Gluconobacter engineering strain, construction method and application thereof

Abstract

The application provides a torulopsis pullulans engineering strain and a construction method and application thereof, and belongs to the field of microorganisms and food biotechnology. The application provides a torulopsis pullulans DL-XKX01 engineering strain with high ergothioneine production capacity. The RNA interference technology is used to inhibit the carotenoid synthesis pathway which competes with ergothioneine for substrates, so as to improve the ergothioneine production capacity. The ergothioneine level of the torulopsis pullulans DL-XKX01 provided by the application can reach 116.24+ / -4.33 mg / L in a flask fermentation, and can reach 858.18+ / -5.23 mg / L in a fermenter. The torulopsis pullulans DL-XKX01 can be used to improve the ergothioneine content in fermented food and improve the food quality, and has a good industrial application prospect.

Description

Technical Field

[0001] This invention relates to engineered strains of Rhodotorula glutinis, their construction methods, and applications, belonging to the fields of microbial genetic engineering and food biotechnology. Background Technology

[0002] Ergothioneine (EGT) is a unique natural antioxidant with powerful antioxidant, anti-inflammatory, and immunomodulatory functions. It cannot be synthesized by the human body and is mainly obtained through the consumption of specific foods such as edible fungi and animal liver.

[0003] Ergothioneine has received "Generally Recognized As Safe" (GRAS) certification from the U.S. Food and Drug Administration (FDA) and has been approved by the European Union for use in infant and pregnant women's foods. This certification demonstrates ergothioneine's broad application potential in organ transplantation, cell preservation, pharmaceuticals, food and beverages, functional foods, animal feed, cosmetics, and biotechnology. For example, literature (Pérez-Matute et al., 2023) reports that ergothioneine can effectively scavenge free radicals and reduce oxidative stress, thereby protecting cells from damage. Literature (Zhang et al., 2023) points out that ergothioneine has a positive impact on the prevention of chronic diseases such as cardiovascular diseases and neurodegenerative diseases. Literature (García et al., 2022) shows that ergothioneine significantly delays lipid oxidation and microbial growth, and enhances the sensory properties and nutrient retention of products. Currently, the market size of ergothioneine is rapidly expanding and is expected to reach US$171.9 million by 2028.

[0004] The main methods for synthesizing ergothioneine include chemical synthesis and microbial fermentation. While chemical synthesis offers high-efficiency production, its complex reaction steps and toxic reagents negatively impact the environment, and the difficult-to-remove byproducts reduce product purity and safety. In contrast, microbial fermentation, due to its environmental friendliness and sustainability, is gradually becoming the primary synthetic route. Furthermore, the application of genetically engineered strains has further improved ergothioneine production efficiency. By optimizing microbial metabolic pathways, substrate utilization and product synthesis capabilities are increased, thereby enhancing yield and purity while reducing production costs, opening up broader prospects for its application in the pharmaceutical and food industries.

[0005] Invention patent CN107250347B discloses an ergothioneine yield of 438 mg / L from a genetically engineered Aspergillus oryzae strain and an ergothioneine yield of up to 640 mg / L from a genetically engineered Escherichia coli strain; literature (BioRxiv, 2019: 667592) reports an ergothioneine yield of 630 mg / L from a metabolically engineered Saccharomyces cerevisiae; literature (Scientific Reports 2019, 9(1):1895-1895) reports the highest ergothioneine yield among genetically engineered bacteria to date: by continuously supplementing the precursor substance histidine, Escherichia coli underwent metabolic engineering, and after 216 h (9 d) of fermentation, the final ergothioneine yield reached 1.3 g / L. However, as an opportunistic pathogen and due to the toxicity limitations of the expression inducer IPTG (isopropyl-β-D-thiogalactoside), Escherichia coli and its transgenic strains cannot be used in traditional fermented foods, which makes the application prospects of their products worrying. Therefore, finding microorganisms with natural sources and food safety, modifying their metabolic pathways, and increasing their ergothioneine production are particularly important for achieving efficient and safe ergothioneine production. Summary of the Invention

[0006] To address this, the present invention employs genetic engineering technology to modify food-derived natural red yeast (Rhodotorula glutinis). Rhodotorula mucilaginous Genetic modification was performed to construct a high-yield ergothionein strain. Specifically, this invention includes the following technical solutions: This invention provides engineered Rhodotorula glutinis strains, which reduce the degradation of the original strain. Ministry of Interior Gene expression intensity; the Ministry of Interior The nucleotide sequence of the gene is shown in SEQ ID NO:16.

[0007] In one implementation, using red yeast ( Rhodotorula mucilaginosa DL-XSY01 is the starting strain; the preservation number of the Rhodotorula glutinis DL-XSY01 is CGMCC No. 23534, which has been disclosed in patent CN115322912B.

[0008] In one embodiment, the Ministry of Interior The nucleotide sequence of the gene is shown in SEQ ID NO: 16.

[0009] In one embodiment, the engineered strain of Rhodotorula glutinis is Rhodotorula glutinis DL-XKX01, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 64934; the deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0010] This invention also provides a method for increasing ergothioneine production in Rhodotorula glutinis, the method being to inhibit the production of ergothioneine in the Rhodotorula glutinis genome. Ministry of Interior Gene expression; the inhibition is achieved using an RNA interference vector containing the sequence shown in SEQ ID NO.3.

[0011] In one embodiment, the method includes: (1) Construct an expression vector containing a specific RNA interference sequence targeting the MVD gene of Rhodotorula glutinis; (2) The expression vector is transformed into Agrobacterium to obtain engineered Agrobacterium bacteria; (3) The Agrobacterium engineered strain is used to transform Rhodotorula glutinis, so that the RNA interference sequence is expressed in Rhodotorula glutinis, thereby inhibiting the transcription or translation of the MVD gene.

[0012] In one embodiment, the RNA interference vector is PZPK-pGPD-RmMVD-HSPt, containing the promoter pGPD shown in SEQ ID NO:18.

[0013] The present invention also provides a method for preparing ergothionein, wherein the engineered strain of Rhodotorula glutinis is fermented in a culture medium.

[0014] In one embodiment, the culture medium includes, but is not limited to, YPD culture medium.

[0015] In one implementation, glucose and histidine are fed during fermentation.

[0016] In one embodiment, the fermentation is carried out at 28-30°C.

[0017] The present invention also provides an antioxidant prepared using the engineered Rhodotorula glutinis strain.

[0018] In one embodiment, the antioxidant contains metabolites of the engineered Rhodotorula glutinis.

[0019] In one embodiment, the antioxidant is a crude extract of the fermentation supernatant of the engineered Rhodotorula glutinis after centrifugation and filtration.

[0020] The present invention also provides the application of the engineered Rhodotorula glutinis or the antioxidant in the food field.

[0021] In one implementation, the application includes, but is not limited to, participating in the fermentation of red wine, white wine, and cider.

[0022] In one embodiment, the application includes, but is not limited to, adding the antioxidant to fruit juice or flavoring.

[0023] Beneficial effects: (1) This invention uses genetic engineering to genetically modify natural red yeast rice, and uses RNA interference technology to reduce the expression of the MVD gene, thereby upregulating the expression levels of the key ergothioneine synthesis genes EGT1 and EGT2 by 3.43 times and 4.44 times, respectively. Fermentation verification showed that the intracellular ergothioneine yield of this strain reached 116.24 mg / L in shake flask fermentation, which was significantly higher than that of the wild type; and the ergothioneine yield reached 858.18±5.23 mg / L after 96 h of fermentation in a 5L fermenter.

[0024] (2) The modified food-derived Rhodotorula glutinis strain constructed in this invention R. mucilaginosa DL-XKX01 is sensitive to common fungal antibiotics and has good safety. It can be used as a fermentation strain in fermented food systems to increase the ergothioneine content and antioxidant properties of the product.

[0025] (3) The strain constructed in this invention uses the strain isolated from vinegar mash as the host, and has good alcohol and acid resistance, and is more adapted to the complex fermented food environment.

[0026] (4) The strain constructed in this invention has a strong free radical scavenging ability. When DL-XKX01 or its extract is added to various foods such as red wine, white wine, cider, fruit juice, soy sauce and vinegar, its ABTS and DPPH free radical scavenging abilities are significantly higher than those of the traditional preservative sulfur dioxide, and it can be used as an antioxidant for food safety.

[0027] (5) The present invention adds the constructed strain DL-XKX01 to alcoholic beverages, which can increase the brightness of red wine from 16.5 to 30.5.

[0028] In summary, the strain constructed in this invention has good food safety and the ability to improve the quality of fermented foods, and has good potential for industrial application.

[0029] Preservation of biological materials Red yeast rice ( Rhodotorula mucilaginosa DL-XKX01, categorized and named Rhodotorula mucilaginous It was deposited on August 1, 2024 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCCNo: 64934. The deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0030] Figure 1 This is a colony photograph of the YPD medium of Rhodotorula glutinis DL-XKX01 of this invention.

[0031] Figure 2This is a schematic diagram of the spectral structure of the knockdown vector (PZPK-pGPD-RmMVD-HSPt) constructed in this invention.

[0032] Figure 3 The yield of ergothionein during shake-flask fermentation of the starting strain DL-XSY01 and the genetically engineered strain DL-XKX01.

[0033] Figure 4 The yield of ergothionein during fed-batch fermentation of the starting strain DL-XSY01 and the genetically engineered strain DL-XKX01 in a 5-L bioreactor was determined. Detailed Implementation

[0034] The following embodiments are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. The present invention mainly describes the strains and the application ideas based on the strains. The substitution of simple parameters in the embodiments cannot be described one by one in the embodiments. Any other changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be regarded as equivalent substitutions. Any person skilled in the art should be covered within the scope of protection of the present invention within the scope of the technology disclosed in the present invention.

[0035] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field; unless otherwise specified, the reagents and materials used in the following embodiments are all commercially available.

[0036] All culture media used in this invention were prepared using conventional methods. Unless otherwise specified, the molecular biology operations involved in the examples refer to Sambrook J et al., eds., Science Press, 2002, Molecular Cloning: A Laboratory Manual (3rd Edition); or to the product instruction manual.

[0037] The culture medium used in the following examples was prepared as follows: YPD liquid culture medium: 20.0 g peptone, 10.0 g yeast extract, 20.0 g glucose, distilled water to 1L, adjust pH to 7.0, autoclave for 15 min.

[0038] YPD solid medium: 20.0 g peptone, 10.0 g yeast extract, 20.0 g glucose, 15 g agar, distilled water to 1 L, adjust pH to 7.0, autoclave for 15 min, and then pour into plates.

[0039] LB liquid medium: 10.0 g tryptone, 5.0 g yeast extract, 10.0 g sodium chloride, distilled water to 1 L, pH adjusted to 7.0, autoclave for 15 min.

[0040] LB solid medium: 10.0 g tryptone, 5.0 g yeast extract, 10.0 g sodium chloride, 15 g agar, distilled water to 1 L, pH adjusted to 7.0, autoclaved for 15 min, and then poured into plates.

[0041] His-YPD liquid culture medium: histidine 3.0 g, peptone 20.0 g, yeast extract 10.0 g, glucose 20.0 g, distilled water to 1 L, adjust pH to 7.0, autoclave for 15 min.

[0042] The raw materials used in the following examples are as follows: The commercial brewing yeast used was Angel Yeast Wine-Fruit Wine Special Yeast, the pectinase (30000 U / g) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., the soy sauce koji was Aspergillus oryzae Hu Niang 3.042, and the vinegar koji was provided by Zhenjiang Liu Hengji Food Co., Ltd.

[0043] The ergothioneine detection method involved in the following examples: Ergothioneine standard, purchased from Macklin.

[0044] The HPLC detection conditions were as follows: Agilent HPLC system 1260 Infinity II, Elite ODS-BP column, column temperature 40℃, mobile phase: A, ammonium dihydrogen phosphate (preparation method: weigh 1.1503 g ammonium dihydrogen phosphate, add 400 mL purified water, adjust pH to 5.0 with ammonia, then add 100 mL purified water); B, acetonitrile. A:B = 99:1, flow rate 1 mL / min, injection volume 10 μL, detection wavelength 258 nm.

[0045] Detection of extracellular ergothionein: The fermentation broth was centrifuged at 10,000 rpm for 4 min, the supernatant was collected, filtered through a 0.22 μm filter, and the sample was then analyzed by HPLC.

[0046] Intracellular ergothionein detection: The fermentation broth was centrifuged at 10,000 rpm for 4 min to collect the cells. 100 mg of wet cells was weighed and transferred to a clean 1.5 mL centrifuge tube. 1 mL of sterile water was added, the cells were vortexed, and then incubated at 90℃ for 30 min. After the incubation, the cells were centrifuged at 12,000 rpm for 10 min. The supernatant was collected, filtered through a 0.22 μm filter, and the sample was then analyzed by HPLC.

[0047] The following examples illustrate the methods for detecting carotenoids: Fermentation broth was subjected to 4000× g Centrifuge for 10 min, discard the supernatant, add 5 mL DMSO, and incubate at room temperature for 1 h to disrupt the cell wall. Then, centrifuge at 4000 × 10⁻⁶. g Centrifuge at room temperature for 10 min and collect the supernatant. Repeat the extraction three times, and combine the supernatants to obtain the pigment extract. The pigment extract has a maximum absorbance at 478 nm. The carotenoid content in the extract is calculated according to Formula 1: Carotenoid content (μg / g) = ( 478 ×V) / (0.16×W); In the formula A 478 ε is the absorbance of the sample at 478 nm, V is the volume of solvent added (mL), 0.16 is the absorption coefficient, and W is the cell dry weight (g).

[0048] The following examples illustrate the methods for determining the ABTS and DPPH free radical scavenging rates: ABTS free radical scavenging rate test: ABTS (14 mM) and potassium persulfate (5 mM) were dissolved in 0.1 M potassium phosphate buffer (pH 7.4) at a 1:1 ratio and reacted at 25°C for 12–16 h, and named the solution ABTS. 100 μL of bacterial culture DL-XKX01 (10 7 (CFU / mL) was added to 900 μL of ABTS solution and reacted in the dark at 25°C for 15 min. After centrifugation (14000×g, 10 min), the absorbance of the supernatant was measured at 734 nm.

[0049] Calculation of ABTS radical scavenging rate: ; Where A1 is the absorbance of 100 μL of the test bacterial culture with 900 μL of ABTS solution; A2 is the absorbance of 1 mL of ABTS solution.

[0050] DPPH free radical scavenging rate test: Dissolve 0.0078 g of DPPH in anhydrous ethanol and bring the volume to 100 ml to prepare a 0.2 mmol / L DPPH solution, named DPPH solution. Store in the dark and use immediately. The above 10 7CFU / mL DL-XKX01 bacterial culture was mixed with DPPH solution at a volume ratio of 1:1 and reacted in the dark at 25°C for 30 min. After centrifugation (14000×g, 1 min), the supernatant was collected and the absorbance was measured at 517 nm.

[0051] Calculation of DPPH free radical scavenging rate: ; Where A3 is the absorbance of 1 mL of the test bacterial solution plus 1 mL of DPPH solution; A4 is the absorbance of 1 mL of the test bacterial solution plus 1 mL of anhydrous ethanol; and A5 is the absorbance of 1 mL of PBS plus 1 mL of DPPH solution.

[0052] The following examples illustrate the methods for determining the color of fruit wines and juices: The CIELAB spatial method was used, with a D65 light source and a 10° observer, to evaluate the color rendering index. Wine samples were filtered through a 0.45 μm filter and placed in quartz cuvettes with a 2 mm optical path. Distilled water was used as a control. The visible light absorption spectrum of the wine in the 400–700 nm range was measured at 1 nm intervals using a wine colorimeter. Brightness, red / green channel, yellow / blue channel, and color saturation are expressed as follows: L *、 a *、 b *、 C * ab .

[0053] Example 1: Construction of the knockdown vector (PZPK-pGPD-RmMVD-HSPt) 1. Extraction of Rhodotorula glutinis genome Rhodotorula glutinis DL-XSY01 (accession number CGMCC No. 23534) is disclosed in patent publication number CN115322912B. The genome of Rhodotorula glutinis DL-XSY01 was extracted using the glass bead method described in "A Concise Guide to Molecular Biology Experiments" and stored at -20°C.

[0054] 2. Construction of the PZPK-pGPD-RmMVD-HSPt vector Target fragment amplification: R. mucilaginosa Using genomic DNA as a template, PCR amplification of the required positive strand of the RNAi hairpin fragment (SEQ ID NO:1, MVD-248 bp) was performed using the high-fidelity DNA polymerase Prime STAR MAX. Figure 2 ) and antisense strand (SEQ ID NO:2, MVD-203 bp, Figure 2Primers SEQ ID NO: 4 and SEQ ID NO: 5 were used to amplify the sense strand, and primers SEQ ID NO: 6 and SEQ ID NO: 7 were used to amplify the antisense strand. The composition of the 50 μL PCR system and the PCR program are shown in Table 1 and Table 2, respectively.

[0055] Table 1 PCR system

[0056] Table 2 PCR Procedure

[0057] Target fragment digestion: The PCR amplified fragment was recovered using the PCR product or a gel extraction kit, and then double-digested to separate the fragment and the vector. The digestion system was 100 μL: 10×H buffer, DNA amplified fragment ≤1 μg, 30-45 units of each of the two enzymes (EcoRV / NcoI, NcoI / SpeI, EcoRV / SpeI), and sterile water was added to bring the volume to 100 μL. Reaction conditions: 37°C, 2 h. The digested fragment was recovered. Post-digestion ligation: Fragment-to-fragment ligation system (60 μL): Equal molar mass of the two fragments, with the same volume of ligase solution II added, mixed, and then an equal volume of solution I added. The ligation procedure is shown in Table 3.

[0058] Table 3 Fragment Connection Conditions

[0059] The successfully ligated fragment was recovered after electrophoresis. The fragment-vector ligation system (10 μL): Solution I 5 μL, fragment: pZPK molar ratio 1:3, sterile water added to bring the volume to 10 μL. The ligation procedure was the same as in Table 3, but the endpoint temperature was changed to 4°C. The resulting MVD gene RNAi vector PZPK-pGPD-RmMVD-HSPt with the nucleotide sequence shown in SEQ ID NO: 3 was used for E. coli transformation.

[0060] Example 2: Preparation of Agrobacterium engineered strain (1) Preparation of Agrobacterium AGL1 competent cells According to the method described in the reference (Lin Xinping. Construction and application of genetic manipulation platform for oil-producing fungus *Rhodotorula glutinis* [D]. Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 2014.), competent *Agrobacterium* cells were prepared. Ice residue from the frozen *Agrobacterium* AGL1 strain was picked and inoculated into 10 mL of LB medium, and activated at 30°C and 200 rpm for 36 h. Then, it was inoculated into 10 mL of fresh LB medium at a ratio of 1% (v / v) and cultured for 20 h. Finally, it was transferred to 500 mL of fresh LB medium at a ratio of 1% (v / v) and cultured for 7-8 h until OD600 = 0.5-0.8. The cells were transferred to centrifuge cups, incubated on ice for 10 min, and then centrifuged at 4000×g, 4°C for 10 min, discarding the supernatant. The cells were then resuspended in 50 mL of ice-cold sterile water, aliquoted into 50 mL centrifuge tubes, centrifuged at 4000×g, 4°C for 10 min, and the supernatant was discarded. Resuspend the bacterial cells in 50 mL of ice-cold 10% (v / v) glycerol, centrifuge at 2500 rpm and 4°C for 10 min, and discard the supernatant. Add 1 mL of 10% (v / v) glycerol to each tube to resuspend the bacterial cells, aliquot 50 μL of competent cells into 1.5 mL centrifuge tubes, flash freeze in liquid nitrogen, and store at -80°C for later use.

[0061] (2) Plasmid transformation into Agrobacterium AGL1 Take 50 μL of Agrobacterium AGL1 competent cells and add 1 μL of correctly sequenced plasmid. Gently mix, incubate on ice for 10 min, then transfer the mixture to a 0.2 cm pre-chilled electroporation cuvette for electroporation transformation at 2.5 kV for 4-5 ms. Immediately after electroporation, add 1 mL of ice-cold LB medium and incubate at 30°C with shaking at 200 rpm for 1 h to rejuvenate. Take 100 μL of the rejuvenated bacterial culture and spread it on a plate containing Kans and Ampicillin, and incubate at 30°C for about 48 h until transformants grow.

[0062] Example 3: Agrobacterium-mediated transformation of Rhodotorula glutinis ( R. mucilaginosa ) Pick Rhodotorula glutinis DL-XSY01 from the activated agar plate and inoculate into 5 mL of YPD liquid medium. Incubate at 30°C and 200 rpm for 15–16 h. After washing with sterile water, measure the OD value, calculate the dilution factor, and adjust the OD. 600 nm The value was approximately 0.6. Simultaneously, the *Agrobacterium* engineered strain was picked from the activated plate and inoculated into 5 mL of LB medium (containing the antibiotics Amp / Kan), and cultured at 30°C and 200 rpm for 15-16 h. After washing once with sterile water, the OD value was measured, the dilution factor was calculated, and the OD value was adjusted accordingly. 600 nmThe concentration was approximately 0.6. Take 100 μL each of the diluted yeast and Agrobacterium suspensions, mix well, and directly spread onto an IM induction plate with a filter paper membrane, face up. Use yeast culture without Agrobacterium as a negative control. Incubate at 24°C for 2-3 days; a slightly reddish bacterial growth will appear on the filter membrane. Transfer the filter paper membrane from the IM plate directly to a YPD selection plate containing the corresponding resistance to the expression cassette, and incubate at 30°C until transformants appear. Name the correctly verified transformants *Rhodotorula glutinis* (Yeast). R. mucilaginosa DL-XKX01.

[0063] Example 4: Genetically engineered Rhodotorula glutinis ( R. mucilaginosa )DL-XKX01 Ministry of Interior , EGT 1. EGT 2-gene RT-qPCR validation (1) RNA extraction The bacterial strain was cultured in YPD liquid medium at 30°C and 200 rpm for 16–20 h. Two mL of the bacterial culture was centrifuged at 8000 × g for 5 min and then washed with ultrapure water. Glass beads were added, and the cells were disrupted using a FastPrep cell disruptor: shaking at 4 m / s for 1 min, followed by an ice bath for 1 min, repeated twice. RNA was extracted following the instructions of the Eastep Super Total RNA Extraction Kit.

[0064] (2) Reverse transcription PCR The extracted RNA was reverse transcribed according to the FastKing gDNA Dispelling RT SuperMix kit instructions. The reaction volume (20 μL) and reaction procedure are shown in Tables 4 and 5, respectively. Table 4 Composition of the reverse transcription system

[0065] After obtaining the cDNA product, the target gene sequence was amplified by RT-PCR using primers with sequences SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, with the GAPDH gene (SEQ ID NO: 19) as an internal control.

[0066] Table 5 Reverse transcription reaction conditions

[0067] 3. Quantitative Real-Time PCR Using the obtained cDNA product as a template, real-time PCR was performed according to the SuperReal PreMix Plus (SYBR Green) kit instructions. The reaction system (50 μL) and reaction procedure are shown in Tables 6 and 7, respectively. Table 6 Composition of the Real-Time PCR System

[0068] Table 7. Reaction conditions for quantitative real-time PCR

[0069] The results showed that in the genetically engineered strain DL-XKX01 constructed using RNA interference (RNAi) technology, the target gene ( Ministry of Interior The expression level of the strain (nucleotide sequence shown in SEQ ID NO:16) was significantly downregulated, decreasing by 52.35% compared to the wild-type strain, indicating a good effect of RNAi-mediated gene silencing. Meanwhile, EGT The expression level of gene 1 increased by 3.43 times. EGT The expression level of gene 2 increased by 4.44 times.

[0070] The above results indicate that Ministry of Interior Suppressing the expression of genes related to ergothionein synthesis promotes their expression. EGT 1 and EGT The expression for 2 is upregulated.

[0071] Example 5: Shake-flask fermentation of engineered Rhodotorula glutinis strain DL-XKX01 A single colony of *Rhodotorula glutinis* DL-XKX01 constructed in Example 3 was inoculated into 10 mL of YPD liquid medium (50 mL centrifuge tube) and cultured at 28°C and 200 rpm for 24 h to prepare a primary seed culture. The primary seed culture was then transferred to 50 mL of YPD liquid medium (250 mL Erlenmeyer flask) at a 10% (V / V) inoculation ratio and cultured at 28°C and 200 rpm for 24 h to prepare a secondary seed culture. The secondary seed culture was then transferred to 50 mL of YPD liquid medium (250 mL Erlenmeyer flask) and 50 mL of His-YPD liquid medium (250 mL Erlenmeyer flask) at a 10% (V / V) inoculation ratio, respectively, and cultured at 28°C and 200 rpm for 120 h. Each group was divided into three replicates. After culture, intracellular and extracellular ergothionein levels were measured.

[0072] The results showed that the carotenoid production of the genetically engineered strain DL-XKX01 decreased to 115.25±8.14 µg / g compared to the wild-type strain, while the ergothioneine production significantly increased, with intracellular ergothioneine production reaching 116.24±4.33 mg / L and extracellular ergothioneine production at 1.00±0.13 mg / L. (Original strain) R. mucilaginosa DL-XSY01 (CGMCC No. 23534, Ergothioneine High-Yielding Strains and Their Screening Methods and Applications CN115322912B) possesses the metabolic capacity to simultaneously synthesize ergothioneine and carotenoids. Carotenoid biosynthesis relies entirely on the mevalonate (MVA) pathway, with MVD, as a key terminal enzyme in this pathway, catalyzing the decarboxylation of mevalonate-5-bisphosphate (MVAPP) to generate isopentenyl pyrophosphate (IPP), providing the essential C5 unit precursor for carotenoid synthesis. Based on the results of quantitative real-time PCR in Example 4, specifically, ergothioneine production is increased by inhibiting the carotenoid synthesis pathway that competes with ergothioneine for substrates (FoodBioscience, 2023, 53: 102745). This regulatory mechanism may be related to the regulation of intracellular redox balance. Ministry of Interior Inhibition may lead to the accumulation of intermediate products of the MVA pathway (such as isopentenyl pyrophosphate) or changes in metabolic flux. These changes may affect the cellular redox sensing mechanism, thereby inducing the expression of genes related to EGT synthesis and ultimately increasing ergothionein production.

[0073] Example 6: Fermentation of engineered Rhodotorula glutinis strain DL-XKX01 in a fermenter and preparation of antioxidant crude extract. Ergothioneine fed-batch fermentation was conducted in a T&j-Atype 5L bioreactor. The initial culture medium was 3.0 L His-YPD liquid medium, with seed culture prepared according to the method in Example 5 at an initial inoculation ratio of 10% (v / v). Temperature, agitation, aeration, and pH were monitored and controlled using a D2MSNative system. The fermentation temperature was set at 28°C, the fermenter agitation speed at 600 rpm, dissolved oxygen coupling, dissolved oxygen control at 40%, air flow rate at 3 L / min, and pH control at 6.5 for a total fermentation time of 96 h. Feeding was performed between 24 and 72 h, with an automatic peristaltic pump adding 500 g / L glucose and 3 g / L histidine solution at a rate of 5 mL / h. Intracellular and extracellular ergothioneine levels were measured separately.

[0074] After fermentation, 2 L of fermentation broth was collected, centrifuged at 4000 rpm for 10 min to collect the cells, 100 mL of sterile water was added, the cells were vortexed and resuspended, and then incubated at 90℃ for 30 min. After incubation, the cells were centrifuged at 12000 rpm for 10 min, the supernatant was collected, and filtered through a 0.22 μm filter to obtain the crude antioxidant extract, which was used as an antioxidant for subsequent experiments.

[0075] The results showed that the genetically engineered red yeast DL-XXK01 of the present invention, fermented in His-YPD liquid medium and a 5L fermenter for 96 h, produced ergothioneine at a yield of 858.18±5.23 mg / L.

[0076] Table 8. Intracellular and extracellular ergothionein content of engineered Rhodotorula glutinis strain DL-XKX01

[0077] Example 7: Antibiotic susceptibility test of engineered Rhodotorula glutinis strain DL-XKX01 The obtained red yeast was constructed using the paper disc diffusion method. R. mucilaginosa The antibiotic susceptibility spectrum of DL-XKX01 was characterized.

[0078] Single colonies of DL-XKX01 were picked from YPD solid medium and transferred to YPD liquid medium for culture to obtain a bacterial suspension (approximately 1 × 10⁻⁶). 7 (CFU / mL); 200 μL of DL-XKX01 bacterial suspension was spread on YPD solid medium, and antimicrobial susceptibility test discs for five antibiotics, namely fluconazole (25 μg), ketoconazole (15 μg), miconazole (10 μg), voriconazole (1 μg), and itraconazole (10 μg), were placed on the medium, with a spacing of not less than 24 mm between each disc; the medium with the discs was incubated at 28℃ for 24 h, and the diameter of the inhibition zone was measured and counted to analyze its resistance (R ≤ 14 mm), intermediate I (14-20 mm) or sensitive S (≥ 20 mm). Each group was set up in triplicate. The antibiotic susceptibility of DL-XKX01 is shown in Table 9.

[0079] Table 9. Antibiotic susceptibility analysis of engineered Rhodotorula glutinis strain DL-XKX01

[0080] The results showed that Rhodotorula glutinis ( R. mucilaginosa DL-XKX01 is sensitive to five common fungal antibiotics and does not exhibit antibiotic resistance, indicating good strain safety.

[0081] Example 8: Application of engineered Rhodotorula glutinis strain DL-XKX01 in red wine fermentation The fermentation of red wine is carried out in accordance with GB / T 23543-2009 Good Manufacturing Practices for Wine Enterprises. Fresh Pinot Noir grapes are used as raw materials. After harvesting, sorting, destemming, crushing, and pressing, secondary seed liquid yeast (…) is added… R. mucilaginous DL-XKX01 engineered strain at 10 7 CFU / mL and commercial brewing yeast were simultaneously inoculated into the grape juice at a ratio of 0.2% (w / w) and fermented for 14 days. After aging, clarification, filtration, and sterilization, the wine was produced. Subsequent tests were conducted using samples from the fermentation endpoint. During the clarification stage, pectinase (4 mg / kg) was added and the wine was clarified at 22°C for 48 hours. This product was named Example 8.

[0082] Example 9: Application of engineered Rhodotorula glutinis strain DL-XKX01 in white wine fermentation The fermentation of white wine was carried out in accordance with GB / T 23543-2009 Good Manufacturing Practices for Wine Enterprises. Fresh Grenache grapes were used as raw material; after harvesting, sorting, destemming, crushing, and pressing, secondary seed liquid yeast (…) was added… R. mucilaginous DL-XKX01 engineered strain at 10 7 CFU / mL and commercial brewing yeast were simultaneously inoculated into the grape juice at a ratio of 0.2% (w / w) and fermented for 14 days. After aging, clarification, filtration, and sterilization, the wine was produced. Subsequent tests were conducted using samples from the fermentation endpoint. During the clarification stage, pectinase (4 mg / kg) was added and the wine was clarified at 18°C ​​for 24 hours. This product was named Example 9.

[0083] Example 10: Application of Rhodotorula glutinis engineered strain DL-XKX01 in cider fermentation Cider fermentation was conducted according to NY / T 4707-2025, Technical Specifications for Fruit Wine Processing. Fresh Fuji apples were used as raw material; after washing, crushing, and juicing, pectinase (10 mg / kg) was added, and the mixture was clarified at 50°C for 4 hours. Subsequently, it was fermented according to a 10... 7 Inoculate secondary seed culture with Rhodotorula glutinis at a ratio of CFU / mL. R. mucilaginosa The DL-XKX01 engineered strain was pasteurized in apple juice, and then commercial brewing yeast was added to the apple juice at a ratio of 0.2 g / L. Fermentation was carried out at 20°C for 7 days. After clarification and sterilization, cider was produced. Subsequent tests were conducted using samples from the fermentation endpoint. The above product was named Example 10.

[0084] Example 11: Application of antioxidants from engineered Rhodotorula glutinis strain DL-XKX01 in apple juice processing Apple juice was prepared according to GB / T 31121-2014, the standard for fruit and vegetable juices and their beverages. Fresh Guoguang apples were used as raw material. After harvesting, sorting, washing, crushing, and juicing, pectinase (10 mg / kg) was added, along with an antioxidant containing ergothioneine prepared in Example 6 at a ratio of 1% (v / v). The mixture was clarified at 50°C for 4 h. After pasteurization, the apple juice was prepared, and subsequent tests were performed using samples from the fermentation endpoint. The obtained sample was named Example 11.

[0085] Example 12: Application of Rhodotorula glutinis engineered strain DL-XKX01 in pear juice processing Pear juice was prepared according to GB / T 31121-2014 standard for fruit and vegetable juices and their beverages. Fresh Akizuki pears were used as raw material; after harvesting, sorting, washing, crushing, and juicing, pectinase (10 mg / kg) was added and mixed with water according to a 10... 7 Inoculate secondary seed culture with Rhodotorula glutinis at a ratio of CFU / mL. R. mucilaginosa The DL-XKX01 engineered strain was clarified at 50°C for 4 hours. After pasteurization, pear juice was prepared, and samples from the fermentation endpoint were used for subsequent tests. The obtained sample was named Example 12.

[0086] Example 13: Application of Rhodotorula glutinis engineered strain DL-XKX01 in soybean paste Fermentation of soybean paste is carried out according to GB / T 24399-2009. Process flow: Soaking soybeans → Steaming → Cooling → Draining → Crushing → Inoculating with soy sauce koji → Making paste blocks → Making koji → Forming koji → Washing paste blocks → Cutting into small pieces → Adding salt to a final concentration of 10% → Stirring and skimming off foam → Pressing 10 8 CFU / mL viable count was inoculated into secondary seed culture of Rhodotorula glutinis (CFU / mL) R. mucilaginous The DL-XKX01 engineered strain was used for fermentation, packaging, sterilization, and final product processing. After fermentation, the flavor compound content, biogenic amine content, and E. coli content of the finished product were determined, and samples from the fermentation endpoint were used for subsequent testing.

[0087] Example 14: Application of Rhodotorula glutinis engineered strain DL-XKX01 in Zhenjiang vinegar The fermentation of Zhenjiang vinegar is carried out according to GB / T 18623-2011, a geographical indication product. Using glutinous rice as the raw material, after washing, soaking, steaming, and saccharification, traditional commercially available vinegar starter is added to the glutinous rice at a ratio of 0.2% (w / w), and then fermented at a ratio of 10... 8 Add the brewer's yeast solution at a ratio of CFU / mL, and at 10... 8 Inoculate secondary seed culture with Rhodotorula glutinis at a ratio of CFU / mL. R. mucilaginousThe DL-XKX01 engineered strain was fermented for 30 days, and after sterilization, it was made into Zhenjiang vinegar. Subsequent tests were conducted using samples from the fermentation endpoint. The above product was named Example 14.

[0088] Comparative Example 1: Application of Sulfur Dioxide in Red Wine Fermentation The engineered Rhodotorula glutinis strain DL-XKX01 in Example 8 was replaced with potassium metabisulfite at a final concentration of 20 mg / L, and other conditions were the same as in Example 8.

[0089] Comparative Example 2: Application of Rhodotorula glutinis DL-XSY01 in Red Wine Fermentation The engineered Rhodotorula glutinis strain DL-XKX01 in Example 8 was replaced with Rhodotorula glutinis strain DL-XSY01, and other conditions were the same as in Example 8.

[0090] Comparative Example 3: Application of Sulfur Dioxide in White Wine Fermentation The engineered Rhodotorula glutinis strain DL-XKX01 in Example 9 was replaced with 20 mg / L potassium metabisulfite, and other conditions were the same as in Example 9.

[0091] Comparative Example 4: Application of Rhodotorula glutinis DL-XSY01 in white wine fermentation The engineered strain DL-XKX01 of *Rhodotorula glutinis* in Example 9 was replaced with *Rhodotorula glutinis*. R. mucilaginous The DL-XSY01 strain was used, and other conditions were the same as in Example 9.

[0092] Comparative Example 5: Application of Sulfur Dioxide in Cider Fermentation The engineered Rhodotorula glutinis strain DL-XKX01 in Example 10 was replaced with 20 mg / L potassium metabisulfite, and other conditions were the same as in Example 10.

[0093] Comparative Example 6: Application of Rhodotorula glutinis DL-XSY01 in Cider Fermentation The engineered Rhodotorula glutinis strain DL-XKX01 in Example 10 was replaced with Rhodotorula glutinis strain DL-XSY01, and other conditions were the same as in Example 10.

[0094] Comparative Example 7: Application of Sulfur Dioxide in Apple Juice Processing The antioxidant containing ergothioneine prepared from Rhodotorula glutinis strain DL-XKX01 in Example 11 was replaced with 20 mg / L potassium metabisulfite, and other conditions were the same as in Example 11.

[0095] Comparative Example 8: Application of Rhodotorula glutinis DL-XSY01 in Apple Juice Processing The antioxidant containing ergothioneine prepared from Rhodotorula glutinis strain DL-XKX01 in Example 11 was replaced with Rhodotorula glutinis strain DL-XSY01, and other conditions were the same as in Example 11.

[0096] Comparative Example 9: Application of Sulfur Dioxide in Pear Juice Processing The engineered Rhodotorula glutinis strain DL-XKX01 in Example 12 was replaced with 20 mg / L potassium metabisulfite, and other conditions were the same as in Example 12.

[0097] Comparative Example 10: Application of Rhodotorula glutinis DL-XSY01 in pear juice processing The engineered Rhodotorula glutinis strain DL-XKX01 in Example 12 was replaced with Rhodotorula glutinis strain DL-XSY01, and other conditions were the same as in Example 12.

[0098] Comparative Example 11: Application of Rhodotorula glutinis DL-XSY01 in Soybean Paste Fermentation The engineered Rhodotorula glutinis strain DL-XKX01 in Example 13 was replaced with Rhodotorula glutinis strain DL-XSY01, and other conditions were the same as in Example 13.

[0099] Comparative Example 12: Application of Rhodotorula glutinis DL-XSY01 in the fermentation of Zhenjiang vinegar The engineered Rhodotorula glutinis strain DL-XKX01 in Example 14 was replaced with Rhodotorula glutinis strain DL-XSY01, and other conditions were the same as in Example 14.

[0100] The samples prepared in the examples and comparative examples were tested, specifically including: 1. Determination of ABTS and DPPH free radical scavenging rates Samples from Examples 8-14 and Comparative Examples 1-12 at the fermentation endpoint were measured. The ABTS free radical scavenging rate results are shown in Table 10, and the DPPH free radical scavenging rate results are shown in Table 11.

[0101] Table 10 Results of ABTS free radical scavenging rate

[0102] Table 11 Results of DPPH free radical scavenging rate

[0103] The results showed that the ABTS free radical scavenging rates in various fermented foods with added Rhodotorula glutinis engineered strain DL-XKX01 were as follows: 94.91±1.59% in red wine, 73.38±4.41% in white wine, 58.34±1.94% in cider, 65.04±2.32% in apple juice, 60.23±1.31% in pear juice, 79.02±1.17% in soybean paste, and 84.27±2.74% in Zhenjiang vinegar. The DPPH free radical scavenging rates of various fermented foods with added Rhodotorula glutinis engineered strain DL-XKX01 were as follows: 89.85±5.49% in red wine, 89.48±1.98% in white wine, 79.04±5.37% in cider, 63.63±4.93% in apple juice, 48.38±2.47% in pear juice, 76.47±4.73% in soybean paste, and 82.38±1.71% in Zhenjiang vinegar. This indicates that Rhodotorula glutinis engineered strain DL-XKX01 can impart good antioxidant activity to fermented foods.

[0104] 2. Determination of dissolved oxygen content The dissolved oxygen content of samples from Examples 8-14 and Comparative Examples 1-12 at the fermentation endpoint was measured, and the results are shown in Table 12.

[0105] Table 12 Dissolved Oxygen Content

[0106] The results showed that the addition of *Rhodotorula glutinis* (Yeast) R. mucilaginosa The dissolved oxygen content of strain DL-XKX01 was 6.0±0.2 mg / L in red wine, 5.9±0.1 mg / L in white wine, 10.6±0.1 mg / L in cider, 15.7±0.1 mg / L in apple juice, 15.4±0.2 mg / L in pear juice, 0.4±0.0 mg / L in soybean paste, and 0.4±0.0 mg / L in Zhenjiang vinegar. This indicates that Rhododendron mucilaginosa DL-XKX01 has good antioxidant activity to maintain product quality stability.

[0107] (3) Colorimetric determination The colorimetric results of samples from Examples 8-14 and Comparative Examples 1-12 at the fermentation endpoint are shown in Table 13.

[0108] Table 13 Colorimetric parameters

[0109] The results showed that the L* value decreased (from 30.5±2.5 to 16.5±2.0 for red wine) while the C*ab value increased (from 22.3±2.5 to 26.6±2.7 for Zhenjiang vinegar), indicating decreased brightness, increased saturation, and a richer overall color. Fermented alcoholic beverages and vinegar products showed the most significant changes, while white wine and pear juice maintained relatively lighter colors. This demonstrates the effectiveness of adding red yeast rice (…). R. mucilaginous The DL-XKX01 strain has the best color-protecting effect, with the addition of red yeast ( ). R. mucilaginosa The DL-XSY01 strain was the second best, and both were superior to the group with added sulfur dioxide.

[0110] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Engineered Rhodotorula glutinis, characterized in that, The strain was reduced based on the original strain. MVD Gene expression intensity; the MVD The nucleotide sequence of the gene is shown in SEQ ID NO:

16.

2. The engineered Rhodotorula glutinis strain according to claim 1, characterized in that, With red yeast ( Rhodotorula mucilaginosa DL-XSY01 is the starting strain; the preservation number of the Rhodotorula glutinis DL-XSY01 is CGMCC No. 23534.

3. Rhodotorula glutinis DL-XKX01 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 1, 2024, with accession number GDMCC No: 64934.

4. A method for increasing the yield of ergothionein in Rhodotorula glutinis, characterized in that, Inhibit the genome of Rhodotorula glutinis MVD Gene expression; the inhibition is achieved using an RNA interference vector containing the sequence shown in SEQ ID NO.

3.

5. The method according to claim 4, characterized in that, The method includes: (1) Construct an expression vector containing a specific RNA interference sequence targeting the MVD gene of Rhodotorula glutinis; (2) The expression vector is transformed into Agrobacterium to obtain engineered Agrobacterium bacteria; (3) The Agrobacterium engineered strain is used to transform Rhodotorula glutinis, so that the RNA interference sequence is expressed in Rhodotorula glutinis, thereby inhibiting the transcription or translation of the MVD gene.

6. A method for preparing ergothioneine, characterized in that, Ferment the engineered Rhodotorula glutinis according to any one of claims 1 to 2 or the Rhodotorula glutinis DL-XKX01 according to claim 3 in a culture medium.

7. The method according to claim 6, characterized in that, During fermentation, glucose and histidine are added as feed.

8. The antioxidant prepared using the Rhodotorula glutinis DL-XKX01 according to claim 3, characterized in that, The antioxidant contains metabolites of the engineered Rhodotorula glutinis.

9. The application of the engineered Rhodotorula glutinis strain according to any one of claims 1 to 2, the Rhodotorula glutinis DL-XKX01 according to claim 3, or the antioxidant according to claim 8 in the food field.

10. The application according to claim 9, characterized in that, The applications include, but are not limited to, participating in the fermentation of red wine, white wine, and cider, or preparing fruit juice or condiments.

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