Method for improving heterologous synthesis of pregnenolone and progesterone by using yarrowia lipolytica
By knocking out the MHY1 gene in Yarrowia lipolytica strain SyBE_Yl2090002 and introducing 3β-hydroxysteroid dehydrogenase, the fermentation liquid viscosity problem caused by the increase in mycelium morphology was solved, the yield of pregnenolone and progesterone was improved, the regulation of bacterial morphology was optimized, and the efficient synthesis of steroid hormones was achieved.
Patent Information
- Application Number
- CN202510414593.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-03
- Publication Date
- 2025-07-04
AI Technical Summary
In the prior art, the increase in the number of Yarrow's hyphae of lipolytica leads to an increase in the viscosity of the fermentation broth, and the difficulty of cells to obtain nutrients and oxygen, which affects bacterial growth and low output rate of steroid products.
By knocking out the MHY1 gene in the recombinant Yarrowia lipolytic strain SyBE_Yl2090002, strain Yl001 was constructed, and 3β-hydroxysteroid dehydrogenase was introduced in strain Yl102, the bacterial morphology was regulated to optimize the synthesis pathway of pregnenolone and progesterone.
The production of pregnenolone by 52.2% and progesterone by 23.7% were improved, the regulation of bacterial morphology was optimized, and the synthesis efficiency of steroid hormones was improved.
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Figure CN120249351A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and in particular to a method for improving the heterologous synthesis of pregnenolone and progesterone in Yarrowia lipolytica. Background Art
[0002] Steroid hormones are a type of tetracyclic aliphatic hydrocarbon with a cyclopentanoperhydrophenanthrene nucleus, and are one of the important components of organisms, commonly present in animals, plants, and some microorganisms. As the second largest category of chemical drugs after antibiotics, steroid hormones are an important part of the pharmaceutical market. Among them, progesterone, as a progestogen necessary for maintaining pregnancy, not only has various physiological activities such as anti-abortion, anti-inflammatory, and immunomodulatory effects, but can also be used as a precursor to produce steroid drugs such as testosterone and hydrocortisone, and is widely used in the medical field. Currently, fully synthetic routes and semi-synthetic methods using diosgenin, ergosterol, etc. as raw materials have been reported. However, due to their complex structures and poor synthetic selectivity, chemical synthesis is relatively difficult. The microbial side-chain degradation route starting from plant and animal sterols is a classic route for the artificial production of steroid hormones. However, many genes in this side-chain degradation pathway are not yet clear, which limits the further development of this route. In contrast, researchers have relatively thoroughly analyzed the genes of the de novo synthesis pathway of steroid hormones in higher biological cells. This synthesis pathway includes: a sterol synthesis pathway using simple carbon sources as substrates; a side-chain cleavage reaction in which sterols are synthesized into the key steroid pregnenolone through the P450 side-chain cleavage enzyme (P450scc) system; and a downstream steroid hormone synthesis pathway highly regulated by a series of P450 oxidases and hydroxysteroid dehydrogenases (HSDs) with non-specific substrate catalytic activities, starting from pregnenolone as the initial substrate. Therefore, using engineered strains to ferment and produce progesterone has become a potentially new manufacturing method, providing an efficient, stable, and sustainable technical route for the green synthesis industry of steroid hormones.
[0003] Yarrowia lipolytica is considered a potential advantageous host for supporting the expression of multi-P450 oxidase pathways due to the sufficient supply of intracellular acetyl-CoA. And Yarrowia lipolytica has dimorphic characteristics, that is, it can switch between the yeast form and the mycelial form. The regulation of cell morphology is also of great significance in the microbial fermentation process, especially crucial for enhancing the efficiency of fungi in industrial fermentation. The appearance of the mycelial form not only changes the physical properties of the fermentation broth but also affects the nutrient absorption and oxygen transfer mechanism. As the number of mycelia increases, the viscosity of the fermentation broth increases, making it more difficult for cells to obtain nutrients and oxygen, ultimately having an adverse impact on cell growth and the production of target products. Therefore, exploring and optimizing the regulation of cell morphology is of great significance for promoting the optimization of the fermentation process of dimorphic fungi. The endogenous gene MHY1 (YALI0_B21582g) of Yarrowia lipolytica is a transcription factor encoding a protein kinase A (PKA) pathway in cAMP and is a key regulator of dimorphic transformation. Knocking out MHY1 can effectively control the morphology of Yarrowia lipolytica in the yeast form, so it can be used as a subsequent screening target. Summary of the Invention
[0004] To overcome the deficiencies in the prior art that the increase in the number of mycelia increases the viscosity of the fermentation broth, making it more difficult for cells to obtain nutrients and oxygen, and having an adverse impact on cell growth and the production of steroid products, the main object of the present invention is to provide a method for improving the heterologous synthesis of pregnenolone and progesterone in Yarrowia lipolytica. By optimizing the regulation of cell morphology, the problem of low production rate of steroid products is solved.
[0005] To achieve the aforementioned invention object, the technical solutions adopted by the present invention include:
[0006] A method for improving the heterologous synthesis of pregnenolone in Yarrowia lipolytica, based on the recombinant Yarrowia lipolytica strain SyBE_Yl2090002, knocking out the MHY1 gene to construct strain Yl001.
[0007] A method for improving the heterologous synthesis of progesterone in Yarrowia lipolytica, based on the recombinant Yarrowia lipolytica strain SyBE_Yl2090002, introducing 3β-hydroxysteroid dehydrogenase and knocking out the MHY1 gene to construct strain Yl102.
[0008] A recombinant Yarrowia lipolytica strain, the construction method thereof is: based on the recombinant Yarrowia lipolytica strain SyBE_Yl2090002, knocking out the MHY1 gene, and the strain number is Yl001.
[0009] A recombinant Yarrowia lipolytica strain, the construction method thereof is: based on the recombinant Yarrowia lipolytica strain SyBE_Yl2090002, introducing 3β-hydroxysteroid dehydrogenase and knocking out the MHY1 gene, and the strain number is Yl102.
[0010] Application of the recombinant Yarrowia lipolytica strain Yl001 in increasing the production of pregnenolone.
[0011] Application of the recombinant Yarrowia lipolytica strain Yl102 in increasing the production of progesterone.
[0012] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0013] By knocking out the MHY1 gene in the strain for de novo synthesis of pregnenolone or progesterone, the present invention increases the production of pregnenolone or progesterone by the recombinant Yarrowia lipolytica strain. The newly constructed strain Yl001 has a 52.2% increase in pregnenolone production compared to the control strain, reaching 2.95 mg / L at the end of fermentation; the newly constructed strain Yl102 has a 23.7% increase in progesterone production compared to the control strain, reaching 6.57 mg / L at the end of fermentation. The influence of regulating the morphological changes of Yarrowia lipolytica on the heterologous synthesis pathway is explored, providing a research direction for subsequent increasing the production of pregnenolone and progesterone. Brief Description of the Drawings
[0014] Figure 1 is a pathway diagram for the synthesis of pregnenolone and progesterone by recombinant Yarrowia lipolytica;
[0015] Figure 2 is a schematic diagram of the plasmid designed to knock out the MHY1 gene;
[0016] Figure 3 is a result diagram of the influence of regulating cell morphology on pregnenolone and progesterone. (A) Cell morphology characterization diagram of the strain at the end of fermentation, i.e., after 168 h of shake-flask culture (B) Influence of regulating cell morphology on pregnenolone production (C) Influence of regulating cell morphology on progesterone production. Detailed Embodiments
[0017] The technical solutions of the present invention will be described clearly and completely below. Apparently, the described embodiments are part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.
[0018] The list of strains used in this experiment is shown in Table 1.
[0019] Example 1 Regulation of the morphology of Yarrowia lipolytica strain to increase the production of pregnenolone
[0020] 1. Obtaining of the recombinant Yarrowia lipolytica strain producing pregnenolone
[0021] The recombinant Yarrowia lipolytica strain for pregnenolone was provided by the Yuan Yingjin research group, and the strain number is SyBE_Yl2090002. The recombinant Yarrowia lipolytica strain SyBE_Yl2090002 uses the wild Yarrowia lipolytica strain ATCC201249 as the starting strain, with the genotype ERG5::URA3, EXP1p-Xl_DHCR7-XPR2t, IntD::Hph, TEF1p-Bt_mAdR-LIP2t-GPDp-Bt_mAdx-OCTt, pBR322::LEU2, TEF1p-Ss_mCYP11A1-LIP2t. It is mentioned in the literature "Pregnenolone Overproduction in Yarrowia lipolytica by Integrative Components Pairing of the Cytochrome P450scc System". Among them, ATCC201249 can be obtained from the American Type Culture Collection, with the number 201249. As Figure 1 shown, the strain SyBE_Yl2090002 successfully introduced the heterologous synthesis pathway of pregnenolone into Yarrowia lipolytica, knocked out the endogenous ERG5 and integrated the DHCR7 gene, shifted the metabolic pathway from the synthesis of endogenous ergosterol to the synthesis of campesterol, and catalyzed the production of pregnenolone from campesterol through the P450scc system.
[0022] 2. Construction of the morphology-regulating strain
[0023] A knockout expression cassette was designed for the endogenous gene MHY1 (SEQ ID NO.1). The knockout expression cassette is a plasmid obtained by ligating the upstream and downstream arms of the gene to be knocked out and the screening tag into the same vector, and ligated in vitro by the seamless cloning method. The norstatin fragment (NoR) of the antibiotic was amplified and ligated into the promoter LEUp-terminator CYCt to obtain the antibiotic screening marker expression cassette. Then, this expression cassette was ligated into the plasmid designed with the upstream and downstream arms of the MHY1 homologous arm, as Figure 2 shown, to obtain the plasmid pUC57-MHY1(UP)-LEUp-NoR-CYCt-MHY1(down).
[0024] The constructed plasmid was transferred into TOP10 competent cells. Transformants were selected for large-scale culture to extract plasmids for sequencing. The knockout fragment containing MHY1(UP)-LEUp-NoR-CYCt-MHY1(down) was amplified and transformed into Yarrowia lipolytica SyBE_Yl2090002 using the Frozen-EZ kit. After transformation, screening was carried out using a YPD solid plate containing NoR. The YPD solid plate containing NoR was prepared with 20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract powder, and 2% agar powder. After autoclaving, a mother liquor of NoR with a final concentration of 200 mg / L was added. The selected transformants were boiled in 20 mM NaOH solution for PCR verification. The single colonies with correct verification were isolated and purified for fermentation detection, and the single-gene knockout strain Yl001 was obtained.
[0025] 3. Application of the recombinant Yarrowia lipolytica strain Yl001 in improving the production of pregnenolone
[0026] The single-gene knockout strain Yl001 and the control strain SyBE_Yl2090002 were respectively inoculated into 5 mL of 2% glucose YPD medium and incubated at 30 °C and 220 rpm for 24 h to obtain primary seeds. The primary seeds were transferred to fresh 5 mL of 2% glucose YPD medium at a volume ratio of 1 / 25 and cultured for another 16 h to obtain secondary seeds. The secondary seeds were inoculated into 50 mL of 5% glucose YPD fermentation medium at an initial concentration of OD 600 = 0.1 and cultured at 28 °C and 220 rpm for 168 h.
[0027] When extracting pregnenolone, 1 mL of the fermentation medium was collected in a 2 mL centrifuge tube and centrifuged at 12000 rpm for 1 min to collect the cells. The cells were resuspended with 1 mL of distilled water, centrifuged at 12000 rpm for 1 min, and the supernatant was discarded. 1 mL of 3 M hydrochloric acid was added to resuspend the cells, and the mixture was boiled in a 100 °C water bath for 15 min and then cooled in an ice bath for 5 min. After the suspension cooled to room temperature, it was centrifuged at 12,000 rpm for 1 min to discard the supernatant. 1 mL of distilled water was added to resuspend the cell debris, and the supernatant was discarded after centrifugation. The cell debris was resuspended with 300 μL of 2 M potassium hydroxide-methanol solution, the edge of the centrifuge tube mouth was sealed with a sealing film, and the reaction was carried out in a 55 °C metal bath for more than 6 h. The saponification reaction suspension was transferred to a new centrifuge tube with a tightly capped tube lid. After the saponification reaction system cooled to room temperature, 500 μL of n-hexane was added, and vortex oscillation was carried out for 10 min for extraction. The upper n-hexane phase was collected into a new centrifuge tube. The lower saponification solution was repeatedly extracted with 500 μL of fresh n-hexane once, and the two n-hexane phases were combined and vacuum centrifuged and dried for 20 min to obtain a dried steroid sample.
[0028] The dried steroid sample was dissolved in 100 μL of the derivatization reagent N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) and incubated in a 37°C incubator for 2 hours. After adding 100 μL of n-hexane, the sample was filtered through a 2 μm organic filter. The pregnenolone standard was dissolved in MSTFA and incubated in a 37°C incubator for 4 hours to ensure complete steroid derivatization. The sample was mixed every 30 minutes during the incubation. After filtering through a 2 μm organic filter, it can be diluted with chromatographic grade n-hexane and a standard curve can be prepared.
[0029] In the GC / MS detection of this study, a DB-5MS gas chromatography column was used, high-purity helium was used as the carrier gas, the injection volume was 1 μL, and the split ratio was 50: 1. The injection port temperature was 290°C, the column temperature was maintained at 70°C for 1 min, increased to 250°C at 30°C / min and maintained for 2 min, and then increased to 280°C at 20°C / min and maintained for 20 min.
[0030] The results are as follows Figure 3 As shown in A, at 168 h of fermentation, the cells of the strain after MHY1 gene knockout showed obvious morphological differences, with the overall mycelial morphology decreasing and the yeast morphology increasing. Figure 3 As shown in B, after knocking out the MHY1 gene, the biomass increased by 13.9%, and the pregnenolone production increased by 52.2%, reaching 2.95 mg / L at the end of fermentation. This indicates that knocking out the C2H2-type zinc finger protein MHY1 can not only eliminate the formation of hyphal morphological cells, but also increase biomass accumulation and pregnenolone production.
[0031] Example 2 Recombinant Yarrowia lipolytica strains to increase progesterone production
[0032] 1. Obtaining a recombinant Yarrowia lipolytica strain that produces progesterone
[0033] The strain number is SyBE_Yl2090002 provided by Yuan Yingjin's research group. The recombinant Yarrowia lipolytica strain SyBE_Yl2090002 is based on the wild Yarrowia lipolytica strain ATCC201249, and the genotype is ERG5::URA3, EXP1p-Xl_DHCR7-XPR2t, IntD::Hph, TEF1p-Bt_mAdR-LIP2t-GPDp-Bt_mAdx-OCTt, pBR322::LEU2, TEF1p-Ss_mCYP11A1-LIP2t. Figure 1 As shown, in this study, SyBE_Yl2090002 was used as the chassis strain, and bovine 3β-hydroxysteroid dehydrogenase (Bt_3βHSD, SEQ ID NO.2) was introduced to successfully catalyze pregnenolone to produce progesterone in Yarrowia lipolytica, introduce the progesterone heterologous synthesis pathway, and obtain the progesterone de novo synthesis strain Yl101.
[0034] 2. Construction of Morphology-Regulating Strains
[0035] The construction of the knockout expression cassette of the endogenous gene MHY1 was the same as in Example 1. The knockout fragment containing MHY1(UP)-LEUp-NoR-CYCt-MHY1(down) was amplified and transformed into the strain Yl101. The verified correct single colonies were isolated and purified for fermentation detection, and the single-gene knockout strain Yl102 was obtained.
[0036] 3. Application of Recombinant Yarrowia lipolytica Strain Yl102 in Improving Progesterone Production
[0037] The single-gene knockout strain Yl102 and the control strain Yl101 were respectively inoculated into 5 mL of 2% glucose YPD medium and cultured for 24 h (30 °C, 220 rpm) to obtain the primary seed liquid, and then transferred at 1 / 25 volume to 5 mL of 2% glucose YPD medium and continued to be cultured for 16 h to obtain the secondary seed. The secondary seed was inoculated into 50 mL of 5% glucose YPD fermentation medium at an initial concentration of OD 600 = 0.1 and cultured at 28 °C and 220 rpm for 192 h.
[0038] The extraction and detection methods of pregnenolone were the same as in Example 1.
[0039] The extraction and detection methods of progesterone were as follows: When extracting progesterone, 1 mL of the fermentation medium was collected in a 2 mL centrifuge tube, about 200 μg of glass beads with a diameter of 425 - 600 μm were added, 700 μL of ethyl acetate was added, vortexed for 10 min, centrifuged at 12,000 rpm for 5 min, and the upper organic phase was collected. The lower fermentation broth was repeatedly extracted once with 700 μL of fresh ethyl acetate, and the two ethyl acetate phases were combined and vacuum centrifuged and dried for 40 - 60 min to obtain a dry steroid sample. The dry steroid sample was dissolved in 100 μL of the derivatization reagent MSTFA and incubated in an incubator at 37 °C for 2 h. After adding 100 μL of n-hexane, it was filtered and loaded onto the column with a 2 μm organic filter membrane. The progesterone standard was dissolved in MSTFA and incubated in an incubator at 37 °C for 2 h to ensure complete steroid derivatization. During the incubation, it was mixed every 30 min. After filtering with a 2 μm organic filter membrane, it could be diluted with chromatographic grade n-hexane to prepare a standard curve.
[0040] In the GC / MS detection of this study, a DB-5MS gas chromatographic column was used, high-purity helium was used as the carrier gas, the injection volume was 1 μL, and the split ratio was 50:1. The injection port temperature was 290 °C, the column temperature was maintained at 70 °C for 1 min, increased to 250 °C at a rate of 30 °C / min and maintained for 2 min, and then increased to 280 °C at a rate of 20 °C / min and maintained for 20 min.
[0041] The results were asFigure 3 As shown in C, knocking out the MHY1 gene is beneficial to increasing progesterone production, which is increased by 23.7% compared with the control strain. The progesterone production at the end of fermentation is 6.57 mg / L, and only extremely low levels of pregnenolone can be detected intracellularly. Therefore, knocking out the MHY1 gene can not only make yeast cells in the yeast form, but also increase the flux of heterologous steroid synthesis.
[0042] The above-mentioned embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.
[0043] Table 1 List of strains used in this experiment
[0044]
Claims
1. A method for improving the heterologous synthesis of pregnenolone in Yarrowia lipolytica, characterized in that, Based on the recombinant Yarrowia lipolytica strain SyBE_Yl2090002, the MHY1 gene was knocked out to construct strain Yl001.
2. A method for improving the heterologous synthesis of progesterone by Yarrowia lipolytica, characterized in that, Based on the recombinant Yarrowia lipolytica strain SyBE_Yl2090002, 3β-hydroxysteroid dehydrogenase was introduced and the MHY1 gene was knocked out to construct strain Yl102.
3. A recombinant Yarrowia lipolytica strain, characterized in that, The construction method is as follows: Based on the recombinant Yarrowia lipolytica strain SyBE_Yl2090002, the MHY1 gene was knocked out, and the strain number is Yl001.
4. A recombinant Yarrowia lipolytica strain, characterized in that, The construction method is as follows: Based on the recombinant Yarrowia lipolytica strain SyBE_Yl2090002, 3β-hydroxysteroid dehydrogenase was introduced and the MHY1 gene was knocked out, and the strain number is Yl102.
5. Use of the recombinant Yarrowia lipolytica strain described in claim 3 in increasing the production of pregnenolone.
6. Use of the recombinant Yarrowia lipolytica strain described in claim 4 in increasing the production of progesterone.