CN113736677A - Recombinant yarrowia lipolytica for high yield of tocotrienol, construction method and application thereof - Google Patents
Recombinant yarrowia lipolytica for high yield of tocotrienol, construction method and application thereof Download PDFInfo
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Abstract
The invention provides a recombinant yarrowia lipolytica for high yield of tocotrienols, which contains a key gene for accumulation of geranylgeranyl diphosphate (GGPP), which is a substrate for tocotrienol synthesis, and a codon-optimized tocotrienol synthesis gene, wherein proteins encoded by the codon-optimized tocotrienol synthesis gene are 2-methyl-6-geranylgeranyl benzoquinone methyltransferase, tocopherol cyclase and gamma-tocopherol methyltransferase. Experiments confirm that the yield of the total tocotrienols can reach 2423.7 mu g/g DCW when the obtained recombinant strain is subjected to fermentation culture, wherein the content of gamma-tocotrienols is 1675.2 mu g/g DCW, the content of alpha-tocotrienols is 748.5 mu g/g DCW, and the yield is obviously higher than the highest yield of the existing research.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to recombinant yarrowia lipolytica for high yield of tocotrienols, and a construction method and application of the strain.
Technical Field
Vitamin E is an amphiphilic molecule and is also a strong oxidant for prenequinols. Vitamin E comprises two different types of tocopherols and tocotrienols, both of which have the structure of chroman derivatives, consisting of a polar aromatic ring head on the cell membrane and a hydrophobic tail associated with membrane lipids. The main difference is that to the extent that their carbohydrate isoprenoid side chain tail structures are saturated, tocotrienols (tocotrienols) each have a trans double bond at carbon 3,7 and 11, while tocopherols (tocopherols) are saturated. Each type has 4 different components, namely four components of alpha, beta, gamma and delta, according to the difference of the number and the positions of methyl groups on the aromatic ring.
Recent studies have shown that tocotrienols may have better cholesterol lowering, diabetes prevention, bone resorption promotion (unique to tocotrienols), antioxidant, anticancer, anti-inflammatory, cardioprotective, neuroprotective, etc. effects. Although tocotrienols account for half of the VE family members, they have been poorly studied. The tocotrienol product is mainly extracted from crude palm oil in the fruit of oil palm (Elaeis guineensis), the source of the tocotrienol product is rare, the market price is high at present, related products are few, and the market demand of the tocotrienol is increased along with the increase of the cognition degree of people on the strong functions of the tocotrienol. The research on the tocotrienols is carried out, and the improvement on the content and the components of the plant tocotrienols is beneficial to further exerting the efficacy. Among them, the improvement of genetic engineering is one of the methods for efficiently synthesizing tocotrienols. Researchers at Stuttgart, Germany, metabolically engineered E.coli by introducing tocotrienol synthesis pathway genes from photosynthetic organisms to obtain engineered strains producing 15 μ g/g (dry cell weight) of delta-tocotrienol [ ALBERMANN C, GHANGEGAONKAR S, LEMUTH K, et al, biosyntheses of the vitamin E compound delta-tocotrienol in recombinant Escherichia coli cells [ J ]. Chembeiochem, 2008,9(15):2524-33 ]. In 2013, the subject group constructed an engineering strain [ GHANGEGAONKAR S, CONRAD J, BEIFUSS U, et al. the engineering strain of the in vivo protocol of the biochemical compounds of 2-methyl-6-gernylgeranyl-benzoquinone [ J ]. J Biotechnology, 2012,164(2):238-47 ] for the heterologous synthesis of the heterologous synthesis through genome integration and enhanced MEP pathway, which produced 1425. mu.g/g (cell dry weight) of tocotrienol common precursor MGGBQ. In addition, in the research of hong Wei team at home [ Shen. research of heterologous synthesis of vitamin E (tocotrienol) in Saccharomyces cerevisiae [ D ]. Zhejiang university, 2019 ], a metabolic pathway of heterologous synthesis of vitamin E (tocotrienol) is constructed by combining an endogenous shikimic acid pathway and an MVA pathway in Saccharomyces cerevisiae, and the saccharomyces cerevisiae is used as a research platform, key genes from arabidopsis thaliana are cloned and codon optimization is combined to obtain the yield of 244.2 mu g/g dry weight, wherein the yield of gamma-tocotrienol is 172.9 mu g/g dry weight, and the yield of alpha-tocotrienol is 71.3 mu g/g dry weight.
Aiming at the problems of unclear existing tocotrienol synthesis method and low yield, the invention takes yarrowia lipolytica as a research platform according to the currently clear synthetic route of tocotrienols, carries out gene mining on key genes of 2-methyl-6-geranylgeranylbenzoquinone methyltransferase, tocopherol cyclase and gamma-tocopherol methyltransferase for the synthesis of tocotrienols, combines codon optimization, and overexpresses key genes of BrHPPD, GaHPT, SmMPBQMT, DcCTC and Bo gamma _ TMT in the synthetic route of tocotrienols and key genes of HMG1 and CrtE accumulated in geranylgeranyl diphosphate (GGPP) as substrates for the synthesis of tocotrienols on the basis of the gene mining, the tocopherol cyclase and the gamma-tocopherol methyltransferase, so as to further improve the yield of tocotrienols.
Disclosure of Invention
The object of the present invention is to further improve the production of tocotrienols by selecting more optimal tocotrienol synthesis key genes 2-methyl-6-geranylgeranylbenzoquinone methyltransferase, tocopherol cyclase and gamma-tocopherol methyltransferase while overexpressing key genes BrHPPD, GaHPT, SmMPBQMT, DcTC and Bo γ _ TMT in the tocotrienol synthesis pathway and key genes HMG1 and CrtE accumulated in geranylgeranyl diphosphate (GGPP) which is a substrate for tocotrienol synthesis. Provides a recombinant yarrowia lipolytica with high yield of tocotrienols.
In order to achieve the above objects, the present invention provides a recombinant yarrowia lipolytica that produces tocotrienols in high yield, said recombinant yarrowia lipolytica comprising a recombinant plasmid containing a key gene for accumulation of geranylgeranyl diphosphate (GGPP), which is a tocotrienol synthesis substrate, and a codon-optimized tocotrienol synthesis gene, for accumulation of geranylgeranyl diphosphate (GGPP), which is a tocotrienol synthesis substrate, HMG1 and CrtE, said codon-optimized tocotrienol synthesis gene encoding proteins of 2-methyl-6-geranylgeranyl benzoquinone methyltransferase, tocopherol cyclase and γ -tocopherol methyltransferase.
In the present invention, the 2-methyl-6-geranylgeranylbenzoquinone methyltransferase is from walnut, peanut, wasabi, pigeon pea or selaginella; the tocopherol cyclase is from shepherd's purse, wasabi, cabbage, rape, citrus or dendrobium; the gamma-tocopherol methyltransferase is from shepherd's purse, wasabi, cabbage, and rape.
According to a particularly preferred embodiment, the 2-methyl-6-geranylgeranylbenzoquinone methyltransferase is from selaginella tamariscina, whose codon-optimized sequence is shown in SEQ ID NO. 1; the tocopherol cyclase is from dendrobe, and the sequence is shown as SEQ ID NO. 2; the gamma-tocopherol methyltransferase is from cabbage and has a sequence shown in SEQ ID NO. 3.
Based on the above, the invention also provides a construction method of the recombinant yarrowia lipolytica, which comprises the following steps:
(1) construction of yarrowia lipolytica engineering bacterium containing HPPD gene and HPT gene
Obtaining and synthesizing HPPD gene (BrHPPD) from rape and HPT gene (GaHPT) sequence from blue algae, carrying out enzyme digestion connection with a vector ploxpura3loxp to obtain a recombinant plasmid ploxpura3loxp-BrHPPD-GaHPT, carrying out enzyme digestion linearization on the recombinant plasmid, transferring the recombinant plasmid into engineering bacteria polf of yarrowia lipolytica by adopting a yeast transformation kit method to obtain a recombinant strain polf-BHGH, and carrying out colony PCR verification on the obtained recombinant strain to be correct;
(2) construction of recombinant plasmid containing 2-methyl-6-geranylgeranylbenzoquinone methyltransferase Gene
Obtaining and synthesizing 2-methyl-6-geranylgeranyl benzoquinone methyltransferase gene sequences from walnut (JrMPBQMT), peanut (AhmPBQMT), wasabi (EsmMPBQMT), pigeon pea (CcMPBQMT) and selaginella tamariscina (SmMPBQMT), respectively taking Nde I/SpeI as enzyme cutting sites to obtain double enzyme cutting vectors ploxpura3loxp and 2-methyl-6-geranylgeranyl benzoquinone methyltransferase gene, respectively connecting to obtain recombinant plasmids ploxpura3loxp-JrMPBQMT, ploxpura3 loxp-AhmPBT, ploxpura3 loxp-EsMPQMT, ploxpura3 loxp-CcBQMT and ploxpura3loxp-SmMPBQMT, linearizing the obtained recombinant plasmids by SmaI, transferring the recombinant plasmids into lipolysis yeast engineering strains of yarrowia, respectively obtaining recombinant strains of 2, 5 and 4 through PCR, and verifying the obtained recombinant strains to obtain correct colonies;
(3) construction of recombinant plasmid containing tocopherol cyclase Gene
Obtaining and synthesizing tocopherol cyclase gene sequences from shepherd's purse (CrTC), wasabi (EsTC), cabbage (BoTC), rape (BnTC), orange (CcTC) and dendrobium (DcTC), respectively connecting double enzyme digestion vectors ploxpura3loxp and the tocopherol cyclase gene with Nde I/SpeI as enzyme digestion sites to obtain recombinant plasmids ploxpura3loxp-CrTC, ploxpura3loxp-EsTC, ploxpura3loxp-BoTC, ploxpura3loxp-BnTC, ploxpura3loxp-CcTC and ploxpura3loxp-DcTC, carrying out enzyme digestion linearization on the obtained recombinant plasmids by using SmaI respectively, transferring the plasmids into recombinant strains 1-5 obtained in the step (1) through a yeast transformation kit method to obtain recombinant strains 6, 7, 8, …, 34 and 35, and verifying the correct colonies of the obtained recombinant strains through PCR;
(4) construction of recombinant plasmid containing gamma-tocopherol methyltransferase Gene
Acquiring and synthesizing a gamma-tocopherol methyltransferase gene sequence from shepherd's purse (Cr gamma-TMT), wasabi (Es gamma-TMT), cabbage (Bo gamma-TMT) and rape (Bn gamma-TMT), respectively connecting double enzyme digestion vectors ploxpura3loxp and gamma-tocopherol methyltransferase genes with Nde I/SpeI as enzyme digestion sites to obtain recombinant plasmids ploxpura3loxp-Cr gamma-TMT, ploxpura3loxp-Es gamma-TMT, ploxpura3loxp-Bo gamma-TMT and ploxpura3loxp-Bn gamma-TMT, respectively carrying out enzyme digestion linearization on the obtained recombinant plasmids by using a restriction enzyme SmaI, and transferring the recombinant plasmids into the recombinant strains 6 to 35 in the step (3) by using a yeast transformation kit method to obtain the recombinant lipolysis rhodotorula torula benthamoides with high yield of tocotrienols.
As a particularly preferred embodiment, the recombinant yarrowia lipolytica is constructed by the following steps:
(1) construction of yarrowia lipolytica engineering bacterium containing HPPD gene and HPT gene
Obtaining and synthesizing HPPD gene (BrHPPD) from rape and HPT gene (GaHPT) sequence from blue algae, carrying out enzyme digestion connection with a vector ploxpura3loxp to obtain a recombinant plasmid ploxpura3loxp-BrHPPD-GaHPT, carrying out enzyme digestion linearization on the recombinant plasmid, transferring the recombinant plasmid into engineering bacteria polf of yarrowia lipolytica by adopting a yeast transformation kit method to obtain a recombinant strain polf-BHGH, and carrying out colony PCR verification on the obtained recombinant strain to be correct;
(2) construction of recombinant plasmid containing 2-methyl-6-geranylgeranylbenzoquinone methyltransferase Gene
Obtaining and synthesizing a 2-methyl-6-geranylgeranylgeranylbenzoquinone methyltransferase gene sequence from selaginella tamariscina (SmMPBQMT), connecting a double enzyme digestion carrier ploxpura3loxp and the 2-methyl-6-geranylgeranylgeranylbenzoquinone methyltransferase gene with Nde I/Spe I as an enzyme digestion site respectively to obtain a recombinant plasmid ploxpura3loxp-SmMPBQMT, carrying out enzyme digestion linearization on the obtained recombinant plasmid by using a restriction enzyme SmaI, transferring the recombinant plasmid into a yarrowia lipolytica engineering bacterium polf-BHGH by adopting a yeast transformation kit method to obtain a recombinant strain 5, and verifying the correctness of the obtained recombinant strain by colony PCR;
(3) construction of recombinant Strain containing Tocopherol cyclase Gene
Obtaining and synthesizing a tocopherol cyclase gene sequence from dendrobe (DcTC), connecting a double enzyme digestion vector ploxpura3loxp and the tocopherol cyclase gene by taking Nde I/SpeI as an enzyme digestion site, obtaining a recombinant plasmid ploxpura3loxp-DcTC, carrying out enzyme digestion linearization on the obtained recombinant plasmid by using a restriction enzyme SmaI, respectively transferring the recombinant plasmid into the recombinant strain 5 obtained in the step (2) by a yeast transformation kit method, obtaining a recombinant strain 35, and verifying the correctness of the obtained recombinant strain through PCR;
(4) construction of recombinant Strain containing Gamma-Tocopherol methyltransferase Gene
Acquiring and synthesizing a gamma-tocopherol methyltransferase gene sequence from cabbage (Bo gamma-TMT), connecting to obtain a recombinant plasmid ploxpura3loxp-Bo gamma-TMT by taking Nde I/SpeI as enzyme cutting sites and double enzyme cutting carriers ploxpura3loxp and gamma-tocopherol methyltransferase genes respectively, carrying out enzyme cutting linearization on the obtained recombinant plasmid respectively by using a restriction enzyme SmaI, transferring into the recombinant strain 35 in the step (2) by adopting a yeast transformation kit method to obtain a recombinant strain 38, and verifying the accuracy of the obtained recombinant strain by colony PCR;
(5) construction of recombinant Strain containing HMG1 and CrtE genes
Obtaining and synthesizing HMG1 and CrtE gene sequences, designing primers to amplify a target fragment from the genomic DNA of yarrowia lipolytica engineering bacteria po1f, connecting the obtained target fragment to a multienzyme cutting site of a vector pJN44, and respectively obtaining plasmids pJN44-HMG1 and pJN 44-CrtE; the obtained plasmids are respectively transferred into a bacterial strain 38 to obtain a recombinant bacterial strain 40, and the obtained recombinant bacterial strain is verified to be correct through colony PCR;
(6) overexpression of the Gene of interest
Respectively connecting the target genes BrHPPD, GaHPT, SmMPBQMT, DcTC and Bo gamma _ TMT obtained in the previous step to a multi-enzyme cutting site of a vector pJN44 to respectively obtain plasmids pJN44-BrHPPD, pJN44-GaHPT, pJN44-SmMPBQMT, pJN44-DcTC and pJN44-Bo gamma _ TMT; and (3) transferring the obtained plasmid into the recombinant strain 40 in the step (5) after enzyme digestion verification to obtain the recombinant yarrowia lipolytica with high tocotrienol yield, and verifying the accuracy of the obtained recombinant strain through colony PCR.
In the present invention, as a preferred embodiment, the method further comprises the step (7) of removing the ura3 label:
and culturing the obtained recombinant yarrowia lipolytica on an SD-LEU culture medium, wherein a single colony growing on the SD-LEU culture medium is the recombinant yarrowia lipolytica with high tocotrienol yield.
In the present invention, the ploxpura3loxp vector is described in "research on the synthesis of campesterol by recombinant yarrowia lipolytica" (Tanshina), and the pJN44 vector is described in "research on the synthesis of microbial oil by recombinant yarrowia lipolytica" (Donggui sha).
In the present invention, the reaction system for colony PCR verification is as follows:
reaction system:
PCR procedure:
the recombinant yarrowia lipolytica provided by the invention is used for producing tocotrienols, and has significant advantages in the production of alpha-tocotrienol and gamma-tocotrienol.
According to the invention, codon-optimized recombinant plasmids of tocotrienol synthetic genes 2-methyl-6-geranylgeranylbenzoquinone methyltransferase, tocopherol cyclase and gamma-tocopherol methyltransferase from different sources are respectively constructed; and integrating the recombinant plasmid into an engineering bacterium genome, screening a single colony growing on an SD-LEU culture medium, and removing the ura3 mark to obtain the recombinant bacterium for improving the yield of the tocotrienol. On this basis, key genes BrHPPD, GaHPT, SmMPBQMT, DcTC and Bo gamma _ TMT in the synthesis pathway of tocotrienols and key genes tHMG1 and CrtE accumulated in geranylgeranyl diphosphate (GGPP) as a substrate for the synthesis of tocotrienols are overexpressed. Through comparison, the recombinant strain 41 is fermented and cultured to produce tocotrienols, the total tocotrienol yield can reach 2423.7 mu g/g DCW at most, wherein the content of gamma-tocotrienol is 1675.2 mu g/g DCW, the content of alpha-tocotrienol is 748.5 mu g/g DCW, and the highest yield is 2085.3 mu g/g DCW in the existing research.
[ description of the drawings ]
FIG. 1 a tocotrienol biosynthetic pathway;
FIG. 2 comparison of gamma-tocotrienol content for each recombinant strain;
FIG. 3 comparison of the tocotrienol content of each recombinant strain.
[ detailed description ] embodiments
The following examples serve to illustrate the technical solution of the present invention without limiting it.
In the present invention, "%" used for specifying concentrations is, unless otherwise specified, "%" used for specifying the ratio of amounts ": all the terms "are mass ratios.
The present invention relates to the following media:
SD-LEU medium: glucose 2%, (NH)4)2SO40.5 percent of agar, 0.17 percent of YNB, 0.2 percent of Drop-out mix synthetic minus leucoil w/o yeast nitro gene base, 2.5 percent of agar powder is additionally added into the solid culture medium, and the mixture is sterilized by high-pressure steam at 121 ℃ for 20 min.
YPD liquid medium: 2% of glucose, 1% of yeast powder, 2% of peptone and 0.1% of tyrosine, and sterilizing for 20min by high-pressure steam at 121 ℃.
The present invention relates to the following genes:
TABLE 12-methyl-6-geranylgeranylbenzoquinone methyltransferase genes and sources thereof
TABLE 2 tocopherol cyclase genes and their sources
TABLE 3 Gamma-tocopherol methyltransferase and its source
The invention relates to the following primers:
the reaction system for colony PCR verification provided by the invention comprises the following steps:
reaction system:
PCR procedure:
EXAMPLE 1 construction of a recombinant plasmid containing the 2-methyl-6-geranylgeranylbenzoquinone methyltransferase (MPBQMT) Gene
Obtaining HPPD Gene (BrHPPD) from rape (Gene ID: 103843547) and HPT Gene (GaHPT) sequence (GenBank: MBR8827918.1) from NCBI, feeding into a carrier ploxpura3loxp after optimized synthesis, carrying out enzyme digestion and connection with the carrier ploxpura3loxp to obtain recombinant plasmid ploxpura3loxp-BrHPPD-GaHPT, carrying out enzyme digestion linearization on the recombinant plasmid, transferring into engineering bacteria polf of yarrowia lipolytica by adopting a yeast transformation kit method to obtain recombinant strain polf-BHGH, and adopting a colony PCR (polymerase chain reaction) mode as a verification method.
According to the description in table 1, MPBQMT gene sequences derived from walnuts (jrmmpbqmt), peanuts (AhMPBQMT), wasabi (EsMPBQMT), pigeon peas (CcMPBQMT) and selaginella tamariscina (SmMPBQMT) were obtained from NCBI and sent to industrial optimization synthesis.
The vector ploxpura3loxp and the gene MPBQMT are digested separately with Nde I/Spe I as the digestion site, and the recombinant plasmids ploxpura3loxp-JrMPBQMT, ploxpura3loxp-AhMPBQMT, ploxpura3loxp-EsMPBQMT, ploxpura3loxp-CcMPBQMT and ploxpura3loxp-SmMPBQMT are obtained by ligation. The recombinant plasmids are linearized by restriction enzyme SmaI, transferred into yarrowia lipolytica engineering bacteria polf-BHGH by a yeast transformation kit method to obtain recombinant strains 1, 2, 3, 4 and 5, and the correctness is verified by a colony PCR method.
The 1-5 genotypes of the recombinant strains are as follows:
example 2 construction of recombinant plasmid containing Tocopherol Cyclase (TC) Gene
According to the record in table 2, the TC gene sequences derived from capsella bursa-pastoris (CrTC), docosanthes japonica (EsTC), cabbage (BoTC), rape (BnTC), citrus (CcTC) and dendrobium nobile (DcTC) are obtained from NCBI, and are subjected to industrial optimization synthesis. Nde I/Spe I are respectively used as enzyme cutting sites to carry out double enzyme cutting on a vector ploxpura3loxp and a gene TC and are connected to obtain recombinant plasmids ploxpura3loxp-CrTC, ploxpura3loxp-EsTC, ploxpura3loxp-BoTC, ploxpura3loxp-BnTC, ploxpura3loxp-CcTC and ploxpura3loxp-DcTC, the recombinant plasmids are subjected to enzyme cutting linearization by using a restriction enzyme SmaI, the recombinant plasmids are respectively transferred into the recombinant strains by adopting a yeast transformation kit method to obtain recombinant strains 6, 7, 8 … 34 and 35, and the correctness is verified by a colony PCR method.
The recombinant strain 6-35 has genotype as follows:
and screening the recombinant strain with high yield of the gamma-tocotrienol by detecting the content of the gamma-tocotrienol in the fermentation liquor. The detection method comprises the following steps:
the detection sample is prepared by placing 1ml SD fermentation liquid in 1.5ml centrifuge tube, centrifuging at 12000rpm for 5min, placing the supernatant in a new centrifuge tube, adding 40ml glacial acetic acid, and filtering with 0.22um water system pinhole type filter head.
HPLC detection condition is that the mobile phase is 0.01M KH2PO4Solution (a) and methanol (B); the proportion of 90 percent of A to 10 percent of B; the flow rate is 0.8 ml/min; the detection wavelength is 290 nm; column YMC-Pack ODS-AQ (4.6X 250 mm).
The standard curve was prepared as follows:
preparation of a gamma-tocotrienol standard curve: weighing 10mg of gamma-tocotrienol by a ten-thousandth balance, dissolving the gamma-tocotrienol in 10ml of pure methanol solution, determining the mother solution with the concentration of 1g/L, sequentially diluting the mother solution by the pure methanol solution in a gradient way to obtain gamma-tocotrienol solutions with the concentrations of 250mg/L, 100mg/L,50mg/L, 20mg/L and 5mg/L respectively, diluting the gamma-tocotrienol solutions by the same method to obtain two batches of gamma-tocotrienol solutions with the same concentration, filtering all samples by a 0.22-micrometer organic system pinhole filter head, and analyzing by HPLC.
As a result, as shown in FIG. 2, the tocotrienol content of each recombinant strain was compared, and it was found that the gamma-tocotrienol content of the recombinant strain 35 was the highest, 2014.5. mu.g/g DCW, and thus the recombinant strain 35(polf-BHGH-SmMPBQMT-DcTC) constructed from the MPBQMT gene of Selaginella tamariscina and the TC gene of Dendrobii nobile was selected for the subsequent study.
EXAMPLE 3 construction of recombinant strains expressing a Gene encoding a Gamma-tocopherol methyltransferase
The gamma-TMT gene sequences derived from Capsella bursa-pastoris (Cr gamma-TMT), Wasabia japonica Matsum (Es gamma-TMT), Brassica oleracea (Bo gamma-TMT) and Brassica campestris (Bn gamma-TMT) are obtained from NCBI according to the records in Table 3 and sent to the industrial optimization synthesis. Nde I/SpeI is respectively used as an enzyme cutting site for double enzyme cutting carriers ploxpura3loxp and a gene TC and is connected to obtain recombinant plasmids ploxpura3loxp-Cr gamma-TMT, ploxpura3loxp-Es gamma-TMT, ploxpura3loxp-Bo gamma-TMT and ploxpura3loxp-Bn gamma-TMT, the recombinant plasmids are subjected to enzyme cutting linearization by using a restriction enzyme SmaI, the recombinant plasmids are respectively transferred into a recombinant strain 35(polf-BHGH-SmMPBQMT-DcTC) by adopting a yeast transformation kit method to obtain recombinant strains 36, 37, 38 and 39, and the obtained strains are verified to be correct by PCR.
The genotypes of the recombinant strains 36-39 are:
and (3) screening the recombinant strain with the highest tocotrienol content by detecting the content of alpha-tocotrienol in the fermentation liquor, wherein the HPLC detection method is the same as the gamma-tocotrienol method, and the standard curve preparation method is the same as the gamma-tocotrienol standard curve preparation method.
The content detection method of the alpha-tocotrienol comprises the following steps:
culturing each recombinant strain in 50ml YPD liquid culture medium added with 0.1% (w/v) tyrosine in a constant temperature shaking table at 30 ℃ at 220rpm for 96h, then centrifuging 5ml fermentation liquor, collecting thalli, grinding and breaking cells, extracting twice by using 2ml acetone, centrifuging, taking supernatant, filtering, and carrying out HPLC analysis. Extracting tocotrienol from the fermentation broth with acetone as extraction solvent.
TABLE 4 content of various tocotrienols in fermentation broths of various recombinant strains
As shown in FIG. 3 and Table 4, the recombinant strain 38 (polf-BHGH-SmMPBQMT-DcTC-Bo. gamma. -TMT) had the highest content of α -tocotrienol, reaching 453.6 μ g/g DCW and the γ -tocotrienol content was 1260.9 μ g/g DCW. Therefore, the yield of the tocotrienols obtained by the recombinant strain constructed by the MPBQMT gene (shown in SEQ ID NO. 1) derived from selaginella tamariscina, the TC gene (shown in SEQ ID NO. 2) derived from dendrobium nobile and the gamma-TMT gene (shown in SEQ ID NO. 3) derived from cabbage is the highest, the total amount reaches 1714.5 mu g/g DCW, and the method is remarkably superior to the records in the prior art.
Example 4 overexpression of Key genes
Primers were designed based on the HMG1 and CrtE gene sequences (GeneID:854900, GeneID:37543877) in the NCBI database to amplify the fragment of interest from genomic DNA of Y.lipolytica po1f strain. The desired fragment was ligated to the multiple enzyme cleavage site of vector pJN44 to obtain plasmids pJN44-HMG1 and pJN44-CrtE, respectively. The verified expression plasmids pJN44-HMG1 and pJN44-CrtE were transferred into strain 38, respectively, to obtain recombinant strain 40, which was verified to be correct by means of colony PCR.
The target genes BrHPPD, GaHPT, SmMPBQMT, DcTC and Bo gamma _ TMT obtained in the previous step are connected to a multi-enzyme cutting site of the vector pJN44 to obtain plasmids pJN44-BrHPPD, pJN44-GaHPT, pJN44-SmMPBQMT, pJN44-DcTC and pJN44-Bo gamma _ TMT respectively. The obtained recombinant expression plasmid is transferred into a strain 40 after enzyme digestion verification to obtain a recombinant strain 41 with high yield of tocotrienols, which is named as recombinant yarrowia lipolytica polf- (MTT)+HC-ura3, verified by colony PCR.
The genotypes of the recombinant strains 40 and 41 for high-yield tocotrienol are as follows:
culturing the recombinant strain 41 on an SD-LEU culture medium to remove ura3 mark, wherein a single colony growing on the SD-LEU culture medium is the recombinant yarrowia lipolytica with high tocotrienol yield and is named polf- (MTT)+HC。
For the obtained recombinant strain polf- (MTT) for high-yield tocotrienol+HC was fermented 4 times, and the contents of alpha-tocotrienol and gamma-tocopherol were measured on the results of the 4 fermentations, as shown in Table 5, which shows the recombinant strain polf- (MTT)+The content of alpha-tocotrienol of HC reaches 748.5ug/g DCW to the maximum, the content of gamma-tocotrienol is 1675.2 mu g/g DCW, and the content of total tocotrienol is 2423.7 mu g/g DCW. Its average total tocotrienol productionReaching 2286.8 mug/g DCW, which is obviously improved compared with the prior art. Therefore, the key genes in the synthesis pathway of the overexpressed tocotrienols have obvious effect on the improvement of the yield of the tocotrienols on the basis of gene mining.
TABLE 5 recombinant Strain polf- (MTT)+Content of various tocotrienols in HC fermentation broth
Sequence listing
<110> Seawa biosciences, Inc
<120> recombinant yarrowia lipolytica for high yield of tocotrienol, construction method and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1047
<212> DNA
<213> 2-methyl-6-geranylgeranylgeranylbenzoquinone methyltransferase gene (Selaginella sp.)
<400> 1
atggccatgg ccatggctgc cgccgccccc cgaaccgtcc aggccctcga gtgcggcgcc 60
tccggctccg agctgccccg aatcgcccga atttctgccc gacttccctc ccgaaagggc 120
ttccgaggcc tggccgtcgc cgcccgaatg cgacgaaact ccccgctggt gcgatgcgcc 180
gccgcccagt ccgcctccgc ctccccccga cccgccatgc agccccgatt catccagcac 240
aagcaggagg ccttctggtt ctaccgattc ctgtctatca tctacgacca catcatcaac 300
cccggtcact ggactgagga catgcgtgac gatgccctgg agcccgccga cctgtctgac 360
cgaaacctgg tcgtcgtcga cgtcggcggc ggcaccggct tcaccaccct gggcatcgtg 420
aagcacgtcg acgcccgaaa cgtgaccatt ctggaccagt ccccccacca gctggccaag 480
gccaaggaga aggagcccct gaaggagtgc aagatcatcg agggtgacgc tgaggacctg 540
ccctttgaga ctgattacgc tgacagatac gtttctgctg gttccatcga gtactggcct 600
gatccccagc gaggaattaa ggaggcttac agagtgctga agaagggcgg caaggcctgc 660
ctgatcggcc ccgtgcaccc caccttctgg ctgtcccgat tcttcgccga catgtggatg 720
ctgttcccca aggaggagga gtacattgac tggttcacca aggccggctt ccaggacgtc 780
cagctgaagc gaatcggtcc aaagtggtac cgaggtgtgc gtagacatgg tcttattatg 840
ggttgtagtg tgactggtgt taagcctgag gccggcgact ctcccctgga cctgggcccc 900
aaggccgagg acgtccaggc cccctctaac cccctgacct tcttcttccg attcctggtc 960
ggcggcatcg cctccctgta cttcgtcctg gtccccatct acatgtggct gaaggacctg 1020
atcaccccca agggccagcc catctaa 1047
<210> 2
<211> 1458
<212> DNA
<213> tocopherol cyclase Gene (Dendrobium sp.)
<400> 2
atggatcgat ctggttcctc tgccctgctg cgacagcccc tgaccgccgt ctgccccctg 60
cccccccgat tcctgatctt ccgaaaggtc cctattgcca acgtcgagtt tcgacgacga 120
ccccccaagc ccaaggccgc cacctcttcc tctgaggccg aaaagaccgg cgccgtgtcc 180
tcttcccccg tgtacatccc cacccctcga gaccgagagc tgcgaacccc ccactccggc 240
taccacttcg acggcaccgc tcgagttttc ttcgagggtt ggtacttcaa ggtctctatc 300
ccccactgtc gacagtcctt ctgcttcatg tactccgttg agaaccccgc cttttccgac 360
ggtatgggtg tactggaccg agccatgtac ggtcaccgat tcaccggcgt cggcgcccag 420
atcctgggcg ctgacgacaa gtacatctgc cagttctctg agaagtctaa gaacttctgg 480
ggttctcgac acgagctgat gctgggcaac actttcatcg ctggcaacaa ctccgccccc 540
cctaacggcg agctgccccc ccaggagttc cgaaagcgag tgctggaggg tttccaggtt 600
tctcccttct ggcaccaggg cttcattcgt gacgacggcc gatctaccta cgtccagacc 660
gtcaagaccg cccgatggga gtactccacc cgacctgtct acggttgggg cgacttcaag 720
tctaaacaga agtccaccgc cggttggctg gccgccttcc ccgtctttga gccccactgg 780
cagatttgca tggccggcgg cctgtccacc ggctggattg agtgggacgg cgagcgattc 840
gagttcgaga acgccccctc gtactccgag aagaactggg gcggcggctt cccccgaaag 900
tggttctggg tccagtgtaa cgtgttcaag ggtgcttccg gcgaggtggc tctcaccgcc 960
gccggtggtc tccgaaagct ggccggcctc gccgacaact acgagaacgc cgccctggtg 1020
ggcgtccatt acggcggcaa gttctacgag ttcgtcccct ggaacggttc tgtgtcctgg 1080
gagatctccc agtggggcta ctggcacatc tccgccgaga acaaccagaa catggtcgag 1140
ctggtcgcta ccgtcgagga gcccggtacc cccctgagag ctcctacaca ggaagcagga 1200
cttgttactg cttgtaagga tacttgttac ggtgacctta ccctgcaact atgggagaaa 1260
acttccgacg gtaaaaaggg aaagcttatc ctggaggcca cctctaacat ggccgccgtt 1320
gaagtcggag gtggcccctg gttttcgact tggaagggaa ctacctctat gcccgagctg 1380
gtctcctccg cccttcaggt tcctattgac gttgagtcct tcttccccgt cccactgttt 1440
aagccccctg gactgtaa 1458
<210> 3
<211> 1044
<212> DNA
<213> gamma-tocopherol methyltransferase gene (Brassica oleracea)
<400> 3
atgaaggcca ccctggcccc cccctcctcc ctgatctccc tgccccgaca caaggtctcc 60
tccctgcgat ccccctccct gctgctccag tcccagcgac cctcctccgc cctgatgaca 120
accaccgcca cccgaggctc cgtcgccgtc accgccgccg ccacctcctc tgccgaggcc 180
ctgcgagagg gcatcgccga gttctacaac gagacctccg gcctgtggga ggagatctgg 240
ggcgaccaca tgcaccacgg tttctacgac cccgactcct ctgtccagct gtctgactcc 300
ggacaccgag aggcccagat ccgaatgatc gaggagtccc tgcgattcgc cggcgtgacc 360
gaggaggaga agaagatcaa gcgagttgtt gacgtcggct gtggtatcgg tggatcttct 420
cgatacattg cttctaagtt cggagctgag tgtattggca tcaccctgtc ccccgtgcag 480
gccaagcgag ctaacgacct ggccgctgcc cagtccctgt ctcacaaggt gtccttccag 540
gtcgccgacg ccctggacca gcccttcgag gacggaatct tcgacctcgt ctggtccatg 600
gagtctggtg agcacatgcc cgacaaggcc aagttcgtga aggagctggt ccgagtgacc 660
gcccccggcg gtcgaatcat catcgtgacc tggtgccacc gaaacctgtc tcagggtgag 720
gagtctctgc agccctggga gcagaacctg ctggaccgaa tctgtaagac cttctacctg 780
cccgcctggt gctccacctc cgactacgtc gagctgctgc agtctctctc cctccaggac 840
attaagtgcg ccgactggtc tgagaacgtc gcccccttct ggcccgccgt gatccgaacc 900
gccctgacct ggaagggact ggtctccctg ctgcgatccg gtatgaagtc cattaagggc 960
gccctgacca tgcccctgat gatcgagggc tacaagaagg gcgtcatcaa gttcggcatc 1020
atcgcctgcc agaagcccct gtaa 1044
Claims (9)
1. A recombinant yarrowia lipolytica for high production of tocotrienols, characterized in that said recombinant yarrowia lipolytica comprises a recombinant plasmid comprising a key gene for accumulation of geranylgeranyl diphosphate (GGPP) which is a substrate for tocotrienol synthesis and a codon-optimized tocotrienol synthesis gene encoding 2-methyl-6-geranylgeranyl benzoquinone methyltransferase, tocopherol cyclase and γ -tocopherol methyltransferase, HMG1 and CrtE which are key genes for accumulation of geranylgeranyl diphosphate (GGPP) which is a substrate for tocotrienol synthesis.
2. The recombinant yarrowia lipolytica yeast according to claim 1, characterized in that said 2-methyl-6-geranylgeranylbenzoquinone methyltransferase is from walnut, peanut, wasabi, pigeon pea or selaginella; the tocopherol cyclase is from shepherd's purse, wasabi, cabbage, rape, citrus or dendrobium; the gamma-tocopherol methyltransferase is from shepherd's purse, wasabi, cabbage, and rape.
3. The recombinant yarrowia lipolytica yeast according to claim 1, characterized in that said 2-methyl-6-geranylgeranylbenzoquinone methyltransferase is from selaginella tamariscina, the codon-optimized sequence of which is shown in SEQ ID No. 1; the tocopherol cyclase is from dendrobe, and the sequence is shown as SEQ ID NO. 2; the gamma-tocopherol methyltransferase is from cabbage and has a sequence shown in SEQ ID NO. 3.
4. The method of constructing recombinant yarrowia lipolytica of claim 1 or 2, comprising the steps of:
(1) construction of yarrowia lipolytica engineering bacterium containing HPPD gene and HPT gene
Obtaining and synthesizing HPPD gene (BrHPPD) from rape and HPT gene (GaHPT) sequence from blue algae, carrying out enzyme digestion connection with a vector ploxpura3loxp to obtain a recombinant plasmid ploxpura3loxp-BrHPPD-GaHPT, carrying out enzyme digestion linearization on the recombinant plasmid, transferring the recombinant plasmid into engineering bacteria polf of yarrowia lipolytica by adopting a yeast transformation kit method to obtain a recombinant strain polf-BHGH, and carrying out colony PCR verification on the obtained recombinant strain to be correct;
(2) construction of recombinant plasmid containing 2-methyl-6-geranylgeranylbenzoquinone methyltransferase Gene
Obtaining and synthesizing 2-methyl-6-geranylgeranyl benzoquinone methyltransferase gene sequences from walnut (JrMPBQMT), peanut (AhmPBQMT), wasabi (EsmMPBQMT), pigeon pea (CcMPBQMT) and selaginella tamariscina (SmMPBQMT), respectively taking Nde I/SpeI as enzyme cutting sites to obtain double enzyme cutting vectors ploxpura3loxp and 2-methyl-6-geranylgeranyl benzoquinone methyltransferase gene, respectively connecting to obtain recombinant plasmids ploxpura3loxp-JrMPBQMT, ploxpura3 loxp-AhmPBT, ploxpura3 loxp-EsMPQMT, ploxpura3 loxp-CcBQMT and ploxpura3loxp-SmMPBQMT, linearizing the obtained recombinant plasmids by SmaI, transferring the recombinant plasmids into lipolysis yeast engineering strains of yarrowia, respectively obtaining recombinant strains of 2, 5 and 4 through PCR, and verifying the obtained recombinant strains to obtain correct colonies;
(3) construction of recombinant plasmid containing tocopherol cyclase Gene
Obtaining and synthesizing tocopherol cyclase gene sequences from shepherd's purse (CrTC), wasabi (EsTC), cabbage (BoTC), rape (BnTC), orange (CcTC) and dendrobium (DcTC), respectively connecting double enzyme digestion vectors ploxpura3loxp and the tocopherol cyclase gene with Nde I/SpeI as enzyme digestion sites to obtain recombinant plasmids ploxpura3loxp-CrTC, ploxpura3loxp-EsTC, ploxpura3loxp-BoTC, ploxpura3loxp-BnTC, ploxpura3loxp-CcTC and ploxpura3loxp-DcTC, carrying out enzyme digestion linearization on the obtained recombinant plasmids by using SmaI respectively, transferring the plasmids into recombinant strains 1-5 obtained in the step (1) through a yeast transformation kit method to obtain recombinant strains 6, 7, 8, …, 34 and 35, and verifying the correct colonies of the obtained recombinant strains through PCR;
(4) construction of recombinant plasmid containing gamma-tocopherol methyltransferase Gene
Acquiring and synthesizing a gamma-tocopherol methyltransferase gene sequence from shepherd's purse (Cr gamma-TMT), wasabi (Es gamma-TMT), cabbage (Bo gamma-TMT) and rape (Bn gamma-TMT), respectively connecting double enzyme digestion vectors ploxpura3loxp and gamma-tocopherol methyltransferase genes with Nde I/SpeI as enzyme digestion sites to obtain recombinant plasmids ploxpura3loxp-Cr gamma-TMT, ploxpura3loxp-Es gamma-TMT, ploxpura3loxp-Bo gamma-TMT and ploxpura3loxp-Bn gamma-TMT, respectively carrying out enzyme digestion linearization on the obtained recombinant plasmids by using a restriction enzyme SmaI, and transferring the recombinant plasmids into the recombinant strains 6 to 35 in the step (3) by using a yeast transformation kit method to obtain the recombinant lipolysis rhodotorula torula benthamoides with high yield of tocotrienols.
5. The method for constructing recombinant yarrowia lipolytica according to claim 3, comprising the steps of:
(1) construction of yarrowia lipolytica engineering bacterium containing HPPD gene and HPT gene
Obtaining and synthesizing HPPD gene (BrHPPD) from rape and HPT gene (GaHPT) sequence from blue algae, carrying out enzyme digestion connection with a vector ploxpura3loxp to obtain a recombinant plasmid ploxpura3loxp-BrHPPD-GaHPT, carrying out enzyme digestion linearization on the recombinant plasmid, transferring the recombinant plasmid into engineering bacteria polf of yarrowia lipolytica by adopting a yeast transformation kit method to obtain a recombinant strain polf-BHGH, and carrying out colony PCR verification on the obtained recombinant strain to be correct;
(2) construction of recombinant plasmid containing 2-methyl-6-geranylgeranylbenzoquinone methyltransferase Gene
Obtaining and synthesizing a 2-methyl-6-geranylgeranylgeranylbenzoquinone methyltransferase gene sequence from selaginella tamariscina (SmMPBQMT), connecting a double enzyme digestion carrier ploxpura3loxp and the 2-methyl-6-geranylgeranylgeranylbenzoquinone methyltransferase gene with Nde I/Spe I as an enzyme digestion site respectively to obtain a recombinant plasmid ploxpura3loxp-SmMPBQMT, carrying out enzyme digestion linearization on the obtained recombinant plasmid by using a restriction enzyme SmaI, transferring the recombinant plasmid into a yarrowia lipolytica engineering bacterium polf-BHGH by adopting a yeast transformation kit method to obtain a recombinant strain 5, and verifying the correctness of the obtained recombinant strain by colony PCR;
(3) construction of recombinant Strain containing Tocopherol cyclase Gene
Obtaining and synthesizing a tocopherol cyclase gene sequence from dendrobe (DcTC), connecting a double enzyme digestion vector ploxpura3loxp and the tocopherol cyclase gene by taking Nde I/SpeI as an enzyme digestion site, obtaining a recombinant plasmid ploxpura3loxp-DcTC, carrying out enzyme digestion linearization on the obtained recombinant plasmid by using a restriction enzyme SmaI, respectively transferring the recombinant plasmid into the recombinant strain 5 obtained in the step (2) by a yeast transformation kit method, obtaining a recombinant strain 35, and verifying the correctness of the obtained recombinant strain through PCR;
(4) construction of recombinant Strain containing Gamma-Tocopherol methyltransferase Gene
Acquiring and synthesizing a gamma-tocopherol methyltransferase gene sequence from cabbage (Bo gamma-TMT), connecting to obtain a recombinant plasmid ploxpura3loxp-Bo gamma-TMT by taking Nde I/SpeI as enzyme cutting sites and double enzyme cutting carriers ploxpura3loxp and gamma-tocopherol methyltransferase genes respectively, carrying out enzyme cutting linearization on the obtained recombinant plasmid respectively by using a restriction enzyme SmaI, transferring into the recombinant strain 35 in the step (2) by adopting a yeast transformation kit method to obtain a recombinant strain 38, and verifying the accuracy of the obtained recombinant strain by colony PCR;
(5) construction of recombinant Strain overexpressing HMG1 and CrtE genes
Obtaining and synthesizing HMG1 and CrtE gene sequences, designing primers to amplify a target fragment from the genomic DNA of yarrowia lipolytica engineering bacteria po1f, connecting the obtained target fragment to a multienzyme cutting site of a vector pJN44, and respectively obtaining plasmids pJN44-HMG1 and pJN 44-CrtE; the obtained plasmids are respectively transferred into a bacterial strain 38 to obtain a recombinant bacterial strain 40, and the obtained recombinant bacterial strain is verified to be correct through colony PCR;
(6) overexpression of the Gene of interest
Respectively connecting the target genes BrHPPD, GaHPT, SmMPBQMT, DcTC and Bo gamma _ TMT obtained in the previous step to a multi-enzyme cutting site of a vector pJN44 to respectively obtain plasmids pJN44-BrHPPD, pJN44-GaHPT, pJN44-SmMPBQMT, pJN44-DcTC and pJN44-Bo gamma _ TMT; the obtained plasmid is transferred into the recombinant strain 40 in the step (5) after enzyme digestion verification, and the recombinant yarrowia lipolytica 41(polf- (MTT) with high yield of tocotrienol is obtained+HC-ura3), the resulting recombinant strain was verified to be correct by colony PCR.
6. The method of constructing a recombinant yarrowia lipolytica of claim 5, further comprising the step of (7) removing the ura3 marker:
the resulting recombinant yarrowia lipolytica 41(polf- (MTT)+HC-ura3) in SD-LEU medium, wherein the single colony grown on SD-LEU medium is recombinant yarrowia lipolytica polf- (MTT) with high tocotrienol yield+HC。
7. The method according to any one of claims 4 to 6, wherein the colony PCR-verified reaction system is as follows:
reaction system:
PCR procedure:
8. use of the recombinant yarrowia lipolytica of any one of claims 1-4 for the production of tocotrienols.
9. Use of the recombinant yarrowia lipolytica obtained by the construction method according to any one of claims 5 to 8 for the production of tocotrienols.
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| CN114958627A (en) * | 2022-05-05 | 2022-08-30 | 陕西海斯夫生物工程有限公司 | Construction method and application of recombinant schizochytrium limacinum engineering bacterium for high yield of tocopherol |
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