Saccharomyces cerevisiae engineering strain and application thereof
Patent Information
- Application Number
- CN202311197845.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Priority Date
- 2023-08-08
- Filing Date
- 2023-09-18
- Publication Date
- 2026-10-30
- Estimated Expiration
- 2043-09-18
AI Technical Summary
[0004]本发明的目的在于提供能够显著提升将柚皮素转化为二氢山奈酚效率的酵母工程菌及其构建方法，解决现有酿酒酵母不能以柚皮素为底物，合成二氢山奈酚，及合成转化率低下的不足
[0024]本发明对表达黄烷酮-3-羟化酶酵母工程菌进行了针对LncRNA靶点的改造，构建得到高产二氢山奈酚的工程菌对底物柚皮素转化率显著提升，均超过50.2％。其中，Q3M菌株转化率可达75.9％，相比对照菌株Q0M二氢山奈酚的产量提高了50％以上。不同于提高重组F3H表达质粒拷贝数等传统方法，该酵母工程菌仅从出发菌底盘细胞自身出发，围绕LncRNA相关靶点的敲除而展开。创新性地改造长非编码RNA靶点，有效的解决了酿酒酵母表达黄烷酮-3-羟化酶对底物转化率不高的首要问题。酵母工程菌用于提高其他高经济价值植物外源酶有较强的潜力，具备广阔的应用前景和重要的实践意义。
Smart Images
Figure CN117384775B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to an engineered strain of Saccharomyces cerevisiae and its applications. Background Technology
[0002] Dihydrokaempferol is widely found in plant-based foods such as legumes, broccoli, cabbage, kiwifruit, grapes, Brussels sprouts, strawberries, tea, and tomatoes. It possesses various health benefits, including antioxidant, antibacterial, anti-inflammatory, lipid-lowering, and anti-cancer effects, but it is expensive. Dihydrokaempferol is primarily obtained through isolation and purification from plants. Traditional phytochemical extraction methods often yield mixtures of dihydrokaempferol, and downstream purification processes are cumbersome and environmentally unfriendly. Furthermore, due to the low dihydrokaempferol content in these plants and the variations in their geographical location and climate, using plants as a source for dihydrokaempferol production is limited by their slow growth rate and non-uniform composition, making extraction difficult.
[0003] Flavanone-3-hydroxylase (F3H) plays a crucial role in the biosynthesis of flavonoids, primarily catalyzing the hydroxylation of naringenin at the 3-position of its C-ring to form dihydrokaempferol (DHK). F3H from different plant sources, such as tea (Camellia sinensis), ginkgo (Ginkgobiloba), soybean (Glycine max), strawberry (Fragaria x ananassa), and crabapple (Malus x domestica), exhibits similar catalytic properties. Compared to traditional plant extraction methods, studies have explored the use of recombinant microbial cells to synthesize DHK with less time and energy consumption. For example, expressing F3H genes from different plant sources in Saccharomyces cerevisiae has shown that naringenin (NAR) can be converted to DHK. However, the conversion rate is low and requires further improvement. Therefore, modifying yeast strains through genetic engineering and metabolic engineering to increase the yield and productivity of dihydrokaempferol is of great significance and practical application value. Summary of the Invention
[0004] The purpose of this invention is to provide an engineered yeast strain and its construction method that can significantly improve the efficiency of converting naringenin to dihydrokaempferol, overcoming the shortcomings of existing brewing yeast strains that cannot synthesize dihydrokaempferol using naringenin as a substrate and suffer from low conversion rates. By identifying effective long non-coding RNA (lncRNA) targets, the conversion ability of brewing yeast to naringenin is enhanced, thereby increasing the yield and efficiency of dihydrokaempferol. This invention knocks out key lncRNA targets in F3H-expressing brewing yeast, improving the conversion efficiency of naringenin in yeast cells, overcoming the deficiencies of existing technologies, and achieving a significant increase in the ability to synthesize dihydrokaempferol.
[0005] The objective of this invention is achieved through the following technical solution:
[0006] A strain of Saccharomyces cerevisiae has the following characteristics: at least one of LncRNA SUT067, LncRNA SUT433 and LncRNA CUT782 is deleted or mutated; and it contains a nucleic acid encoding flavanone-3-hydroxylase.
[0007] The starting strain of the engineered Saccharomyces cerevisiae is preferably a strain with a CEN.PK background. This strain is a commonly used yeast strain and is widely used in metabolic engineering and systems biology research in industry and academia (Microb CellFact, 2012, 11, 36), with good representativeness and universality; preferably Saccharomyces cerevisiae CEN.PK 113-5D.
[0008] The deletion is preferably removed without scarring by homologous recombination or CRISPR / Cas9 gene editing technology.
[0009] The mutation described above renders the corresponding LncRNA ineffective and can be obtained through homologous recombination or CRISPR / Cas9 gene editing technology.
[0010] The flavanone-3-hydroxylase is a plant-derived flavanone-3-hydroxylase; preferably strawberry flavanone-3-hydroxylase and crabapple flavanone-3-hydroxylase.
[0011] The nucleic acid encoding flavanone-3-hydroxylase is preferably obtained through the following steps: based on the amino acid sequence of flavanone-3-hydroxylase, codon optimization is performed to find a suitable expression method for Saccharomyces cerevisiae, and the optimized nucleic acid encoding flavanone-3-hydroxylase is cloned into an expression vector to obtain the nucleic acid encoding flavanone-3-hydroxylase.
[0012] When the flavanone-3-hydroxylase is strawberry flavanone-3-hydroxylase, the optimized nucleic acid sequence is preferably as shown in SEQ ID No. 1.
[0013] When the flavanone-3-hydroxylase is begonia flavanone-3-hydroxylase, the optimized nucleic acid sequence is preferably as shown in SEQ ID No. 2.
[0014] The preferred expression vectors are p416GPD, p416TEF, p416ADH, p416CYC1, p426GPD, p426TEF, p426ADH, or p426CYC1.
[0015] The engineered strain of *Saccharomyces cerevisiae* has a high conversion efficiency for naringenin, thus it can be used to prepare dihydrokaempferol.
[0016] A method for preparing dihydrokaempferol includes the following steps:
[0017] 1) Inoculate the above-mentioned engineered strain of Saccharomyces cerevisiae into the fermentation medium and culture it;
[0018] 2) Next, add the substrate naringenin, continue fermentation, and collect the fermentation product to obtain dihydrokaempferol.
[0019] The fermentation medium mentioned in step 1) is preferably YPD medium.
[0020] The preferred culture conditions described in step 1) are 28–32°C and 150–250 rpm for 20–30 h; more preferably, 30°C and 200 rpm for 24 h.
[0021] The amount of naringenin added in step 2) is preferably 150-300 mg / L; more preferably 200 mg / L.
[0022] The fermentation conditions described in step 2) are preferably 28–32°C and 150–250 rpm for 80–120 h; more preferably 30°C and 200 rpm for 96 h.
[0023] The present invention has the following advantages and effects compared with the prior art:
[0024] This invention modifies a yeast strain expressing flavanone-3-hydroxylase targeting a lncRNA, resulting in a high-yield dihydrokaempferol engineered strain that significantly improves the conversion rate of the substrate naringenin, exceeding 50.2%. Specifically, the Q3M strain achieves a conversion rate of 75.9%, more than 50% higher than the control strain Q0M. Unlike traditional methods such as increasing the copy number of recombinant F3H expression plasmids, this yeast engineered strain focuses solely on the knockout of lncRNA-related targets within the starting cell chassis itself. This innovative modification of long non-coding RNA targets effectively solves the primary problem of low substrate conversion rates for flavanone-3-hydroxylase expression in Saccharomyces cerevisiae. Yeast engineered strains have strong potential for improving exogenous enzymes in other high-economic-value plants, possessing broad application prospects and significant practical value. Attached Figure Description
[0025] Figure 1 This is a schematic diagram of the composition of recombinant expression plasmids p416-FaF3H and p416-MdF3H carrying the URA3 selection marker.
[0026] Figure 2 This is an agarose gel electrophoresis image of recombinant expression plasmids p416-FaF3H and p416-MdF3H verified by restriction endonucleases SacI and BamHI; lane M is the DNA molecular weight marker.
[0027] Figure 3 This is an agarose gel electrophoresis image of PCR identification of the knockout strains SUT067, SUT433, and CUT782, where lane M is the DNA molecular weight marker.
[0028] Figure 4 This is an agarose gel electrophoresis image showing the successful transformation of p416-FaF3H and p416-MdF3H plasmids into strains ΔSUT067, ΔSUT433, and ΔCUT782 by PCR; lane M is the DNA molecular weight marker.
[0029] Figure 5 This is a graph showing the yield of dihydrokaempferol (DHK) synthesized by in vitro fermentation of Q0F, Q1F, Q2F, and Q3F yeast engineered strains in Example 4, as well as the conversion rate of the substrate naringenin (NAR); Q0F serves as the control.
[0030] Figure 6 This is a graph showing the yield of dihydrokaempferol (DHK) synthesized by in vitro fermentation of Q0M, Q1M, Q2M, and Q3M yeast engineered strains in Example 4, as well as the conversion rate of the substrate naringenin (NAR); Q0M serves as the control. Detailed Implementation
[0031] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.
[0032] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.
[0033] Unless otherwise specified, experimental methods in the following examples are generally performed under conventional conditions, such as those described in Molecular Cloning: A Laboratory Manual (Beijing: Science Press, 2017) and Yeast Genetics: A Laboratory Manual (Beijing: Science Press, 2016).
[0034] The CRISPR technology used in the following examples is described in the prior art (Zhang et al. A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomycescerevisiae. Nat Commun 2019, 10(1):1053). Furthermore, the sequences of the psgtRNA, pCas, and pScURA plasmid fragments used in the following examples are disclosed in the appendix to the document "Zhang et al. AgRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomycescerevisiae. Nat Commun 2019, 10(1):1053". The pScURA plasmid fragment can be obtained through artificial synthesis.
[0035] To better understand the content of this invention, the background strain of Saccharomyces cerevisiae CEN.PK 113-5D (which can be obtained from EUROSCARF) is used as the starting strain for further explanation of specific embodiments.
[0036] The culture media involved in the following examples are as follows:
[0037] LB liquid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, solvent is deionized water; solid medium is supplemented with 2% agar powder.
[0038] LB / AMP medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, solvent is deionized water; solid medium with 2% agar powder added; after sterilization, cool to about 40°C and add 100 μg / mL ampicillin (filtered for sterilization).
[0039] YPD medium: 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose (sterilized separately and then added), solvent is deionized water; solid medium is supplemented with 2% agar powder.
[0040] SC-Ura auxotrophic medium: 0.77 g / L CSM-Ura, 1.7 g / L YNB w / o AA & w / o (NH4)2SO4 (Yeast Nitrogen Base without Amino acids and without Ammonium sulphate), 5.0 g / L (NH4)2SO4, 20 g / L glucose (Note: glucose is sterilized separately), adjust pH to 5.5-6.0; add 2% agar powder to solid medium; solvent is deionized water.
[0041] The methods involved in the following embodiments are as follows:
[0042] Plasmid construction:
[0043] (1) Gibson assembly method, the specific operation shall be carried out in accordance with the NEB Gibson Assembly Cloning kit (part number E2611, NEB) instruction manual;
[0044] (2) Golden gate assembly method, specific operation according to Follow the instructions for use of the Golden Gate Assembly Kit (Catalog No. E1602L, NEB);
[0045] (3) Transform 5 μL of the assembly system into 50 μL of E. coli DH5α competent cells, spread them on LB / AMP solid medium and culture overnight;
[0046] (4) Positive clones were obtained by screening, and plasmids were extracted after amplification and culture. The specific extraction process was carried out in accordance with the instructions of HiPure Plasmid Micro Kit (product number P1001-03, Magen).
[0047] Yeast strain transformation:
[0048] Unless otherwise specified, the lithium acetate conversion method shall be used. For specific operation procedures, please refer to the relevant standards and specifications.
[0049] Recombinant Saccharomyces cerevisiae strain OD600nm Detection methods:
[0050] A single colony of *Saccharomyces cerevisiae* was inoculated into 3 mL of fermentation medium and cultured on a shaker at 30°C and 200 rpm. The fermentation broth was then diluted appropriately and its OD (exponential growth factor) was measured using a UV spectrophotometer. 600nm .
[0051] Method for detecting dihydrokaempferol:
[0052] Naringenin (NAR) and dihydrokaempferol (DHK) standards were purchased from Herbest (Baoji, China). The content was determined using Shimadzu high-performance liquid chromatography (HPLC) with a Hypers11 ODS C18 column (250 × 4.6 mm), a detection wavelength of 290 nm, a flow rate of 1 mL / min, and a column temperature of 40 °C. The elution program is shown in Table 1 below.
[0053] Table 1
[0054] 0 10％ 90％ 9 60％ 40％ 19 40％ 60％ 21 10％ 90％
[0055] The primer sequences involved in the following examples are shown in Table 2:
[0056] Table 2: Primer sequences (5'-3')
[0057]
[0058]
[0059] Example 1: Obtaining the gene encoding flavanone 3-hydroxylase in strawberry and crabapple
[0060] Based on the amino acid sequences of flavanone 3-hydroxylase (Q66MF0) from strawberry (Fragaria ananassa) and flavanone 3-hydroxylase (Q06942) from crabapple (Malus domestica) published in the UniProt database, and after codon optimization for Saccharomyces cerevisiae, Nanjing GenScript Biotech Co., Ltd. synthesized DNA coding sequences (as shown in SEQ ID No. 1 and SEQ ID No. 2, both encoding 364 amino acids) and cloned them into the vector pUC57, obtaining plasmids pUC57-FaF3H and pUC57-MdF3H.
[0061] Example 2: Construction of an expression vector for strawberry flavanone 3-hydroxylase
[0062] The structure of the expression vector p416-FaF3H for strawberry flavanone 3-hydroxylase is as follows: Figure 1 As shown in (A), the specific construction process is as follows:
[0063] (1) Using primers p416-KF / p416-KR, the plasmid framework was amplified using p416-GPD plasmid as a template; PCR amplification included routine denaturation, annealing and extension steps (the same below); the annealing temperature was 56℃, the extension temperature was 72℃ for 300s, and 30 cycles of amplification were performed.
[0064] (2) Using the primer pair Primer-A1F / Primer-A1R, the PDC1 promoter fragment with homologous arms was amplified using the genome of Saccharomyces cerevisiae CEN.PK 113-5D as a template; the annealing temperature of PCR amplification was 56℃, the extension temperature was 72℃ for 20s, and 30 cycles were performed.
[0065] (3) Using the primer pair Primer-A2F / Primer-A2R, the FaF3H fragment with homologous arms was amplified using pUC57-FaF3H as a template; the annealing temperature for PCR amplification was 56℃, the extension temperature was 72℃ for 20s, and 30 cycles were performed.
[0066] (4) Using the primer pair Primer-A3F / Primer-A3R, the PGK1 terminator fragment with homologous arms was amplified using the genome of Saccharomyces cerevisiae CEN.PK 113-5D as a template; the annealing temperature of PCR amplification was 56℃, the extension temperature was 72℃ for 20s, and 30 cycles were performed.
[0067] (5) Using primer pair FaF3H-F / FaF3H-R, the FaF3H fragment with homologous arms was amplified using pUC57-FaF3H plasmid as template; the annealing temperature of PCR amplification was 56℃, the extension temperature was 72℃ for 60s, and 30 cycles were performed.
[0068] (6) Using Gibson assembly technology, the above four fragments were spliced together and transformed into Escherichia coli DH5α.
[0069] Sequencing and enzyme digestion (Agarose gel electrophoresis image after SacI and BamHI digestion, as shown below) Figure 2 As shown in the figure, the plasmid construction was successfully verified and named p416-FaF3H.
[0070] Example 3: Construction of an expression vector for flavanone 3-hydroxylase from crabapple
[0071] The structure of the expression vector p416-MdF3H for the flavanone 3-hydroxylase of Begonia is as follows: Figure 1 As shown in (B) above, the specific construction process is as follows:
[0072] (1) Using primers p416-KF / p416-KR, the plasmid framework was amplified using p416-GPD plasmid as a template; the annealing temperature for PCR amplification was 56℃, the extension temperature was 72℃ for 300s, and 30 cycles were performed.
[0073] (2) Using the primer pair Primer-A1F / Primer-A1R, the PDC1 promoter fragment with homologous arms was amplified using the genome of Saccharomyces cerevisiae CEN.PK 113-5D as a template; the annealing temperature of PCR amplification was 56℃, the extension temperature was 72℃ for 20s, and 30 cycles were performed.
[0074] (3) Using the primer pair Primer-A2F / Primer-A2R, the MdF3H fragment with homologous arms was amplified using pUC57-MdF3H as a template; the annealing temperature of PCR amplification was 56℃, the extension temperature was 72℃ for 20s, and 30 cycles were performed.
[0075] (4) Using the primer pair Primer-A3F / Primer-A3R, the PGK1 terminator fragment with homologous arms was amplified using the genome of Saccharomyces cerevisiae CEN.PK 113-5D as a template; the annealing temperature of PCR amplification was 56℃, the extension temperature was 72℃ for 20s, and 30 cycles were performed.
[0076] (5) Using primer pair MdF3H-F / MdF3H-R, the MdF3H fragment with homologous arms was amplified using pUC57-MdF3H plasmid as template; the annealing temperature of PCR amplification was 56℃, the extension temperature was 72℃ for 60s, and 30 cycles were performed.
[0077] (6) Using Gibson assembly technology, the above four fragments were spliced together and transformed into Escherichia coli DH5α.
[0078] Sequencing and enzyme digestion (Agarose gel electrophoresis image after SacI and BamHI digestion, as shown below) Figure 2 As shown in the figure, the plasmid construction was successfully verified and named p416-MdF3H.
[0079] Example 4: Construction of an engineered Saccharomyces cerevisiae strain expressing strawberry flavanone 3-hydroxylase
[0080] 4.1 Knockout of SUT067 to achieve high production of dihydrokaempferol
[0081] The non-coding RNA SUT067 knockout process is as follows:
[0082] (1) Construction of plasmid pCas9-SUT067
[0083] The nucleotide sequence of the non-coding RNA SUT067 is shown in SEQ ID NO.3.
[0084] Using plasmid psgtRNA as a template, a gRNA sequence targeting SUT067 was obtained by amplification with primer pair Primer-A4F / Primer-A4R. The annealing temperature of PCR amplification was 56℃, followed by extension at 72℃ for 20s, and 30 amplification cycles were performed.
[0085] Using the pScURA plasmid fragment as a template, a 1127bp plasmid framework fragment with the nutrient marker URA3 was amplified by primer pair Primer-A5F / Primer-A5R; the annealing temperature for PCR amplification was 56℃, followed by extension at 72℃ for 30s, and 30 amplification cycles were performed.
[0086] Using the Golden Gate assembly technique, the two fragments mentioned above were spliced together with a pCas plasmid containing a Cas9 plasmid fragment, and the resulting plasmid was named pCas9-SUT067.
[0087] (2) PCR amplification of the SUT067 repair fragment
[0088] A 59bp primer pair, Primer-A6F / Primer-A6R, that perfectly matched the upstream and downstream of SUT067 was synthesized. The targeted repair fragment with 50bp homologous arms to the upstream and downstream of SUT067 was obtained by PCR amplification and gel recovery. The annealing temperature of PCR amplification was 56℃, followed by extension at 72℃ for 20s, and 30 cycles were performed.
[0089] (3) The plasmid pCas9-SUT067, along with the SUT067 repair fragment, was transformed into strain CEN.PK 113-5D using the lithium acetate transformation method. The transformed strains were plated on SC-Ura solid culture media and incubated at 30℃ for 3–4 days. Single-colony PCR was performed using primer pairs Primer-A7F / Primer-A7R to verify the transformations and screen for positive transformants (results). Figure 3 As shown in the figure, plasmid pCas9-SUT067 was removed, and the resulting Saccharomyces cerevisiae strain was named Q1.
[0090] Transform plasmid p416-FaF3H into strain Q1 to express strawberry flavanone 3-hydroxylase:
[0091] Saccharomyces cerevisiae Q1 was prepared into competent cells, and the strawberry flavanone 3-hydroxylase expression plasmid p416-FaF3H was transformed into these competent cells via lithium acetate conversion. The cells were then plated onto SC-Ura solid medium and cultured at 30℃ for 3-4 days. Single-colony PCR was performed using primers Primer-A8F / Primer-A8R to verify the colonies and screen for positive strains. (Results) Figure 4 As shown in the figure, it was named Saccharomyces cerevisiae Q1F.
[0092] Transform plasmid p416-MdF3H into strain Q1 to express begonia flavanone 3-hydroxylase:
[0093] Saccharomyces cerevisiae Q1 was prepared into competent cells, and the expression plasmid p416-MdF3H of flavanone 3-hydroxylase was transformed into these competent cells via lithium acetate transformation. The cells were then plated onto SC-Ura solid medium and cultured at 30℃ for 3-4 days. Single-colony PCR was performed using primers Primer-A8F / Primer-A8R to verify the colonies and screen for positive strains. (Results) Figure 4 As shown in the figure, it was named Saccharomyces cerevisiae Q1M.
[0094] 4.2 Knockout of SUT433 to achieve high production of dihydrokaempferol
[0095] The non-coding RNA SUT433 knockout process is as follows:
[0096] (1) Constructing plasmid pCas9-SUT433
[0097] The nucleotide sequence of the non-coding RNA SUT433 is shown in SEQ ID NO.4.
[0098] Using plasmid psgtRNA as a template, a gRNA sequence targeting SUT433 was obtained by amplification with primer pair Primer-A9F / Primer-A4R. The annealing temperature of PCR amplification was 56℃, extension at 72℃ for 20s, and amplification was performed for 30 cycles.
[0099] Using the pScURA plasmid fragment as a template, a 1127bp plasmid framework fragment with the nutrient marker URA3 was amplified by primer pair Primer-A5F / Primer-A5R. The annealing temperature for PCR amplification was 56℃, the extension temperature was 72℃ for 30s, and the amplification was performed for 30 cycles.
[0100] Using the Golden Gate assembly technique, the two fragments mentioned above were spliced together with a pCas plasmid containing a Cas9 plasmid fragment, and the resulting plasmid was named pCas9-SUT433.
[0101] (2) PCR amplification of SUT433 repair fragment
[0102] A 59bp primer pair, Primer-A10F / Primer-A10R, that was perfectly matched to the upstream and downstream of SUT433 was synthesized. The targeted repair fragment with 50bp homologous arms to the upstream and downstream of SUT433 was obtained by PCR amplification and gel recovery. The annealing temperature of PCR amplification was 56℃, extension temperature was 72℃ for 20s, and amplification was performed for 30 cycles.
[0103] (3) The plasmid pCas9-SUT433, along with the SUT433 repair fragment, was transformed into strain CEN.PK 113-5D using the lithium acetate transformation method. The transformed strains were plated on SC-Ura solid culture media and incubated at 30℃ for 3-4 days. Single-colony PCR was performed using primer pairs Primer-A11F / Primer-A11R to verify the transformations and screen for positive transformants (results). Figure 3 As shown in the figure, plasmid pCas9-SUT433 was removed, and the resulting Saccharomyces cerevisiae strain was named Q2.
[0104] Transform plasmid p416-FaF3H into strain Q2 to express strawberry flavanone 3-hydroxylase:
[0105] Saccharomyces cerevisiae Q2 was prepared into competent cells, and the strawberry flavanone 3-hydroxylase expression plasmid p416-FaF3H was transformed into these competent cells via lithium acetate conversion. The cells were then plated onto SC-Ura solid medium and cultured at 30℃ for 3-4 days. Single-colony PCR was performed using primers Primer-A8F / Primer-A8R to verify the colonies and screen for positive strains. (Results) Figure 4 As shown in the figure, it was named Saccharomyces cerevisiae Q2F.
[0106] Transform plasmid p416-MdF3H into strain Q2 to express begonia flavanone 3-hydroxylase:
[0107] Saccharomyces cerevisiae Q2 was prepared into competent cells, and the expression plasmid p416-MdF3H of flavanone 3-hydroxylase was transformed into these competent cells using the lithium acetate transformation method. The cells were then plated on SC-Ura solid medium and cultured at 30℃ for 3-4 days. Single-colony PCR was performed using primers Primer-A8F / Primer-A8R to verify the colonies and screen for positive strains. (Results) Figure 4 As shown in the figure, it was named Saccharomyces cerevisiae Q2M.
[0108] 4.3 Knockout of CUT782 to achieve high production of dihydrokaempferol
[0109] The non-coding RNA CUT782 knockout process is as follows:
[0110] (1) Constructing plasmid pCas9-CUT782
[0111] The nucleotide sequence of the non-coding RNA CUT782 is shown in SEQ ID NO.5.
[0112] Using plasmid psgtRNA as a template, a gRNA sequence with a 20bp target CUT782 was amplified by primer pair Primer-A12F / Primer-A4R. The annealing temperature for PCR amplification was 56℃, the extension temperature was 72℃ for 20s, and the amplification was repeated for 30 cycles.
[0113] Using the pScURA plasmid fragment, a 1127bp plasmid framework fragment with the nutrient marker URA3 was amplified by primer pair Primer-A5F / Primer-A5R. The PCR amplification temperature was 56℃, extension temperature was 72℃ for 30s, and amplification was performed for 30 cycles.
[0114] Using the Golden Gate assembly technique, the two fragments mentioned above were spliced together with a pCas plasmid containing a Cas9 plasmid fragment, and the resulting plasmid was named pCas9-CUT782.
[0115] (2) PCR amplification of the CUT782 repair fragment
[0116] A 59bp primer pair, Primer-A13F / Primer-A13R, that was perfectly matched with the upstream and downstream of CUT782 was synthesized. The targeted repair fragment with 50bp homologous arms with CUT782 was obtained by PCR amplification and gel recovery. The PCR amplification annealing temperature was 56℃, extension temperature was 72℃ for 20s, and amplification was performed for 30 cycles.
[0117] (3) The plasmid pCas9-CUT782, along with the CUT782 repair fragment, was transformed into strain CEN.PK 113-5D using the lithium acetate transformation method. The transformed strains were plated on SC-Ura solid culture media and incubated at 30℃ for 3-4 days. Single-colony PCR was performed using primer pairs Primer-A14F / Primer-A14R to verify the transformations and screen for positive transformants (results). Figure 3 As shown in the figure, plasmid pCas9-CUT782 was removed, and the resulting Saccharomyces cerevisiae strain was named Q3.
[0118] Transform plasmid p416-FaF3H into strain Q3 to express strawberry flavanone 3-hydroxylase:
[0119] Saccharomyces cerevisiae Q3 was prepared into competent cells, and the strawberry flavanone 3-hydroxylase expression plasmid p416-FaF3H was transformed into these competent cells via lithium acetate conversion. The cells were then plated onto SC-Ura solid medium and cultured at 30℃ for 3-4 days. Single-colony PCR was performed using primers Primer-A8F / Primer-A8R to verify the colonies and screen for positive strains. (Results) Figure 4 As shown in the figure, it was named Saccharomyces cerevisiae Q3F.
[0120] Transform plasmid p416-MdF3H into strain Q3 to express begonia flavanone 3-hydroxylase:
[0121] Saccharomyces cerevisiae Q3 was prepared into competent cells, and the expression plasmid p416-MdF3H of flavanone 3-hydroxylase was transformed into these competent cells via lithium acetate conversion. The cells were then plated onto SC-Ura solid medium and cultured at 30℃ for 3-4 days. Single-colony PCR was performed using primers Primer-A8F / Primer-A8R to verify the colonies and screen for positive strains. (Results) Figure 4 As shown in the figure, it was named Saccharomyces cerevisiae Q3M.
[0122] Example 5: Fermentation production of dihydrokaempferol using engineered strains
[0123] (1) The above-mentioned Saccharomyces cerevisiae strains Q0F, Q1F, Q2F, Q3F, Q0M, Q1FM, Q2M, and Q3M were inoculated from plates into 2.5 mL of YPD fermentation medium and cultured at 30℃ and 200 rpm for 24 h. Then, naringenin (NAR) was added to a final concentration of 200 mg / L. Q0F and Q0M were used as control strains. The preparation process of Q0F is as follows: Saccharomyces cerevisiae CEN.PK 113-5D was prepared into competent cells, and the strawberry flavanone 3-hydroxylase expression plasmid p416-FaF3H was transformed into the competent cells by lithium acetate conversion method. The cells were plated on SC-Ura solid medium and cultured at 30℃ for 3-4 days. Single colony PCR verification was performed using primers Primer-A8F / Primer-A8R to screen for positive strains (results). Figure 4 (As shown), it was named *Saccharomyces cerevisiae* Q0F. The preparation process of Q0M is as follows: *Saccharomyces cerevisiae* CEN.PK 113-5D was prepared into competent cells, and the *Pyrospermum jasminoides* 3-hydroxylase expression plasmid p416-MdF3H was transformed into the competent cells by lithium acetate transformation. The cells were plated on SC-Ura solid medium and cultured at 30℃ for 3-4 days. Single colony PCR verification was performed using primers Primer-A8F / Primer-A8R, and positive strains were screened (results). Figure 4 As shown in the figure, it was named Saccharomyces cerevisiae Q0M.
[0124] (2) After fermentation for 96 hours, the fermentation broth was diluted 50 times and the OD was measured using a UV spectrophotometer. 600nm .
[0125] (3) After centrifuging 1 mL of fermentation broth, take 600 μL of the supernatant and add 600 μL of methanol.
[0126] (4) Shake to mix the mixture, centrifuge, take 500 μL of supernatant and filter.
[0127] (5) Determine the content of dihydrokaempferol and calculate the conversion rate.
[0128] Conversion rate = (Dihydrokaempferol yield * (Narcissin molecular weight / Dihydrokaempferol molecular weight) / Added narcissin substrate concentration) * 100%.
[0129] The detection results of yeast expressing strawberry flavanone 3-hydroxylase are as follows: Figure 5 As shown in (A), the dihydrokaempferol yield (92.8 mg / L) of strains Q1F, Q2F, and Q3F was increased by 30.4%, 30.2%, and 59.8% respectively compared with strain Q0F. Figure 5 (B) shows that strain Q0F has a conversion rate of 43.8% for the substrate naringenin, strains Q1F and Q2F have a conversion rate of over 57%, and strain Q3F has a conversion rate of over 70%.
[0130] The detection results of yeast expressing flavanone 3-hydroxylase are as follows: Figure 6 As shown in (A), strains Q1M, Q2M, and Q3M increased the yield of dihydrokaempferol (106.3 mg / L) by 10.4%, 28.2%, and 51.2%, respectively, compared to strain Q0M. Figure 6 (B) shows that strain Q0M achieved a conversion rate of 50.2% for the substrate naringenin, while strains Q1M and Q2M achieved conversion rates of over 55%, with Q3M reaching 75.9%, indicating broad prospects for industrial application.
[0131] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.
Claims
1. An engineered strain of *Saccharomyces cerevisiae*, characterized in that... It has the following characteristics: deletion or mutation of any one of LncRNA SUT067, LncRNA SUT433 and LncRNA CUT782; and contains a nucleic acid encoding flavanone-3-hydroxylase; The nucleotide sequence of the LncRNA SUT067 is shown in SEQ ID NO.3; The nucleotide sequence of the LncRNA SUT433 is shown in SEQ ID NO.4; The nucleotide sequence of the LncRNA CUT782 is shown in SEQ ID NO.5; The aforementioned deletion or mutation renders the corresponding LncRNA no longer functional.
2. The engineered strain of *Saccharomyces cerevisiae* according to claim 1, characterized in that: The starting strain of the engineered Saccharomyces cerevisiae is a strain with a CEN.PK background.
3. The engineered strain of *Saccharomyces cerevisiae* according to claim 2, characterized in that: The starting strain of the engineered Saccharomyces cerevisiae strain is Saccharomyces cerevisiae CEN.PK 113-5D.
4. The engineered strain of *Saccharomyces cerevisiae* according to claim 1, characterized in that: The flavanone-3-hydroxylase mentioned is strawberry flavanone-3-hydroxylase or crabapple flavanone-3-hydroxylase.
5. The engineered strain of *Saccharomyces cerevisiae* according to claim 1, characterized in that: The nucleic acid encoding flavanone-3-hydroxylase is obtained through the following steps: based on the amino acid sequence of flavanone-3-hydroxylase, codons suitable for expression in Saccharomyces cerevisiae are optimized, and the optimized nucleic acid encoding flavanone-3-hydroxylase is cloned into an expression vector to obtain the nucleic acid encoding flavanone-3-hydroxylase.
6. The engineered strain of *Saccharomyces cerevisiae* according to claim 5, characterized in that: When the flavanone-3-hydroxylase is strawberry flavanone-3-hydroxylase, the optimized nucleic acid sequence is shown in SEQ ID No. 1; When the flavanone-3-hydroxylase is begonia flavanone-3-hydroxylase, the optimized nucleic acid sequence is shown in SEQ ID No.
2.
7. The engineered strain of *Saccharomyces cerevisiae* according to claim 5, characterized in that: The expression vector is p416GPD, p416TEF, p416ADH, p416CYC1, p426GPD, p426TEF, p426ADH or p426CYC1.
8. The use of the engineered strain of Saccharomyces cerevisiae according to any one of claims 1 to 7 in the preparation of dihydrokaempferol.
9. A method for preparing dihydrokaempferol, characterized in that... Includes the following steps: 1) The engineered strain of *Saccharomyces cerevisiae* according to any one of claims 1 to 7 is inoculated into a fermentation medium and cultured. 2) Next, add the substrate naringenin, continue fermentation, and collect the fermentation product to obtain dihydrokaempferol.
10. The method for preparing dihydrokaempferol according to claim 9, characterized in that: The fermentation medium mentioned in step 1) is YPD medium; The culture conditions described in step 1) are: culture at 28–32°C and 150–250 rpm for 20–30 h; The amount of naringenin added in step 2) is calculated as 150-300 mg / L; The fermentation conditions described in step 2) are 28–32°C and 150–250 rpm for 80–120 h.