Production method of tryptophol and new application of tryptophol

Abstract

The invention discloses a production method and novel application of tryptophol. Streptomyces longsheng and candida albicans are co-cultured on a solid culture medium to obtain tryptophol. The compound has remarkable anti-tumor activity, IC50 of the compound to human lung cancer cells, mouse colon cancer cells, human liver cancer cells and human breast cancer cells is 154.5 mu g / mL, 144.3 mu g / mL, 122.6 mu g / mL and 110.7 mu g / mL respectively, and meanwhile the compound has important value as a key intermediate of multiple drugs. According to the method, the tryptophol is produced by utilizing a microbial synthesis method, the yield of the tryptophol is increased, the tryptophol is successfully expressed by introducing a tryptophol synthesis gene into escherichia coli C41, and the tryptophol yield is increased to 149.76 mg / L after a tryptophol synthesis path is further optimized. According to the invention, microbial co-culture and genetic engineering technologies are combined for tryptophol synthesis, and the method has profound significance for producing tryptophol through microbial fermentation and promoting the conversion of tryptophol synthesis to a green and energy-saving direction.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to a method for producing croterol and its novel uses. Background Technology

[0002] Bacteria (mainly actinomycetes) and fungi possess a vast library of natural products. These microbial natural products, as a key source of novel bioactive small molecule drugs, have immense development and application value, finding wide application in clinical, agricultural, and livestock fields. With the rapid development of third-generation whole-genome sequencing technology, researchers have discovered the powerful potential of microbial communities to synthesize a variety of secondary metabolites. Genome mining technology has further revealed numerous uncharacterized or unexpressed "silent" metabolic pathways in bacteria and fungi; their products are termed silent secondary metabolites. As the number of newly discovered antibiotics decreases and bacterial resistance increases, these pathways are gradually becoming a valuable resource for discovering new drugs. [1] Therefore, activating microbial secondary metabolic pathways has become a core task in discovering novel secondary metabolites.

[0003] Microbial co-culture is an effective approach for discovering bioactive natural products, and its application in evaluating product activity and increasing the yield of valuable products is of significant scientific importance. In natural environments, the ecological relationships among microorganisms are complex, involving various mechanisms such as coexistence, mutualism, symbiosis, and antagonism. Standard laboratory cultures often overlook this complexity, leading to reduced yields of secondary metabolites. Co-culture technology, however, can simulate natural ecosystems and stimulate microorganisms to secrete bioactive metabolites, thus becoming a cutting-edge strategy for discovering new molecules. [2] Its activation mechanism is also quite complex, including: ① nutrient acquisition: diffused small molecules act as precursors or substrates to induce another strain to produce new metabolites; ② competition and signal transduction: exogenous molecules act as chemical defense mechanisms, producing antibiotics or signaling molecules to participate in the competition; ③ physical contact: direct intercellular contact can activate the expression of silent gene clusters, although the specific mechanism is still unclear. [3] .

[0004] Studies show that interactions between bacteria and fungi can be broadly categorized into three types: ① Bacterial-bacterial co-culture activates natural products: When Mycobacterium flavum and Streptomyces cerevisiae are co-cultured under iron-limited conditions, the competition for iron triggers the synthesis of actinorhodin in Streptomyces cerevisiae. [4] ② Fungal-fungus co-culture is an important source of novel bioactive metabolites. For example, Pham et al. used basidiomycetes... Phellinus orientoasiaticus and Xylodon flaviporus Co-culturing and targeted isolation yielded three novel sesquiterpenes (Cyclohumulanoid Sesquiterpenes) and five known analogues.[5] ③ The co-culture of fungi and bacteria is more complex. The co-culture of Fusarium tumefaciens and Streptomyces listeri on solid rice culture medium can induce the production of four novel naphthoquinone dimers (fusatricinones AD) and a new safflower pyranone derivative, dihydrolateropyrone. [6] . Summary of the Invention

[0005] The purpose of this invention is to provide a method for producing crcotol through microbial co-culture.

[0006] Another objective of this invention is to provide an engineered strain of *Escherichia coli* that produces croterol, its construction method, and its application in croterol fermentation production.

[0007] Another object of the present invention is to provide new uses for colorimetric alcohols.

[0008] To achieve the objective of this invention, in a first aspect, this invention provides a method for producing chromol through microbial co-culture, comprising co-culturing *Streptomyces longshengensis* and *Candida albicans*, wherein *Streptomyces longshengensis* (… Streptomyces longshengensis ) induces Candida albicans ( Candida albicans The silent chromolate synthesis gene in the body produces chromolate.

[0009] Furthermore, croterol was obtained by co-culturing Streptomyces lanceolata and Candida albicans on a solid culture medium.

[0010] Preferably, the solid culture medium is MS medium (mannitol 20 g / L, soybean powder 20 g / L, agar powder 1.5%-2% (w / w)).

[0011] Preferably, the *Streptomyces longshengensis* strain has the accession number CGMCC 4.1101, and the *Candida albicans* strain has the accession number CGMCC 2.4159. Both strains were purchased from the China General Microbiological Culture Collection Center (CGMCC).

[0012] Secondly, this invention provides engineered Escherichia coli that produces crcotol, derived from Candida albicans. Candida albicans The SC5314 croterol synthesis gene, after codon optimization, was introduced into E. coli via plasmid or integrated into E. coli through genetic engineering. Escherichia coli ) on chromosomes; Among them, from Candida albicans The SC5314's tryptol synthesis gene is aro9CA (XP_716545.2) aro10CA (XP_711076.1) and adh1CA (XP_721905.2).

[0013] Furthermore, the optimized genes aro9CA , aro10CA and adh1CA The nucleotide sequences are shown in SEQ ID NO:1-3, respectively.

[0014] Furthermore, the optimized genes aro9CA , aro10CA and adh1CA The cells were sequentially tandemly constructed into a pET expression vector and introduced into E. coli.

[0015] Preferably, the expression vector is pET28a.

[0016] Preferably, the starting strain is Escherichia coli C41.

[0017] Thirdly, the present invention provides the application of the engineered bacteria in the production of croterol through fermentation.

[0018] Fourthly, the present invention provides a method for producing croterol, wherein the engineered bacteria are used as fermentation bacteria, fermentation is carried out in a culture medium supplemented with tryptophan, and croterol is separated from the fermentation product.

[0019] Fifthly, the present invention provides a novel use of tryptol in the preparation of antitumor drugs.

[0020] The tumors mentioned include, but are not limited to, lung cancer, liver cancer, colon cancer, and breast cancer. For example, human lung cancer cells (A549), mouse colon cancer cells (MC38), human liver cancer cells (HepG2), and human breast cancer cells (MCF-7), etc.

[0021] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: This invention involves co-culturing *Streptomyces longshengensis* and *Candida albicans* to discover a novel product originating from a non-single, complex pathway. The product's structure was analyzed by mass spectrometry and nuclear magnetic resonance, identifying it as indole-3-ethanol (i.e., croterol). It exhibits significant antitumor activity, with an IC50 concentration against human lung cancer cells (A549), mouse colon cancer cells (MC38), human liver cancer cells (HepG2), and human breast cancer cells (MCF-7). 50The concentrations were 154.5 μg / mL, 144.3 μg / mL, 122.6 μg / mL, and 110.7 μg / mL, respectively, and the drug also holds significant value as a key intermediate in many pharmaceuticals. Considering the problems of high energy consumption and severe environmental pollution associated with chemical synthesis methods, this invention utilizes a microbial synthesis method to produce and increase the yield of tryptophan. The tryptophan synthesis gene was successfully introduced into *E. coli* C41, resulting in successful expression of tryptophan. Further optimization of the tryptophan production pathway and tryptophan synthesis significantly increased the tryptophan yield to 149.76 mg / L. This invention combines microbial co-culture and genetic engineering techniques for tryptophan synthesis, which has profound significance for the microbial fermentation production of tryptophan and for promoting its synthesis towards a green and energy-saving direction, providing theoretical and practical basis for industrial production and sustainable development. Attached Figure Description

[0022] Figure 1 The above are standard curves for chromols in a preferred embodiment of the present invention. A is a low-concentration standard curve for chromols; B is a high-concentration standard curve for chromols.

[0023] Figure 2 In a preferred embodiment of the present invention, a new product characteristic peak was obtained by co-culturing *Streptomyces longshengensis* and *Candida albicans*. A: Co-culture chromatogram of *Streptomyces longshengensis* and other bioactive detection bacteria such as *Candida albicans* using a binary method; B: HPLC detection and analysis of the co-culture product of *Streptomyces longshengensis* and *Candida albicans*.

[0024] Figure 3 This invention provides a preferred embodiment of the isolation, purification, and structural identification of a novel characteristic product from the co-culture of *Streptomyces longshengensis* and *Candida albicans*. A: Three rounds of isolation and purification process for preparing LSBN-280; B: Positive ion mode mass spectrometry of LSBN-280; C: LSBN-280... 1 H NMR spectral data; D: LSBN-280 13 C NMR spectral data.

[0025] Figure 4 This invention illustrates the inhibitory effects of different concentrations of crcotol on A549, MC38, HepG2, and MCF-7 cells in a preferred embodiment. AD: Concentrations of crcotol that inhibited the growth of A549 lung cancer cells, MC38 colon cancer cells, HepG2 liver cancer cells, and MCF-7 breast cancer cells, respectively; E: Antibacterial activity assay of crcotol.

[0026] Figure 5 This invention provides a preferred embodiment of the synthetic pathway of troponin and the construction of its heterologous expression vector. A: Biosynthetic pathway of troponin, with highly homologous synthases identified in Candida albicans marked in red; B: pET28a::P for expression in Escherichia coli. T7 -aro9CA-aro10CA-adh1CA Plasmid map.

[0027] Figure 6 This invention provides a preferred embodiment of the heterologous expression and yield enhancement of chromols. A: HPLC analysis of chromol expression in different *E. coli* strains; 3A represents the vector pET28a::P T7 -aro9CA-aro10CA-adh1CA B: HPLC analysis of tryptophan expression in different Escherichia coli strains and under different treatment methods; C: Effect of different concentrations of tryptophan and IPTG addition on tryptophan yield; D: Tryptophan yield at different fermentation volumes. Detailed Implementation

[0028] This invention involves co-culturing *Streptomyces longshengensis* and *Candida albicans* to discover novel products from non-single complex pathways. The structure of these products is analyzed using mass spectrometry and nuclear magnetic resonance data, and their antibacterial and antitumor activities are evaluated. The products are then heterologously expressed to enhance metabolic pathways and increase yield.

[0029] Specifically, this invention relates to the co-culture of Streptomyces longshengensis and Candida albicans to activate the production of a new characteristic peak, which was identified as chromol. Subsequently, its anti-tumor activity against human lung cancer, liver cancer, and breast cancer was studied, and a new method for producing chromol was provided, and its potential application value was discovered.

[0030] This invention involves co-culturing *Streptomyces longshengensis* and *Candida albicans*, and HPLC analysis revealed a newly activated product peak. Mass spectrometry and NMR confirmed that this product is derived from tryptophan in *Candida albicans*. Tryptophan, a widely distributed indole derivative, is closely linked to the tryptophan metabolic pathway and possesses numerous biological activities, including promoting sleep, inhibiting pathogenic fungal growth, and inducing apoptosis. It has broad application prospects in the pharmaceutical and food industries and is also an important intermediate for pharmaceutically active molecules. For example, the anti-inflammatory and analgesic drug etoposide, the tryptophan-based anti-migraine drug sumatriptan, and the anti-adrenergic drug indolamin are all synthesized using tryptophan as an intermediate. [7] Currently, the main synthesis route for tryptols is through chemical synthesis, which is highly polluting and energy-intensive. Therefore, there is a desire to increase their yield through microbial synthesis. *Escherichia coli* possesses advantages such as lower cost, simple cultivation, rapid growth, and ease of genetic manipulation, making it of significant application value in the field of biotechnology, especially in tryptol synthesis.

[0031] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0032] Unless otherwise specified, the percentage sign "%" used in this invention refers to mass percentage. However, the percentage of a solution, unless otherwise specified, refers to the number of grams of solute contained in 100 mL of solution.

[0033] The experimental strains, cells, and plasmids involved in the following examples: Escherichia coli JM109 is used for plasmid construction.

[0034] Escherichia coli C41 is used for croterol expression.

[0035] Longsheng Streptomyces Streptomyces longshengensis (Accession number CGMCC 4.1101) and Candida albicans Candida albicans (Accession number CGMCC 2.4159) Used for co-culture.

[0036] Human lung cancer cells (A549), mouse colon cancer cells (MC38), human liver cancer cells (HepG2), and human breast cancer cells (MCF-7) were used for the detection of croterol activity.

[0037] The plasmids involved in this invention are as follows:

[0038] Example 1: Co-culture of *Streptomyces longshengensis* and *Candida albicans*, and isolation, purification, and identification of the products.

[0039] I. Experimental Methods 1. Strains culture Co-culture conditions of *Streptomyces longshengensis* and *Candida albicans*: Single colonies of *Candida albicans* were inoculated into 3.5 mL of PDA medium (potato 200 g / L, glucose 20 g / L, where the potato was the filtrate) and incubated overnight at 37℃ and 220 r / min. The next day, the colonies were transferred to 10 mL PDA tubes for mass culture. *Streptomyces longshengensis* was activated on MS plates, and after confluent spores were observed, it was co-cultured with *Candida albicans* on MS medium (mannitol 20 g / L, soybean flour 20 g / L, agar powder 20 g / L). Large-scale co-culture fermentation conditions for product isolation and identification: Prepare 10 L of solid culture medium, using disposable petri dishes of 90 mm × 15 mm, pour 25 mL of solid culture medium into each plate, and prepare the seed liquid of Candida albicans and the spore suspension of Streptomyces lanceolatum. Take 20 μL of Candida albicans and spread it on one half of the MS solid plate, and take 50 μL of Streptomyces lanceolatum spore suspension and spread it on the other half. Incubate at 28℃ for 6-8 days.

[0040] Culture conditions for *E. coli* growth: 1% *E. coli* C41 was inoculated into a modified M9 medium (yeast extract 5 g / L, glucose 20 g / L, disodium hydrogen phosphate 6.78 g / L, potassium dihydrogen phosphate 3 g / L, ammonium chloride 1 g / L, sodium chloride 0.5 g / L, magnesium chloride 0.12 g / L, calcium chloride 0.11 g / L, vitamin B1 10 mg / L, 1 mL / L of 1000× trace metal solution (1000× trace metal solution consisted of H3BO3 2.86 g / L, MnCl·4H2O 1.81 g / L, ZnSO4·7H2O 0.222 g / L, NaMoO4·2H2O 0.39 g / L, CuSO4·5H2O 0.079 g / L, Co(NO3)2·6H2O 49.4 g / L). In a solution of mg / L, incubate at 37°C for 2-3 h. OD 600 When the concentration is 0.6-0.8, IPTG with a final concentration of 0.1-1 mM is added to induce the expression of the relevant protein.

[0041] 2. Product separation, purification and identification (1) Concentration and extraction: The fermentation product and culture medium were cut into small pieces and collected in a 3 L wide-mouth bottle. The same volume of ethyl acetate was added and ultrasonically extracted for 1 h. The crude extract was concentrated using a rotary evaporator. (2) Preliminary silica gel column separation: The crude extract was passed through a reduced pressure chromatography column (diameter: 40 mm, length: 300 mm, packing material: 200-300 mesh silica gel), and linear gradient elution was performed with acetonitrile aqueous solution at a concentration of 10% ~ 90% (v / v). Subsequently, the fraction containing the target compound was detected by HPLC and dried. (3) Semi-preparative column separation: Separation was performed using a semi-preparative HPLC column (ZOBAX SB-C18, 5 μm, 9.4 mm × 250 mm), with a flow rate of 3 mL / min, a column temperature of room temperature, and the injection volume was increased to the maximum volume according to the experimental conditions. The two HPLC separation conditions of the semi-preparative column are shown in Table 1. (4) Analytical column separation: The optimized conditions for the final purification of the fraction were 60 min, using a C18 analytical column, isocratic elution with 17% (v / v) reagent B, and a flow rate of 1 (5) NMR analysis: The obtained pure product was dissolved in deuterated reagent DMSO, transferred to an NMR tube, and data was collected using a nuclear magnetic resonance spectrometer. The structure was determined by high-resolution mass spectrometry (HRESI-MS), 1H and 13C NMR spectra.

[0042] Table 1. Linear elution ratios of acetonitrile and water during HPLC semi-preparative column separation.

[0043] 3. Quantitative detection of chromols and preparation of standard curves To obtain the optimal detection conditions for chromols, we explored various options and ultimately found the optimal HPLC detection conditions as follows: The organic phase was methanol:water = 50%:50% (v / v), the flow rate was 0.5 mL / min, the detection wavelength was 280 nm, and the column used was a Shimadzu C18 column (Shim-pack GIST C18, 5 μm, 4.6 x 150 mm). First, the standard was analyzed, and a peak eluted at approximately 10.50 min, showing good and stable peak shape, which was used for the subsequent determination of chromol content.

[0044] Precisely prepared 1 mg / mL chromol standard was diluted to different concentrations. To ensure greater accuracy in the standard curve determination for chromol quantification, two sets of standard curves were prepared: one for low concentrations and one for high concentrations. The low-concentration standard curves had chromol concentrations of 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, and 60 μg / mL, while the high-concentration standard curves had chromol concentrations of 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, and 600 μg / mL. Before loading, the standard was filtered through a 0.22 μm filter, and 10 μL was taken for HPLC analysis. The peak areas obtained after the analysis were used to plot and analyze the standard curve. If R... 2 A value >0.99 indicates that the standard curve is highly feasible and can be used to calculate the chromol content in subsequent experimental samples. The x-axis (X-axis, μg / mL) of the curve represents the chromol standard content, and the y-axis (Y-axis, mAU) represents the peak area. The chromol standard curve is shown below. Figure 1 As shown: The formula for the low-concentration croteric alcohol standard curve is: y = 47.839x + 6.5723, R0 2 =0.9904 The formula for the high-concentration crtanol standard curve is: y = 41.655x + 307.45, R0 2 =0.9988 II. Experimental Results 1. Determination of co-cultivation conditions We selected actinomycetes and bioactivity-detecting bacteria for co-culture on solid culture medium: 50 μL of freshly activated actinomycete spore suspension and 20 μL of bioactivity-detecting bacteria seed solution were respectively spread on both sides of an MS plate, each occupying half the area, and incubated at 28℃ for 6-8 days until the actinomycetes produced mature spores. After fermentation, the solid culture medium was cut into pieces, extracted with ethyl acetate, sonicated, filtered and concentrated, and then filtered through a 0.22 μm filter before HPLC analysis. We found that *Streptomyces longshengensis* and *Candida albicans* produced new characteristic peaks at 280 nm and 310 nm. After multiple repetitions, the new characteristic peak at 280 nm was the most stable. Figure 2 ).

[0045] 2. Structural identification of products co-cultured with Streptomyces longshengensis and Candida albicans To identify the structure of this compound, three large-scale fermentations were performed, each using approximately 10 L of solid culture medium, with each fermentation lasting 6-8 days. The solid culture medium was cut into small pieces and placed in large glass beakers or shake flasks, and extracted three times with ethyl acetate to submerge the solids. The extracts were combined and concentrated using a rotary evaporator. The compound was then purified three times by HPLC using a semi-preparative column and an analytical column. The difference peak observed under 280 nm UV absorption was named LSBN-280. The first and second rounds used a semi-preparative reversed-phase Zorbax SB C18 column (9.4 × 250 mm, 5 μm) at a flow rate of 3 mL / min. The third round of purification used an analytical Zorbax SB-C18 column (4.6 × 250 mm, 5 μm particle size). Finally, 2 mg of compound LSBN-280 was obtained. Figure 3 A). High-resolution liquid chromatography-mass spectrometry analysis revealed that the mass-to-charge ratio of compound LSBN-280 in positive ion mode ( m / z The result is: HRESI-MS m / z = 162.0896 [M+H] + ( Figure 3 B), its relative molecular mass is estimated to be 161.08. After dissolving compound LSBN-280 in the deuterated reagent dimethyl sulfoxide (DMSO), its proton nuclear magnetic resonance spectrum was measured (NMR spectrum). 1 H-NMR) Figure 3 C) Carbon nuclear magnetic resonance spectroscopy ( 13 C-NMR) Figure 3 The compound LSBN-280 was identified as tryptophol (TOL) using high-resolution mass spectrometry (HRMS) to resolve its two-dimensional structure.

[0046] Example 2: Activity Evaluation of Chromols To investigate the bioactivity of tryptol against bacteria, its antibacterial activity against Streptococcus pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, and Micrococcus luteus was tested. The results showed that no significant antibacterial activity was found in any of them.

[0047] Cancer cell culture and Cell Counting Kit-8 (CCK-8) assay for cytotoxicity: To investigate the inhibitory activity of tryptol on different cancer cells, we used the CCK-8 assay to detect its inhibitory effect on human lung cancer cells (A549), mouse colon cancer cells (MC38), human liver cancer cells (HepG2), and human breast cancer cells (MCF-7).

[0048] Cell Culture and Passaging: During cell resuscitation, A549, MC38, HepG2, and MCF7 cells were removed from the liquid nitrogen container and quickly thawed in a 37°C water bath while simultaneously preheating the corresponding culture medium. The cryovials were centrifuged at 1000 rpm for 3 min, the supernatant was discarded, and the cells were resuspended by pipetting with 1 mL of culture medium and transferred to a 25 cm³ culture tube. 2 Add 3-5 mL of the appropriate culture medium to each culture flask and incubate at 37℃ with 5% CO2 for 2-4 days. When passaged, when the cells have adhered to the wall and grown to more than 80%, wash them 2-3 times with PBS in a clean bench, add 0.05% trypsin solution and digest them in the incubator for 10-15 min. When a large number of cells detach and become round in the field of view, add an equal volume of culture medium to stop the trypsin effect. Collect the cell suspension in a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, and seed them into new culture dishes at a 1:3 ratio, and continue to incubate at 37℃ with 5% CO2. The main components of the A549 cell culture medium are DMEM (high glucose) + 10% FBS (premium fetal bovine serum) + 1% P / S (penicillin-streptomycin); the main components of the HepG2 cell culture medium are MEM + 10% FBS + 1% P / S; the main components of the MC38 cell culture medium are DMEM + 10% FBS + 1% MEM NEAA (non-essential amino acids) + 1% Sodium Pyruvate + 1% GlutaMAX-1 (L-glutamine) + 1% HEPES + 1% P / S; and the main components of the MCF-7 cell culture medium are MEM + 10% FBS + 1% P / S.

[0049] CCK-8 assay for cellular IC50 50Experiment: The CCK-8 assay is an important experimental method widely used for detecting cell proliferation and cytotoxicity. Its core principle is based on the water-soluble tetrazolium salt (WST-8) contained in the CCK-8 reagent. Under the catalysis of the electron carrier 1-methoxy-5-methylphenazineonium sulfate dimethyl ester (1-Methoxy PMS), WST-8 can be reduced by dehydrogenases in living cells to a highly water-soluble yellow formazan dye. The relative number of living cells can be reflected by measuring the absorbance at 450 nm using a microplate reader. After counting using an automated cell analyzer, the cell suspension is diluted to 5 × 10⁻⁶ cells / mL. 4 / mL or 1×10 5 100 μL of DMSO was added to each well of a 96-well plate and cultured at 37°C and 5% CO2 for 24 h for adherent cell culture. The platinum alcohol was prepared as an 80 mg / mL stock solution using DMSO and then serially diluted. 100 μL of different concentrations of platinum alcohol solution was added to each well to bring the final DMSO concentration to 0.5%. Cells were cultured for 48 h or 72 h depending on their growth rate. CCK-8 (containing WST-8) solution was added to each well, and the cells were cultured for another 30 min–1 h. The absorbance of each well was measured at 450 nm using a microplate reader to construct an IC50 assay. 50 curve.

[0050] Different concentrations of tryptol dissolved in DMSO were added to cultured A549, MC38, HepG2, and MCF-7 cells and incubated for a period of time. The inhibitory effect of tryptol on cell proliferation was then studied. The results are as follows: Figure 4 As shown, tryptol exhibited varying degrees of inhibitory effects on lung cancer cells and liver cancer cells, showing a dose-dependent effect. With increasing tryptol concentration, the cell viability of A549, MC38, HepG2, and MCF-7 cells all showed significant inhibitory effects, with IC50... 50 The values ​​were 154.5 μg / mL, 144.3 μg / mL, 122.6 μg / mL and 110.7 μg / mL, respectively, which shows potential application prospects in terms of antitumor activity.

[0051] Example 3: Biosynthetic pathway and heterologous expression of tryptophan. The tryptophan synthesis pathway consists of two parts: the tryptophan synthesis pathway and the tryptophan utilization pathway. In the first part, glucose is degraded via the central carbon metabolism pathway into phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P), which then condense to form 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP). DAHP then passes through the shikimic acid pathway to produce shikimic acid and cladonic acid, which subsequently enters the L-tryptophan metabolism pathway to generate tryptophan. In the second part, tryptophan enters the Ehrlich pathway and, catalyzed by aromatic amino acid transaminases (such as Aro8 and Aro9), is converted into indole-3-pyruvate. Indole-3-acetaldehyde (IAAld) is decarboxylated by pyruvate decarboxylases (such as Aro10, Pdc1, Pdc5, and Pdc6) to form indole-3-acetaldehyde (IAAld). Finally, through NAD(P)H-dependent reduction by various dehydrogenases (such as Adh1-5, Sfa1, and Aads), it is converted into chromol. [8] .

[0052] Homology comparisons were performed between the genes encoding key enzymes in the troponosynthetic pathway and the genomes of *Streptomyces longshengensis* and *Candida albicans*, respectively. The results showed that troponosynthetic acid is a metabolite produced by the induction of silent gene expression in *Candida albicans* by *Streptomyces longshengensis*, but the specific activation mechanism remains unclear. Table 2 shows the homology comparison results of key enzymes in the troponosynthetic pathway in *Candida albicans*. Since *Candida albicans* is a pathogenic bacterium, direct modification and yield enhancement within this bacterium are not feasible. Therefore, we selected *Escherichia coli*, which has a short generation time, simple genetic manipulation, and a simple background, for heterologous expression and yield enhancement. We selected a model strain with a complete genome sequence. Candida albicans Sequence alignment and analysis were performed on SC5314, first identifying the key gene for the production of crtanol in Candida albicans. aro9CA (XP_716545.2) aro10CA (XP_711076.1) and adh1CA (XP_721905.2) performs codon optimization (optimized gene) aro9CA , aro10CA and adh1CA The nucleotide sequences are shown in SEQ ID NO:1-3, respectively, and the expression vector pET28a::P is constructed. T7 - aro9CA - aro10CA - adh1CA(abbreviated as 3A) Figure 5 (A and B).

[0053] Carrier pET28a::P T7 - aro9CA - aro10CA - adh1CA The commonly used inducible T7 promoter of E. coli was employed, and the T7 promoter was combined with a transaminase-encoding gene derived from Candida albicans and optimized with codons. aro9CA Decarboxylase gene aro10CA and dehydrogenase encoding genes adh1CA By linking them together, the three enzymes can be co-expressed in E. coli.

[0054] Table 2. Homology comparison of key enzymes in the crombol synthesis pathway in Candida albicans.

[0055] This invention attempts to enhance tryptophan synthesis from various dimensions, including tryptophan synthesis pathway, tryptophan conversion pathway, IPTG induction concentration, exogenous tryptophan addition concentration, chassis cell selection, and compound extraction method. To test the optimal conditions for enzyme expression induction, different chassis strains, different tryptophan addition concentrations, and different IPTG induction concentrations were used. First, the constructed plasmid pET28a::P... T7 - aro9CA - aro10CA - adh1C The cells were transferred into Arctic, Rossetta, C41, and BL21 E. coli chassis cells, respectively. All of them produced troponin, and HPLC analysis showed that C41 and BL21 produced higher levels of troponin. Figure 6 A). Three different methods were used to treat the fermentation samples: the supernatant of the untreated fermentation broth, a sample after mixing methanol and fermentation broth at a 1:1 (volume ratio), and a sample after mixing ethanol and fermentation broth at a 1:1 (volume ratio). It was found that treatment with ethanol significantly increased the yield of *E. coli* C41. Figure 6 B). Subsequent yield improvement experiments will be conducted by comparing and screening E. coli C41 and BL21.

[0056] Considering that tryptophan production may be affected by the supply of its precursor tryptophan and the concentration of IPTG, thus influencing the expression of key proteins along the pathway, we used orthogonal experiments to explore optimal conditions by combining different concentrations of tryptophan (0.5 g / L, 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L) and IPTG (0.1 mM, 0.5 mM, 1 mM). Based on previous observations of the influence of *E. coli* itself on tryptophan expression, we used *E. coli* strain C41, which exhibited the highest production, for subsequent experiments. The strain was fermented in small quantities on 2 mL wells of liquid medium at 220 rpm and 30 °C. HPLC analysis showed that the tryptophan production capacity of strain C41 increased with increasing tryptophan concentration. No tryptophan was produced with 0 g / L tryptophan, but the production gradually increased with the addition of 0.5–2.5 g / L tryptophan. Similarly, tryptophan production increased with increasing IPTG concentration. When the tryptophan concentration was 2.5 g / L and the IPTG concentration was 1 mM, the tryptophan yield reached its maximum of 126.71 μg / mL. Figure 6 C). After expanded culture, under the same conditions, the tryptophan yield in 100 mL of culture medium could reach 149.76 μg / mL (C). Figure 6 D). This invention provides important guidance for the efficient synthesis of tryptophan in Escherichia coli and lays the foundation for the industrial production of tryptophan in microbial cell factories.

[0057] Tritonols have demonstrated significant application potential and commercial value across multiple fields. Possessing an indole ring, they belong to the indole derivative class and thus exhibit some of the properties of indole compounds. Studies have confirmed that indole compounds can combat drug-resistant cancer cells and pathogens, and possess various effects including inhibiting bacteria, fungi, and viruses, as well as antioxidant, anti-inflammatory, and anti-tumor properties. In the pharmaceutical field, triptonols and their analogues are common intermediates for many important pharmaceutical molecules. [7] Meanwhile, tryptol also has antibacterial activity. Schirmer et al. found that it can inhibit the production of cytokines TNFα and IFNγ induced by pathogens such as Candida albicans. [9] Elleuch et al.'s research also confirmed that tryptol has... Fusarium sp. and S. enterica ATCC43972 both have antibacterial effects.

[10] This invention also demonstrates its inhibitory effects on human liver cancer (HepG2), lung cancer (A549), breast cancer (MCF7), and mouse colon cancer cells (MC38), but it has no effect on mouse colon cancer cells (Caco2), indicating that its inhibitory effect on pathogens has relatively strict selectivity, and there is still much room for optimization in the inhibitory concentration it achieves on these cancer cells. As a natural small molecule, crromol has the advantages of low toxicity and easy structural modification. Its anti-cancer cell proliferation activity also supports its potential as a candidate molecule for anti-cancer drug development. It is believed that in the future, crromol as an intermediate or derivative of the parent nucleus structure will have more opportunities to enter clinical treatment and benefit human health.

[0058] The discovery of crantrol in this invention was achieved through the co-culture of *Streptomyces lanceolata* and *Candida albicans* on a solid culture medium. Its yield was higher than that obtained through liquid co-culture and more closely resembled the natural soil or its habitat. The mechanisms by which microbial interactions activate previously silent products can be diverse, such as signal molecule exchange or stress response. *Streptomyces lanceolata* provided certain precursors for crantrol production, and the competition between *Streptomyces lanceolata* and *Candida albicans* for limited resources stimulated the tryptophan metabolic pathway or the crantrol synthesis pathway. This invention further increased crantrol yield by optimizing the substrate and inducer ratio in *Escherichia coli* and enhancing the crantrol synthesis pathway, laying the foundation for subsequent process development. However, there is still significant room for improvement in the synthesis of crantrol in microorganisms, for example, by further enhancing tryptophan and crantrol synthesis pathways through CRISPR gene editing, adding substrates during production, and adjusting pH. Since crantrol and its important precursor indole-3-acetaldehyde are volatile, physical and chemical factors need to be considered during production to reduce volatility and enhance timeliness.

[0059] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

[0060] References: [1]BARAL B, AKHGARI A, METSÄ-KETELÄ M. Activation of microbialsecondary metabolic pathways: Avenues and challenges[J]. Synthetic andSystems Biotechnology, 2018, 3(3): 163-178. [2]WANG G, RAN H, FAN J, KELLER NP, LIU Z, WU F, YIN WB. Fungal-fungal cocultivation leads to widespread secondary metabolite alterationrequiring the partial loss-of-function VeA1 protein[J]. Science Advances,2022, 8: eabo6094. [3]PENG XY, WU JT, SHAO CL, LI ZY, CHEN M, WANG CY. Co-culture:stimulate the metabolic potential and explore the molecular diversity ofnatural products from microorganisms[J]. Marine Life Science & Technology,2021, 3(3): 363-374. [4]LEE N, KIM W, CHUNG J, LEE Y, CHO S, JANG KS, KIM SC, PALSSON B,CHO BK. Iron competition triggers antibiotic biosynthesis in Streptomyces coelicolor during coculture with Myxococcus xanthus [J]. The ISME Journal,2020, 14(5): 1111-1124. [5]PHAM HT, DOAN TP, KIM HW, KIM TW, PARK SY, KIM H, LEE M, KIM KH,OH WK, LIM YW, KANG KB. Cyclohumulanoid sesquiterpenes induced by thenoncompetitive coculture of Phellinus orientoasiaticus and Xylodon flaviporus [J]. Journal of Natural Products, 2022, 85(3): 511-518. [6]MOUSSA M, EBRAHIM W, BONUS M, GOHLKE H, MÁNDI A, KURTÁN T, HARTMANN R, KALSCHEUER R, LIN W, LIU Z, PROKSCH P. Co-culture of the fungus Fusarium tricinctum with Streptomyces lividans induces production of crypticnaphthoquinone dimers[J]. RSC Advances, 2019, 9(3), 1491–1500. [7]GUTMANN B, GOTTSPONER M, ELSNER P, CANTILLO D, ROBERGE DM, KAPPECO. On the fischer indole synthesis of 7-ethyltryptophol—mechanistic and process intensification studies under continuous flow conditions[J]. OrganicProcess Research & Development, 2013, 17(2): 294-302. [8] Wu Heng, Zhao Wei, Zhao Yang, Xiao Yongyin, Yang Liu, Qin Yunhua, Du Chao, Luo Yiyong, Gong Xiaowei. Research progress on biosynthetic pathway and molecular regulatory mechanism of tryptamine in Saccharomyces cerevisiae, Journal of Kunming University of Science and Technology (Natural Science Edition), 2024, 49: 137-146. WU H, ZHAO W, ZHAO Y, XIAO YY, YANG L, QIN YH, DU C, LUO YY, GONG XW.Research progress on the biosynthesis pathways and molecular regulatory mechanism of tryptophol in Saccharomyces cerevisiae [J]. Journal of KunmingUniversity of Science and Technology(Natural Science), 2024, 49(1): 137-146 (inChinese). [9]SCHIRMER M, SMEEKENS SP, VLAMAKIS H, JAEGER M, OOSTING M, FRANZOSAEA, TER HORST R, JANSEN T, JACOBS L, BONDER MJ, KURILSHIKOV A, FU J, JOOSTENLAB, ZHERNAKOVA A, HUTTENHOWER C, WIJMENGA C, NETEA MG, XAVIER RJ. Linkingthe human gut microbiome to inflammatory cytokine production capacity[J].Cell, 2016, 167(4): 1125-1136.

[10] ELLEUCH L, SHAABAN M, SMAOUI S, MELLOULI L,KARRAY-REBAI I,FOURATI-BEN FGUIRA L, SHAABAN KA, LAATSCH H. Bioactive secondary metabolitesfrom a new terrestrial Streptomyces sp. TN262[J]. Applied Biochemistry andBiotechnology, 2010, 162: 579-593.

Claims

1. A method for producing crombols through microbial co-culture, characterized in that, Streptomyces longshengensis and Candida albicans were co-cultured, and Streptomyces longshengensis ( Streptomyces longshengensis ) induces Candida albicans ( Candida albicans The silent chromolate synthesis gene in the body produces chromolate.

2. The method according to claim 1, characterized in that, Indomethacin was obtained by co-culturing Streptomyces lanceolata and Candida albicans on a solid culture medium; Preferably, the solid culture medium is MS medium.

3. The method according to claim 1 or 2, characterized in that, The accession number of the *Streptomyces longshengensis* is CGMCC4.1101, and the accession number of the *Candida albicans* is CGMCC 2.4159.

4. A engineered strain of *Escherichia coli* that produces cronol, characterized in that... Will originate from Candida albicans Candida albicans The SC5314 croterol synthesis gene, after codon optimization, was introduced into E. coli via plasmid or integrated into E. coli through genetic engineering. Escherichia coli ) on chromosomes; Among them, from Candida albicans The SC5314's tryptol synthesis gene is aro9CA , aro10CA and adh1CA .

5. The engineered bacteria according to claim 4, characterized in that, Optimized genes aro9CA , aro10CA and adh1CA The nucleotide sequences are shown in SEQ ID NO:1-3, respectively.

6. The engineered bacteria according to claim 5, characterized in that, Optimized genes aro9CA , aro10CA and adh1CA The components were sequentially tandemly constructed into a pET expression vector and introduced into E. coli. Preferably, the expression vector is pET28a.

7. The engineered bacteria according to any one of claims 4-6, characterized in that, The starting strain was Escherichia coli C41.

8. The application of the engineered bacteria according to any one of claims 4-7 in the production of croterol through fermentation.

9. A method for producing color alcohol, characterized in that, Using the engineered bacteria described in any one of claims 4-7 as the fermentation bacteria, fermentation is carried out in a culture medium supplemented with tryptophan, and tryptophan alcohol is separated from the fermentation product.

10. Novel applications of tryptophan in the preparation of antitumor drugs; The tumors include lung cancer, liver cancer, colon cancer, and breast cancer.

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