 1 μL RT Primer Mix. The reaction program was: 37℃ for 15 min, 85℃ for 5 s, and finally stored at -20℃.

[0047] The nucleotide sequences of candidate genes were analyzed using various software to remove introns and splice them to obtain the coding region sequence of the candidate genes. The cDNA obtained after reverse transcription was used as a template, and the coding gene of the candidate genes was amplified using designed primers. The target fragment was recovered and sent to a sequencing company for sequencing and alignment to obtain the coding region sequence of the candidate genes.

[0048] 2) Construction of yeast heterologous expression vector Using gene cloning technology, the plasmid pJET-pTDH3-cDNA was constructed using the pJET vector (pJET1.2 vector), the TDH3 promoter pTDH3, and the cDNA of the candidate gene. The specific method is as follows: With brewer's yeast S.cerevisiae Using BJ5464-NpgA as a template, the TDH3 promoter was amplified. When synthesizing primers, a 15-20 bp homologous region of the pJET vector was added before the TDH3 promoter, and a 15-20 bp homologous region of the candidate gene cDNA was added after the TDH3 promoter, with a fragment length of 0.6 kb. Using the candidate gene cDNA as a template, the candidate gene cDNA used to construct the vector was amplified. When synthesizing primers, a 15-20 bp homologous region of the pJET vector was added after the candidate gene cDNA.

[0049] The PCR reaction system consisted of: 25 μL of 2×Phanta Max Master Mix, 20 μL of ddH2O, 2 μL each of upstream and downstream primers, and 1 μL of template.

[0050] PCR program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension at a rate of 30 s / kb, with the extension time determined by the fragment size, 35 cycles; 72℃ final extension for 5 min, and storage at 4℃.

[0051] After recovering the pJET linear vector fragment and two PCR product fragments, the fragments were fused using the In-Fusion Kit and transformed into competent E. coli DH5α cells using the heat shock method. Ampr antibody screening and colony PCR identification were performed. Positive clone plasmids were extracted and sent to a sequencing company for sequencing. Correctly sequenced plasmids were stored at -20°C.

[0052] Using the constructed plasmid as a template, the pTDH3-cDNA fragment is amplified. When synthesizing primers, 50 bp upstream and downstream of the yeast DPP1 gene needs to be added before and after the primers so that the subsequent fragment can be integrated into the yeast genome.

[0053] 3) Yeast transformation The pTDH3-cDNA fragment and the pCas9-DPP1 plasmid were transformed using the CRISPR-Cas9 method via electroporation. The specific procedure is as follows: Preparation of yeast electrocompetent cells: a. Inoculate a single colony of the yeast strain BJ5464 for transformation into 4 ml of 2% YPD medium and incubate overnight at 30°C until saturation (OD=3.0). b. Inoculate the bacterial culture in a 150 ml sterile flask containing 20 ml of YPD medium (inoculation amount of 0.15 OD / ml) and incubate at 30℃ and 220 rpm for 7 h; c. Collect cells by centrifugation at 3000 rpm for 3 min at 4℃, and resuspend the cells in 20 ml of pre-cooled sterile water; d. Collect cells by centrifugation at 3000 rpm for 3 min at 4℃, and resuspend the cells in 20 ml of pre-cooled 1M Sorbitol; e. Collect cells by centrifugation at 3000 rpm for 3 min at 4°C. Resuspend cells in 16 ml of pre-chilled 1M Sorbitol, add 2 ml of 10x TE buffer (pH=7.5), shake well, then add 2 ml of 10x lithium acetate, and incubate at 30°C with shaking at 250 rpm for 30 min. f. Add 200 μl of 1 M DTT, continue shaking at 30°C and 250 rpm for 15 min; g. Collect cells by centrifugation at 3000 rpm for 3 min at 4℃. Wash cells twice with 20 ml of pre-chilled 1M Sorbitol. h. Resuspend the yeast cells in 0.4 ml of pre-chilled 1 M Sorbitol. The final OD600 should be approximately 200. Aliquot 100 μl per tube and store at -80°C.

[0054] Electrocution Transformation: a. Set the yeast electroporation program (0.2cm): voltage 1500 V, perform electroporation (capacitor 25μF, resistor 200Ω, electroporation time 4 ms); b. Place the pre-prepared competent cells on ice. After the competent cells thaw, add the DNA to be transformed (1 μg / kb) and incubate on ice for 5 min. c. Transfer the mixture to an electroporation vessel for electroporation; d. After electroporation, add 2 ml (1M Sorbitol: 2% YPD=1:1) to resuspend the cells, transfer the transformed cells from the electroporation cuvette to a 12 ml shaking tube, and incubate at 30°C and 250 rpm for 5 h. e. Centrifuge at 3000 rpm for 3 minutes at room temperature, then discard the supernatant; f. Wash twice with 1 mL of SCura-liquid medium; g. Discard the supernatant and resuspend the cells in 4 ml of SCura- liquid medium, incubate at 30°C and 250 rpm for 24 h; h. Concentrate the yeast to 200 μl and spread it on SC ura- medium; after 5 days, single yeast colonies will grow on the plate. Perform colony PCR to check whether pTDH3-cDNA has been inserted at the correct genomic location in the yeast. Store the positive yeast cells at -80°C.

[0055] Example 3 Evaluation of yeast fermentation and squalene yield Yeast fermentation: First, an appropriate amount of positive yeast transformants were inoculated into 4 mL of 2% YPD medium and cultured overnight as a seed culture. Then, the seed culture was inoculated into 50 mL of 2%+1 / 10 PBS liquid medium with an initial OD of 0.1 and cultured at 30℃ and 220 rpm for 7 days.

[0056] Evaluation of squalene production: Yeast extraction method: a. Pipette 5 mL of bacterial culture into a threaded tube and centrifuge at 12,000 rpm for 1 min (centrifugation can be done in batches). After centrifugation, wash twice with water to remove as much water as possible.

[0057] b. Add 200 μL H2O, 2 5 mm styrax beads, 3 3 mm styrax beads, or 0.5 mm styrax beads (the volume of one PCR tube) to each tube, and finally add 1 mL of ethyl acetate.

[0058] c. Grind twice with a grinder at 70 Hz for 90 s, and then vibrate at 200 rpm for 2 min with a vibrator. Repeat this step 3 times.

[0059] d. Oscillate at 200 rpm for 30 minutes.

[0060] Centrifuge at 8000 rpm for 3 min and collect the supernatant in a 1.5 mL EP tube.

[0061] f. Dry with nitrogen, reconstitute with 1 mL of ethyl acetate, and detect by GCMS.

[0062] The GCMS detection conditions were as follows: Squalene was detected using an Agilent 8890-5977B gas chromatograph-mass spectrometer with a DB-5MS UI capillary column (30 m × 0.25 mm, 0.25 μm; Agilent Technologies, USA) and helium gas (1 mL / min). -1 The carrier gas was 1 mL / min. The crusher voltage was maintained at 70 eV (EI), the inlet temperature was 250 °C, and the flow rate was 1 mL / min. -1 The pressure was 9.3825 psi. Data acquisition was performed in full scan mode (m / z 50-650), and the molecular weight of squalene was 410.7.

[0063] Quantitative methods: Standards with concentrations of 100, 200, 300, and 400 mg / L were prepared using pure squalene. Peak areas were determined using GC-MS, and standard curves were plotted. (See table below.) Figure 3 .

[0064] Experimental results and analysis: Table 3 shows the squalene yield of yeast strains overexpressing each candidate gene under 7-day fermentation in 2% YPD + 1 / 10 PBS liquid medium. It can be seen that the yeast strain overexpressing S3G4685 in this invention significantly increases the squalene yield, reaching 73.98 mg / L, which is 128.09% of the yield of the original strain.

[0065] Table 3. Squalene yield after overexpression of each candidate gene in yeast

[0066] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The S3G4685 gene, characterized by, The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The S3G4685 protein, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A recombinant vector containing the S3G4685 gene as described in claim 1.

4. A recombinant strain containing the recombinant vector of claim 3, characterized in that, The strain is either Saccharomyces cerevisiae or a filamentous fungus.

5. The application of the S3G4685 gene as described in claim 1 in the synthesis of triterpenoid bioactive products.

6. The application of the S3G4685 gene according to claim 5 in the synthesis of triterpenoid bioactive products, characterized in that, The triterpenoid active product is squalene.

7. A method for increasing the yield of terpenoid compounds in Saccharomyces cerevisiae or filamentous fungi, characterized in that, The S3G4685 gene of claim 1 is expressed in Saccharomyces cerevisiae or filamentous fungi.

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