CN114606150B - Gene engineering strain for producing gamma-linolenic acid, construction method and application thereof - Google Patents
Gene engineering strain for producing gamma-linolenic acid, construction method and application thereof Download PDFInfo
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- CN114606150B CN114606150B CN202210402994.4A CN202210402994A CN114606150B CN 114606150 B CN114606150 B CN 114606150B CN 202210402994 A CN202210402994 A CN 202210402994A CN 114606150 B CN114606150 B CN 114606150B
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- linolenic acid
- yarrowia lipolytica
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- VZCCETWTMQHEPK-QNEBEIHSSA-N gamma-linolenic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/CCCCC(O)=O VZCCETWTMQHEPK-QNEBEIHSSA-N 0.000 title claims abstract description 75
- 235000020664 gamma-linolenic acid Nutrition 0.000 title claims abstract description 75
- VZCCETWTMQHEPK-UHFFFAOYSA-N gamma-Linolensaeure Natural products CCCCCC=CCC=CCC=CCCCCC(O)=O VZCCETWTMQHEPK-UHFFFAOYSA-N 0.000 title claims abstract description 74
- 229960002733 gamolenic acid Drugs 0.000 title claims abstract description 74
- 238000010276 construction Methods 0.000 title claims abstract description 35
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 27
- 239000008157 edible vegetable oil Substances 0.000 claims abstract description 23
- 108010037138 Linoleoyl-CoA Desaturase Proteins 0.000 claims abstract description 20
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Abstract
The application discloses a genetic engineering strain for producing gamma-linolenic acid, a construction method and application thereof, and belongs to the technical field of bioengineering. The application takes yarrowia lipolytica Yarrowia lipolytica as an original strain, expresses delta 6 desaturase gene and simultaneously overexpresses PEX4 gene and PEX5 gene of beta-oxidation pathway; the engineering strain constructed by the application can synthesize gamma-linolenic acid on a culture medium which takes waste edible oil as a unique carbon source. The engineering bacteria of the application have good stability, high content of intracellular grease gamma-linolenic acid, no limitation of seasons, stable yield, low cost of fermentation substrate and resource utilization of waste edible oil. The application is suitable for constructing the genetic engineering strain for producing the gamma-linolenic acid, and the constructed engineering strain is further applied to producing the gamma-linolenic acid.
Description
Technical Field
The application belongs to the technical field of bioengineering, and relates to a yarrowia lipolytica engineering strain, in particular to a genetic engineering strain for producing gamma-linolenic acid, a construction method and application thereof.
Background
Gamma-linolenic acid (GLA, C18:3) Δ6,Δ9,Δ12 ) Has wide physiological activity and obvious pharmacological action, and is widely applied in the fields of medical treatment, beauty treatment, cosmetics and the like. At present, gamma-linolenic acid produced at home and abroad mainly comes from evening primrose (Oenothera biennis) of the salicaceae, but the extraction of gamma-linolenic acid from plants is limited by a plurality of factors, for example, the growth period of the plants is long, the occupied area is large, the natural condition is limited, the oil content is unstable and the like, so that the increasing market demands of people are difficult to meet.
Microbial fermentation is not limited by seasons, can be used for large-scale production, has the advantages of stable yield, reliable quality, wide sources of materials used for fermentation, low price and the like, and yarrowia lipolytica (yarrowia lipolytica) is currently used as a research hotspot for polyunsaturated fatty acid production. Since the naturally occurring fatty acid in yarrowia lipolytica is octadecadienoic acid (LA, C18:2), the fermentative production of gamma-linolenic acid with yarrowia lipolytica requires exogenous introduction of the Δ6 desaturase gene, the mechanism of which is shown in FIG. 1.
Conventional yarrowia lipolytica fermentations to produce polyunsaturated fatty acids typically use glucose as a carbon source, which is costly, limiting their use in industrial production. The waste edible oil contains long-chain fatty acids such as palmitic acid, stearic acid, oleic acid, linoleic acid and the like, and the current main stream treatment mode of the waste edible oil generally has negative influence on the environment and water quality and causes resource waste. Yarrowia lipolytica can however utilize the in vivo beta-oxidation pathway to degrade long chain fatty acids into medium chain fatty acids (e.g., caproic acid, caprylic acid, capric acid, etc.) and be further utilized as a source of fermentative carbon.
In the culture medium taking the waste edible oil as the sole carbon source, the yeast growth amount is low, the yield of fermentation products is low, and the requirement of industrial production is difficult to meet, so that the engineering yarrowia lipolytica which can grow well in the culture medium taking the waste edible oil as the sole carbon source and has high gamma-linolenic acid yield is constructed, and the engineering yarrowia lipolytica has good application prospect.
Disclosure of Invention
The application aims at providing a genetic engineering strain for producing gamma-linolenic acid, which provides an effective way for the commercial production of gamma-linolenic acid by using yarrowia lipolytica;
the application further aims at providing a construction method of the genetically engineered strain for producing gamma-linolenic acid, so as to achieve the aim of simply, effectively and accurately constructing the strain;
the application also aims to provide an application of the genetically engineered strain for producing the gamma-linolenic acid, and the waste edible oil is used as a sole carbon source, so that the aim of synthesizing the gamma-linolenic acid more healthily without being limited by seasons and with relatively low cost is fulfilled; meanwhile, the corrosion and the blockage of municipal sewage pipe networks are reduced, the harm caused by the inflow of waste grease into society is prevented, and the improvement of the recycling economy level is promoted.
In order to achieve the above purpose, the technical scheme adopted by the application is as follows:
a genetically engineered strain for producing gamma-linolenic acid uses yarrowia lipolytica as an original strain, expresses delta 6 desaturase gene and simultaneously overexpresses PEX4 gene and PEX5 gene, and can grow by taking waste edible oil as a unique carbon source.
As a limitation of the application, the delta 6 desaturase gene is derived from mortierella alpina Mortierella alpina, and the delta 6 desaturase gene sequence is shown in SEQ ID No.1, specifically:
atggctgctgctcccagtgtgaggacgtttactcgggccgagattttgaatgccgaggccctgaatgagggcaagaaggatgccgaggcaccctttctgatgatcattgacaacaaggtgtacgatgtccgcgagtttgtccctgatcatcccggtggaagtgtgattctcacgcacgttggcaaggacggcactgacgtctttgacactttccaccccgaggctgcttgggagactcttgccaacttttacgttggtgatattgatgagagcgatcgtgccatcaagaatgatgactttgcggccgaggttcgcaagctgcgcaccttgttccagtcccttggctactacgactcgtccaaggcatactatgccttcaaggtctcgttcaacctctgcatctggggcttgtcgactttcattgttgccaagtggggccagacctcgaccctcgccaacgtgctctcggctgcgctcttgggtctcttctggcagcagtgcggatggttggcgcacgactttttgcaccaccaggtcttccaggaccgtttctggggtgatcttttcggcgccttcttgggaggtgtctgccagggtttctcgtcctcctggtggaaggacaagcacaacactcaccacgctgctcccaacgtccacggcgaggatcccgacattgacactcaccctctgttgacctggagtgagcatgctctggagatgttctcggatgttcctgacgaggagctgacccgtatgtggtcgcgcttcatggtcctcaaccagacctggttctacttccccattctctcgtttgcccgtctgtcctggtgcctccagtccattatgtttgttctgcccaacggtcaggcccacaagccctctggagcgcgtgtgcccatttcgttggtcgagcagctgtctctggctatgcactggacctggtacctcgccaccatgttcctgttcattaaggatcccgtcaacatgattgtgtactttttggtgtcgcaggctgtttgcggcaacttgttggcgattgtgttctcgctcaaccacaacggcatgcctgtgatctccaaggaggaagcggtcgatatggacttcttcaccaagcagatcatcacgggtcgtgatgttcaccctggtctgtttgccgactggttcacgggtggattgaactaccagattgagcaccacttgttcccttcgatgccccgccacaacttttcaaagatccagcctgctgtcgagactttgtgcaaaaagtacggtgtccgataccataccactggtatgatcgagggaactgcagaggtctttagccgtttgaacgaggtctccaaggcggcctccaagatgggtaaggcgcagtaa；
the PEX4 gene and the PEX5 gene are derived from yarrowia lipolytica, and the PEX4 gene sequence is shown in SEQ ID NO.4, specifically:
atgatcaccccaaaccccgctaacgacattgtccatgacggcaagctctacgacaccttcactgagccccccaagctgatggctcaggagcgagctcagctggacttcgaccctagagacatcacctactttctggatggctctaaggaggagaccgagctgctggagtcgctcatgctcatgtacgagcgagaccctctcttcaacaaccagaacgagtacgatgaatcgtttgaaacactgcgagagcgatctgtgaagcgaattttccagctgtccaagtccatcgccatggaccccgagcccatgtctttccgaaagattgggttcctgggtattcttgacatgggaacgtatgctcgactgggagtccactacgcgctcttctgtaactccatccggggccagggaacccccgatcagctcatgtactggctggaccagggagccatggtcatcaagggcttctacggctgttttgccatgaccgaaatgggccatggatctaacctgtcgcgtctggaaaccatcgccactttcgacaaagagaccgacgaatttatcattaacacgccccacgttggagccacaaagtggtggattggaggagccgcccacactgctactcacacacttgcctttgcccgtcttcaagtagacggaaaggactacggtgtgaaatcgtttgtcgtacctctccgaaacctggacgaccattcgctgcgtcctggaatcgccacaggtgatattggtaagaagatgggtcgagatgccgttgacaacggctggattcagttcaccaacgtccgagtgccccgaaactacatgctcatgaagcataccaaggttcttcgagacggtaccgtcaagcagccgcctttggcccaactgacttacggatctctcatcactggacgagtccagatgaccactgactctcacaatgtgtccaaaaagttcctcaccattgccctgagatacgccaccatccgacgacagttctcgtcaactccaggagagcccgaaacccgactaattgactacctgtaccaccaaagacgactcctgcctcttatggcttactcttacgccatgaaactagctggagatcacgtccgagagctgttctttgcatcccaggagaaggctgagagcctcaaggaggacgacaaagccggagttgagtcttacgtccaggatatcaaggagctcttctctgtttctgctggtctcaaggctgccactacatgggcttgtgctgacatcattgacaaggcccgacaggcgtgtggaggccacggatactctgcctacaacggctttggacaggccttccaggactgggttgtccagtgcacttgggagggtgacaatactgttctgactctatctgccggccgagctctgatccaatctgctctcgtctaccgaaaggagggcaaactaggtaacgccacgaagtacctctctcggtccaaggagcttgccaacgccaagagaaacggacgatccctggaagaccccaagctgctcgtggaggcatgggaggctgtctctgccggtgctatcaacgctgctactgacgcttacgaggagctctccaagcagggagtttctgttgacgagtgctttgagcaggtgtcccaggagcgattccaggctgcccgaatccacactcgacgagctcttatcgaggccttctactcacgaatcgccactgctgatgagaaggtgaagcctcatctgatccctctggccaacctgtttgccctgtggtccattgaggaggactctgctctgttcctggctgagggctactttgagcctgaggatatcattgaggtgacttctcttgtcaacaagtactgcggaattgttcgaaagaacgttattggatacaccgatgccttcaacctgtccgactacttcatcaacgctgccattggacgatacgacggagacgtgtacaagaactactttgagaaggtcaaacagcagtaccctcctgagggtggcaagcctcactactacgaggatgtcatgaagcc；
the PEX5 gene sequence is shown as SEQ ID NO.5, and concretely comprises the following steps:
atgaacaacaaccccaccaacgtgatccttggaggcaaggagtacgacaccttcaccgagcctccggcccagatggagctggagcgagccaagacacaattcaaggtccgagacgtgaccaacttcctcacaggcagcgagcaggagacactgctgaccgagcgaatcatgcgggagattgagcgagatcccgttctcaacgtcgccggcgactacgacgccgatcttcccaccaagcgacgacaagctgttgagcgaatcggggctctggcccgatacctgcccaaggattccgagaaggaggccattttgcgaggccagctgcatggtattgtggacatgggtacccgaacccgaatcgccgttcactacggtctgtttatgggcgccattcgtggctcaggaaccaaggagcagtacgattactgggtcgccaagggcgccgctactctgcacaaattctatggctgctttgccatgactgagctgggtcacggatctaacgtggccggtctcgagaccaccgccacccttgataaggacaccgacgagttcatcatcaacacccccaactcgggagccacaaagtggtggattggaggagctgcccactctgctacccacacggcttgtcttgcccgactcattgttgatggcaaggactatggtgttaagatcttcattgttcagctgcgagacctcaactcccactctctactcaacggtattgccattggagatatcggcaagaagatgggccgagatgccattgataatggttggatccagttcacagacgtccgaattccccgacagaacatgctcatgcgatacgaccgggtgtctcgagacggcgaggttaccacctccgagcttgcccagctcacctacggagcacttctgtctggccgagtgaccatgattgccgagtctcacctcctgtctgctcggttcctcaccattgctcttcggtacgcctgtatccgtcgacagttcggagctgtgcctgacaagcccgagactaagctcatcgactacccctaccaccaacgacgtctgctgcctcttctggcctacacctacgccatgaagatgggcgccgacgaggcccagcagcagtacaactcctcctttggcgctcttctcaagctcaaccccgtcaaggacgctgagaagtttgctgtcgccactgccgacctcaaggctctgtttgcctcttctgccggaatgaaggccttcaccacctgggctgccgccaagatcattgacgagtgccgacaggcctgtggtggccatggctactccggctacaacggtttcggtcaggcttacgccgactgggtcgtccaatgcacttgggagggtgacaacaacgtgctgtgtctgtccatgggtcgatcgctcatccagtcgtgcattgccatgagaaagaagaagggccatgtcggcaagtcggtcgagtacctgcagcgacgagacgagctgcagaatgcccgagttgacaacaagcctctcactgaccctgctgtgctcatcactgcatgggagaaggttgcctgcgaggccatcaacagagccactgactccttcatcaagctcacccaggagggtctgtctcctgaccaggcctttgaggagctgtctcaacagagatttgagtgtgcgcgaatccacacccgaaagcatctgatcacctcgttctacgctcgaatctccaaggccaaggcccgagtcaagccccaccttactgttcttgccaacctctttgccgtctggtccatcgaggaggactctggtctcttccttcgggagggctgcttcgagcctgccgagatggacgagatcaccgctctggtcgacgagctgtgctgcgaggctcgagagcaggtcattggattcaccgacgccttcaacctgtccgacttcttcattaacgcccccattggccgattcgacggagacgcctacaagcactacatggacgaggtcaaggctgccaacaaccctcgtaacacccatgctccttactacgagaccaagctgcgacc。
the application also provides a construction method of the strain, which comprises the following steps:
(1) Construction of yarrowia lipolytica engineering strain Polf-001
Respectively constructing uracil auxotroph yarrowia lipolytica Polf and a recombinant plasmid PUC-inTF 3-delta 6DEs, and transferring the recombinant plasmid PUC-inTF 3-delta 6DEs into the uracil auxotroph yarrowia lipolytica Polf to obtain a yarrowia lipolytica engineering strain Polf-001;
(2) Preparation of yarrowia lipolytica engineering strain Polf-002
Inoculating the yarrowia lipolytica engineering strain Polf-001 into a YPD-5FOA solid culture medium, and after single colony grows out, scribing and activating again in the YPD solid culture medium to obtain the yarrowia lipolytica engineering strain Polf-002;
(3) Construction of recombinant plasmid PUC-SCP2-PEX4-5
Taking a PUC-57-SCP2 plasmid as a framework, and inserting a PEX4 gene expression cassette and a PEX5 gene expression cassette into the plasmid framework to obtain a recombinant plasmid PUC-SCP2-PEX4-5;
(4) Construction of genetically engineered strains producing gamma-linolenic acid
Transferring the recombinant plasmid PUC-SCP2-PEX4-5 into a yarrowia lipolytica engineering strain Polf-002 to obtain the gene engineering strain for producing gamma-linolenic acid.
As one limitation, the uracil auxotroph yarrowia lipolytica Polf of step (1) is constructed by knocking out the uracil-encoding gene in yarrowia lipolytica Po1f by CRISPR/Cas9 technology;
the construction method of the recombinant plasmid PUC-inTF 3-delta 6DEs in the step (1) is that the PUC-57-inTF3 plasmid with uracil genetic marker is taken as a skeleton, and a delta 6 desaturase gene expression cassette is inserted into the plasmid skeleton.
By way of further limitation, in step (1),the delta 6 desaturase gene expression cassette promoter is P derived from yarrowia lipolytica YAT Promoter, P TEF Promoters or P FBAin One of the promoters, the terminator is T from yarrowia lipolytica XPR2T Terminator, T lip2t Terminator and T CYC1t One of the terminators.
In the step (3), the PEX4 gene expression cassette promoter is P derived from yarrowia lipolytica YAT Promoter, P TEF Promoter and P FBAin One of the promoters, the terminator is T from yarrowia lipolytica XPR2T Terminator, T lip2t Terminator and T CYC1t One of the terminators;
in the step (3), the PEX5 gene expression cassette promoter is P derived from yarrowia lipolytica YAT Promoter, P TEF Promoter and P FBAin One of the promoters, the terminator is T from yarrowia lipolytica XPR2T Terminator, T lip2t Terminator and T CYC1t One of the terminators.
The application also provides an application of the genetically engineered strain for producing the gamma-linolenic acid, and the genetically engineered strain for producing the gamma-linolenic acid can ferment and produce the gamma-linolenic acid on a culture medium which takes waste edible oil as a unique carbon source.
As a limitation to the application of genetically engineered strains producing gamma-linolenic acid, the fermentation comprises the steps of:
(1) ' pretreatment of waste edible oil
Filtering the waste edible oil, removing solid impurities, washing with distilled water for 3-4 times, layering, taking upper-layer oil, and sterilizing;
(2) Preparation of' seed liquid
Activating the genetically engineered strain for producing gamma-linolenic acid, inoculating the genetically engineered strain into YPD liquid culture medium, and culturing the genetically engineered strain to a logarithmic growth phase to obtain seed liquid;
(3) ' fermentation production of gamma-linolenic acid
Inoculating the seed solution into YNB fermentation medium with waste oil as the sole carbon source according to the volume ratio of 5-10%, and carrying out ventilation culture for 92-100 h at the temperature of 25-30 ℃ and at the speed of 200-240 rpm to obtain a fermentation product gamma-linolenic acid.
As a further limitation to the above-mentioned fermentation,
in the step (1)' the sterilization temperature is 105-115 ℃ and the sterilization time is 25-30 min;
in the step (2)' the number of viable bacteria in the seed liquid is 1.2X10 8 ～2.4×10 8 CFU/mL；
In the step (3)' the amount of waste oil added in the fermentation culture is 20-30 g/L, and the amount of YNB added is 1.0-2.5 g/L.
By adopting the technical scheme, compared with the prior art, the application has the following technical progress:
(1) according to the application, two recombinant plasmids, namely PUC-inTF 3-delta 6DEs and PUC-SCP2-PEX4-5, containing uracil genetic markers are respectively constructed and then are introduced into uracil auxotroph yarrowia lipolytica strains, so that the stability of exogenous genes in the insertion process is improved, and the stability of engineering strains is further improved;
(2) according to the application, the intermediate of the engineering strain is inoculated in a solid culture medium containing 5FOA for screening, uracil is removed by cell loss, and the intermediate of the engineering strain is changed into a uracil-deficient strain, so that the recycling of uracil screening markers is realized;
(3) the yarrowia lipolytica engineering strain constructed by the application can synthesize gamma-linolenic acid in a culture medium taking waste oil as a sole carbon source, and provides a gamma-linolenic acid synthesis way with the advantages of no limitation of seasons, large-scale production, stable yield, reliable quality, wide fermentation substrate sources, low cost and the like;
(4) when the engineering strain constructed by the application is used for fermentation, waste edible oil is used as a fermentation substrate, so that the production efficiency of microbial oil can be improved, and the oil production cost can be obviously reduced; the waste can be changed into valuables, the corrosion and blockage of the municipal sewage pipe network by the waste edible oil are reduced, the harm caused by the inflow of the waste grease into society is prevented, the recycling of the waste edible oil is realized, and the improvement of the recycling economy level is promoted.
In summary, the genetically engineered strain for producing gamma-linolenic acid, the construction method and the application thereof provided by the application express delta 6 desaturase genes from Mortierella alpina in yarrowia lipolytica, overexpress beta-oxidation pathway PEX4 genes and PEX5 genes, can synthesize gamma-linolenic acid on a culture medium with waste edible oil as a unique carbon source, has good engineering bacteria stability and high gamma-linolenic acid content of intracellular grease, has a synthetic pathway which is not limited by seasons, stable yield and low cost of fermentation substrates, and can recycle the waste edible oil.
The application is suitable for constructing the genetic engineering strain for producing the gamma-linolenic acid, and the constructed engineering strain is further applied to producing the gamma-linolenic acid.
Drawings
The application will be described in more detail below with reference to the accompanying drawings and specific examples.
FIG. 1 is a diagram showing a mechanism for producing gamma-linolenic acid in the background of the application;
FIG. 2 is a backbone diagram of the recombinant plasmid PUC-inTF3- Δ6DEs in example 1 of the present application;
FIG. 3 is a diagram showing the skeleton of recombinant plasmid PUC-SCP2-PEX4-5 in example 1 of the present application;
FIG. 4 is a graph showing growth of the engineering strain Polf-001 and the engineering strain Polf-003 in example 2 of the present application;
FIG. 5 is a standard graph of gamma-methyl linolenate in example 3 according to the present application;
FIG. 6 is a gas chromatogram of oil obtained after fermentation of the engineering strain Polf-003 in example 3 of the present application;
FIG. 7 is a graph showing the oil content of the fermented broth after fermentation of two strains, namely, engineering strain Polf-001 and engineering strain Polf-003, in example 3 of the present application;
FIG. 8 is a graph showing the content of gamma-linolenic acid in fermentation broth after fermentation of two strains, namely, engineering strain Polf-001 and engineering strain Polf-003, in example 3 of the present application.
Detailed Description
The application is further illustrated by the following examples. It should be understood that the described embodiments are only for explaining the present application and do not limit the present application.
Materials, reagents, and the like used in the examples of the present application are commercially available unless otherwise specified. The experimental methods for which specific conditions are not specified in the examples are generally conducted under conventional conditions or under conditions recommended by the manufacturer.
In this example, although yarrowia lipolytica was used as the starting strain, conventional uracil auxotroph yarrowia lipolytica was used as the starting strain for transformation to perform the test in the example.
Yarrowia lipolytica used in the present application was capable of synthesizing C16 and C18 long chain fatty acids by itself, and was constructed for this purpose by converting Δ6 desaturase from Mortierella alpina Mortierella alpina into uracil auxotrophic yarrowia lipolytica Po1 f.
The content of linoleic acid in the waste edible oil used in the application is 4.9%.
EXAMPLE 1 construction of genetically engineered Strain for production of gamma-linolenic acid
1. Construction of uracil auxotrophs yarrowia lipolytica Polf
The part is uracil auxotroph yarrowia lipolytica Polf construction, and the construction method comprises the following steps: the gene sequence of uracil-encoding yarrowia lipolytica Polf in uracil auxotrophs is obtained by knocking out the gene sequence of uracil-encoding yarrowia lipolytica Po1f by using a CRISPR/Cas9 technology by taking 1000bp upstream and downstream of the gene sequence of uracil-encoding gene as homology arms.
Specific construction methods are described in journal Journal of Agricultural and Food Chemistry, 2021, volume 69, 46, pages 13831-13837, under the paper designation "Harnessing Yarrowia lipolytica Peroxisomes as a Subcellular Factory for. Alpha. -Humulene Overproduction".
2. Construction of recombinant plasmid PUC-inTF 3-delta 6DEs
S1, synthesizing an optimized delta 6 desaturase gene from Mortierella alpina Mortierella alpina to obtain a codon optimized delta 6 desaturase gene, wherein the nucleotide sequence of the codon optimized delta 6 desaturase gene is shown as a sequence table SEQ ID No. 1.
S2, amplification of target genes
S21, performing PCR amplification by using the delta 6 desaturase gene synthesized in the step S1 as a template and using Des6-F (the nucleotide sequence is shown as SEQ ID NO. 6) and Des6-R (the nucleotide sequence is shown as SEQ ID NO. 7) as primers to obtain an amplified fragment Des6-A;
s22, carrying out PCR amplification by taking plasmid PUC-HUH-FY as a template and YAT-F and YAT-R genes as primers to obtain a promoter PYAT, wherein the gene sequence of the promoter PYAT is shown as SEQ ID NO. 2;
s23, carrying out PCR amplification by taking plasmid PUC-HUH-FY as a template and CY-F and CY-R groups as primers to obtain a terminator TCYC1t, wherein the gene sequence of the terminator TCYC1t is shown as SEQ ID NO. 3;
the procedure for PCR was as follows: denaturation at 98℃for 10s, annealing at 56℃for 10s, and extension at 72℃for 1min (extension time=target fragment length/1 kb, unit min), 35 cycles were repeated.
S3, enzyme cutting is carried out on the PUC-57-inTF3 plasmid with uracil genetic marker through BamH1 enzyme, the three amplified genes are connected into an enzyme-cut PUC-57-inTF3 plasmid skeleton, and one-step cloning is carried out through ClonExpress MultiS One Step Cloning Kit, so that a recombinant plasmid PUC-inTF 3-delta 6DEs with uracil genetic marker is constructed;
s4, purifying and recovering each fragment by AxyPrepTM DNA GelExtraction Kit (purchased from Corning life sciences Co., ltd.);
s5, transforming the circular recombinant plasmid into escherichia coli DH5a competent cells, screening by an ampicillin resistance flat plate, and verifying by colony PCR and sequencing to obtain a positive recombinant plasmid PUC-inTF 3-delta 6DEs, wherein a skeleton diagram of the recombinant plasmid PUC-inTF 3-delta 6DEs is shown in figure 2.
Wherein (1) Des6-F gene sequence is shown in SEQ ID NO.6, specifically:
gagtataagaatcattcaaaatggctgctgctcccagtgt
(2) the Des6-R gene sequence is shown as SEQ ID NO.7, and specifically comprises the following steps:
Tgacataactaattacatgattactgcgccttacccatct
③P YAT the gene sequence of the promoter is shown as SEQ ID NO.2, and specifically comprises the following steps:
gtacgtagcaacaacagtgtacgcagtactatagaggaacaattgccccggagaagacggccaggccgcctagatgacaaattcaacaactcacagctgactttctgccattgccactaggggggggcctttttatatggccaagccaagctctccacgtcggttgggctgcacccaacaataaatgggtagggttgcaccaacaaagggatgggatggggggtagaagatacgaggataacggggctcaatggcacaaataagaacgaatactgccattaagactcgtgatccagcgactgacaccattgcatcatctaagggcctcaaaactacctcggaactgctgcgctgatctggacaccacagaggttccgagcactttaggttgcaccaaatgtcccaccaggtgcaggcagaaaacgctggaacagcgtgtacagtttgtcttagcaaaaagtgaaggcgctgaggtcgagcagggtggtgtgacttgttatagcctttagagctgcgaaagcgcgtatggatttggctcatcaggccagattgagggtctgtggacacatgtcatgttagtgtacttcaatcgccccctggatatagccccgacaataggccgtggcctcatttttttgccttccgcacatttccattgctcggtacccacaccttgcttctcctgcacttgccaaccttaatactggtttacattgaccaacatcttacaagcggggggcttgtctagggtatatataaacagtggctctcccaatcggttgccagtctcttttttcctttctttccccacagattcgaaatctaaactacacatcacacaatgcctgttactgacgtccttaagcgaaagtccggtgtcatcgtcggcgacgatgtccgagccgtgagtatccacgacaagatcagtgtcgagacgacgcgttttgtgtaatgacacaatccgaaagtcgctagcaacacacactctctacacaaactaacccagctcttc
④T CYC1t the gene sequence of the terminator is shown in SEQ ID NO.3, and specifically comprises the following steps:
Tcatgtaattagttatgtcacgcttacattcacgccctccctccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgc；
wherein the PCR enzyme used in the PCR reaction is PrimeSTAR MaxDNA polymerase of TAKARA, the PCR amplification system is shown in Table 1, the BamH1 cleavage system is shown in Table 2, and the one-step cloning system is shown in Table 3.
TABLE 1 PCR amplification System
| Reagent(s) | Usage amount |
| PrimeSTAR Max(2×) | 25μL |
| Primer Des6-F | 2μL |
| Primer Des6-R | 2μL |
| Template | 1μL |
| ddH2O | 20μL |
TABLE 2 PUC-57-inTF3 plasmid enzyme digestion system
| Component (A) | Recombination reactions |
| Plasmid vector | 2μg |
| BamH1 enzyme | 2μL |
| 10xQCut Buffer | 5μL |
| ddH2O | To50μL |
TABLE 3 one-step cloning System
| Component (A) | Recombination reactions |
| Linearization carrier | XμL |
| N inserts | Y1+Y2+…YnμL |
| 2×ClonExpress Mix | 5μL |
| ddH2O | To 10μL |
X= (0.02 Xbase pair of cloning vector) ng (0.03 pmol)
Y= (0.02. Times.base pair per fragment) ng (0.03 pmol)
3. Construction of yarrowia lipolytica engineering strain Polf-001
The construction method of the yarrowia lipolytica genetic engineering strain expressing delta 6 desaturase gene comprises the following steps in sequence:
s1 activation of uracil auxotrophs yarrowia lipolytica Po1f
5 mu L of uracil auxotroph engineering strain obtained in the 2 nd part of the example is streaked on YPD solid medium for activation, and single colony is grown;
s2 preparation of uracil auxotroph yarrowia lipolytica Po1f competence
Selecting single colony to 5mL YPD liquid culture medium, culturing in a shaking table at 28 ℃ and 220rpm until the absorption value of bacterial liquid at 600nm is 0.8-1.0;
taking 1mL of bacterial liquid, centrifuging at 4 ℃ and 4000rpm for 3min, collecting bacterial cells, and preparing competence according to the operation method of a yeast transformation kit Frozen-EZ Y east Transformation IITM, wherein the specific method comprises the following steps: re-suspending with 1mL EZ1, centrifuging at 4000rpm at 4 ℃ for 3min, and collecting the thalli again; discarding supernatant, suspending thallus with 100 μl EZ2, packaging 50 μl each, and preserving at-80deg.C to obtain yarrowia lipolytica Po1f competent cells;
s3 construction of yarrowia lipolytica Po1f-001
Taking out yarrowia lipolytica Po1f competent cells stored at-80deg.C, and placing on ice for 10min; 1 mug of recombinant plasmid PUC-inTF 3-delta 6DEs with uracil genetic marker obtained in example 2 is added into yarrowia lipolytica Po1f competent cells, gently blown and mixed uniformly, then 500 mug EZ3 is added and mixed uniformly, incubated in a shaker at 28 ℃ and 220rpm for 60min, coated on YNB solid medium for screening, positive strains are selected as engineering strains Polf-001,
wherein, the proportion of YPD solid culture medium is shown in Table 4;
the proportion of YPD liquid culture medium is shown in Table 5;
the proportions of YNB solid medium are shown in Table 6.
TABLE 4 YPD solid Medium ratio Table
| Name of the name | Yeast paste | Peptone | Glucose | Agar-agar |
| Content (g/L) | 10 | 20 | 20 | 20 |
TABLE 5 YPD liquid Medium ratio Table
| Name of the name | Yeast paste | Peptone | Glucose |
| Content (g/L) | 10 | 20 | 20 |
TABLE 6 YNB solid Medium proportioning Table
| Name of the name | YNB (without amino acid and ammonium sulfate) | Ammonium sulfate | Glucose | Agar-agar |
| Content (g/L) | 1.7 | 5 | 20 | 20 |
4. Construction of recombinant plasmid PUC-SCP2-PEX4-5
S1, amplification of target gene fragment
S11, extracting whole genome DNA of yarrowia lipolytica Po1f by referring to a method in a yeast genome DNA extraction kit instruction (the kit is purchased from Shanghai Limited of bioengineering);
s12, carrying out PCR (polymerase chain reaction) amplification by taking genomic DNA as a template and using primers PEX4-F and PEX4-R as primers to obtain an amplified fragment PEX4;
s13, carrying out PCR (polymerase chain reaction) amplification by taking genomic DNA as a template and using primers PEX5-F and PEX5-R as primers to obtain an amplified fragment PEX5;
s14, performing PCR amplification by taking plasmid PUC-HUH-FY as a template and YAT-F and YAT-R groups as primers to obtain a promoter P YAT ；
S15, performing PCR amplification by taking yarrowia lipolytica genome as a template and CY-F and CY-R groups as primers to obtain a terminator T CYC1t ；
Wherein the enzymes and systems used for PCR amplification are as shown in Table 1, and the procedure for PCR is as follows: denaturation at 98℃for 10s, annealing at 56℃for 10s, and extension at 72℃for 1min (extension time=target fragment length/1 kb, unit min), 35 cycles were repeated.
S2, construction of recombinant plasmid PUC-SCP2-PEX4
S21, carrying out enzyme digestion on plasmid PUC-57-SCP2 with uracil genetic marker by ECoR1 enzyme, connecting amplified fragments of PEX4, a promoter PYAT and a codon TCYC1t into an enzyme-digested plasmid skeleton of the PUC-57-SCP2, and carrying out one-step cloning by adopting ClonExpress MultiS One Step Cloning Kit to construct recombinant plasmid PUC-SCP2-PEX4;
s22, purifying and recovering each fragment by AxyPrepTM DNA Gel Extraction Kit (purchased from Corning life sciences Co., ltd.);
s23, transforming the annular recombinant plasmid into escherichia coli DH5a competent cells, screening by an ampicillin resistance flat plate, and verifying by colony PCR and sequencing to obtain a positive recombinant plasmid PUC-SCP2-PEX4.
S3, construction of recombinant plasmid PUC-SCP2-PEX4-5
S31, carrying out enzyme digestion on a recombinant plasmid PUC-SCP2-PEX4 through PacI enzyme, connecting amplified fragments of PEX5, a promoter PYAT and a codon TCYC1t into a plasmid skeleton of the enzyme-digested PUC-SCP2-PEX4, and carrying out one-step cloning by adopting ClonExpress MultiS One Step Cloning Kit to construct the recombinant plasmid PUC-SCP2-PEX4-5;
s32, purifying and recovering each fragment by AxyPrepTM DNA Gel Extraction Kit (purchased from Corning life sciences Co., ltd.);
s33, transforming the annular recombinant plasmid into escherichia coli DH5a competent cells, screening by an ampicillin resistance flat plate, and verifying by colony PCR and sequencing to obtain a positive recombinant plasmid PUC-SCP2-PEX4-5, wherein the recombinant plasmid is provided with uracil genetic markers, and a skeleton diagram of the PUC-SCP2-PEX4-5 plasmid is shown in figure 3.
Wherein, the gene sequence of (1) PEX4-F is shown in SEQ ID NO.8, and is specifically as follows:
ccctgattgactggaacagcatgatcaccccaaaccccgc
(2) the gene sequence of PEX4-R is shown as SEQ ID NO.9, and specifically comprises the following steps:
tgacataactaattacatgaggcttcatgacatcctcgta
(3) the gene sequence of PEX5-F is shown as SEQ ID NO.10, and specifically comprises the following steps:
gctcgaaggctttaatttgcatgaacaacaaccccaccaa
(4) the gene sequence of PEX5-R is shown as SEQ ID NO.11, and specifically comprises the following steps:
Acaagttccgtagttggatcggtcgcagcttggtctcgta；
wherein, the system of one-step cloning is shown in Table 3, the system of ECoR1 cleavage of plasmid PUC-57-SCP2 is shown in Table 7, the system of Pac1 cleavage of recombinant plasmid PUC-SCP2-PEX4 is shown in Table 8, and the specific steps are the same as the construction of recombinant plasmid PUC-inTF 3-delta 6 DEs.
TABLE 7 plasmid PUC-57-SCP2 cleavage System
TABLE 8 cleavage System for recombinant plasmid PUC-SCP2-PEX4
| Component (A) | Recombination reactions |
| Plasmid vector | 2μg |
| Pac1 enzyme | 2μL |
| 10x QCut Buffer | 5μL |
| ddH 2 O | To 50μL |
5. Construction of genetically engineered strains for the production of gamma-linolenic acid
The part is a construction method of a genetic engineering strain for producing gamma-linolenic acid, which comprises the following steps in sequence:
s1, activation of engineering strain Polf-001
Taking 5 mu L of the engineering strain Polf-001 to be streaked on a YPD-5FOA solid culture medium, removing uracil by cells under the action of 5FOA, recycling a screening mark, and streaking and activating again in the YPD solid culture medium after a single colony grows out to obtain the engineering strain Polf-002;
s2, preparation of engineering strain Polf-002 competence
Selecting single colony to 5mL YPA liquid culture medium, and culturing in a shaking table at 28 ℃ and 220rpm until the absorption value of bacterial liquid at 600nm is 0.8-1.0;
taking 1mL of bacterial liquid, centrifuging at 4 ℃ and 4000rpm for 3min, collecting bacterial cells, and preparing competence according to the operation method of a yeast transformation kit Frozen-EZ Y east Transformation IITM, wherein the specific method comprises the following steps: re-suspending with 1mL EZ1, centrifuging at 4000rpm at 4 ℃ for 3min, and collecting the thalli again; removing supernatant, suspending thallus with 100 μl EZ2, packaging 50 μl each, and preserving at-80deg.C to obtain engineering strain Polf-002 competent cells;
s3 construction of yarrowia lipolytica engineering strain Po1f-003
Taking out competent cells of yarrowia lipolytica Polf-002 stored at-80deg.C, and placing on ice for 10min; adding 1 mug of recombinant plasmid PUC-SCP2-PEX4-5 with uracil genetic marker obtained in the 4 th part into competent cells of yarrowia lipolytica Po1f-002, gently blowing and mixing, adding 500 mug of EZ3 and mixing, incubating in a shaker at 28 ℃ and 220rpm for 60min, coating on YNB solid medium, screening, and selecting positive strains to obtain engineering strains Polf-003, wherein the engineering strains Polf-003 are genetic engineering strains for producing gamma-linolenic acid.
Wherein, the proportion of YPD-5FOA fermentation medium is shown in Table 9.
TABLE 9 proportion Table of YPD-5FOA solid Medium
Example 2 growth of yarrowia lipolytica Polf, engineering Strain Polf-001 and engineering Strain Polf-003 on waste oil as sole carbon Source Medium
This example examined the growth of yarrowia lipolytica Polf, the engineered strain Polf-001 constructed in example 1 and the engineered strain Polf-003 on a medium with waste oil as the sole carbon source, comprising the following steps in order:
s1, activation
Taking 1mL of viable bacteria with the number of 1.0 x 10 8 CFU/mL yarrowia lipolytica Polf was inoculated into 10mL of conventional YPD solid medium and cultured in a shaker at 28℃and 220rpm for 24h;
taking 1mL of viable bacteria with the number of 1.0 x 10 8 CFU/mL engineering strain Po1f-001 is inoculated into 10mL of conventional YPD solid culture medium, and cultured for 24h at 28 ℃ in a shaking table at 220 rpm;
taking 1mL of viable bacteria with the number of 1.0 x 10 8 The CFU/mL engineering strain Po1f-003 is inoculated into 10mL of conventional YPD solid culture medium, and cultured for 24 hours at 28 ℃ in a shaking table at 220 rpm;
s2, determination of growth conditions
The three strains after activation are respectively inoculated into 5mL YPD liquid culture medium, cultured to logarithmic phase, seed liquid is obtained, 500 mu L seed liquid is respectively inoculated into fermentation culture medium with waste oil as the only carbon source, the growth OD value is tested, and the measurement result is shown in figure 4.
As can be seen from fig. 4, in the growth condition, in the culture medium with the waste oil as the sole carbon source, both the engineering strain Po1f-003 and the engineering strain Po1f-001 can grow normally, and the growth condition of the engineering strain Po1f-003 is better than that of the wild strain, which proves that the engineering strain Po1f-003 can grow normally on the waste oil as the sole carbon source.
Example 3 Synthesis of gamma-linolenic acid by engineering Strain Polf-001 and engineering Strain Polf-003 on culture Medium with waste oil as sole carbon Source
This example examined the conditions of the engineering strains Polf-001 and Polf-003 constructed in example 1 for synthesizing gamma-linolenic acid, aiming at examining the effect of engineering strains overexpressing PEX4 gene and PEX5 gene of beta-oxidation pathway on increasing oil content when fermenting with waste oil as the sole carbon source medium, the method comprising the following steps sequentially carried out:
s1, pretreatment of waste edible oil
Taking 1kg of waste edible oil, carrying out rough filtration by a 100-mesh screen, carrying out microfiltration by a 2.0 mu m filter membrane, removing solid impurities therein, washing with distilled water for 3 times, removing water-soluble impurities therein, layering, taking upper-layer oil, and carrying out damp-heat sterilization for 0.5h at 115 ℃.
S2, preparation of seed liquid
Inoculating single colonies of the engineering strain Polf-001 and the engineering strain Polf-003 into 10mL of conventional YPD solid culture medium, and culturing for 24h to obtain first-stage seeds; inoculating the first seed into 10mLYPD culture medium, culturing again for 24 hr to obtain second seed fermentation broth with viable count of 1.2X10 8 ～2.4×10 8 CFU/mL。
S3, fermenting engineering strains
Respectively taking seed fermentation liquid of the two engineering strains, inoculating the seed fermentation liquid into 1000mL YNB fermentation medium with waste oil as a unique carbon source according to the inoculum size of 5-10% by volume ratio, ensuring that the number of viable bacteria inoculated into the culture medium is the same, and carrying out ventilation culture for 96 hours at 28 ℃ and 220rpm respectively to obtain a fermentation product.
The ratio of YNB fermentation medium using waste oil as the only carbon source is shown in Table 10.
TABLE 10 YNB fermentation Medium ratio Table Using waste oil as sole carbon Source
| Name of the name | Yeast powder | YNB | Waste oil |
| Content (g/L) | 2.5 | 1.7 | 25 |
S4, analysis of fermentation culture products
Respectively carrying out solid-liquid separation on the obtained fermentation products, collecting solids to obtain grease thalli, adding 0.5% (v/v) of muramidase, carrying out enzymolysis for 1h at 55 ℃, adding 30mL of n-hexane for extraction, standing for layering, taking upper organic phase liquid, repeatedly extracting, volatilizing a solvent, and respectively measuring total grease;
taking 30 mu L of grease obtained in the previous step, and carrying out methyl esterification on fatty acid: 500. Mu.L of 1mol/L NaOH in methanol was added and the mixture was centrifuged at 1200rpm for 30min at room temperature; then 40. Mu.L of sulfuric acid and 500. Mu.L of chromatographic grade n-hexane are added to extract fatty acid methyl ester; and finally centrifuging at 8000rpm for 2min, removing impurities from the upper layer through a microporous filter membrane, injecting into a gas chromatograph, and analyzing and calculating the content of gamma-linolenic acid.
The specific detection method of the gas chromatograph is as follows:
detection conditions: the temperature of the sample inlet is 100 ℃, the sample inlet volume is 1.0 mu L, and the split ratio is 69.8:1;
chromatographic column: DB-23 (60.0mX0.25mm X0.25μm);
chromatographic conditions: the initial temperature was 100deg.C, and 25 deg.C/min was raised to 196 deg.C. Then, heating to 220 ℃ at 2 ℃/min, and preserving heat for 6min;
detector temperature: 280 ℃.
Qualitative and quantitative determination is carried out by using a gamma-methyl linolenate standard, and a gamma-methyl linolenate standard curve is shown in figure 5;
the gas chromatogram of the oil after the fermentation of the engineering strain Polf-003 is shown in figure 6;
the diagram of the oil content in the fermentation broth after fermentation of the engineering strain Polf-001 and the engineering strain Polf-003 is shown in figure 7;
the graph of the gamma-linolenic acid content in the fermentation broth after fermentation of the engineering strain Polf-001 and the engineering strain Polf-003 is shown in figure 8.
As can be seen from fig. 7, the engineering strain Po1f-003 is significantly superior to the engineering strain Po1f-001 in terms of total oil content of the fermentation product; from FIG. 8, it is clear that the engineering strain Po1f-003 is significantly higher than the engineering strain Po1f-001 in terms of the gamma-linolenic acid content of the fermentation product, and the gamma-linolenic acid content in the fermentation broth of the engineering strain Po1f-003 is about 2 times the gamma-linolenic acid content in the fermentation broth of the engineering strain Po1 f-001. Therefore, the engineering strain Po1f-003 is more suitable as a strain for producing gamma-linolenic acid than the engineering strain Po1 f-001.
According to calculation, the oil content of the engineering strain Po1f-003 in 1L fermentation liquid reaches 1.2g, and gamma-linolenic acid accounts for 52.3% of total fatty acid, namely 38.5g of oil containing 52.3% of gamma-linolenic acid can be prepared per 1L of kitchen waste oil.
Examples 4-8 use of genetically engineered strains producing gamma-linolenic acid in the Synthesis of gamma-linolenic acid
Examples 4-8 are the same as example 3 in the steps of the method for synthesizing gamma-linolenic acid by using the engineering strain Polf-003 as the fermentation strain, and are different in the proportion of each component of YNB fermentation medium with waste oil as the sole carbon source and different in the technological parameters, and the proportion of YNB fermentation medium with waste oil as the sole carbon source is shown in Table 11 in detail, and the technological parameters of different preparation steps are shown in Table 12.
TABLE 11 YNB fermentation Medium ratios Table in examples 4-8 with waste oil as the sole carbon source
Table 12 table of process parameters for the steps of examples 4-8
Examples 4-8 the oil content of the engineering strain and the ratio of gamma-linolenic acid to total fatty acid when synthesizing gamma-linolenic acid using the genetically engineered strain producing gamma-linolenic acid are summarized in Table 13 below.
TABLE 13 summary of the fat content and the ratio of gamma-linolenic acid to total fatty acids
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From the experimental results, when 25g/L of waste oil is used as a substrate, the oil yield and the proportion of gamma-linolenic acid to total fatty acid are both good. The sterilization temperature and the sterilization time have less influence on the final content of the experiment; the culture temperature and the rotation speed have a certain influence on the growth of the strain, and the yarrowia lipolytica has the optimal growth state at the rotation speed of 30 ℃ and 220 rpm.
It is to be understood that the above examples of the present application are provided by way of illustration only and not by way of limitation of the embodiments of the present application. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. Not all embodiments are exhaustive. All obvious changes or modifications which come within the spirit of the application are desired to be protected.
SEQUENCE LISTING
<110> university of Nanjing teachers and students
<120> a genetically engineered strain for producing gamma-linolenic acid, construction method and application thereof
<130> 10
<160> 11
<170> PatentIn version 3.3
<210> 1
<211> 1374
<212> DNA
<213> Mortierella alpina (Mortierella alpina)
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atggctgctg ctcccagtgt gaggacgttt actcgggccg agattttgaa tgccgaggcc 60
ctgaatgagg gcaagaagga tgccgaggca ccctttctga tgatcattga caacaaggtg 120
tacgatgtcc gcgagtttgt ccctgatcat cccggtggaa gtgtgattct cacgcacgtt 180
ggcaaggacg gcactgacgt ctttgacact ttccaccccg aggctgcttg ggagactctt 240
gccaactttt acgttggtga tattgatgag agcgatcgtg ccatcaagaa tgatgacttt 300
gcggccgagg ttcgcaagct gcgcaccttg ttccagtccc ttggctacta cgactcgtcc 360
aaggcatact atgccttcaa ggtctcgttc aacctctgca tctggggctt gtcgactttc 420
attgttgcca agtggggcca gacctcgacc ctcgccaacg tgctctcggc tgcgctcttg 480
ggtctcttct ggcagcagtg cggatggttg gcgcacgact ttttgcacca ccaggtcttc 540
caggaccgtt tctggggtga tcttttcggc gccttcttgg gaggtgtctg ccagggtttc 600
tcgtcctcct ggtggaagga caagcacaac actcaccacg ctgctcccaa cgtccacggc 660
gaggatcccg acattgacac tcaccctctg ttgacctgga gtgagcatgc tctggagatg 720
ttctcggatg ttcctgacga ggagctgacc cgtatgtggt cgcgcttcat ggtcctcaac 780
cagacctggt tctacttccc cattctctcg tttgcccgtc tgtcctggtg cctccagtcc 840
attatgtttg ttctgcccaa cggtcaggcc cacaagccct ctggagcgcg tgtgcccatt 900
tcgttggtcg agcagctgtc tctggctatg cactggacct ggtacctcgc caccatgttc 960
ctgttcatta aggatcccgt caacatgatt gtgtactttt tggtgtcgca ggctgtttgc 1020
ggcaacttgt tggcgattgt gttctcgctc aaccacaacg gcatgcctgt gatctccaag 1080
gaggaagcgg tcgatatgga cttcttcacc aagcagatca tcacgggtcg tgatgttcac 1140
cctggtctgt ttgccgactg gttcacgggt ggattgaact accagattga gcaccacttg 1200
ttcccttcga tgccccgcca caacttttca aagatccagc ctgctgtcga gactttgtgc 1260
aaaaagtacg gtgtccgata ccataccact ggtatgatcg agggaactgc agaggtcttt 1320
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gtacgtagca acaacagtgt acgcagtact atagaggaac aattgccccg gagaagacgg 60
ccaggccgcc tagatgacaa attcaacaac tcacagctga ctttctgcca ttgccactag 120
gggggggcct ttttatatgg ccaagccaag ctctccacgt cggttgggct gcacccaaca 180
ataaatgggt agggttgcac caacaaaggg atgggatggg gggtagaaga tacgaggata 240
acggggctca atggcacaaa taagaacgaa tactgccatt aagactcgtg atccagcgac 300
tgacaccatt gcatcatcta agggcctcaa aactacctcg gaactgctgc gctgatctgg 360
acaccacaga ggttccgagc actttaggtt gcaccaaatg tcccaccagg tgcaggcaga 420
aaacgctgga acagcgtgta cagtttgtct tagcaaaaag tgaaggcgct gaggtcgagc 480
agggtggtgt gacttgttat agcctttaga gctgcgaaag cgcgtatgga tttggctcat 540
caggccagat tgagggtctg tggacacatg tcatgttagt gtacttcaat cgccccctgg 600
atatagcccc gacaataggc cgtggcctca tttttttgcc ttccgcacat ttccattgct 660
cggtacccac accttgcttc tcctgcactt gccaacctta atactggttt acattgacca 720
acatcttaca agcggggggc ttgtctaggg tatatataaa cagtggctct cccaatcggt 780
tgccagtctc ttttttcctt tctttcccca cagattcgaa atctaaacta cacatcacac 840
aatgcctgtt actgacgtcc ttaagcgaaa gtccggtgtc atcgtcggcg acgatgtccg 900
agccgtgagt atccacgaca agatcagtgt cgagacgacg cgttttgtgt aatgacacaa 960
tccgaaagtc gctagcaaca cacactctct acacaaacta acccagctct tc 1012
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tagtattaag aacgttattt atatttcaaa tttttctttt ttttctgtac agacgcgtgt 180
acgcatgtaa cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgaaggctt 240
taatttgc 248
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<213> yarrowia lipolytica (Yarrowia lipolytica)
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atgatcaccc caaaccccgc taacgacatt gtccatgacg gcaagctcta cgacaccttc 60
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atcacctact ttctggatgg ctctaaggag gagaccgagc tgctggagtc gctcatgctc 180
atgtacgagc gagaccctct cttcaacaac cagaacgagt acgatgaatc gtttgaaaca 240
ctgcgagagc gatctgtgaa gcgaattttc cagctgtcca agtccatcgc catggacccc 300
gagcccatgt ctttccgaaa gattgggttc ctgggtattc ttgacatggg aacgtatgct 360
cgactgggag tccactacgc gctcttctgt aactccatcc ggggccaggg aacccccgat 420
cagctcatgt actggctgga ccagggagcc atggtcatca agggcttcta cggctgtttt 480
gccatgaccg aaatgggcca tggatctaac ctgtcgcgtc tggaaaccat cgccactttc 540
gacaaagaga ccgacgaatt tatcattaac acgccccacg ttggagccac aaagtggtgg 600
attggaggag ccgcccacac tgctactcac acacttgcct ttgcccgtct tcaagtagac 660
ggaaaggact acggtgtgaa atcgtttgtc gtacctctcc gaaacctgga cgaccattcg 720
ctgcgtcctg gaatcgccac aggtgatatt ggtaagaaga tgggtcgaga tgccgttgac 780
aacggctgga ttcagttcac caacgtccga gtgccccgaa actacatgct catgaagcat 840
accaaggttc ttcgagacgg taccgtcaag cagccgcctt tggcccaact gacttacgga 900
tctctcatca ctggacgagt ccagatgacc actgactctc acaatgtgtc caaaaagttc 960
ctcaccattg ccctgagata cgccaccatc cgacgacagt tctcgtcaac tccaggagag 1020
cccgaaaccc gactaattga ctacctgtac caccaaagac gactcctgcc tcttatggct 1080
tactcttacg ccatgaaact agctggagat cacgtccgag agctgttctt tgcatcccag 1140
gagaaggctg agagcctcaa ggaggacgac aaagccggag ttgagtctta cgtccaggat 1200
atcaaggagc tcttctctgt ttctgctggt ctcaaggctg ccactacatg ggcttgtgct 1260
gacatcattg acaaggcccg acaggcgtgt ggaggccacg gatactctgc ctacaacggc 1320
tttggacagg ccttccagga ctgggttgtc cagtgcactt gggagggtga caatactgtt 1380
ctgactctat ctgccggccg agctctgatc caatctgctc tcgtctaccg aaaggagggc 1440
aaactaggta acgccacgaa gtacctctct cggtccaagg agcttgccaa cgccaagaga 1500
aacggacgat ccctggaaga ccccaagctg ctcgtggagg catgggaggc tgtctctgcc 1560
ggtgctatca acgctgctac tgacgcttac gaggagctct ccaagcaggg agtttctgtt 1620
gacgagtgct ttgagcaggt gtcccaggag cgattccagg ctgcccgaat ccacactcga 1680
cgagctctta tcgaggcctt ctactcacga atcgccactg ctgatgagaa ggtgaagcct 1740
catctgatcc ctctggccaa cctgtttgcc ctgtggtcca ttgaggagga ctctgctctg 1800
ttcctggctg agggctactt tgagcctgag gatatcattg aggtgacttc tcttgtcaac 1860
aagtactgcg gaattgttcg aaagaacgtt attggataca ccgatgcctt caacctgtcc 1920
gactacttca tcaacgctgc cattggacga tacgacggag acgtgtacaa gaactacttt 1980
gagaaggtca aacagcagta ccctcctgag ggtggcaagc ctcactacta cgaggatgtc 2040
atgaagcc 2048
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aacttcctca caggcagcga gcaggagaca ctgctgaccg agcgaatcat gcgggagatt 180
gagcgagatc ccgttctcaa cgtcgccggc gactacgacg ccgatcttcc caccaagcga 240
cgacaagctg ttgagcgaat cggggctctg gcccgatacc tgcccaagga ttccgagaag 300
gaggccattt tgcgaggcca gctgcatggt attgtggaca tgggtacccg aacccgaatc 360
gccgttcact acggtctgtt tatgggcgcc attcgtggct caggaaccaa ggagcagtac 420
gattactggg tcgccaaggg cgccgctact ctgcacaaat tctatggctg ctttgccatg 480
actgagctgg gtcacggatc taacgtggcc ggtctcgaga ccaccgccac ccttgataag 540
gacaccgacg agttcatcat caacaccccc aactcgggag ccacaaagtg gtggattgga 600
ggagctgccc actctgctac ccacacggct tgtcttgccc gactcattgt tgatggcaag 660
gactatggtg ttaagatctt cattgttcag ctgcgagacc tcaactccca ctctctactc 720
aacggtattg ccattggaga tatcggcaag aagatgggcc gagatgccat tgataatggt 780
tggatccagt tcacagacgt ccgaattccc cgacagaaca tgctcatgcg atacgaccgg 840
gtgtctcgag acggcgaggt taccacctcc gagcttgccc agctcaccta cggagcactt 900
ctgtctggcc gagtgaccat gattgccgag tctcacctcc tgtctgctcg gttcctcacc 960
attgctcttc ggtacgcctg tatccgtcga cagttcggag ctgtgcctga caagcccgag 1020
actaagctca tcgactaccc ctaccaccaa cgacgtctgc tgcctcttct ggcctacacc 1080
tacgccatga agatgggcgc cgacgaggcc cagcagcagt acaactcctc ctttggcgct 1140
cttctcaagc tcaaccccgt caaggacgct gagaagtttg ctgtcgccac tgccgacctc 1200
aaggctctgt ttgcctcttc tgccggaatg aaggccttca ccacctgggc tgccgccaag 1260
atcattgacg agtgccgaca ggcctgtggt ggccatggct actccggcta caacggtttc 1320
ggtcaggctt acgccgactg ggtcgtccaa tgcacttggg agggtgacaa caacgtgctg 1380
tgtctgtcca tgggtcgatc gctcatccag tcgtgcattg ccatgagaaa gaagaagggc 1440
catgtcggca agtcggtcga gtacctgcag cgacgagacg agctgcagaa tgcccgagtt 1500
gacaacaagc ctctcactga ccctgctgtg ctcatcactg catgggagaa ggttgcctgc 1560
gaggccatca acagagccac tgactccttc atcaagctca cccaggaggg tctgtctcct 1620
gaccaggcct ttgaggagct gtctcaacag agatttgagt gtgcgcgaat ccacacccga 1680
aagcatctga tcacctcgtt ctacgctcga atctccaagg ccaaggcccg agtcaagccc 1740
caccttactg ttcttgccaa cctctttgcc gtctggtcca tcgaggagga ctctggtctc 1800
ttccttcggg agggctgctt cgagcctgcc gagatggacg agatcaccgc tctggtcgac 1860
gagctgtgct gcgaggctcg agagcaggtc attggattca ccgacgcctt caacctgtcc 1920
gacttcttca ttaacgcccc cattggccga ttcgacggag acgcctacaa gcactacatg 1980
gacgaggtca aggctgccaa caaccctcgt aacacccatg ctccttacta cgagaccaag 2040
ctgcgacc 2048
<210> 6
<211> 40
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 6
gagtataaga atcattcaaa atggctgctg ctcccagtgt 40
<210> 7
<211> 40
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 7
tgacataact aattacatga ttactgcgcc ttacccatct 40
<210> 8
<211> 40
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 8
ccctgattga ctggaacagc atgatcaccc caaaccccgc 40
<210> 9
<211> 40
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 9
tgacataact aattacatga ggcttcatga catcctcgta 40
<210> 10
<211> 40
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 10
gctcgaaggc tttaatttgc atgaacaaca accccaccaa 40
<210> 11
<211> 40
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 11
acaagttccg tagttggatc ggtcgcagct tggtctcgta 40
Claims (8)
1. A genetic engineering strain for producing gamma-linolenic acid is characterized in that yarrowia lipolytica Yarrowia lipolytica is taken as an original strain, delta 6 desaturase gene is expressed, PEX4 gene and PEX5 gene are simultaneously and excessively expressed, the strain can grow by taking waste edible oil as a unique carbon source, and the delta 6 desaturase gene is called delta 6DEs;
the delta 6 desaturase gene is derived from mortierella alpina Mortierella alpina, and the delta 6 desaturase gene sequence is shown in SEQ ID No. 1;
the PEX4 gene and the PEX5 gene are derived from yarrowia lipolytica, the PEX4 gene sequence is shown in SEQ ID NO.4, and the PEX5 gene sequence is shown in SEQ ID NO. 5.
2. A method of constructing a genetically engineered strain for producing gamma-linolenic acid according to claim 1, comprising the steps of:
(1) Construction of yarrowia lipolytica engineering strain Polf-001
Construction of uracil auxotrophs yarrowia lipolytica Polf and recombinant plasmid PUC-inTF3-
Delta 6DEs, transferring the recombinant plasmid PUC-inTF 3-delta 6DEs into uracil auxotroph yarrowia lipolytica Polf to obtain a yarrowia lipolytica engineering strain Polf-001;
(2) Preparation of yarrowia lipolytica engineering strain Polf-002
Inoculating the yarrowia lipolytica engineering strain Polf-001 into a YPD-5FOA solid culture medium, and after single colony grows out, scribing and activating again in the YPD solid culture medium to obtain the yarrowia lipolytica engineering strain Polf-002;
(3) Construction of recombinant plasmid PUC-SCP2-PEX4-5
Taking a PUC-57-SCP2 plasmid as a framework, and inserting a PEX4 gene expression cassette and a PEX5 gene expression cassette into the plasmid framework to obtain a recombinant plasmid PUC-SCP2-PEX4-5;
(4) Construction of genetically engineered strains producing gamma-linolenic acid
Transferring the recombinant plasmid PUC-SCP2-PEX4-5 into a yarrowia lipolytica engineering strain Polf-002 to obtain the gene engineering strain for producing gamma-linolenic acid;
the uracil auxotroph yarrowia lipolytica Polf construction method in the step (1) is that the uracil encoding gene in the yarrowia lipolytica Po1f is knocked out by CRISPR/Cas9 technology;
the construction method of the recombinant plasmid PUC-inTF 3-delta 6DEs in the step (1) is that the PUC-57-inTF3 plasmid is taken as a framework, and a delta 6 desaturase gene expression cassette is inserted into the plasmid framework.
3. The construction method according to claim 2, wherein,
in step (1), the Δ6 desaturase gene expression cassette promoter is P derived from yarrowia lipolytica YAT Promoter, P TEF Promoters or P FBAin One of the promoters, the terminator is T from yarrowia lipolytica XPR2T Terminator, T lip2t Terminator and T CYC1t One of the terminators;
in the step (3), the PEX4 gene expression cassette promoter is P derived from yarrowia lipolytica YAT Promoter, P TEF Promoter and P FBAin One of the promoters, the terminator is T from yarrowia lipolytica XPR2T Terminator, T lip2t Terminator and T CYC1t One of the terminators;
in the step (3), the PEX5 gene expression cassette promoter is P derived from yarrowia lipolytica YAT Promoter, P TEF Promoter and P FBAin One of the promoters, the terminator is T from yarrowia lipolytica XPR2T Terminator, T lip2t Terminator and T CYC1t One of the terminators.
4. The use of the genetically engineered strain for producing gamma-linolenic acid according to claim 1, wherein the genetically engineered strain for producing gamma-linolenic acid can ferment on a culture medium with waste edible oil as the sole carbon source to produce gamma-linolenic acid.
5. The use of a genetically engineered strain for the production of gamma-linolenic acid according to claim 4, wherein the fermentation comprises the steps of:
(1) ' pretreatment of waste edible oil
Filtering the waste edible oil, removing solid impurities, washing with distilled water for 3-4 times, layering, taking upper-layer oil, and sterilizing;
(2) Preparation of' seed liquid
Activating the genetically engineered strain for producing gamma-linolenic acid, inoculating the genetically engineered strain into YPD liquid culture medium, and culturing the genetically engineered strain to a logarithmic growth phase to obtain seed liquid;
(3) ' fermentation production of gamma-linolenic acid
Inoculating the seed solution into YNB fermentation medium with waste oil as the sole carbon source according to the volume ratio of 5-10%, and carrying out ventilation culture for 92-100 h at the temperature of 25-30 ℃ and at the speed of 200-240 rpm to obtain a fermentation product gamma-linolenic acid.
6. The use of the genetically engineered strain for producing gamma-linolenic acid according to claim 5, wherein in the step (1)' the sterilization temperature is 105-115 ℃ and the sterilization time is 25-30 min.
7. The use of the genetically engineered strain for producing gamma-linolenic acid according to claim 5, wherein in step (2)' the viable count in the seed fluid is 1.2X10 8 ～2.4×10 8 CFU/mL。
8. The use of a genetically engineered strain for producing gamma-linolenic acid according to any one of claims 5 to 7, wherein in step (3)' the amount of waste oil added in the fermentation culture is 20 to 30g/L and the amount of YNB added is 1.0 to 2.5g/L.
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