Glycosyltransferase for catalyzing glycosylation of No. 7 and No. 8 hydroxyls of gossypol and application of glycosyltransferase

Abstract

The invention relates to glycosyltransferase for catalyzing glycosylation of hydroxyl groups at No.7 and No.8 sites of gossyn and application of the glycosyltransferase, and belongs to the technical field of enzyme engineering. The invention aims to excavate key glycosyl transferase UGT capable of catalyzing gossypol glycosylation, and provides a saccharomyces cerevisiae engineering bacterium capable of synthesizing gossypol glycoside derivatives by taking quercetin as a substrate, especially 7 # and 8 # hydroxyl glycosylated derivatives. Specifically, the glucoside transferase for efficiently synthesizing the gossydin-7-O-glucoside and the gossydin-8-O-glucoside is determined by performing tissue collection, RNA (Ribonucleic Acid) extraction, gene amplification and bioinformatics analysis, preliminary verification of cotton cotyledons and fermentation verification of saccharomyces cerevisiae on various plant materials rich in flavone. And then integrating F8H hydroxylase required by cotton element biosynthesis and UGT with a specific catalytic function in the saccharomyces cerevisiae to construct the saccharomyces cerevisiae engineering bacterium synthesized by the cotton element derivative.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of enzyme engineering, and particularly relates to a glycosyltransferase for catalyzing hydroxyl group glycosylation at positions 7 and 8 of gossypetin and application thereof. BACKGROUND

[0002] Gossypetin is a flavonoid compound with six hydroxyl groups, which is widely present in Malvaceae plants. Gossypetin and its glycoside derivatives have strong biological activities, including antioxidant, anti-inflammatory, antibacterial, immunomodulatory, neuroprotective and other functions, and have potential application value in the fields of medicine, food, cosmetics and the like. Compared with free gossypetin, glycoside products (i.e., gossypetin glycosides) have better water solubility, bioavailability and chemical stability due to the introduction of glycosyl structure, and are suitable for developing oral preparations or external functional products. Current studies have shown that gossypetin glycosides exhibit target and multi-target mechanism in the prevention and treatment of various diseases, especially in cardiovascular system diseases (such as atherosclerosis), and exhibit potential value in drug development through mechanisms such as scavenging of active oxygen, improvement of endothelial function and inhibition of vascular smooth muscle cell proliferation. In addition, glycosylation modification can reduce the toxic and side effects of natural products and improve the safety of their use in functional foods and health products. In particular, gossypetin-7-O-glucoside and gossypetin-8-O-glucoside formed after glycosylation of hydroxyl groups at positions 7 and 8 of gossypetin have strong biological activities in inhibiting adenosine deaminase activity, neuroprotection, antibacterial and anti-infection, and have strong potential in drug development.

[0003] At present, gossypol glycoside compounds are mainly derived from natural plant extraction, such as Gossypium and Hibiscus. However, the content of gossypol in these plants is low, and the types and yield of glycosylation products are limited by the plant's own metabolic capacity, growth cycle, climate conditions and other factors, resulting in low production efficiency and high cost, which is difficult to meet the large-scale industrialization demand. Traditional extraction process also has problems such as complex process flow, difficult purity control, and environmental unfriendliness. In recent years, relying on the development of synthetic biology and metabolic engineering, the construction of a microbial synthesis platform to realize the large-scale production of natural products has become a new research hotspot. Using engineered Saccharomyces cerevisiae as a chassis cell, the controllable synthesis of target compounds in the fermentation system can be realized by expressing related biosynthetic enzymes. UDP-glycosyltransferase (UGT) is a class of enzymes that can catalyze the combination of small molecules and sugar donors (such as UDP-Glc) to generate glycosylation products, and is widely present in plants, which is a key functional enzyme for glycosylation modification of natural products. Currently, several UGTs have been reported to catalyze the glycosylation modification of flavonoids, isoflavones and other substances, but there have been no systematic screening and functional verification of UGTs for specific glycosylation modification of gossypol, especially UGTs that can catalyze the glycosylation of hydroxyl groups at positions 7 and 8 of gossypol, and the related metabolic pathways have not been established. SUMMARY

[0004] Therefore, the technical problem to be solved by the present application is to overcome the lack of glycosyltransferases that can catalyze the glycosylation of hydroxyl groups at positions 7 and 8 of gossypol in the prior art, and to provide a new glycosyltransferase and an engineered strain constructed based on the enzyme. A new method for efficient, green and controllable synthesis of gossypol glycoside derivatives is developed based on the expression system of engineered microorganisms, which has important technical significance and industrial value, especially in the synthesis of gossypol-7-O-glucoside and gossypol-8-O-glucoside.

[0005] The present application provides a recombinant Saccharomyces cerevisiae strain, which at least includes the following modifications: overexpression of flavone-8-hydroxylase encoding gene, and overexpression of glycosyltransferase encoding gene represented by any one of SEQ ID NO. 1-5.

[0006] Further, the nucleotide sequence of the flavone-8-hydroxylase encoding gene is shown in SEQ ID NO. 8.

[0007] Furthermore, the host strain of the recombinant Saccharomyces cerevisiae strain includes S. cerevisiae CEN PK2-1CMATa; ura3-52; trp1-289; leu2-3,112; his3Δ 1; MAL2-8C; SUC2.

[0008] Furthermore, the overexpression can be either free expression or integrated expression. When integrated expression occurs, the integration site includes, but is not limited to, the 308a site on the genome.

[0009] Furthermore, the flavonoid-8-hydroxylase encoding gene and the glycosyltransferase encoding gene are respectively activated by promoter P. gal7 and promoter P gal1 Start the expression.

[0010] A second objective of this invention is to provide the application of the recombinant Saccharomyces cerevisiae strain in the preparation of gossypol derivatives, the gossypol derivatives including gossypol-7-O-glucoside and gossypol-8-O-glucoside.

[0011] Furthermore, when preparing gossypol-7-O-glucoside, the recombinant Saccharomyces cerevisiae strain overexpresses the flavonoid-8-hydroxylase encoding gene with the nucleotide sequence shown in SEQ ID NO.8 and the glycosyltransferase encoding gene with the nucleotide sequence shown in SEQ ID NO.2;

[0012] And / or, when preparing gossypol-8-O-glucoside, the recombinant Saccharomyces cerevisiae strain overexpresses the flavonoid-8-hydroxylase encoding gene with the nucleotide sequence as shown in SEQ ID NO.8 and the glycosyltransferase encoding gene with the nucleotide sequence as shown in SEQ ID NO.4;

[0013] And / or, when preparing gossypol-7-O-glucoside and gossypol-8-O-glucoside, the recombinant Saccharomyces cerevisiae strain overexpresses the flavonoid-8-hydroxylase encoding gene with the nucleotide sequence shown in SEQ ID NO.8 and the glycosyltransferase encoding gene with the nucleotide sequence shown in any one of SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.4.

[0014] Furthermore, the gossypol derivative was prepared using quercetin as a substrate.

[0015] A third object of the present invention is to provide the use of glycosyltransferases with nucleotide sequences such as any one of SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.4 in catalyzing the synthesis of gossypol derivatives, said gossypol derivatives including gossypol-7-O-glucoside and gossypol-8-O-glucoside.

[0016] A fourth object of the present invention is to provide the application of a glycosyltransferase with a nucleotide sequence as shown in SEQ ID NO.2 in catalyzing the synthesis of a gossypol derivative, wherein the gossypol derivative is gossypol-7-O-glucoside.

[0017] A fifth object of the present invention is to provide a system for synthesizing gossypol derivatives (such as a carrier system, a cell system, etc.), comprising:

[0018] (1) The gene encoding flavonoid-8-hydroxylase with nucleotide sequence as shown in SEQ ID NO.8 and the gene encoding glycosyltransferase with nucleotide sequence as shown in SEQ ID NO.2;

[0019] Or (2) the flavonoid-8-hydroxylase encoding gene with a nucleotide sequence as shown in SEQ ID NO.8 and the glycosyltransferase encoding gene with a nucleotide sequence as shown in any one of SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.4.

[0020] A sixth object of the present invention is to provide recombinant cells containing the system, wherein the host cell is a non-plant cell, such as a microorganism, more preferably a brewer's yeast.

[0021] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0022] This invention first screens plants for glycosyltransferases that may catalyze the synthesis of gossypol derivatives. Through tissue collection, RNA extraction, gene amplification, and bioinformatics analysis of various flavonoid-rich plant materials, a batch of candidate UGT genes with conserved glycosyltransferase domains, namely CiUGT11, CiUGT123, CiUGT29, CiUGT31, CiUGT32, AtUGT89C1, and PaUGT1, were screened and cloned. Subsequently, candidate UGT genes capable of producing cotton derivatives were screened by transient expression in cotton cotyledons. Metabolomics analysis confirmed that all candidate UGT genes can produce gossypol derivatives (such as gossypol-3-O-glucoside, gossypol-7-O-glucoside, and gossypol-8-O-glucoside). Finally, through fermentation verification with recombinant Saccharomyces cerevisiae, the following systems were identified for efficient synthesis of gossypol-7-O-glucoside, efficient synthesis of gossypol-8-O-glucoside, and simultaneous synthesis of both gossypol-7-O-glucoside and gossypol-8-O-glucoside. The recombinant Saccharomyces cerevisiae provided by this invention can achieve the synthesis of gossypol glycoside derivatives, laying the foundation for metabolic engineering modification of Saccharomyces cerevisiae to produce gossypol glycoside derivatives. The construction method of the recombinant Saccharomyces cerevisiae provided by this invention is simple, easy to use, and has excellent application prospects. Attached Figure Description

[0023] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0024] Figure 1 This is a flowchart illustrating the technical approach of the present invention.

[0025] Figure 2 The distribution of different candidate genes in the phylogenetic tree.

[0026] Figure 3 The structural formulas are those of quercetin, gossypol, and gossypol glycoside derivatives.

[0027] Figure 4 Results of gossypol derivative yields from different engineered strains. Detailed Implementation

[0028] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0029] The solution involved in this invention is as follows:

[0030] Flavonoids are widely found in plants and have attracted considerable attention due to their bioactivities, including antioxidant, anti-inflammatory, anticancer, antibacterial, and cardiovascular protective effects. In recent years, with the continuous development of natural product structure modification strategies, functional modification of flavonoids through enzymatic catalysis has become an effective way to enhance their application value. Glycosylation is one of the most common and effective modification methods, significantly improving the water solubility, stability, and bioavailability of flavonoids, thereby expanding their application potential in food, pharmaceuticals, and daily chemicals. UDP-glycosyltransferases (UGTs) catalyze the formation of glycosidic bonds between glycosyl donors (such as UDP-glucose) and substrate molecules, playing a crucial role in the synthesis of flavonoid glycosides. Currently, numerous studies have reported the glycosylation activities of UGTs from different sources on flavonoid substrates. For example, AtUGT78D2, AtUGT89C1, and AtUGT73C5 in Arabidopsis thaliana have been shown to catalyze the glycosylation of quercetin, kaempferol, and anthocyanins at the 3-O or 7-O positions, respectively. Furthermore, some studies have also explored UGTs from traditional Chinese medicinal plants such as licorice and scutellaria baicalensis, and attempted to reconstruct the biosynthetic pathways of flavonoid glycosides in microbial chassis (such as Escherichia coli or Saccharomyces cerevisiae).

[0031] However, the following problems still exist: (1) Lack of specificity studies on gossypetin substrates: Gossypetin is more complex in structure than quercetin, kaempferol, etc. due to the presence of six hydroxyl sites in its molecule, and its glycosylation modification requires higher enzyme selectivity. However, no UGT has been reported in the existing literature and databases to achieve efficient and specific glycosylation reactions for gossypetin substrates. (2) Limited substrate range and low catalytic efficiency of known UGTs: Most known plant UGTs have a certain degree of substrate specificity for flavonoid substrates, but the conversion efficiency is low, and most can only modify a few sites such as 3-OH or 7-OH, which is difficult to meet the requirements of multi-site structural modification of gossypetin. In addition, some UGTs have strict restrictions on substrate selection, which limits their engineering applications. (3) Lack of systematic in vivo functional verification methods, especially the lack of application in target plants (such as cotton): Most current UGT studies are limited to the in vitro enzyme activity assay of prokaryotic expression systems, and an efficient and reliable in vivo plant verification platform has not yet been established. In particular, there is no systematic verification method for UGT function in cotton, making it difficult to evaluate the actual function of candidate enzymes in the context of natural substrates. (4) The microbial expression system pathway is incomplete and cannot achieve one-step synthesis of target compounds: Although some studies have constructed microbial synthesis pathways for flavonoids such as quercetin and kaempferol, they have not integrated the F8H hydroxylase and UGT combination expression module required for gossypol biosynthesis. The existing system cannot directly synthesize gossypol and its glycosylated products from the substrate quercetin, which restricts its application potential in the development of fermentation products. (5) Insufficient reserves of functional UGT resources and narrow species sources: Most of the currently disclosed UGT genes are from model plants such as Arabidopsis thaliana and rice. There is a lack of in-depth exploration of functionally diverse UGTs in medicinal plants and bryophytes, which limits the diversity of glycosylation sites and product structures. In summary, existing technologies have not yet solved key technical problems such as how to screen and verify functional UGT genes that can be used for gossypol modification, how to construct effective expression systems suitable for cotton plants and microbial platforms, and how to achieve the integrated synthesis of quercetin derivatives. These technical deficiencies are precisely the focus of this invention, demonstrating its innovative significance and practical value in metabolic engineering, functional enzyme screening, and green manufacturing pathway construction.

[0032] In the field of microbial synthesis, existing studies have utilized engineered strains such as *Saccharomyces cerevisiae* or *Escherichia coli* to successfully synthesize flavonoid glycosides with relatively simple structures, such as isorhamnetin-3-O-glucoside and apigenin-7-O-glucoside. For example, Wang et al. (2020) co-expressed FNSI and UGT73C6 in *Saccharomyces cerevisiae* to achieve the synthesis of quercetin-3-O-glucoside. Patent literature has also disclosed technical schemes for synthesizing kaempferol glycosides using a three-gene module. These studies have provided a foundation for the biosynthesis of flavonoid glycosides. However, currently disclosed technical schemes mostly focus on the glycosylation of common flavonoids such as quercetin, kaempferol, and apigenin, rarely involving the synthetic pathways of gossypetin and its glycoside derivatives. Gossypetin is a special flavonoid structure with six phenolic hydroxyl groups, and its glycosylation modification faces higher site selectivity challenges. Due to the numerous and densely distributed hydroxyl sites, existing UGTs are unable to effectively identify and specifically catalyze them, resulting in unstable or mixed glycosylation products, which restricts their functional development and application expansion.

[0033] Currently, there is a lack of reports on specific glycosyltransferases that can be used to catalyze the glycosylation of gossypol, as well as a lack of plant expression validation methods for these enzymes or research on their reconstruction in heterologous microbial systems. In particular, there is a lack of complete biosynthetic platforms for the efficient and targeted synthesis of specific derivatives such as gossypol-7-O-glucoside and gossypol-8-O-glucoside. Therefore, developing a novel UGT with specific glycosyltransfer activity and integrating it with the flavonoid synthesis pathway into a microbial cell factory to construct an efficient and controllable synthetic system for gossypol glycoside derivatives would not only expand the application boundaries of gossypol-like substances but also provide new ideas and strategies for the green synthesis of high-value-added plant flavonoids.

[0034] Saccharomyces cerevisiae, with its high safety, low pathogenicity, high stress resistance, and low probability of phage contamination, has been considered a superior host for constructing engineered strains that efficiently synthesize natural products. However, Saccharomyces cerevisiae lacks the metabolic pathway for the synthesis of gossypol glycoside derivatives. Therefore, to obtain engineered strains capable of producing gossypol glycoside derivatives, we used quercetin as a substrate and introduced a recombinant expression plasmid carrying key genes in the metabolic pathway catalyzing the synthesis of gossypol glycoside derivatives from quercetin into engineered yeast. Structural analysis revealed that the hydrogen at position 8 of quercetin was replaced by a hydroxyl group, yielding gossypol. Simultaneously, glycosyltransferases with different functions catalyzed the glycosylation of the hydroxyl groups at positions 7 and 8 of gossypol, yielding gossypol-7-O-glucoside and gossypol-8-O-glucoside. Glycosyltransferases (UGTs) have the potential to catalyze the synthesis of gossypol derivatives from quercetin. Therefore, we screened eight UGTs from different sources through sequence alignment analysis for functional verification. We found that CiUGT11, CiUGT123, CiUGT29, and CiUGT31 can mediate the synthesis of gossypol-7-O-glucoside, with CiUGT123 being the most effective. Furthermore, CiUGT11, CiUGT29, CiUGT31, and CiUGT32 can also mediate the synthesis of gossypol-8-O-glucoside, with CiUGT31 being the most effective. In addition, because excessive accumulation of flavonoids is cytotoxic, we selected the β-galactose and glucose dual-regulated promoter P... gal1 and P gal7 This will separate cell growth from the production of gossypol and its derivatives, thereby achieving the effect of highly efficient synthesis of gossypol and its derivatives.

[0035] In summary, this invention explores the following steps (see flowchart). Figure 1 ):

[0036] (1) Screening for candidate genes for glycosyltransferases in plants;

[0037] (2) Verify the function of glycosyltransferase in cotton;

[0038] (3) Metabolomics analysis;

[0039] (4) Recombinant brewing yeast fermentation to synthesize gossypol derivatives.

[0040] The above method provides an engineered Saccharomyces cerevisiae strain capable of producing gossypol derivatives. Using recombinant Saccharomyces cerevisiae CEN PK2-1C MATa; ura3-52; trp1-289; leu2-3,112; his3Δ 1; MAL2-8C; SUC2 as the host, key genes of the gossypol derivative synthesis pathway are integrated into the Saccharomyces cerevisiae genome using the Cre-LoxP method, resulting in a Saccharomyces cerevisiae strain capable of synthesizing gossypol derivatives.

[0041] In one embodiment of the present invention, the key gene flavonoid-8-hydroxylase, named GaF8H, which is derived from Asian cotton (Gossypium arboreum) and is used to catalyze the one-step synthesis of gossypol from quercetin.

[0042] In one embodiment of the present invention, when emphasizing the ability to synthesize gossypol-7-O-glucoside, recombinant Saccharomyces cerevisiae overexpressing flavonoid-8-hydroxylase with nucleotide sequence as shown in SEQ ID NO.8 and glycosyltransferase CiUGT123 with nucleotide sequence as shown in SEQ ID NO.2 is preferred; when emphasizing the ability to synthesize gossypol-8-O-glucoside, recombinant Saccharomyces cerevisiae overexpressing flavonoid-8-hydroxylase with nucleotide sequence as shown in SEQ ID NO.8 and glycosyltransferase CiUGT31 with nucleotide sequence as shown in SEQ ID NO.4 is preferred; when emphasizing the ability to simultaneously synthesize gossypol-7-O-glucoside and gossypol-8-O-glucoside, recombinant Saccharomyces cerevisiae overexpressing flavonoid-8-hydroxylase with nucleotide sequence as shown in SEQ ID NO.8 and glycosyltransferase CiUGT31 with nucleotide sequence as shown in SEQ ID NO.4 is preferred.

[0043] In one embodiment of the present invention, the construction of engineered brewer's yeast includes the following steps:

[0044] 1) Select the P promoter, which is dually regulated by β-galactose and glucose. gal7 and P gal1 Gene integration frames P for flavonoid-8-hydroxylases GaF8H, CiUGT11, and CiUGT31 were constructed using overlap extension PCR. gal7 -GaF8H- P gal1 -CiUGT11、P gal7 -GaF8H-P gal1 -CiUGT31.

[0045] 2) The integration frame of the key gene for the synthesis of gossypol derivatives was integrated into the 308a site of the Saccharomyces cerevisiae chromosome using the Cre-LoxP method.

[0046] The materials and methods involved in the embodiments of the present invention are as follows:

[0047] (1) Method for producing gossypol and its derivatives by fermentation of recombinant Saccharomyces cerevisiae: Seeds cultured at 30℃ and 220 rpm for 18-20 h were transferred to fermentation medium at an inoculation rate of 5% and cultured at 30℃ and 220 rpm for 24 h. The cells were then transferred to a complete nutrient medium (containing β-galactose to induce Gal promoter expression, and 100 mg / L quercetin was added as a substrate for synthesizing gossypol glycoside derivatives) and cultured at 30℃ and 220 rpm for another 96 h.

[0048] (2) Culture medium for the fermentation of recombinant Saccharomyces cerevisiae to produce gossypol and its derivatives: (1) Seed culture medium: Yeast Nitrogen Base (without amino acids and ammonium sulfate) 13.4 g / L, Histidine (His) 10 mg / L, Tryptophan (Trp) 10 mg / L, Leucine (Leu) 10 mg / L, Ura 20 mg / L; (2) Fermentation culture medium: YPD peptone 15-25 g / L, yeast extract 5-15 g / L and glucose 20-25 g / L; (expansion culture for 24 h) (3) Complete nutrient culture medium: Yeast Nitrogen Base (without amino acids and ammonium sulfate) 13.4 g / L, Histidine (His) 10 mg / L, Tryptophan (Trp) 10 mg / L, Leucine (Leu) 10 mg / L, Ura 20 mg / L, D(+)-galactose 25 g / L, glucose 5-10 g / L, quercetin 100 mg / L.

[0049] (3) Detection method of gossypol and its derivatives in recombinant brewing yeast: Take 1 mL of fermentation broth, centrifuge at 5000 rpm for 1 min, add glass beads for homogenization, take 500 µL of the ground sample, add an equal volume of acetonitrile, mix well, centrifuge and take the supernatant, and use liquid chromatography-mass spectrometry to perform qualitative analysis on whether there is gossypol and its glycoside derivatives in the fermentation broth (using the peak time and mass spectrum of the standards of gossypol, gossypol-7-O-glucoside and gossypol-8-O-glucoside as the standard).

[0050] (4) Detection method involved: High performance liquid chromatography-mass spectrometry (LC-MS) detection method: Waters (UPLC-Q-TOF-MS, Waters Synapt), BEH-C18 column, mobile phase acetonitrile and 0.1% formic acid aqueous solution, column temperature 45℃, injection volume 1 μL.

[0051] Example 1: Screening for candidate glycosyltransferase genes in plants

[0052] This embodiment aims to screen candidate glycosyltransferase (UGT) genes that can catalyze the glycosylation modification reaction of gossypetin. The specific steps are as follows:

[0053] 1. Sources of plant materials and samples

[0054] The following plant species, which are representative in phylogenetic analysis and are known to be rich in flavonoids, were selected as gene donors:

[0055] Asteraceae: Wild Chrysanthemum (Chrysanthemum indicum), collect flowers and tender leaves during peak bloom.

[0056] Brassicaceae: Arabidopsis thaliana, Colombian ecotype, harvest the entire above-ground parts.

[0057] Merantiaceae: Plagiochasma appendiculatum, collect the whole gametophyte.

[0058] Ericaceae: Blueberry (Vaccinium corymbosum), harvest the peel of ripe fruit.

[0059] Lamiaceae family: Salvia miltiorrhiza, roots collected.

[0060] Leguminosae: Licorice (Glycyrrhiza uralensis), roots collected.

[0061] All plant samples were immediately flash-frozen in liquid nitrogen after collection and stored in an ultra-low temperature freezer at -80°C for later use.

[0062] 2. Total RNA extraction and cDNA synthesis

[0063] Approximately 100 mg of each plant tissue was weighed and rapidly ground into a fine powder in a mortar pre-cooled with liquid nitrogen. Total RNA was extracted using a modified TRIzol method (Invitrogen, USA). DNase I (TaKaRa, Japan) was used for digestion to remove genomic DNA contamination. RNA integrity was assessed by 1.5% agarose gel electrophoresis, and its concentration and purity (A260 / A280 ratios between 1.8 and 2.1, A260 / A230 > 2.0) were determined using a NanoDrop 2000 micro-spectrophotometer (Thermo Fisher Scientific, USA). 1 μg of high-quality total RNA was used for reverse transcription using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, Japan) with Oligo(dT)18 primers to synthesize the first strand of cDNA, which was then stored at -20°C.

[0064] 3. Cloning and amplification of the UGT gene

[0065] Degenerate primers were designed based on the highly conserved C-terminal PSPG box sequence of the UGT gene family. The primer sequences are as follows:

[0066] Upstream: 5'-ATGGG(AT)GG(AT)TG(CT)AA(CT)GG(AT)AC-3'

[0067] Downstream: 5'-TC(AG)AA(AG)TT(CT)TC(AG)TC(AG)TC(AG)TA(AG)TC-3'

[0068] High-fidelity PCR amplification was performed using the synthesized cDNA as a template. The reaction program was: 94°C pre-denaturation for 5 min; 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 90 s; extension at 72°C for 10 min. The PCR product was recovered by 1.0% agarose gel electrophoresis, and the target band (approximately 1.4 kb) was cloned into the pMD19-T simple vector (TaKaRa, Japan). This vector was then transformed into *E. coli* DH5α competent cells. Positive clones were sequenced using blue-white screening and colony PCR verification.

[0069] 4. Bioinformatics Analysis

[0070] (1) Sequence alignment and phylogenetic analysis:

[0071] Correctly sequenced gene sequences were translated into amino acid sequences and subjected to multiple sequence alignment using ClustalW. Functionally identified flavonoid UGT sequences (such as AtUGT78D2 and VvGT1, which catalyze quercetin glycosylation) were downloaded from the NCBI database. A phylogenetic tree was constructed using the neighbor-joining method with MEGA 11.0 software, and the bootstrap test was repeated 1000 times to assess the confidence of each branch. The selection criteria were: sequences clustered in the same high-confidence clade (Bootstrap value > 70) with known functional flavonoid UGTs were prioritized as candidate genes.

[0072] Among them, recombinant cloning and expression verification: the candidate UGT gene was cloned into the pET series expression vector, transformed into Escherichia coli BL21(DE3) for induction of expression, and the enzyme protein was purified.

[0073] (2) Sequence similarity analysis and conserved domain prediction:

[0074] The amino acid sequences of candidate genes were compared with the NCBI nr database using the NCBI BLASTP tool to screen for candidate genes with a sequence similarity greater than 60% to known flavonoid glycosyltransferases. Protein functional domains were predicted using the InterPro and SMART databases to ensure that all candidate genes contained complete glycosyltransferase characteristic domains, particularly the C-terminal 44-amino acid PSPG box.

[0075] (3) Homologous modeling of three-dimensional structures and prediction of substrate binding:

[0076] Homology modeling of core candidate genes (such as CiUGT31) was performed using the SWISS-MODEL server, with grape-derived VvGT1 (PDB: 2c1x) as a template. The size, hydrophobicity, and key amino acid residues of their substrate-binding pockets were analyzed using PyMOL and LigPlot+ software to predict their possible binding modes with gossypol molecules and catalytic site preferences (such as 7-O- or 8-O-).

[0077] The results are as follows:

[0078] 1. Cloning and sequence characteristics of candidate UGT genes

[0079] Eight UGT candidate genes were successfully cloned from six plant materials. Their names and sources are as follows:

[0080] Derived from wild chrysanthemum (C. indicum): CiUGT11, CiUGT123, CiUGT29, CiUGT31, CiUGT32

[0081] Derived from Arabidopsis thaliana: AtUGT89C1

[0082] Derived from *P. appendiculatum*: PaUGT1

[0083] Derived from blueberry (V. corymbosum): EdF7GAT

[0084] The coding sequences of all candidate genes are between 1400-1500 bp in length, and the deduced amino acid sequences all contain complete conserved PSPG motifs (the core sequence is HCGWNS), confirming that they are typical members of the UGT family.

[0085] 2. Phylogenetic tree and functional cluster analysis

[0086] Phylogenetic tree analysis results ( Figure 2 The results show that the eight candidate genes are distributed across different branches of the phylogenetic tree:

[0087] CiUGT11, CiUGT123, CiUGT29, CiUGT31, and CiUGT32 cluster in a large evolutionary clade with known flavonoid-7-O-glycosyltransferases (Bootstrap value = 95), suggesting that they may preferentially catalyze the 7-hydroxyl group of flavonoid substrates.

[0088] AtUGT89C1 and PaUGT1 are located in another branch associated with flavonoid-3-O-glycosylation (Bootstrap value = 87).

[0089] EdF7GAT is closely related to known anthocyanin glycosyltransferases.

[0090] 3. Sequence Similarity and Functional Prediction Results

[0091] (1) Sequence similarity: BLASTP alignment results showed that all candidate genes had a sequence similarity of 62% to 85% with the known flavonoid UGT sequence, with CiUGT31 having the highest similarity to AtUGT78D2, reaching 85%.

[0092] (2) Functional prediction: Based on phylogenetic position and sequence alignment, CiUGT11, CiUGT123, CiUGT29, CiUGT31, and CiUGT32 are predicted to have flavonoid-7-O-glycosyltransferase activity; AtUGT89C1 and PaUGT1 are predicted to have 3-O- or 5-O-glycosylation activity; and EdF7GAT is predicted to be preferentially catalyzed for the glycosylation of anthocyanin B ring. Homology modeling shows that CiUGT31 has a wide active pocket entrance and contains multiple hydrophobic residues, which is conducive to the entry of gossypol nucleus. The layout of key residues (such as histidine and aspartic acid) near its 7- and 8-hydroxyl groups suggests that it may have dual catalytic site preference.

[0093] Example 2: Verification of glycosyltransferase function in cotton

[0094] Since cotton contains gossypol, and the glycosyltransferase bacteria we screened were of plant origin, we attempted to use cotton as a host for preliminary functional gene verification. In this embodiment, we screened for functional glycosyltransferases by transiently expressing candidate UGT genes in cotton and then analyzing them using liquid chromatography-mass spectrometry (LC-MS / MS).

[0095] Materials and Methods:

[0096] 1. Construction of expression vectors: The UGT gene screened in Example 1 was subcloned into a transient expression vector driven by the 35S promoter (067 vector [pCAMBIA1300-Ubi-Nos]) to construct expression vectors for CiUGT11, CiUGT123, CiUGT29, CiUGT31, CiUGT32, AtUGT89C1 and PaUGT1.

[0097] 2. Instantaneous transformation of cotton:

[0098] (1) Select upland cotton (Gossypium hirsutum.L.) plants in good growth condition;

[0099] (2) The UGT expression vector was transiently introduced into the leaf injection method mediated by Agrobacterium tumefaciens;

[0100] (3) After being kept away from light for 24 hours, the leaves were placed under normal light for 72 hours, and then the leaves were taken for further testing.

[0101] (4) After the leaves are removed, they should be immediately placed in liquid nitrogen for quick freezing and stored in an ultra-low temperature freezer at -80℃ for later use;

[0102] 3. Extraction and analysis of metabolites:

[0103] (1) Extract the metabolites from the leaves using a 70% methanol aqueous solution;

[0104] (2) The sample was filtered through a 0.22 μm filter membrane and then analyzed by a liquid chromatography-mass spectrometry (LC-MS / MS) system;

[0105] 4. Detection of target metabolites:

[0106] (1) Using gossypetin as a reference substrate, the focus is on detecting its monosaccharide or polysaccharide modified products;

[0107] (2) Screening out key enzymes that can catalyze the glycosylation of gossypol in vivo.

[0108] 5. Preliminary functional verification results are as follows:

[0109] Eight candidate genes were transiently expressed in cotton cotyledons, and metabolomics analysis was performed by LC-MS. The results showed that all candidate genes could successfully catalyze the production of corresponding glucosinolate products from gossypol, preliminarily verifying that they could produce gossypol-3-O-glucoside, gossypol-7-O-glucoside, or gossypol-8-O-glucoside, confirming their basic catalytic activity.

[0110] In summary, through the comprehensive application of phylogenetic analysis, sequence similarity comparison, conserved domain identification, three-dimensional structural simulation, and transient expression validation, we successfully screened a UGT candidate gene library containing eight members with the potential to catalyze gossypol glycosylation. Notably, the bioinformatics predictions and preliminary functional validation results showed a high degree of consistency, particularly in the successful prediction of the 7-O-glycosylation activity of genes such as CiUGT31. This lays a solid foundation for subsequent precise functional identification and combinatorial optimization in multi-module yeast engineered strains.

[0111] Example 3: Metabolomics Analysis Validation

[0112] This embodiment utilizes non-targeted metabolomics technology to further verify the changes in metabolites in plants after UGT gene expression and confirm its catalytic activity.

[0113] 1. Dry sample extraction

[0114] (1) The biological samples were placed in a freeze dryer (Scientz-100F) and freeze-dried under vacuum for 63 h;

[0115] (2) Grind into powder using a grinder (MM 400, Retsch) (30 Hz, 1.5 minutes);

[0116] (3) Weigh 50 mg of sample powder using an electronic balance (MS105DM), add 1200 μL of 70% methanol-water internal standard extraction solution pre-cooled at -20℃ (if less than 50 mg, add 1200 μL of extraction solution for every 50 mg sample). The internal standard extraction solution is prepared by dissolving 1 mg of standard in 1 mL of 70% methanol-water to prepare a 1000 μg / mL standard stock solution, and further diluting the 1000 μg / mL stock solution with 70% methanol to prepare a 250 μg / mL internal standard solution.

[0117] (4) Vortex once every 30 minutes, each time lasting 30 seconds, for a total of 6 vortices;

[0118] (5) After centrifugation (12000 rpm, 3 minutes), aspirate the supernatant, filter the sample with a microporous membrane (0.22 μm pore size), and store it in a sample vial for UPLC-MS / MS analysis.

[0119] 2. Chromatographic and mass spectrometric acquisition conditions

[0120] The data acquisition instrument system mainly includes ultra-high performance liquid chromatography (UPLC) (ExionLC™ AD, https: / / sciex.com.cn / ) and tandem mass spectrometry (MS / MS).

[0121] Liquid phase conditions mainly include:

[0122] (1) Column: Agilent SB-C18 1.8 µm, 2.1 mm * 100 mm;

[0123] (2) Mobile phase: Phase A is ultrapure water (with 0.1% formic acid added), and Phase B is acetonitrile (with 0.1% formic acid added).

[0124] (3) Elution gradient: The proportion of phase B is 5% at 0.00 min, and increases linearly to 95% within 9.00 min and is maintained at 95% for 1 min. From 10.00 to 11.10 min, the proportion of phase B decreases to 5% and is balanced at 5% until 14 min.

[0125] (4) Flow rate 0.35 mL / min; column temperature 40°C; injection volume 2 μL.

[0126] Mass spectrometry conditions mainly include:

[0127] Electrospray ionization (ESI) was performed at 500°C; the ion spray voltage (IS) was 5500 V (positive ion mode) / -4500 V (negative ion mode); ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) were set to 50, 60, and 25 psi, respectively, and the collision-induced ionization parameter was set to high. QQQ scans were performed using MRM mode with the collision gas (nitrogen) set to medium. The DP and CE of each MRM ion pair were optimized through further declustering potential (DP) and collision energy (CE). A specific set of MRM ion pairs was monitored at each epoch based on the metabolites eluted within each epoch.

[0128] 3. Test Results

[0129] The validation results showed that the expression of all candidate UGT genes could promote the accumulation of various gossypol derivatives in cotton to varying degrees, indicating that these enzymes have the potential to participate in the synthesis of gossypol derivatives.

[0130] Example 4: Verification of the synthetic ability of recombinant Saccharomyces cerevisiae to synthesize gossypol derivatives

[0131] This embodiment constructs a recombinant Saccharomyces cerevisiae to catalyze the in vivo synthesis of gossypol and its glycoside derivatives—gossypol-7-O-glucoside and gossypol-8-O-glucoside (structural formulas are shown in [reference]). Figure 3 ).

[0132] The specific method is as follows:

[0133] The key genes in the quercetin-to-gossypol derivative pathway are shown in SEQ ID NO.1: CiUGT11, CiUGT123, CiUGT29, CiUGT31, CiUGT32, PaUGT1, AtUGT89C1, and GaF8H.

[0134] The gene encoding the gossypol derivative synthesis pathway was expressed through genome integration using Cre-LoxP technology, with promoter P gal1 and P gal7To control gene expression in the gossypol synthesis pathway, a gene expression cassette containing the target gene was introduced into *Saccharomyces cerevisiae*, and transformed using the lithium-ion-PEG (LiAc / PEG) method to construct an engineered strain with specific metabolic functions. The promoter P... gal1 and P gal7 Promoters Gal1 and Gal7 are co-induced by β-galactose and glucose.

[0135] Using the original yeast S. cerevisiae CEN PK2-1C as the starting strain, the gene integration frame fragment: 308aUP-P was introduced. gal7 -GaF8H -308aDOWN, strain GTO, which produces gossypol, was obtained as a control. Using the original yeast S. cerevisiae CEN PK2-1C as the starting strain, the gene integration frame fragment: 308aUP-P was introduced. gal7 -GaF8H-P gal1 -CiUGT11-308aDOWN、308aUP-P gal7 -GaF8H-P gal1 -CiUGT123-308aDOWN、308aUP-P gal7 -GaF8H-P gal1 -CiUGT29-308aDOWN、308aUP-P gal7 -GaF8H-P gal1 -CiUGT31-308aDOWN、308aUP-P gal7 -GaF8H-P gal1 -CiUGT32-308aDOWN、308aUP-P gal7 -GaF8H-P gal1 -AtUGT89C1-308aDOWN, 308aUP-P gal7 -GaF8H-P gal1 -PaUGT1-308aDOWN, strains GT1, GT2, GT3, GT4, GT5, GT6, and GT7 with the potential to produce gossypol derivatives were obtained.

[0136] The results show that ( Figure 4The fermentation products of strains GT1, GT2, GT3, and GT4 all showed retention times and molecular weights consistent with the gossypol-7-O-glucoside standard, with yields of 3692.54, 6055.62, 1480.48, and 1170.47 μg / L, respectively. Meanwhile, the fermentation products of strains GT1, GT3, GT4, and GT5 also contained gossypol-8-O-glucoside standard products with retention times and molecular weights consistent, yields of 153.15, 351.35, 1117.12, and 486.49 μg / L, respectively. However, no product peaks corresponding to the gossypol-7-O-glucoside and gossypol-8-O-glucoside standards were detected in strains GT6 and GT7.

[0137] In summary, CiUGT11, CiUGT123, CiUGT29, and CiUGT31 can mediate the synthesis of gossypol-7-O-glucoside, while CiUGT11, CiUGT29, CiUGT31, and CiUGT32 can mediate the synthesis of gossypol-8-O-glucoside. A recombinant *Saccharomyces cerevisiae* strain with the potential to synthesize gossypol derivatives in an industrial fermentation system has been successfully constructed.

[0138] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A recombinant Saccharomyces cerevisiae strain, characterized in that, The recombinant Saccharomyces cerevisiae strain at least includes the following modifications: overexpression of flavone-8-hydroxylase encoding gene, and overexpression of glycosyltransferase encoding gene shown in any one of nucleotide sequences of SEQ ID NO. 1-5.

2. The recombinant Saccharomyces cerevisiae strain of claim 1, wherein, At least one of the following characteristics is included: (1) the nucleotide sequence of the flavone-8-hydroxylase encoding gene is shown in SEQ ID NO. 8; (2) the host strain of the recombinant Saccharomyces cerevisiae strain includes S. cerevisiae CEN PK2-1C MATa; ura3-52; trp1-289; leu2-3,112; his3Δ 1; MAL2-8C; SUC2; (3) the overexpression is free expression or integrated expression; (4) The flavone-8-hydroxylase-encoding gene and the glycosyltransferase-encoding gene are respectively expressed by the promoters P gal7 and the promoter P gal1 .

3. The recombinant Saccharomyces cerevisiae strain of claim 2, wherein, When integrated expression, the integration site includes 308a site on the genome.

4. Use of the recombinant Saccharomyces cerevisiae strain according to any one of claims 1 to 3 for the preparation of a gossypol derivative, characterized in that, The gossypetin derivatives include gossypetin-7-O-glucoside and gossypetin-8-O-glucoside.

5. Use according to claim 4, characterized in that, The gossypetin derivatives are prepared using quercetin as a substrate.

6. Use according to claim 4 or 5, characterized in that, When preparing gossypetin-7-O-glucoside, the recombinant Saccharomyces cerevisiae strain overexpresses flavone-8-hydroxylase encoding gene with nucleotide sequence shown in SEQ ID NO. 8 and glycosyltransferase encoding gene with nucleotide sequence shown in SEQ ID NO. 2; and / or, when preparing gossypetin-8-O-glucoside, the recombinant Saccharomyces cerevisiae strain overexpresses flavone-8-hydroxylase encoding gene with nucleotide sequence shown in SEQ ID NO. 8 and glycosyltransferase encoding gene with nucleotide sequence shown in SEQ ID NO. 4; and / or, when preparing gossypetin-7-O-glucoside and gossypetin-8-O-glucoside, the recombinant Saccharomyces cerevisiae strain overexpresses flavone-8-hydroxylase encoding gene with nucleotide sequence shown in SEQ ID NO. 8 and glycosyltransferase encoding gene with nucleotide sequence shown in any one of SEQ ID NO. 1, SEQ ID NO. 3 and SEQ ID NO.

4.

7. Use of a glycosyltransferase having a nucleotide sequence as set forth in any one of SEQ ID NO. 1, SEQ ID NO. 3 and SEQ ID NO. 4 in catalyzing the synthesis of a gossypoid from a gossypoid, characterized in that, The gossypetin derivatives include gossypetin-7-O-glucoside and gossypetin-8-O-glucoside.

8. Use of a glycosyltransferase having the nucleotide sequence shown in SEQ ID NO. 2 for catalyzing the synthesis of a cottonin derivative from cottonin, characterized in that, The gossypetin derivative is gossypetin-7-O-glucoside.

9. A system for synthesizing a gossypol derivative, comprising: It includes: (1) flavone-8-hydroxylase encoding gene with nucleotide sequence shown in SEQ ID NO. 8 and glycosyltransferase encoding gene with nucleotide sequence shown in SEQ ID NO. 2; or (2) flavone-8-hydroxylase encoding gene with nucleotide sequence shown in SEQ ID NO. 8 and glycosyltransferase encoding gene with nucleotide sequence shown in any one of SEQ ID NO. 1, SEQ ID NO. 3 and SEQ ID NO.

4.

10. A recombinant cell comprising the system of claim 9.

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