Recombinant strain for producing campesterol and application thereof
The recombinant Yarrowia lipolytica strain, engineered with optimized At_Dwf5, At_Dwf1, and At_Dwf7 genes, addresses the low yield and conversion issues in campesterol production, achieving high-yield campesterol synthesis suitable for industrial applications.
Patent Information
- Application Number
- CN202410740887.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-07
- Publication Date
- 2024-12-13
AI Technical Summary
Current methods for producing campesterol in microorganisms, such as yeast, face challenges in achieving high yields and efficient conversion to downstream plant sterols, with existing enzyme activities being insufficient for industrial application.
A recombinant strain of Yarrowia lipolytica is engineered using a gene combination of At_Dwf5, At_Dwf1, and optionally At_Dwf7 and/or erg3, integrated into the yeast chromosome, with codon optimization and enhanced MVA pathway, to enhance campesterol synthesis.
The recombinant strain achieves high-yield production of plant-derived campesterol, providing a chassis for further downstream plant sterol pathways and demonstrating efficient microbial synthesis.
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Abstract
Description
Technical Field
[0001] The invention relates to the field of biotechnology, in particular to a recombinant strain for producing campesterol and application thereof. Background Art
[0002] Steroid hormone drugs are a special class of polycyclic terpenoid compounds widely found in animals and plants in nature. They have a variety of physiological activities and uses, especially in the field of medicine, and are an important class of clinical drugs. There are many kinds of steroid hormone drugs in nature, which are widely distributed in animals and plants. In plants, there are soybean phytosterols and diosgenin; in higher organisms, there are cholesterol, bile acid, vitamin D, adrenocortical hormones, sex hormones; in insects, there are ecdysteroids; in lower eukaryotic organisms, such as fungi and yeast, there are mainly ergosterols. These steroid hormone drugs have various biological activities and physiological functions, and are closely related to the life process. Phytosterols are an important sterol compound that can reduce blood cholesterol concentration, prevent prostate diseases, and have hormone-like effects. Phytosterols have also been reported to be associated with reduced cancer risk, anti-oxidation and anti-osteoarthritis. Common phytosterols include campesterol, rapeseed sterol, stigmasterol and β-sitosterol. Among them, campesterol is a precursor of many drugs, such as progesterone and pregnenolone. In addition, campesterol is also an important precursor for the synthesis of downstream plant steroids, such as the plant hormone brassinolide (BRs) and the medicinally interesting phytosterol lactones. Currently, phytosterols are mainly extracted from vegetable oils, but only a mixture of phytosterols can be obtained, which is difficult to separate further.
[0003] The development of synthetic biology in recent years has made it possible to introduce complete plant pathways into microorganisms to produce phytosterols. Compared with obtaining them from plants, the use of microorganisms to synthesize phytosterols has the advantages of a short microbial growth cycle and growth and reproduction that are not affected by seasons, climate and other conditions, and has great prospects. Yarrowia lipolytica is a typical oil-producing fungus, certified as GRAS (Generally Recognized as Safe) by the US Food and Drug Administration. Its genetic manipulation methods are mature and it can accumulate a large amount of lipids in its cells. Studies by Mlicˇková, Holzschu and others have shown that the accumulation of lipids in cells can provide space for the storage of low-polarity compounds, making it a potential host for many hydrophobic compounds.
[0004] Our laboratory has achieved the bioproduction of campesterol in Saccharomyces cerevisiae and Yarrowia lipolytica by replacing the endogenous C22 sterol desaturase (ERG5) terminal encoding gene with the exogenous 7-dehydrocholesterol reductase (DHCR7) encoding gene. However, the campesterol synthesized by this pathway is difficult to be converted downstream to produce plant sterols. In plants, campesterol is synthesized from ergosterol 7,24(28)-diene-3β-ol through the action of Δ7 sterol-c5-desaturase (DWF7), 7-dehydrocholesterol reductase (DWF5) and Δ24(28)-sterol reductase (DWF1). In 2020, Yanran Li et al. used plant enzymes to transform the production of campesterol in Saccharomyces cerevisiae. They increased the titer of campesterol to ~40 mg / L by upregulating the mevalonate pathway. In 2022, Lirong Yang et al. introduced plant Δ24(28)sterol reductase (DWF1) into Saccharomyces cerevisiae to construct an artificial pathway, and then performed directed enzyme evolution to enable yeast to synthesize 24-epiergosterol from scratch. Although the promiscuousness of enzymes such as plant-derived DWF1 enables the production of phytosterols in yeast, its activity towards the precursor ergo-5,7,22,24(28)-tetraene-3β-ol is still very low, and the production level is far from the level of industrial application, indicating that further modification through protein engineering and metabolic engineering is needed. Therefore, it is of great significance to further explore the synthesis of phytosterols using microbial hosts. Summary of the invention
[0005] In view of this, the present invention provides a recombinant strain for producing campesterol and its application. The present invention realizes the efficient synthesis of plant campesterol in Yarrowia lipolytica for the first time.
[0006] A gene combination, which includes At_Dwf5 gene and At_Dwf1 gene.
[0007] The gene combination provided by the present invention also includes At_Dwf7 gene and / or erg3 gene.
[0008] In some embodiments, in the gene combination of the present invention, the At_Dwf5 gene has:
[0009] 1) the nucleotide sequence shown in SEQ ID NO: 1;
[0010] 2) A nucleotide sequence obtained by replacing, deleting or adding one or more bases in the sequence shown in 1), and having the same or similar function as a);
[0011] 3) a nucleotide sequence having at least 80% homology to the sequence shown in 1) or 2);
[0012] The At_Dwf1 gene has:
[0013] 4) the nucleotide sequence shown in SEQ ID NO: 2;
[0014] 5) A nucleotide sequence obtained by replacing, deleting or adding one or more bases in the sequence shown in 4), and having the same or similar function as a);
[0015] 6) a nucleotide sequence having at least 80% homology with the sequence shown in 4) or 5);
[0016] The At_Dwf7 gene has:
[0017] 7) the nucleotide sequence shown in SEQ ID NO: 3;
[0018] 8) A nucleotide sequence obtained by replacing, deleting or adding one or more bases in the sequence shown in 7), and having the same or similar function as a);
[0019] 9) A nucleotide sequence having at least 80% homology with the sequence shown in 7) or 8).
[0020] The present invention discovered the best gene combination for synthesizing plant-derived campesterol in yeast: At_Dwf5 gene and At_Dwf1 gene from Arabidopsis thaliana, which achieved the production of plant-derived campesterol in yeast with a high yield. On this basis, the production of campesterol was further increased after the integration of At_Dwf7 gene.
[0021] In the present invention, codon optimization was performed on At_Dwf5 gene, At_Dwf1 gene and At_Dwf7 gene, and the optimized sequences are shown in SEQ ID NOs: 1 to 3.
[0022] The present invention provides a biomaterial comprising at least one of the following 1) to III):
[0023] 1), an expression cassette containing at least one gene in the gene combination according to claim 1 or 2;
[0024] II), a recombinant vector containing the expression cassette described in I);
[0025] III), transforming or transfecting the host with the recombinant vector as described in II);
[0026] The host includes yeast, algae, mold or bacteria with enhanced MVA pathway;
[0027] The yeast includes Saccharomyces lipolytica, Saccharomyces kluyveri or Saccharomyces cerevisiae.
[0028] In some embodiments, the host contains an enhanced MVA pathway, the erg4 gene is knocked out, and expresses the gene shown in 1) or 2) below:
[0029] In some embodiments of the present invention, Yarrowia lipolytica is used as an example to carry out genetic modification to obtain a recombinant yeast strain. Specifically, the present invention also provides a recombinant yeast strain, which is a yeast strain containing an enhanced MVA pathway, in which the erg4 gene is knocked out, and expresses the gene shown in 1) or 2) below:
[0030] 1)At_Dwf5, At_Dwf1 and At_Dwf7;
[0031] 2)At_Dwf5, At_Dwf1 and erg3.
[0032] In some embodiments, the At_Dwf7 gene and / or erg3 are integrated into the IntD site of the yeast chromosome.
[0033] The present invention also provides a method for constructing the recombinant yeast strain, comprising: performing the following transformation 1) in the bottom plate bacteria, or further comprising the transformations 2) and / or 3) based on 1):
[0034] 1) The At_Dwf5 and At_Dwf1 genes were used to replace the endogenous Aspergillus fumigatus ergosterol synthesis-related gene erg4 in yeast.
[0035] 2) Overexpression of the endogenous erg3 gene in yeast;
[0036] 3) Introduce the Arabidopsis thaliana At_Dwf7 gene;
[0037] The base bacteria is Yarrowia lipolytica with enhanced MVA pathway.
[0038] In some embodiments, the Arabidopsis thaliana At_Dwf7 gene is integrated into the IntD site of the yeast chromosome.
[0039] In some specific embodiments, the present invention uses the AHH12 strain as the base strain, the starting strain of the AHH12 strain is PO1f, and the E. coli-derived AtoB (ERG10), B. petrii-derived HMGR (ERG13) and endogenous HMGS are overexpressed on the basis of the PO1f strain. See the AHH12 strain reported in the article "Engineering the oleaginous yeast Yarrowia lipolytica for production of α-farnesene. Biotechnol Biofuels (Liu Y, et al. 2019 Dec 23; 12: 296)".
[0040] The present invention also provides the use of any of the following in the preparation of plant-derived campesterol:
[0041] The gene combination of the present invention;
[0042] The biomaterial of the present invention;
[0043] The recombinant yeast strain of the present invention.
[0044] The present invention also provides a method for producing plant-derived campesterol, wherein the recombinant yeast strain of the present invention or the host in the biological material of the present invention is subjected to fermentation culture.
[0045] In some embodiments, the recombinant yeast strain or the host is inoculated into a fermentation medium with an initial bacterial concentration of OD600 = 0.1 for fermentation culture. The fermentation culture medium is a YPD liquid medium; further, the YPD liquid medium includes water and 50 g / L glucose, 20 g / L peptone and 10 g / L yeast extract powder. The fermentation culture conditions are: 28°C, 220 rpm for 96 hours.
[0046] In some embodiments, the fermentation culture further comprises a step of seed culture of the recombinant yeast strain or the host. The medium for the seed culture is a YPD liquid medium; further, the YPD liquid medium comprises water and 20 g / L glucose, 20 g / L peptone and 10 g / L yeast extract powder. The seed culture conditions are 28°C, 220 rpm, and culture for 14-16 hours.
[0047] The present invention uses a strain of Yarrowia lipolytica (strain numbered AHH12) with an enhanced precursor MVA pathway as a chassis, and uses Arabidopsis At_Dwf5 and At_Dwf1 genes to replace the endogenous erg4 gene to obtain a recombinant strain SyBE_Yl8091001 that produces plant-derived campesterol. Further, after the Arabidopsis At_DWF7 gene is integrated into the IntD site of the chromosome of the strain, the obtained strain SyBE_Yl8091002 further improves the synthesis efficiency of plant-derived campesterol. The present invention establishes a plant-derived campesterol synthesis pathway in Yarrowia lipolytica for the first time, providing an excellent chassis for the subsequent construction of a downstream plant sterol pathway, and at the same time providing strong evidence for the artificial synthesis of plant sterols by microorganisms. BRIEF DESCRIPTION OF THE DRAWINGS
[0048] Figure 1 is a roadmap for the synthesis of plant-derived campesterol using recombinant Yarrowia lipolytica;
[0049] Figure 2 It is a diagram of the process of constructing plant heterologous genes by yeast recombination;
[0050] Figure 3 This is the result of knocking out the endogenous erg4 in yeast, introducing the heterologous genes At_Dwf5 and At_Dwf1, introducing the heterologous At_Dwf7, and overexpressing the endogenous Erg3. DETAILED DESCRIPTION
[0051] The present invention provides a recombinant strain for producing campesterol and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.
[0052] Table 1 Gene sequences involved in the present invention
[0053]
[0054]
[0055] The test materials used in the present invention are all common commercial products and can be purchased in the market.
[0056] The present invention will be further described below in conjunction with embodiments:
[0057] Example 1 Construction and fermentation test of recombinant Yarrowia lipolytica producing plant-derived campesterol
[0058] The preliminary construction method of the recombinant Yarrowia lipolytica strain for producing campesterol of the present invention is as follows:
[0059] 1. Obtaining a recombinant Yarrowia lipolytica producing plant-derived campesterol
[0060] The precursor MVA pathway enhanced chassis cells were provided by Hou Jin's research group, with the strain number AHH12. For the genetic modifications contained in the recombinant Yarrowia lipolytica, see the AHH12 strain reported in the article "Engineering the oleaginous yeast Yarrowialipolytica for production of α-farnesene. Biotechnol Biofuels (Liu Y, et al. 2019 Dec 23; 12: 296)".
[0061] (1) First, a 700 bp fragment was amplified downward from the 200 bp position upstream of the endogenous erg4 gene of Saccharomyces lipolytica as the upstream homology arm for gene integration. Similarly, a 700 bp fragment was amplified from the 200 bp downstream of erg4 as the downstream integration homology arm, and a BamHI restriction site was inserted between the two homology arms. After the BglII restriction site was selected for restriction digestion on the PUC57 vector, Escherichia coli transformation was performed, and the strain with the correct colony PCR verification and sequence sequencing was selected for plasmid extraction. After 6 hours of BamHI restriction digestion, the amplified Leu2p-Ura3-XPR2t fragment was inserted into the restriction site, and the BsaI restriction site was introduced at the same time to obtain a plasmid vector containing the erg4 knockout integration homology arm and the Ura auxotrophic screening tag. Secondly, the At_Dwf5 and At_Dwf1 genes were inserted into the existing plasmid vector tool kit in our laboratory (the tool kit consists of 600bp upper and lower integration homology arms of the IntF site of Saccharomyces lipolytica and a nourseoin screening tag, containing two sets of promoters and terminators, promoter 1Hp4d and terminator 1OCTt, with a BamHI restriction site, and a BsmBI restriction site between promoter 2TEFin and terminator 2XPR2t for gene insertion). Among them, At_Dwf5 was inserted between the promoter Hp4d and the terminator OCT, and At_Dwf1 was inserted between the promoter TEFin and the terminator XPR2t. Then, PCR amplification was performed and inserted into the BsaI site of the above-mentioned erg4 knockout tool box to obtain a complete erg4 knockout and integration fragment of At_Dwf5 and At_Dwf1, which was integrated into the chromosome of the chassis AHH12. Ura screening was used, and colony PCR and sequencing were performed to obtain the positive strain SyBE_Yl8091001.
[0062] (2) With the help of the lipolytic yeast IntD tool kit available in our laboratory, the tool kit was digested with BsaI enzyme to obtain a cut in the middle of pTEF-T8 to obtain a linearized vector. The 20 bp homologous arms at the two cuts of the vector were added to the two ends of At_Dwf7 and erg3 respectively through the upper and lower primers (this process was obtained with the help of a PCR instrument) to obtain fragment 1 (containing the homologous arms and the At_Dwf7 gene) and fragment 2 (containing the homologous arms and the erg3 gene). In vitro, fragment 1 and fragment 2 were connected to the linearized vector respectively using the seamless cloning method, and the connection systems were introduced into Top10 respectively. The transformants were selected for amplification and culture to obtain the plasmid for sequencing. Primers were designed at both ends of the homologous arms of the plasmid IntD that was sequenced correctly, and the homologous arms, target genes and nutritional label expression cassettes were amplified with the help of a PCR instrument. Then, they were integrated into the chromosome of the strain SyBE_Yl8091001 through yeast transformation, and strain SyBE_Yl8091002 (At_DWF7) and strain SyBE_Yl8091003 (Yl_Erg3) were obtained respectively.
[0063] Among them, the backbone vector of the IntD toolbox of Saccharomyces lipolytica is the PUC57 vector, which contains 700bp integration homology arms upstream and downstream of the Saccharomyces lipolytica D site, TEFin promoter / T8 terminator and leucine auxotrophic tag. A BsaI restriction site is left between the promoter and terminator for gene insertion. The leucine auxotrophic screening tag is initiated by the TEF promoter and the terminator is XPR2t.
[0064] 2. Fermentation detection of experimental strains producing rapeseed sterol
[0065] Test materials: strain SyBE_Yl8091001-SyBE_Yl8091003
[0066] Test method:
[0067] Seed culture medium: YPD liquid medium (glucose 20 g / L, peptone 20 g / L, yeast extract powder 10 g / L);
[0068] Fermentation medium: YPD liquid medium (glucose 50 g / L, peptone 20 g / L, yeast extract powder 10 g / L).
[0069] The above strains were inoculated into 5 mL of seed culture medium and cultured at 28°C and 220 rpm for 14-16 h. The initial bacterial concentration OD 600 =0.1 were inoculated into 50 mL of fermentation medium, respectively, and cultured at 28°C and 220 rpm for 96 h. The bacterial density (OD600) and the yield of campesterol at the end of fermentation were monitored.
[0070] Quantitative detection method of campesterol:
[0071] Extraction of intracellular rapesterol: Collect 1 mL of fermentation medium in a 2 mL centrifuge tube and centrifuge at 12,000 rpm for 1 min to collect cells. Resuspend the cells with 1 mL of distilled water, centrifuge at 12,000 rpm for 1 min, and discard the supernatant. Add 1 mL of 3M hydrochloric acid to resuspend the cells, boil in 100°C boiling water for 10 min, cool in an ice bath for 5 min, and shake the cell suspension. After the suspension cools to room temperature, centrifuge at 12,000 rpm for 1 min and discard the supernatant (if the cell debris cannot be precipitated, add 1-3 times the volume of distilled water and centrifuge to collect the precipitate), add 1 mL of distilled water to resuspend the cell debris, and centrifuge and discard the supernatant. In the saponification reaction, resuspend the cell debris with 500 μL of 2M potassium hydroxide-methanol solution, seal the edge of the centrifuge tube with a sealing film, and react in a 55°C metal bath for more than 6 hours. Transfer the saponification reaction suspension to a new centrifuge tube with a tight cap. After the saponification reaction system cools to room temperature, add 500 μL of n-hexane, vortex and oscillate for 10 minutes for extraction, and collect the upper n-hexane phase into a new centrifuge tube. Continue to extract the lower saponification solution with 500 μL of fresh n-hexane for 3 times, and combine the 4 n-hexane phases and dry them in a vacuum centrifuge for 30 minutes to obtain a dry steroid sample.
[0072] Extracellular campesterol extraction: 1 mL of fermentation medium was collected in a 2 mL centrifuge tube, and about 200 μg of glass beads with a diameter of 425-600 μm were added, and 700 μL of ethyl acetate was added. The mixture was vortexed for 10 min, and the upper organic phase was collected after centrifugation at 12,000 rpm for 5 min. The lower fermentation broth was repeatedly extracted with 700 μL of fresh ethyl acetate, and the two ethyl acetate phases were combined and vacuum centrifuged for 40-60 min to obtain a dry steroid sample.
[0073] GC / MS detection and analysis: The dried steroid sample was dissolved in 100 μL of the derivatization reagent MSTFA and incubated in a 37°C incubator for 2 h. After adding 100 μL of n-hexane, the sample was filtered with a 2 μm organic filter membrane. A DB-5MS gas chromatography column was used, with high-purity helium as the carrier gas, an injection volume of 1 μL, and a split ratio of 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, maintained for 2 min, and then increased to 280°C at 20°C / min, maintained for 20 min.
[0074] Experimental results: Among the three strains, after statistics on the production of campesterol in each strain, it was found that the strain numbered SyBE_Yl8091002 had the highest production. Compared with the endogenous Δ7 sterol-c5-desaturase (erg3), Arabidopsis thaliana At_Dwf7 was more inclined to synthesize campesterol.
[0075] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. Gene combination, It is characterized in that Including At_Dwf5 gene and At_Dwf1 gene.
2. The gene combination according to claim 1, It is characterized in that Also included are the At_Dwf7 gene and / or the erg3 gene.
3. The gene combination according to claim 2, It is characterized in that The At_Dwf5 gene has: 1) the nucleotide sequence shown in SEQ ID NO: 1; 2) A nucleotide sequence obtained by replacing, deleting or adding one or more bases in the sequence shown in 1), and having the same or similar function as a); 3) a nucleotide sequence having at least 80% homology to the sequence shown in 1) or 2); The At_Dwf1 gene has: 4) the nucleotide sequence shown in SEQ ID NO: 2; 5) A nucleotide sequence obtained by replacing, deleting or adding one or more bases in the sequence shown in 4), and having the same or similar function as a); 6) a nucleotide sequence having at least 80% homology to the sequence shown in 4) or 5); The At_Dwf7 gene has: 7) the nucleotide sequence shown in SEQ ID NO: 3; 8) A nucleotide sequence obtained by replacing, deleting or adding one or more bases in the sequence shown in 7), and having the same or similar function as a); 9) A nucleotide sequence having at least 80% homology with the sequence shown in 7) or 8).
4. Biomaterials, It is characterized in that Including at least one of the following 1) to III): I), an expression cassette containing at least one gene of the gene combination according to any one of claims 1 to 3; II), a recombinant vector containing the expression cassette described in I); III), transforming or transfecting the host with the recombinant vector as described in II); The host includes yeast, algae, mold or bacteria with enhanced MVA pathway; The yeast includes Saccharomyces lipolytica, Saccharomyces kluyveri or Saccharomyces cerevisiae.
5. Recombinant yeast strains, It is characterized in that It is a yeast strain containing an enhanced MVA pathway, in which the erg4 gene is knocked out, and in which the gene shown in 1) or 2) below is expressed: 1)At_Dwf5, At_Dwf1 and At_Dwf7; 2)At_Dwf5, At_Dwf1 and erg3.
6. The recombinant yeast strain according to claim 5, It is characterized in that The At_Dwf7 gene is integrated into the IntD site of the yeast chromosome.
7. A method for constructing a recombinant yeast strain according to claim 5 or 6, It is characterized in that The following transformation as shown in 1) is carried out in the chassis bacteria, or the transformation as shown in 2) and / or 3) is also included on the basis of 1): 1) Replace the endogenous Aspergillus fumigatus ergosterol biosynthesis-related gene erg4 with At_Dwf5 and At_Dwf1 genes; 2) Overexpression of the endogenous erg3 gene in yeast; 3) Introduce At_Dwf7 gene; The base bacteria is Yarrowia lipolytica with enhanced MVA pathway.
8. The construction method according to claim 7, It is characterized in that The MVA pathway-enhanced Yarrowia lipolytica uses PO1f as a starting strain and overexpresses the ERG10 gene from E. coli, the ERG13 gene from B. petrii and the endogenous HMGS gene.
9. Use of any of the following in the preparation of plant-derived campesterol: The gene combination according to any one of claims 1 to 3; The biological material according to claim 4; The recombinant yeast strain according to claim 5 or 6.
10. A method for producing plant-derived campesterol, It is characterized in that The recombinant yeast strain according to any one of claims 5 to 6 or the host in the biological material according to claim 4 is fermented and cultured.