Method for increasing yield of progesterone in yarrowia lipolytica
By constructing a biosensor that responds to steroid hormones and connects DGA1, the key gene for lipid synthesis in yeast yeast, solves the problem of low progesterone production in yeast yeast, and achieves efficient steroid product synthesis.
Patent Information
- Application Number
- CN202510745004.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-05
- Publication Date
- 2025-09-02
AI Technical Summary
The yield of progesterone in the yeast is low, and the lack of specificity and low substrate conversion efficiency in traditional methods lead to low steroid yield.
A biosensor that responds to steroid hormones was constructed. By deeply digging out transcriptomic data under steroid hormone stress conditions, a promoter that can specifically respond to specific steroid compounds was screened out, and it was linked to DGA1, a key gene for lipid synthesis in yeast yeast, to construct a recombinant plasmid, transformed into the recombinant strain yDTY316, and to undergo high-yield progesterone fermentation.
The yield of progesterone was significantly improved, and the problems of insufficient specificity and low substrate conversion efficiency in traditional methods were overcome, and accurate and efficient steroid product synthesis was achieved.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of bioengineering and provides a method for increasing the progesterone production of Yarrowia esterolyticus. Background Art
[0002] Steroid hormones are the second-largest class of drugs globally, after antibiotics. Currently, there are over 400 known steroidal drugs, widely used to treat conditions such as rheumatoid arthritis, bronchial asthma, eczema, and anaphylactic shock. They hold broad market potential. In 2021, the global steroidal drug market reached $147.5 billion, a year-on-year increase of 6.12%. my country is the leading producer of steroidal hormone raw materials and formulations, accounting for approximately 60% of global production. These products primarily utilize the "saponin-diene" process for extracting turmeric saponins from turmeric, as well as microbial degradation of side chains using plant and animal sterols as raw materials. The "saponin-diene" process generates large amounts of acidic chromium-containing wastewater and organic wastewater from the sterol side chain degradation pathway, creating serious environmental challenges and hindering the development of the traditional steroidal API synthesis industry. To address these environmental challenges, the development of biosensors at key nodes in the steroidal synthesis pathway, based on synthetic biology principles, has enabled high-throughput strain screening and dynamic regulation of product synthesis, which is of great significance to the green and sustainable development of the steroidal drug industry.
[0003] Genetically encoded biosensors are considered promising high-throughput analytical approaches due to their ability to sense metabolites and couple detection with actuators, facilitating rapid detection of small molecules at the single-cell level. Juan Ibero et al., through transcriptomics, found that the edc gene cluster in Sphingobacterium neosphingolipids was highly induced in the presence of estradiol (E2). Subsequently, an EdcR-based estrogen biosensor was constructed in Escherichia coli, with an optimized reporter system capable of responding to compounds E1 and E2. Chloé Grazon et al. reported a progesterone sensor based on the bacterial transcription factor aTF. By exposing Pseudomonas cultures to various steroid hormones, RNA sequencing combined with biolayer interferometry confirmed that SRTF1 is the progesterone-responsive transcription factor aTF. Subsequently, a framework (QD-TF-FRET) was developed to couple the molecular transduction mechanism of aTF with light output, enabling sensor visualization. Kun Liu et al. designed a de novo biosensor, DLA (DBD-LBD-AD), based on molecular dynamics simulations. It comprises three domains forming a novel protein. A whole-cell progesterone sensor was constructed in Saccharomyces cerevisiae, expressing DLA using the strong ADH1 promoter and using green fluorescent protein as the output signal. However, compared to the traditional industrial yeast Saccharomyces cerevisiae, Yarrowia lipolytica offers significant advantages in fat metabolism and diverse substrate utilization, making it a potential platform for green biomanufacturing. This is also beneficial for the development of biosensors at key nodes in the steroid biosynthesis pathway, which is of great significance for the green and sustainable development of the steroid drug industry. However, the current progesterone production in Yarrowia lipolytica is low. Summary of the Invention
[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for increasing the progesterone production of Yarrowia esterolyticus.
[0005] The technical solution of the present invention is summarized as follows:
[0006] A method for increasing the production of progesterone in Yarrowia esterolyticus comprises the following steps:
[0007] (1) constructing a biosensor responsive to steroid hormones, wherein the nucleotide sequence of the biosensor responsive to steroid hormones is shown in SEQ ID No. 52;
[0008] (2) A biosensor responsive to steroid hormones was connected to the key gene DGA1 for lipid synthesis in Yarrowia lipolytica, and the resultant ligation was completed with the linearized pYRSB vector to obtain a recombinant plasmid pDTY316. The nucleotide sequence of the gene DGA1 is shown in SEQ ID No. 57; the nucleotide sequence of the plasmid pYRSB is shown in SEQ ID No. 54.
[0009] (3) Transform the recombinant plasmid pDTY316 into the recombinant strain yDTY314 to obtain the recombinant strain yDTY316;
[0010] (4) The recombinant strain yDTY316 was fermented in shake flasks to produce high progesterone.
[0011] Advantages of the present invention:
[0012] Compared with traditional methods that use endogenous metabolic pathways of natural microorganisms to convert steroid substrates, this invention deeply mines transcriptome data under steroid hormone stress conditions to screen out promoters that can specifically respond to specific steroid compounds. By systematically optimizing the response sensitivity and dynamic range of this promoter, we have developed a steroid hormone-responsive biosensor that can accurately and efficiently screen and characterize specific metabolites.
[0013] By utilizing a biosensor that responds to steroid hormones to dynamically regulate the endogenous metabolic pathways of microorganisms, compared with the traditional method of converting steroid substrates by introducing natural endogenous metabolic pathways of microorganisms, this technology not only overcomes the problems of insufficient specificity and low substrate conversion efficiency in traditional methods that lead to low final steroid production, but also significantly increases the production of progesterone. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 This is a volcano plot of differences between genomes under progesterone stress.
[0015] Figure 2 This is an analysis diagram of differentially expressed genes under progesterone stress.
[0016] Figure 3 This is the qPCR analysis diagram of pckA, galK, CDR1, RHOGDI, FOX2 and ACAA1 genes.
[0017] Figure 4 This is a test diagram of the response of galK, CDR1, RHOGDI, and ACAA1 promoters to progesterone.
[0018] Figure 5 This is a diagram showing the response effect of feeding different concentrations of progesterone on the CDR1 promoter.
[0019] Figure 6 Response graphs of CDR1 promoters with different lengths.
[0020] Figure 7 To increase SRE to strengthen CDR1 promoter (P CDR1 )Response dynamic range graph.
[0021] Figure 8 A diagram illustrating the application of effector molecules in a biosensor responsive to steroid hormones.
[0022] Figure 9 It is a biosensor responsive to steroid hormones and is used to enhance progesterone synthesis.
[0023] Figure 10 A biosensor that responds to steroid hormones SRE*3-CDR1(500) Dynamic regulatory maps applied to lipid metabolism and product synthesis. DETAILED DESCRIPTION
[0024] YPD medium: 20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract powder, dilute to volume with distilled water, and sterilize at 115°C for 15 min.
[0025] Y. lipolytica ATCC201249, commercially available. Y. lipolytica Po1f, commercially available.
[0026] pUC57 is commercially available.
[0027] The present invention will be further described below with reference to specific embodiments.
[0028] Example 1 Screening of progesterone-responsive genes
[0029] 1. Acquisition of transcriptome data
[0030] Wild-type Yarrowia lipolytica ATCC201249 was cultured in YPD medium to the mid-logarithmic growth stage, when the strain is sensitive to environmental stress. It was fed with a high concentration (105 mg / L) of progesterone solution (the solvent was ethanol), and the transcriptomes of the samples were analyzed 15 minutes, 30 minutes, and 90 minutes after feeding (early feeding, mid-feeding, and stable period).
[0031] 2. Transcriptome Data Analysis
[0032] The samples obtained in step 1 were analyzed using the BGI interactive reporting system. https: / / report.bgi.com Perform transcriptome data analysis.
[0033] 3. Experimental results
[0034] According to the transcriptome data analysis results at different time points under progesterone stress ( Figure 1 ), the transcriptional response of Yarrowia lipolytica to progesterone stress has obvious time-dependent characteristics: 15-30 minutes show a rapid and strong stress response, while 90 minutes enters a relatively stable adaptation state.
[0035] In order to comprehensively analyze the distribution characteristics of differentially expressed genes (DEGs) under multiple comparison conditions, we used Venn diagrams to visualize the differentially expressed gene data of multiple comparison groups ( Figure 2 ). The screening criteria for differentially expressed genes were
[0036] |log2FC|>1 and Q value<0.05 were used to ensure the statistical significance and biological significance of the results. Based on the results of Venn diagram analysis, we further screened 103 core genes that showed significant differential expression at three time points (15 minutes, 30 minutes, and 90 minutes) and performed differential expression cluster analysis on these genes. Using a hierarchical clustering method, we constructed a heat map of gene expression patterns to intuitively display the expression trends of these core genes at different time points. The cluster analysis results showed that these genes can be divided into several functional clusters based on their expression patterns, each cluster potentially representing a different regulatory pathway or biological process.
[0037] Among the 103 significantly differentially expressed core genes, we screened out the six most significant genes according to the up-regulated expression intensity, including CDR1 (CAG82364.1), pckA (CAG82248.1), ACAA1 (CAG79704.1), galK
[0038] (CAG82094.1), FOX2 (CAG79573.1) and RHOGDI (CAG80931.1). Figure 1 The inter-group difference volcano plot also showed significant differential expression, further confirming that they are key genes in the specific response of Yarrowia lipolytica to progesterone. See Table 1.
[0039] Table 1 Significantly upregulated genes in response to progesterone
[0040]
[0041] Example 2 Testing of the 6 Significant Genes Response to Progesterone Obtained in Example 1
[0042] 1. Obtaining chassis strains
[0043] Wild-type Yarrowia lipolytica strain ATCC201249 and wild-type Yarrowia lipolytica strain po1f were purchased commercially.
[0044] 2. Experimental methods
[0045] 2.1 Real-time quantitative PCR (qPCR)
[0046] Progesterone substrate mother solution 17.5g / L: Accurately weigh 17.5g of progesterone powder (commercial product) and dilute to volume in 1L of anhydrous ethanol.
[0047] Wild-type Yarrowia lipolytica ATCC201249 was inoculated into 3 mL of YPD medium and cultured at 28°C and 220 rpm for 14-16 h. The initial bacterial concentration OD 600=0.2 was inoculated into 3 mL of YPD medium and cultured at 28°C and 220 rpm for 12 h (mid-logarithmic growth phase). The experimental group was supplemented with progesterone substrate stock solution to a final progesterone concentration of 105 mg / L; the control group was supplemented with an equal amount of anhydrous ethanol. After continued shake flask culture for 90 minutes (stability phase), approximately 200 mg of cells were collected by centrifugation, resuspended twice in ultrapure water, centrifuged again, and quickly frozen in liquid nitrogen.
[0048] RNA extraction, reverse transcription and qPCR testing were entrusted to Beijing Qingke Biotechnology Co., Ltd.
[0049] Actin 1 (CAG80754.2) (primer SEQ ID No. 1 / 2) was selected as the internal reference gene.
[0050] qPCR analysis was performed on the following genes: CDR1 (primers SEQ ID No. 3 / 4), pckA (primers SEQ ID No. 5 / 6), ACAA1 (primers SEQ ID No. 7 / 8), galK (primers SEQ ID No. 9 / 10), FOX2 (primers SEQ ID No. 11 / 12), and RHOGDI (primers SEQ ID No. 13 / 14). Expression levels were normalized to those obtained in the control. Results are shown in Figure 3 .
[0051] 2.2 Construction of steroid-responsive gene promoter recombinant plasmid and recombinant strain
[0052] The recombinant plasmid pDTY301 containing the gene CDR1 promoter was constructed using pYRSB (SEQ ID No. 54) as the basic plasmid.
[0053] ① Using the genome of Yarrowia lipolytica strain po1f as a template, amplify the 500bp upstream sequence of gene CDR1;
[0054] ② Amplify the DsRed sequence using plasmid pDsRed (SEQ ID No. 55) as a template;
[0055] ③ Use BamHI and BsaI to digest the plasmid pYRSB to obtain the linearized pYRSB vector;
[0056] ④ By PCR, a BamHI-simulated sticky end cut and a 20 bp homologous sequence to the DsRed sequence were introduced at both ends of the 500 bp sequence upstream of CDR1 to obtain fragment 1 (primers SEQ ID No. 15 / 16). By PCR, a BsaI-simulated sticky end cut and a 20 bp homologous sequence to the CDR1 upstream sequence were introduced at both ends of the DsRed sequence to obtain fragment 2 (primers SEQ ID No. 17 / 18);
[0057] ⑤ Combine the two fragments obtained in step ④ and the linearized pYRSB vector obtained in step ③ in equimolar amounts and complete the splicing using BM seamless cloning enzyme according to the recommended protocol in the corresponding instructions. The resulting complete plasmid is introduced into TOP10 competent cells (commercial) via transformation of E. coli. Spread onto LB+KanR plates and incubate overnight at 37°C.
[0058] ⑥ Colony PCR verified that the recombinant plasmid pDTY301 was successfully constructed (primer SEQ ID No.28 / 29).
[0059] 2.3 Construction process of recombinant plasmid pDTY302.
[0060] The recombinant plasmid pDTY302 containing the gene ACAA1 promoter was constructed using pYRSB (SEQ ID No. 54) as the basic plasmid.
[0061] ① Replace CDR1 in step 2.2① of this example with ACAA1;
[0062] ②, ③ are the same as ②, ③ in step 2.2 of this embodiment;
[0063] ④ By PCR, a BamHI-simulated sticky end cut and a 20 bp homologous sequence to the DsRed sequence were introduced at both ends of the 500 bp upstream sequence of ACAA1 to obtain fragment 3 (primers SEQ ID No. 19 / 20). By PCR, a BsaI-simulated sticky end cut and a 20 bp homologous sequence to the ACAA1 upstream sequence were introduced at both ends of the DsRed sequence to obtain fragment 4 (primers SEQ ID No. 21 / 18).
[0064] ⑤Same as ⑤ in step 2.2 of this embodiment;
[0065] ⑥ Colony PCR verification of the successful construction of recombinant plasmid pDTY302 (primer SEQ ID No. 28 / 29)
[0066] 2.4 Construction process of recombinant plasmid pDTY303.
[0067] The recombinant plasmid pDTY303 containing the gene galK promoter was constructed using pYRSB (SEQ ID No. 54) as the basic plasmid.
[0068] ① Replace CDR1 in step 2.2① of this example with galK;
[0069] ②, ③ are the same as ②, ③ in step 2.2 of this embodiment;
[0070] ④ By PCR, a BamHI-like sticky end cut and a 20 bp homologous sequence to the DsRed sequence were introduced at both ends of the 500 bp upstream sequence of galK to obtain fragment 5 (primers SEQ ID No. 22 / 23). By PCR, a BsaI-like sticky end cut and a 20 bp homologous sequence to the galK upstream sequence were introduced at both ends of the DsRed sequence to obtain fragment 6 (primers SEQ ID No. 24 / 18);
[0071] ⑤Same as ⑤ in step 2.2 of this embodiment;
[0072] ⑥ Colony PCR verification of the successful construction of recombinant plasmid pDTY303 (primer SEQ ID No. 28 / 29)
[0073] 2.5 Construction process of recombinant plasmid pDTY304.
[0074] The recombinant plasmid pDTY304 containing the gene RHOGDI promoter was constructed using pYRSB (SEQ ID No. 54) as the basic plasmid.
[0075] ① Replace CDR1 in step 2.2① of this example with RHOGDI;
[0076] ②, ③ are the same as ②, ③ in step 2.2 of this embodiment;
[0077] ④ By PCR, a BamHI-simulated sticky end cut and a 20 bp homologous sequence to the DsRed sequence were introduced at both ends of the 500 bp upstream sequence of RHOGDI to obtain fragment 7 (primers SEQ ID No. 25 / 26). By PCR, a BsaI-simulated sticky end cut and a 20 bp homologous sequence to the RHOGDI upstream sequence were introduced at both ends of the DsRed sequence to obtain fragment 8 (primers SEQ ID No. 27 / 18);
[0078] ⑤Same as ⑤ in step 2.2 of this embodiment;
[0079] ⑥ Colony PCR verification of the successful construction of recombinant plasmid pDTY304 (primer SEQ ID No. 28 / 29)
[0080] The recombinant plasmids pDTY301-pDTY304 were linearized by NotI digestion and then transformed into Yarrowia lipolytica po1f respectively.
[0081] The Frozen-EZ Yeast Transformation II kit (commercially available) was used to transform Yarrowia lipolytica, and the recombinant strains yDTY301-yDTY304 were obtained after colony PCR verification (primers SEQ ID No. 28 / 29).
[0082] 2.6 Characterization of steroid-responsive gene promoter recombinant strains
[0083] Progesterone substrate mother solution 17.5g / L: Accurately weigh 17.5g of progesterone powder and dilute to volume in 1L of anhydrous ethanol.
[0084] The recombinant strains yDTY301-yDTY304 were inoculated into 3 mL of YPD medium and cultured at 28°C and 220 rpm for 14-16 h. The initial bacterial concentration OD 600 =0.2 were inoculated into 3 mL YPD medium, cultured at 28 ° C, 220 rpm for 12 h, and a certain amount of substrate mother solution was added to continue incubation (adjusted with the feeding concentration, when the final progesterone concentration was 10.5 mg / L, 1.8 uL of mother solution was added, when the final progesterone concentration was 21 mg / L, 3.6 uL of mother solution was added, when the final progesterone concentration was 52.5 mg / L, 9 uL of mother solution was added, when the final progesterone concentration was 70 mg / L, 12 uL of mother solution was added, and when the final progesterone concentration was 105 mg / L, 18 uL of mother solution was added). Samples were taken at regular intervals. 200 uL of bacterial solution was diluted to OD 600 =0.2, the parameters of the microplate reader were set to 510 nm excitation filter and 590 nm emission filter, and the fluorescence value of red fluorescent protein was read.
[0085] 3. Experimental Results
[0086] In order to verify the response sensitivity and dynamic range of the six genes screened in Example 1 to progesterone stress, we performed qPCR analysis on CDR1, pckA, ACAA1, galK, FOX2 and RHOGDI genes ( Figure 3 The results showed that galK, CDR1, RHOGDI, and ACAA1 genes showed significant concentration-dependent upregulation after progesterone addition, indicating that these genes are highly sensitive to progesterone stress. In contrast, the responses of pckA and FOX2 genes were weaker and did not show a clear concentration-dependency.
[0087] Based on the qPCR results, we further selected the promoters of four genes (ACAA1, galK, RHOGDI and CDR1) that showed significant responses as progesterone-responsive sensors for functional verification. The promoter region 500bp before the start site of each gene was intercepted and fused with the fluorescent protein DsRed gene to construct a promoter-reporter gene system. By feeding different concentrations of progesterone and detecting the fluorescence intensity, we evaluated the response performance of these promoters to progesterone. Under the condition of a high concentration of progesterone of 105 mg / L, the promoter of the CDR1 gene showed a significant response, and the relative fluorescence ratio increased by more than 2 times after feeding for 2 hours ( Figure 4), the relative fluorescence ratio is the ratio of the fluorescence intensity of the experimental group to the fluorescence intensity of the control group. A promoter specifically responsive to steroidal compounds was obtained.
[0088] The response of the CDR1 promoter to different concentrations of progesterone (52.5 mg / L to 105 mg / L) was further tested. The results showed that the response fold of the CDR1 promoter to 52.5 mg / L progesterone increased over time, but did not show obvious regularity under different concentration gradients ( Figure 5 Although the CDR1 promoter showed a certain responsiveness under high progesterone concentration conditions, its sensitivity and linear range still need to be further optimized.
[0089] Example 3 Enhancement of Response Sensitivity and Dynamic Range of Promoters Specifically Responding to Steroidal Compounds
[0090] 1. Obtaining chassis strains
[0091] Wild-type Yarrowia lipolytica strain po1f was purchased commercially.
[0092] 2. Experimental methods
[0093] 2.1 Construction of CDR1 gene promoter truncated plasmid (400 bp) and recombinant strain
[0094] The recombinant plasmid pDTY305 containing the gene CDR1 promoter (400 bp) was constructed using pYRSB (SEQ ID No. 54) as the basic plasmid.
[0095] ① Using the yeast genome of Yarrowia lipolytica strain po1f as a template, amplify the 400bp upstream sequence of gene CDR1;
[0096] ② Amplify the DsRed sequence using plasmid pDsRed (SEQ ID No. 55) as a template;
[0097] ③ Use BamHI and BsaI to digest the plasmid pYRSB to obtain the linearized pYRSB vector;
[0098] ④ By PCR, a BamHI-simulated sticky end nick and a 20 bp homologous sequence to the DsRed sequence were introduced at both ends of the 400 bp sequence upstream of CDR1 to obtain fragment 9 (primers SEQ ID No. 30 / 16). By PCR, a BsaI-simulated sticky end nick and a 20 bp homologous sequence to the CDR1 upstream sequence were introduced at both ends of the DsRed sequence to obtain fragment 10 (primers SEQ ID No. 17 / 18).
[0099] ⑤ The two fragments obtained in step ④ and the linearized pYRSB vector obtained in step ③ were mixed in equal moles and then spliced using BM seamless cloning enzyme according to the recommended process in the corresponding instructions. The resulting complete plasmid was introduced into TOP10 competent cells through E. coli transformation. Spread on LB+Kan R Plates were incubated at 37°C overnight.
[0100] ⑥ Colony PCR verified that the recombinant plasmid pDTY305 was successfully constructed (primer SEQ ID No.28 / 29).
[0101] 2.2 Construction of CDR1 gene promoter truncated plasmid (300 bp) and recombinant strain
[0102] The recombinant plasmid pDTY306 containing the gene CDR1 promoter (300 bp) was constructed using pYRSB (SEQ ID No. 54) as the basic plasmid.
[0103] ① Using the yeast genome of Yarrowia lipolytica strain po1f as a template, amplify the 300bp upstream sequence of gene CDR1;
[0104] ②, ③ are the same as ②, ③ in step 2.1 of this embodiment;
[0105] ④ By PCR, a BamHI-simulated sticky end cut and a 20 bp homologous sequence to the DsRed sequence were introduced at both ends of the 300 bp sequence upstream of CDR1 to obtain fragment 11 (primers SEQ ID No. 31 / 16). By PCR, a BsaI-simulated sticky end cut and a 20 bp homologous sequence to the CDR1 sequence upstream were introduced at both ends of the DsRed sequence to obtain fragment 12 (primers SEQ ID No. 17 / 18).
[0106] ⑤Same as ⑤ in step 2.1 of this embodiment;
[0107] ⑥ Colony PCR verification of the successful construction of recombinant plasmid pDTY306 (primer SEQ ID No. 28 / 29)
[0108] 2.3 Construction of CDR1 gene promoter truncated plasmid (200 bp) and recombinant strain
[0109] The recombinant plasmid pDTY307 containing the gene CDR1 promoter (200 bp) was constructed using pYRSB (SEQ ID No. 54) as the basic plasmid.
[0110] ① Using the yeast genome of Yarrowia lipolytica strain po1f as a template, amplify the 200 bp sequence upstream of gene CDR1;
[0111] ②, ③ are the same as ②, ③ in step 2.1 of this embodiment;
[0112] ④ By PCR, a BamHI-simulated sticky end nick and a 20 bp homologous sequence to the DsRed sequence were introduced at both ends of the 200 bp sequence upstream of CDR1 to obtain fragment 13 (primers SEQ ID No. 32 / 16). By PCR, a BsaI-simulated sticky end nick and a 20 bp homologous sequence to the CDR1 sequence upstream were introduced at both ends of the DsRed sequence to obtain fragment 14 (primers SEQ ID No. 17 / 18).
[0113] ⑤Same as ⑤ in step 2.1 of this embodiment;
[0114] ⑥ Colony PCR verification of the successful construction of recombinant plasmid pDTY307 (primer SEQ ID No. 28 / 29)
[0115] 2.4 Construction of CDR1 gene promoter truncated plasmid (100 bp) and recombinant strain
[0116] The recombinant plasmid pDTY308 containing the gene CDR1 promoter (100 bp) was constructed using pYRSB (SEQ ID No. 54) as the basic plasmid.
[0117] ① Using the yeast genome of Yarrowia lipolytica strain po1f as a template, amplify the 100bp upstream sequence of gene CDR1;
[0118] ②, ③ are the same as ②, ③ in step 2.1 of this embodiment;
[0119] ④ By PCR, a BamHI-simulated sticky end nick and a 20 bp homologous sequence to the DsRed sequence were introduced at both ends of the 100 bp sequence upstream of CDR1 to obtain fragment 15 (primers SEQ ID No. 33 / 16). By PCR, a BsaI-simulated sticky end nick and a 20 bp homologous sequence to the CDR1 sequence upstream were introduced at both ends of the DsRed sequence to obtain fragment 16 (primers SEQ ID No. 17 / 18).
[0120] ⑤Same as ⑤ in step 2.1 of this embodiment;
[0121] ⑥ Colony PCR verification of the successful construction of recombinant plasmid pDTY308 (primer SEQ ID No. 28 / 29)
[0122] The recombinant plasmids pDTY305-pDTY308 were linearized by Not I and then transformed into Yarrowia lipolytica po1f respectively.
[0123] Transformation of Yarrowia lipolytica was performed using the Frozen-EZ Yeast Transformation II Kit.
[0124] After colony PCR verification (primer SEQ ID No. 28 / 29), the recombinant strains yDTY305-yDTY308 were obtained.
[0125] 2.5 Construction of SRE-enhanced plasmid (SRE*1) and recombinant strain (yDTY309)
[0126] SRE (CCGAA, SRE*1) (consists of a conserved sequence of 5bp bases)
[0127] The recombinant plasmid pDTY309 containing gene CDR1 promoter (500 bp) and SRE*1 was constructed using pYRSB (SEQ ID No. 54) as the basic plasmid.
[0128] ① Using the yeast genome of Yarrowia lipolytica strain po1f as a template, amplify the 500bp upstream sequence of gene CDR1;
[0129] ② Amplify the DsRed sequence using plasmid pDsRed as a template;
[0130] ③ Use BamHI and BsaI to digest the plasmid pYRSB to obtain the linearized pYRSB vector;
[0131] ④ By PCR, a copy of SRE (CCGAA, SRE*1) (a conserved sequence consisting of 5 bp bases) was added to the 5' end of the 500 bp sequence upstream of CDR1 and a sticky end cut simulating BamHI was introduced. A 20 bp homologous sequence to the DsRed sequence was added to the 3' end to obtain fragment 17 (primers SEQ ID No. 34 / 16). By PCR, sticky end cuts simulating BsaI and a 20 bp homologous sequence to the CDR1 upstream sequence were introduced at both ends of the DsRed sequence to obtain fragment 18 (primers SEQ ID No. 17 / 18);
[0132] ⑤ The two fragments obtained in step ④ and the linearized pYRSB vector obtained in step ③ were mixed in equal moles and then spliced using BM seamless cloning enzyme according to the recommended process in the corresponding instructions. The resulting complete plasmid was introduced into TOP10 competent cells through E. coli transformation. Spread on LB+Kan R Plates were incubated at 37°C overnight.
[0133] ⑥ Colony PCR verified that the recombinant plasmid pDTY309 was successfully constructed (primer SEQ ID No.28 / 29).
[0134] 2.6 Construction of SRE-enhanced plasmid (SRE*2) and recombinant strain (yDTY310)
[0135] The recombinant plasmid pDTY310 containing gene CDR1 promoter (500 bp) and SRE*2 was constructed using pYRSB (SEQ ID No. 54) as the basic plasmid.
[0136] ①, ②, ③ are the same as steps 2.5 ①, ②, ③ of this embodiment;
[0137] ④ By PCR, two SREs (CCGAA, SRE*2) (consisting of 5 bp of conserved sequences) were added to the 5' end of the 500 bp sequence upstream of CDR1, and a sticky end cut simulating BamHI was introduced. A 20 bp homologous sequence to the DsRed sequence was added to the 3' end to obtain fragment 19 (primers SEQ ID No. 35 / 16). By PCR, sticky end cuts simulating BsaI and a 20 bp homologous sequence to the CDR1 upstream sequence were introduced at both ends of the DsRed sequence to obtain fragment 20 (primers SEQ ID No. 17 / 18).
[0138] ⑤Same as step 2.5⑤ of this embodiment;
[0139] ⑥ Colony PCR verified that the recombinant plasmid pDTY310 was successfully constructed (primer SEQ ID No.28 / 29).
[0140] 2.7 Construction of SRE-enhanced plasmid (SRE*3) and recombinant strain (yDTY311)
[0141] The recombinant plasmid pDTY311 containing gene CDR1 promoter (500 bp) and SRE*3 was constructed using pYRSB (SEQ ID No. 54) as the basic plasmid.
[0142] ①, ②, ③ are the same as steps 2.5 ①, ②, ③ of this embodiment;
[0143] ④ By PCR, three copies of SRE (CCGAA, SRE*3) (a conserved sequence consisting of 5 bp bases) were added to the 5' end of the 500 bp sequence upstream of CDR1 and a sticky end cut simulating BamHI was introduced. A 20 bp homologous sequence to the DsRed sequence was added to the 3' end to obtain fragment 21 (primers SEQ ID No. 36 / 16). By PCR, sticky end cuts simulating BsaI and a 20 bp homologous sequence to the CDR1 upstream sequence were introduced at both ends of the DsRed sequence to obtain fragment 22 (primers SEQ ID No. 17 / 18);
[0144] ⑤Same as step 2.5⑤ of this embodiment;
[0145] ⑥ Colony PCR verification of the successful construction of recombinant plasmid pDTY311 (primer SEQ ID No. 28 / 29)
[0146] 2.8 Construction of SRE-enhanced plasmid (SRE*4) and recombinant strain (yDTY312)
[0147] The recombinant plasmid pDTY312 containing gene CDR1 promoter (500 bp) and SRE*4 was constructed using pYRSB (SEQ ID No. 54) as the basic plasmid.
[0148] ①, ②, ③ are the same as steps 2.5 ①, ②, ③ of this embodiment;
[0149] ④ By PCR, four copies of SRE (CCGAA, SRE*4) (a conserved sequence consisting of 5 bp bases) were added to the 5' end of the 500 bp sequence upstream of CDR1 and a sticky end nick mimicking BamHI was introduced. A 20 bp homologous sequence to the DsRed sequence was added to the 3' end to obtain fragment 23 (primer SEQ ID No. 37 / 16). By PCR, sticky end nick mimicking BsaI and a 20 bp homologous sequence to the CDR1 upstream sequence were introduced at both ends of the DsRed sequence to obtain fragment 24 (primers SEQ ID No. 17 / 18);
[0150] ⑤Same as step 2.5⑤ of this embodiment;
[0151] ⑥ Colony PCR verified that the recombinant plasmid pDTY312 was successfully constructed (primer SEQ ID No.28 / 29).
[0152] 2.9 Construction of SRE-enhanced plasmid (SRE*5) and recombinant strain (yDTY313)
[0153] The recombinant plasmid pDTY313 containing gene CDR1 promoter (500 bp) and SRE*5 was constructed using pYRSB (SEQ ID No. 54) as the basic plasmid.
[0154] ①, ②, ③ are the same as steps 2.5 ①, ②, ③ of this embodiment;
[0155] ④ By PCR, five copies of SRE (CCGAA, SRE*5) (a conserved sequence consisting of 5 bp bases) were added to the 5' end of the 500 bp sequence upstream of CDR1 and a sticky end cut simulating BamHI was introduced. A 20 bp homologous sequence to the DsRed sequence was added to the 3' end to obtain fragment 25 (primers SEQ ID No. 38 / 16). By PCR, sticky end cuts simulating BsaI and a 20 bp homologous sequence to the CDR1 upstream sequence were introduced at both ends of the DsRed sequence to obtain fragment 26 (primers SEQ ID No. 17 / 18);
[0156] ⑤Same as step 2.5⑤ of this embodiment;
[0157] ⑥ Colony PCR verified that the recombinant plasmid pDTY313 was successfully constructed (primer SEQ ID No.28 / 29).
[0158] The recombinant plasmids pDTY309-pDTY313 were linearized by Not I and then transformed into Yarrowia lipolytica po1f respectively.
[0159] Transformation of Yarrowia lipolytica was performed using the Frozen-EZ Yeast Transformation II Kit.
[0160] After colony PCR verification (primer SEQ ID No. 28 / 29), the recombinant strains yDTY309-yDTY313 were obtained.
[0161] 2.10 Characterization of Steroid-Response Gene Promoter Recombinant Strains
[0162] For the progesterone substrate mother solution feeding culture and red fluorescence detection method of the recombinant strains yDTY305-yDTY313, see 2.6 of Example 2.
[0163] 3. Experimental Results
[0164] In order to study the response characteristics of CDR1 promoter to progesterone, we first conducted segmented truncation experiments on CDR1 promoter, constructed promoter fragments of 500bp, 400bp, 300bp, 200bp and 100bp, respectively, and fed 10.5mg / L progesterone to test the response effects of these promoter fragments ( Figure 6 The experimental results showed that promoter fragments of 500bp and 400bp showed a clear fluorescence signal under low progesterone concentrations, but as the promoter length was further shortened, the fluorescence signal gradually weakened. In particular, when the promoter length was shortened to 300bp or less, the CDR1 promoter's response to progesterone almost disappeared.
[0165] Analysis of the CDR1 promoter sequence revealed the presence of a steroid-responsive region (SRE) upstream of the CDR1 promoter. The SRE typically contains three sequentially arranged 5-bp conserved segments, with the AAGAA and CCGAA segments being highly conserved. These conserved segments significantly enhance gene expression in response to various steroid hormones. However, as the CDR1 promoter shortens, the SRE gradually disappears, significantly reducing the promoter's responsiveness to progesterone.
[0166] In order to improve the dynamic range and sensitivity of the CDR1 promoter to progesterone, we further rationally designed and modified the CDR1 promoter. CDR1Different numbers of steroid response regions (SREs) were added upstream of the promoter (500 bp) to construct promoter variants with SRE*0, *1, *2, *3, *4, and *5, respectively. Figure 7 By testing the response effects of these variants under different progesterone concentrations, we found that the SRE*3 variant (P SRE*3-CDR1(500) ) has the highest response sensitivity, can produce significant fluorescence signals at a progesterone concentration of 1 mg / L, and its response dynamic range is increased by 2 times. The recombinant strain yDTY311 is the bacterium with the highest response sensitivity among biosensors containing a steroid hormone response.
[0167] The recombinant strain yDTY311 was able to produce a significant fluorescence signal at a progesterone concentration of 1 mg / L, and its response dynamic range was increased by 2 times. SRE*3-CDR1(500) The sequence is a biosensor responsive to steroid hormones, and the nucleotide sequence of the biosensor is shown in SEQ ID No. 52. A plasmid comprising the above-mentioned biosensor responsive to steroid hormones is plasmid pDTY311.
[0168] Example 4 Characterization of the Response of a Steroid Hormone-Responsive Biosensor to Steroid Hormones
[0169] 1. Experimental strains
[0170] The recombinant strain yDTY311 was constructed by the present invention.
[0171] 2. Experimental methods
[0172] Steroidal substrate stock solution: 17.5 g of each of the following steroid hormones were accurately weighed: campesterol, progesterone, pregnenolone, 17-hydroxyprogesterone, 17-hydroxypregnenolone, dehydroepiandrosterone, androstenedione, estradiol, hydrocortisone, 7-dehydrocholesterol, and ergosterol powder were separately diluted to 1 L of anhydrous ethanol.
[0173] The recombinant strain yDTY311 was inoculated into 3 mL of YPD medium and cultured at 28°C and 220 rpm for 14-16 h. The initial bacterial concentration OD 600 =0.2 were inoculated into 3 mL YPD medium, cultured at 28 ° C, 220 rpm for 12 h, and 18 μL steroid substrate mother solution was added to continue incubation, and samples were taken regularly. 200 μL of bacterial solution was diluted to OD 600 =0.2, the parameters of the microplate reader were set to 510 nm excitation filter and 590 nm emission filter, and the fluorescence value of red fluorescent protein was read.
[0174] 3. Experimental results
[0175] We tested the responsiveness of a recombinant strain yDTY311 containing a steroid hormone-responsive biosensor to various steroidal compounds (steroid hormones). In this experiment, we added 11 steroid hormones (pregnenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, hydrocortisone, estradiol, ergosterol, 7-dehydrocholesterol, and campesterol) to the culture medium of the recombinant strain yDTY311 after 12 hours of culture. The response of the biosensor to these steroid hormones was assessed by measuring changes in the fluorescence signal.
[0176] The experimental results show that ( Figure 8 ), in addition to showing a significant response to progesterone, the biosensor of the present invention also has a certain degree of responsiveness to several other steroid hormones. Specifically, the biosensor we constructed that responds to a steroid hormone can respond to dehydroepiandrosterone, androstenedione, estradiol and 7-dehydrocholesterol and drive the enhancement of fluorescence signals. This discovery provides a wider range of possibilities for the application of steroid hormone biosensors. For example, the sensor can be used to detect the presence and concentration changes of multiple steroid compounds in complex biological samples, thereby playing an important role in the fields of steroid hormone metabolism research, drug screening and environmental monitoring. In addition, the experimental results also suggest that by further optimizing the sequence or number of the SRE region, it is possible to further improve the selectivity and sensitivity of the biosensor to specific steroid compounds, thereby meeting the needs of different application scenarios.
[0177] Example 5 A method for increasing the production of progesterone in Yarrowia esterolyticus
[0178] 1. Obtaining chassis strains
[0179] The recombinant Yarrowia lipolytica strain, designated SyBE_Y12091025, was constructed in our laboratory. The specific genotype is shown in Table 2.
[0180] SyBE_Yl2091025 is described as follows: Y. lipolytica ATCC201249, ΔERG5::URA3, P EXP1 -Xl_DHCR7-T XPR2 ,ΔIntD::Hph,P TEF1 -Bt_mAdR-T LIP2 -P GPD -Bt_mAdx-T OCT ,pBR322::LEU2,P TEF1 -Ss_mCYP11A1-T LIP2 -P EXP1 -Bt_3β-HSD-T XPR2(CN115851640A, Module for Mixing Bacteria to Avoid Path Competition and Its Application, December 30, 2022)
[0181] Table 2 Genes and sequences involved in strain SyBE_Y12091025
[0182]
[0183] 2. Acquisition of exogenous functional gene elements
[0184] The gene 3β-HSD (3β-hydroxysteroid dehydrogenase) of the present invention is derived from Vacciniavirus (SEQ ID No. 56).
[0185] 3. Experimental methods
[0186] 3.1 Construction of recombinant plasmids and recombinant strains for steroid synthesis pathway
[0187] The recombinant plasmid pDTY314 containing the sequence shown in SEQ ID No.52 and Vv_3β-HSD (SEQ ID No.56) was constructed using pYRSA (SEQ ID No.53) as the basic plasmid.
[0188] Build process:
[0189] ① Using plasmid pDTY311 as a template, a biosensor P that responds to steroid hormones was amplified. SRE*3-CDR1(500) (SEQID No.52) sequence;
[0190] ② Amplify the Vv_3β-HSD sequence using plasmid pYl-Vv_3β-HSD (SEQ ID No. 56) as a template;
[0191] ③ Use BamHI and BsaI to digest the plasmid pYRSA to obtain the linearized pYRSA vector;
[0192] ④ PCR in a biosensor P that responds to steroid hormones SRE*3-CDR1(500) The ends of the Vv_3β-HSD sequence (SEQ ID No. 52) were introduced with sticky end cuts simulating BamHI and a 20 bp homologous sequence to the Vv_3β-HSD sequence to obtain fragment 27 (primer SEQ ID No. 39 / 40). The sticky end cuts simulating BsaI and a biosensor P that responds to steroid hormones were introduced at both ends of the Vv_3β-HSD sequence by PCR. SRE*3-CDR1(500) (SEQ ID No. 52) sequence 20 bp homologous sequence to obtain fragment 28 (primers SEQ ID No. 41 / 42);
[0193] ⑤ The two fragments obtained in step ④ and the linearized pYRSA vector obtained in step ③ were mixed in equal molar amounts and then spliced using BM seamless cloning enzyme according to the recommended process in the corresponding instructions. The resulting complete plasmid was introduced into TOP10 competent cells through E. coli transformation. Spread on LB+Kan R Plates were plated and cultured overnight at 37° C. Colony PCR was performed to verify the successful construction of the recombinant plasmid pDTY314 (primers SEQ ID No. 43 / 44).
[0194] The recombinant plasmid pDTY314 was linearized by Not I and then transformed into Yarrowia lipolytica SyBE_Y12091025.
[0195] The transformation of Yarrowia lipolytica was performed using the Frozen-EZ Yeast Transformation II kit (commercially available), and the strain yDTY314 was obtained after colony PCR verification (primers SEQ ID No. 43 / 44).
[0196] 3.2 Construction of lipid metabolism recombinant plasmids and recombinant strains
[0197] The recombinant plasmid pDTY315 containing the constitutive promoter expressing gene DGA1 was constructed based on pYRSB (SEQ ID No. 54) as a control strain.
[0198] ① Using the genome of Yarrowia lipolytica strain po1f as a template, the endogenous DGA1 gene sequence (SEQ ID No. 57) was amplified;
[0199] ② Use BsaI to digest the plasmid pYRSB to obtain the linearized pYRSB vector;
[0200] ③ Introducing the sticky end nick fragment 29 (primer SEQ ID No. 49 / 48) that simulates BsaI at both ends of the DGA1 sequence by PCR;
[0201] ④ The fragment obtained in step ③ and the linearized pYRSB vector obtained in step ② were mixed in equal moles and then spliced using BM seamless cloning enzyme according to the recommended process in the corresponding instructions. The resulting complete plasmid was transformed into TOP10 competent cells through E. coli. Spread on LB+Kan R Plates were plated and cultured overnight at 37° C. Colony PCR was performed to verify the successful construction of the recombinant plasmid pDTY315 (primers SEQ ID No. 50 / 51).
[0202] The recombinant plasmid pDTY315 was linearized by Not I and then transformed into the recombinant strain yDTY314.
[0203] Transformation of Yarrowia lipolytica was performed using the Frozen-EZ Yeast Transformation II Kit (commercially available).
[0204] The recombinant strain yDTY315 was obtained after colony PCR verification (primer SEQ ID No.50 / 51).
[0205] 3.3 Construction of recombinant plasmids and recombinant strains for dynamic regulation of steroid synthesis and lipid metabolism pathways
[0206] The recombinant plasmid pDTY316 containing the steroid hormone-responsive biosensor promoter expressing gene DGA1 was constructed using pYRSB (SEQ ID No. 54) as the basic plasmid.
[0207] ① Using plasmid pDTY311 as a template, a biosensor P that responds to steroid hormones was amplified. SRE*3-CDR1(500) (SEQID No.52) sequence;
[0208] ② The endogenous DGA1 gene sequence (SEQ ID No. 57) was amplified using the yeast genome of Yarrowia lipolytica strain po1f as a template;
[0209] ③ Through PCR, a steroid hormone biosensor promoter P SRE*3-CDR1(500) The ends of the sequence (SEQ ID No. 52) were introduced with a BamHI-simulated sticky end cut and a 20 bp homologous sequence to the DGA1 sequence to obtain fragment 30 (primers SEQ ID No. 45 / 46). The ends of the DGA1 sequence were introduced with a BsaI-simulated sticky end cut and a steroid hormone biosensor promoter P by PCR. SRE*3-CDR1(500) (SEQ ID No. 52) sequence 20 bp homologous sequence to obtain fragment 31 (primers SEQ ID No. 47 / 48);
[0210] ④ The two fragments obtained in step ③ and the linearized pYRSB vector obtained by digesting the plasmid pYRSB with BamHI and BsaI were mixed in equal moles and then spliced using BM seamless cloning enzyme according to the recommended process in the corresponding instructions. The resulting complete plasmid was transformed into TOP10 competent cells through E. coli. Spread on LB+Kan R Plates were plated and cultured overnight at 37° C. Colony PCR was performed to verify the successful construction of the recombinant plasmid pDTY316 (primers SEQ ID No. 50 / 51).
[0211] The recombinant plasmid pDTY316 was linearized by Not I and then transformed into the recombinant strain yDTY314.
[0212] Yarrowia lipolytica was transformed using the Frozen-EZ Yeast Transformation II Kit (commercially available), and the recombinant strain yDTY316 was obtained after colony PCR verification (primers SEQ ID No. 50 / 51).
[0213] 3.4 Cultivation of recombinant Yarrowia lipolytica strains
[0214] Seed culture medium: 20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract powder, dilute to volume with distilled water, and sterilize at 115°C for 15 min.
[0215] Fermentation medium: 80 g / L glucose, 20 g / L peptone, 10 g / L yeast extract powder, distilled water to volume, and sterilized at 115°C for 15 min.
[0216] Primary seed culture: Take a single colony of the recombinant strain and inoculate it into 3 mL of fresh seed culture medium and culture it at 28°C and 220 rpm for 24 h.
[0217] Secondary seed culture: Based on the initial OD 600 =0.2. Transfer the primary seeds into 5 mL of fresh seed culture medium and culture at 28°C and 220 rpm for 16-20 h.
[0218] Fermentation: Based on initial OD 600 =0.2 The secondary seeds were transferred to a 250 mL shake flask filled with 50 mL fermentation medium and fermented for 168 h.
[0219] 3.5 Extraction and detection of steroid compounds
[0220] Extraction Method: 1 mL of fermentation broth was placed in a 2 mL centrifuge tube. Approximately 200 mg of glass beads with a particle size of 425-600 μm were added as a dispersion medium. Extraction was then performed with 600 μL of ethyl acetate. The mixture was vortexed for 20 minutes to ensure thorough extraction and then centrifuged at 12,000 rpm for 5 minutes. After centrifugation, the upper organic phase was collected and used for later use. To fully extract the target compound, the above extraction steps were repeated twice on the lower aqueous phase. The organic phases from the three extractions were combined and dried by vacuum centrifugation for 60-90 minutes to obtain a dry steroid sample. The dried steroid sample was dissolved in 200 μL of the derivatization reagent MSTFA (N-methyl-N-trimethylsilyltrifluoroacetamide) and incubated in a 37°C incubator for 2 hours to ensure thorough derivatization. The sample was then diluted with 200 μL of n-hexane and filtered through a 2 μm organic filter before analysis.
[0221] Preparation and handling of standard solution: The steroid (progesterone) standard is dissolved in MSTFA at an initial concentration of 1g / L to prepare a standard stock solution. To ensure sample purity, the standard stock solution must be filtered through a 2μm organic filter membrane. During the incubation process, vortex mix once every 30 minutes to ensure the uniformity of the derivatization reaction. If undissolved particles are present after 2 hours, the incubation time can be extended to 4 hours. To maintain the optimal analytical performance of the standard, it is recommended to prepare a fresh standard stock solution before each test. If temporary storage is required, it can be sealed and stored at 4°C for a short period of time, but should not exceed 24 hours.
[0222] Gas chromatography-mass spectrometry (GC / MS) detection method: A DB-5MS capillary column (30m×0.25mm×0.25μm) was used for separation, with high-purity helium (purity ≥99.999%) as the carrier gas. The specific analysis parameters are as follows: injection volume 1μL, split ratio 50:1, injection port temperature 290°C. Column temperature program: initial temperature 70°C maintained for 1 minute, then increased to 250°C at a rate of 30°C / min and maintained for 2 minutes, then increased to 280°C at a rate of 20°C / min, and finally maintained for 20 minutes. The mass spectrometer detector used an electron impact (EI) ion source with an ion source temperature of 230°C and a scan range of m / z 50-600.
[0223] 4. Experimental results
[0224] The fermentation experiment results showed that ( Figure 9 ), using a biosensor that responds to steroid hormones to regulate the DGA1 gene, the mechanism is as follows Figure 10 As shown, the biosensor was linked to DGA1, a key gene for lipid synthesis in Yarrowia lipolytica. When progesterone is excessive in the cell and lipid droplets are unable to store it, the biosensor responds to the excess progesterone leaking into the cytoplasm, promoting DGA1 expression. This increases the size of intracellular lipid droplets and the amount of progesterone stored. The resulting engineered strain, yDTY316, showed significant improvement in production of the target product, progesterone, compared to recombinant strains yDTY314 and yDTY315, reaching a yield of 12.1 mg / L. Compared to the starting strain, the recombinant strain yDTY316 increased progesterone production by 12.4%. This improvement is not only reflected in the yield value, but also reflects the effectiveness of the metabolic regulation strategy. This biosensor-based precise regulation strategy provides new insights for subsequent metabolic engineering optimization and also demonstrates the feasibility and superiority of dynamic regulation systems for increasing steroid production.
[0225] Table 3: Primers involved in the present invention
[0226]
[0227]
[0228]
Claims
1. A method for increasing the production of progesterone in Yarrowia esterolyticus, characterized in that The following steps are involved: (1) constructing a biosensor responsive to steroid hormones, wherein the nucleotide sequence of the biosensor responsive to steroid hormones is shown in SEQ ID No. 52; (2) A biosensor responsive to steroid hormones was connected to the key gene DGA1 for lipid synthesis in Yarrowia lipolytica, and the resultant ligation was completed with the linearized pYRSB vector to obtain a recombinant plasmid pDTY316. The nucleotide sequence of the gene DGA1 is shown in SEQ ID No. 57; the nucleotide sequence of the plasmid pYRSB is shown in SEQ ID No.
54. (3) Transform the recombinant plasmid pDTY316 into the recombinant strain yDTY314 to obtain the recombinant strain yDTY316; (4) The recombinant strain yDTY316 was fermented in shake flasks to produce high progesterone.