CN116218698B - Pichia pastoris strain for producing ceramide and construction method and application thereof - Google Patents
Pichia pastoris strain for producing ceramide and construction method and application thereof Download PDFInfo
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- ZVEQCJWYRWKARO-UHFFFAOYSA-N ceramide Natural products CCCCCCCCCCCCCCC(O)C(=O)NC(CO)C(O)C=CCCC=C(C)CCCCCCCCC ZVEQCJWYRWKARO-UHFFFAOYSA-N 0.000 title claims abstract description 95
- VVGIYYKRAMHVLU-UHFFFAOYSA-N newbouldiamide Natural products CCCCCCCCCCCCCCCCCCCC(O)C(O)C(O)C(CO)NC(=O)CCCCCCCCCCCCCCCCC VVGIYYKRAMHVLU-UHFFFAOYSA-N 0.000 title claims abstract description 95
- CRJGESKKUOMBCT-VQTJNVASSA-N N-acetylsphinganine Chemical compound CCCCCCCCCCCCCCC[C@@H](O)[C@H](CO)NC(C)=O CRJGESKKUOMBCT-VQTJNVASSA-N 0.000 title claims abstract description 94
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12N9/10—Transferases (2.)
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- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
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- C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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Abstract
The invention relates to the technical field of biology, in particular to a pichia pastoris strain for producing ceramide and a construction method and application thereof. The invention utilizes pichia pastoris strain to simultaneously over-express lcb1 gene, lcb2 gene and XP_002490234 gene for the first time to synthesize ceramide; the pichia pastoris strain with the over-expressed genes can realize high-density culture, and solves the problem of cell oxidative damage during high-density culture of the pichia pastoris strain, thereby improving the yield of ceramide. The total amount of ceramide or the proportion of unsaturated fatty acid ceramide generated by the pichia pastoris strain after genetic modification is improved, wherein the ceramide containing saturated fatty acid accounts for 2.025 percent of total lipid, the ceramide containing unsaturated fatty acid accounts for 0.426 percent, the total amount of the ceramide and the total amount of the ceramide are 2.451 percent, and the ceramide is improved by 82.64 percent compared with the original strain; wherein the proportion of ceramide containing unsaturated fatty acid is 17.38% of the total ceramide, which is 48.55% higher than that of the original strain.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a pichia pastoris strain for producing ceramide and a construction method and application thereof.
Background
Ceramide is a lipid substance widely existing in eukaryotic cells, is an important lipid second messenger in vivo, and is widely involved in physiological and pathological processes such as proliferation, differentiation, apoptosis and injury of cells. Ceramide is formed by covalent condensation of sphingosine and long-chain fatty acid, and shows diversity of ceramide molecules according to different carbon chain lengths of sphingosine, different degrees of unsaturation and different numbers of hydroxyl groups, different carbon chain lengths of fatty acids connected with sphingosine, different numbers of unsaturated bonds, different positions and numbers of hydroxyl groups, and the like.
In recent years, the research shows that the ceramide is an important component of intercellular lipid of skin horny layer, has the functions of maintaining skin barrier, moisturizing, resisting aging and the like, and is greatly applied to the fields of cosmetics and health-care foods, and the raw material market demand of the ceramide is continuously growing at present, so that an economic, safe and efficient means is urgently needed for producing the ceramide. The synthesis of ceramides by microbial transformation has gained increasing attention. Compared with traditional chemical production, the microbial factory has the following advantages: they can utilize renewable raw materials to produce environmentally friendly, and can produce substances based on biosynthesis that are difficult to produce by chemical synthesis. The yeast is taken as a single-cell eukaryote, has the advantages of short production cycle, abundant sources of fermentation production raw materials, easy mass production, clear genetic background, easy genetic operation and favorable metabolic engineering transformation, is an ideal chassis cell, can create more powerful conditions for industrial mass high-density fermentation production products, and brings more economic benefits for enterprises.
The synthesis of sphingolipids begins in the endoplasmic reticulum, where Serine Palmitoyltransferase (SPT) condenses serine with fatty acyl-CoA to form 3-ketodihydrosphingosine (ketosphingosine) and CO 2 Whereas SPT is a heterodimer made from Lcb1 and Lcb2 subunit proteins.
At present, ceramide is mainly prepared and obtained by saccharomyces cerevisiae, and no report on production by pichia pastoris exists. Compared with pichia pastoris, the saccharomyces cerevisiae has lower culture density, and affects the mass production of ceramide; in addition, pichia pastoris has oxidative damage stress under high-density fermentation culture conditions, and Reactive Oxygen Species (ROS) generated in methanol metabolism attack intracellular biomolecules, so that ceramide containing unsaturated fatty acid is reduced.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a pichia pastoris strain for producing ceramide, and a construction method and application thereof. The pichia pastoris strain of the invention simultaneously overexpresses the lcb1 gene, the lcb2 gene and the XP_002490234 gene, can well synthesize the ceramide, and can realize high-density culture, and cells are not easy to oxidize and damage, thereby improving the yield of the ceramide.
In a first object, the present invention provides a pichia pastoris strain for producing ceramide, which simultaneously overexpresses the lcb1 gene, the lcb2 gene and the xp_002490234 gene.
The invention utilizes pichia pastoris strain to simultaneously over-express lcb1 gene, lcb2 gene and XP_002490234 gene for the first time to synthesize ceramide; the pichia pastoris strain with the over-expressed genes can realize high-density culture, and solves the problem of cell oxidative damage during high-density culture of the pichia pastoris strain, thereby improving the yield of ceramide.
Wherein, the Pichia pastoris strain over-expresses XP_002490234 gene, which can regulate the oxidation/peroxidation state of cells.
As a preferred embodiment of the Pichia pastoris strain according to the present invention, the lcb1 gene, lcb2 gene encodes serine palmitoyltransferase subunits.
As a preferred embodiment of the Pichia pastoris strain, the Pichia pastoris strain is Pichia pastoris GS115 and derivative bacteria thereof, and the Pichia pastoris strain is an industrial microorganism host bacteria with application and wide range, and can realize high-density culture.
The second object of the invention is to provide a construction method of the pichia pastoris strain, which is characterized in that expression plasmids of over-expressing lcb1 genes, lcb2 genes and XP_002490234 genes are transformed into pichia pastoris GS115 to carry out inducible expression or constitutive expression, so that the pichia pastoris strain for producing ceramide is obtained.
The specific construction method comprises the following steps:
1) Cloning of fungal-derived serine palmitoyltransferase (rate-limiting enzyme of the ceramide synthesis pathway) subunit genes lcb1 and lcb2;
2) Cloning XP_002490234 gene of Pichia pastoris GS115 source;
3) The gene lcb1, gene lcb2 and XP 002490234 cloned in step 1) -2) are transformed into Pichia pastoris GS115 through expression plasmids to carry out inducible expression or constitutive expression, so as to obtain the Pichia pastoris strain for producing the ceramide.
As a preferred embodiment of the construction method of the Pichia pastoris strain, the expression plasmid comprises pPIC3.5k or pGAPZ. The expression plasmids defined by the invention include not only ppic3.5k or pGAPZ, but also expression plasmids common in the art.
The third object of the invention is to provide the application of the pichia pastoris strain in improving the output of ceramide.
The total amount of the obtained ceramide is higher by the genetically modified Pichia pastoris strain (P.pastoris GS115/pPIC3.5k-lcb1-lcb2-XP_ 002490234), which shows that the total content of the ceramide can be improved by over-expression of genes (lcb 1 gene, lcb2 gene and XP_002490234 gene).
As a preferred embodiment of the use according to the invention, the ceramide is an unsaturated fatty acid-containing ceramide.
The total amount of ceramide or the proportion of unsaturated fatty acid ceramide generated by the modified pichia pastoris strain is improved, wherein the ceramide containing saturated fatty acid accounts for 2.025 percent of total lipid, the ceramide containing unsaturated fatty acid accounts for 0.426 percent, the total amount of the ceramide and the ceramide is 2.451 percent, and the ceramide is improved by 82.64 percent compared with the original strain; wherein the proportion of ceramide containing unsaturated fatty acid is 17.38% of the total ceramide, which is 48.55% higher than that of the original strain.
The fourth object is to provide a preparation method for improving ceramide, which adopts the pichia pastoris strain as a production strain. The pichia pastoris strain transformed by the method can be used as a production strain, and the production of ceramide is effectively improved.
In a fifth object, the present invention provides a ceramide product prepared by using the pichia pastoris strain as a production strain.
As a preferred embodiment of the ceramide product of the present invention, the ceramide product is a ceramide product containing unsaturated fatty acid.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a pichia pastoris strain for producing ceramide and a construction method and application thereof. The invention utilizes pichia pastoris strain to simultaneously over-express lcb1 gene, lcb2 gene and XP_002490234 gene for the first time to synthesize ceramide; the pichia pastoris strain with the over-expressed genes can realize high-density culture, and solves the problem of cell oxidative damage during high-density culture of the pichia pastoris strain, thereby improving the yield of ceramide. The total amount of ceramide or the proportion of unsaturated fatty acid ceramide generated by the pichia pastoris strain (P.pastoris GS115/pPIC3.5k-lcb1-lcb2-XP_ 002490234) after the genetic modification is improved, wherein the proportion of the ceramide containing saturated fatty acid is 2.025 percent of the total lipid, the proportion of the ceramide containing unsaturated fatty acid is 0.426 percent, the total proportion is 2.451 percent, and the proportion is 82.64 percent higher than that of the original strain; wherein the proportion of ceramide containing unsaturated fatty acid is 17.38% of the total ceramide, which is 48.55% higher than that of the original strain.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples.
In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available.
The term "expression" as used herein is used in accordance with its ordinary and customary meaning as understood by one of ordinary skill in the art and is used without limitation to refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid fragment of the present technology. By "overexpression" is meant the production of a gene product in a transgenic or recombinant organism that exceeds the level of production in a normal or non-transformed organism.
"transformation" is used in accordance with its ordinary and customary meaning as understood by those of ordinary skill in the art and is used without limitation to refer to the transfer of a polynucleotide into a target cell. The transferred polynucleotide may be incorporated into the genomic or chromosomal DNA of the target cell, resulting in genetically stable inheritance (genetically stable inheritance), or it may replicate independently of the host chromosome. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" or "recombinant" or "transformed" organisms.
Standard recombinant DNA and molecular cloning techniques used herein are well known in the art.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred materials and methods are described below.
In accordance with the present disclosure, methods have been developed for producing ceramide and pichia pastoris strains with the lcb1 gene, lcb2 gene, and xp_002490234 gene encoding useful for producing ceramide. Surprisingly and unexpectedly, the use of the engineered pichia pastoris strain can increase the ceramide content, especially the yield of ceramide containing unsaturated fatty acids.
Example 1, pichia pastoris Strain for ceramide production and method of constructing the same
The embodiment provides a construction method of a pichia pastoris strain for producing ceramide, which comprises the following steps:
1. cloning of fungal derived serine palmitoyltransferase (rate limiting enzyme of the ceramide synthesis pathway) subunits genes lcb1 and lcb2, lcb1 and lcb2 encoding two subunits of serine palmitoyltransferase. For specific cloning procedures reference is made to [ J. Samsung (JOSEPH SAMBROOK), molecular cloning laboratory Manual (fine editions), 2008, chemical industry Press ]. The primers used for cloning were as follows:
lcb1-up:5'-ATGAGCACCACAACCGTCAC-3' (SEQ ID NO: 1);
lcb1-down:5'-TCAAAGCTGTTGCAAAACATTGTAAATCTCAG-3' (SEQ ID NO: 2);
lcb2-up:5'-ATGTCAAAAACTATCCCAGATGCTCTCATAG-3' (SEQ ID NO: 3);
lcb2-down:5'-TTAGTACATGGCTTTCTTGCAATCCTCC-3' (SEQ ID NO: 4).
2. The XP_002490234 gene derived from Pichia pastoris GS115 was cloned, and specific cloning operations were described in J.Samhobruck (JOSEPH SAMBROOK), molecular cloning laboratory guidelines (fine editions), 2008, chemical industry Press.
3. Three genes of the gene lcb1, the gene lcb2 and the XP_002490234 are converted into pichia pastoris through pPIC3.5k for inducible expression, or are converted into pichia pastoris through pGAPZ for constitutive expression (without adding methanol inducer, and the pichia pastoris grows and is expressed when normal glycerol or glucose is used as a carbon source), and specific operations are described in the molecular biology laboratory Manual (Ma Wenli, 1.6 of 2011, published by people's military medical press).
Electrotransformation conditions used for inducible expression:
1) In an electroporation cuvette, 4. Mu.L (total 50-100 ng) of linearized plasmid DNA was mixed with 40. Mu.L of competent cells and incubated on ice for 2 minutes;
2) The gap between the electric shock cuvette is 2.0 mm; the electric shock voltage is 1500V;
3) Immediately after electroporation, the samples were resuspended in 0.5mL ice-cold 1.0Mmol/L sorbitol and 0.5mL YPD and incubated for 1h at 30℃in a shaker and plated resistance screening plates.
4. After the positive transformant is verified by electrophoresis, the positive transformant is cultured in a shake flask and a fermentation tank to obtain a pichia pastoris strain (P.pastoris GS115/pPIC3.5k-lcb1-lcb2-XP_ 002490234) for producing ceramide, and the specific operation is shown in fermentation engineering experiment (Li Jianghua, 2011, higher education press).
Extraction of ceramide (see lipid extraction method) and mass spectrometry analysis, see previous report: microbiology report, 2003, 43 (2): 189-194; (2) Enzyme and Microbial Technology,2022, https:// doi.org/10.1016/j.enzmictec.2022.110090].
The extraction method of the ceramide comprises the following steps: (1) thawing the sample on ice. Zirconia beads (0.5 mm) with the same volume as the bacterial liquid are added into the tube, and cells are broken for 10min at 4 ℃;
(2) To the cell lysate was added 990. Mu.L of chloroform/methanol (17:1, v/v) to extract lipids. The biphasic aqueous-organic solution was shaken in a cell breaker at 4℃for 30min and then allowed to stand for 120min. Carefully transferring the organic phase into a new centrifuge tube;
(3) 990. Mu.L of chloroform/methanol (2:1, v/v) was added thereto, and the mixture was allowed to stand at 4℃for 120min, and the two organic phases were combined. After nitrogen purging, the mixture was dissolved in 100. Mu.L of chloroform/methanol (1:2, v/v). Note that all organic solvents used in the extraction process need to be cooled in a refrigerator at-20 ℃ before use, and all organic solvents need to be stored at 4 ℃ in the whole experiment process. Equal amounts of 5 replicate samples were formulated as 3 Quality Control (QC) samples.
Liquid phase mass spectrometry method:
(1) Chromatographic conditions:
the chromatographic separation was carried out using a ThermoFisher Ultimate 3000UHPLC system, waters CSH C18 column (2.1 mm. Times.100 mm. Times.1.7 μm). Mobile phase A was acetonitrile-water (60:40, v/v), containing 10 mmol.L-1 ammonium formate; mobile phase B was isopropanol-acetonitrile (90:10, v/v) containing 10 mmol.l-1 ammonium formate. The linear gradient elution procedure was as follows: 0-3min,40% B;3-20min,40% -95% of B;20-22.5min,95% B;22.5-23min,95% -40% B;23-25min,40% B. The column temperature is 45 ℃, the flow rate is 0.3mL/min, and the sample injection amount is 0.5 mu L.
(2) Mass spectrometry conditions:
hybrid quadrupolar orbit rap using ThermoFisher Q ExactivePlus TM Mass spectrometry (QE) was performed. Experiments were performed in the heating electrospray positive (HESI+) and negative (HESI-) modes, respectively. The spray voltage HESI+ was set to 3.5kV and HESI-was set to 2.8kV. The capillary temperature was 325 ℃ and the assist gas temperature was 350 ℃. The sheath gas flow rate was 35 (Arb) and the assist gas flow rate was 15 (Arb). Full sweepThe resolution of the scan was 140000FWHM (m/z=200), ranging from 20-1800m/z.
TABLE 1 comparison of the relative amounts of ceramide of different saturation levels in total lipid
The results are shown in Table 1, P.pastoris GS115 strain itself has a ceramide background expression, in which ceramide containing saturated fatty acid accounts for 1.185% of total lipid, ceramide containing unsaturated fatty acid accounts for 0.157%, and total 1.342%, in which ceramide containing unsaturated fatty acid accounts for 11.70% of all ceramides.
The P.pastoris GS 115/pPICC 3.5k-lcb1-lcb2-XP_002490234 strain subjected to the gene modification has the advantages that the total amount of ceramide and the proportion of unsaturated fatty acid ceramide are both improved, wherein the proportion of ceramide containing saturated fatty acid is 2.025 percent of total lipid, the proportion of ceramide containing unsaturated fatty acid is 0.426 percent, the total proportion of ceramide containing unsaturated fatty acid is 2.451 percent, and the proportion of ceramide containing unsaturated fatty acid is 82.64 percent higher than that of a starting strain; wherein the proportion of ceramide containing unsaturated fatty acid is 17.38% of the total ceramide, which is 48.55% higher than that of the original strain. The pichia pastoris strain is subjected to over-expression of genes (lcb 1 gene, lcb2 gene and XP_002490234 gene), so that the total content of ceramide and the content of ceramide containing unsaturated fatty acid can be improved. Considering that ceramide is a conservation component in cell membranes, the content change of the ceramide is very small, so that the improvement range in the invention is greatly improved, and the ceramide has application prospect.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
Claims (7)
1. A Pichia pastoris strain for producing ceramide, which is characterized in that the pichia pastoris strain is simultaneously over-expressedlcb1Genes (gene),lcb2Gene and geneXP_002490234A gene;
the saidlcb1Gene and genelcb2The gene encodes serine palmitoyltransferase subunit;
the saidlcb1The primer used for gene cloning is shown as SEQ ID NO. 1-2;
the saidlcb2The primers used for gene cloning are shown as SEQ ID NO. 3-4.
2. The pichia pastoris strain of claim 1, wherein the pichia pastoris strain is pichia pastoris GS115.
3. The method for constructing a Pichia pastoris strain according to claim 1 or 2, wherein the expression is to be overexpressedlcb1Genes (gene),lcb2Gene and geneXP_002490234The expression plasmid of the gene is transformed into Pichia pastoris GS115 to carry out inducible expression or constitutive expression, so as to obtain Pichia pastoris strain for producing ceramide;
the saidlcb1Gene and genelcb2The gene encodes serine palmitoyltransferase subunit.
4. The method for constructing a Pichia pastoris strain according to claim 3, wherein the expression plasmid is pPIC3.5k or pGAPZ.
5. Use of a pichia pastoris strain according to claim 1 or 2 for the production of ceramides.
6. The use according to claim 5, wherein the ceramide is an unsaturated fatty acid-containing ceramide.
7. A method for producing ceramide, characterized in that the pichia pastoris strain as defined in claim 1 or 2 is used as a production strain.
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| Jung-Hoon Bae等.Integrative transformation system for the metabolic engineering of the sphingoid base-producing yeast Pichia ciferrii.《Applied and Environmental Microbiology》.2003,第69卷(第2期),第812-819页，参见全文. * |
| Màrius Tomàs-Gamisans等.Redox Engineering by Ectopic Overexpression of NADH Kinase in Recombinant Pichia pastoris (Komagataella phaffii): Impact on Cell Physiology and Recombinant Production of Secreted Proteins.《Applied and Environmental Microbiology》.2020,第86卷(第6期),e02038-19，参见摘要、表S1. * |
| 陈雅维.利用辅因子工程策略提高酿酒酵母中 S-腺苷蛋氨酸的生物合成.《生物工程学报》.2018,第34卷(第2期),第246-254页，参见全文. * |
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