CN111321087A - Yarrowia lipolytica gene engineering bacterium for producing β -carotene and application thereof - Google Patents

Yarrowia lipolytica gene engineering bacterium for producing β -carotene and application thereof Download PDF

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CN111321087A
CN111321087A CN202010107322.1A CN202010107322A CN111321087A CN 111321087 A CN111321087 A CN 111321087A CN 202010107322 A CN202010107322 A CN 202010107322A CN 111321087 A CN111321087 A CN 111321087A
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plasmid
phr
hrgfp
yarrowia lipolytica
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花强
张鑫锎
韦柳静
汪丹妮
陈骏
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East China University of Science and Technology
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Abstract

The invention provides genetically engineered yarrowia lipolytica for producing β -carotene, which is prepared by knocking a carRP gene and a carB gene into chromosomes of uracil and leucine auxotrophic yarrowia lipolytica, knocking GGS1 gene, HMG gene, two copies of carRP gene, 1 copy of carB gene and ERG13 gene, finally, carrying out anaplerosis on two auxotrophic screening markers of uracil and leucine, and finally, continuously feeding and fermenting to ensure that the yield of β -carotene can reach 4.5 g/L.

Description

Yarrowia lipolytica gene engineering bacterium for producing β -carotene and application thereof
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to a yarrowia lipolytica genetic engineering bacterium for producing β -carotene and application thereof.
Background
β -carotene is a natural orange pigment synthesized by plants and microorganisms, and besides playing an important role as a precursor of vitamin A and in photosynthesis, the compound has many physiological effects such as scavenging free radicals, enhancing immune response, and enhancing intercellular junctions, at present, β -carotene has wide applications in the fields of food, aquaculture, cosmetics, and pharmaceuticals, including colorants, food additives, antioxidants, anticancer agents, and prevention of heart diseases.
β the production method of carotene mainly comprises chemical synthesis, natural extraction and microbial fermentation, but the chemical synthesis of β -carotene is complicated and the yield is not high, the extraction of β -carotene from natural sources such as potato, carrot, sea buckthorn and corn is affected by many conditions such as season, climate etc. and the β -carotene content of these plant sources is less, thus resulting in high cost of natural extraction of β -carotene, high energy consumption and low extraction efficiency which hinders the practical use of these methods, therefore the production of β -carotene by microbial fermentation has obvious advantages, especially with the rapid development of biotechnology, the production of β -carotene by microbial fermentation becomes one of the hot spots in the current research.
Currently, β -carotene is generally produced by a biological fermentation method of genes related to synthesis of exogenously introduced β -carotene, compared with saccharomyces cerevisiae, β -carotene production in yarrowia lipolytica is not reported much, but yarrowia lipolytica serving as a lipid yeast can provide a storage space for the carotenoid, has the capability of widely utilizing a substrate and is a well-known safe strain, so that the development of genetic engineering bacteria of yarrowia lipolytica for synthesizing β -carotene is urgently needed.
Disclosure of Invention
The invention can transform yarrowia lipolytica into gene carRP and gene carB to enable the yarrowia lipolytica to produce β -carotene, over express gene GGS1, gene HMG and gene ERG13 on the basis of the gene, and can greatly improve the yield of β -carotene, finally, the uracil and leucine auxotrophy screening marker is complemented to successfully obtain a yarrowia lipolytica production strain producing β -carotene, therefore, the first aim of the invention is to provide a yarrowia lipolytica genetic engineering strain producing β -carotene, the second aim of the invention is to apply the yarrowia lipolytica genetic engineering strain producing β -carotene in the construction of the genetic engineering strain producing carotenoid, and the third aim of the invention is to provide a construction method of the yarrowia lipolytica genetic engineering strain producing β -carotene.
In order to achieve the purpose, the invention provides the following technical scheme:
as a first aspect of the invention, a β -carotene-producing yarrowia lipolytica genetically engineered bacterium XK2 is constructed by knocking a carRP gene and a carB gene into a chromosome of uracil and leucine auxotrophic yarrowia lipolytica, wherein the nucleotide sequence of the carRP gene is shown as SEQ ID NO:55, and the nucleotide sequence of the carB gene is shown as SEQ ID NO: 56.
According to the invention, the two sets of plasmid pairs in which the carRP gene and the carB gene are knocked in are pHR _ XPR2_ carRP and pCRISPRyl _ XPR2 and pHR _ D17_ carB and pCRISPRyl _ D17, respectively.
A gene engineering bacterium XK3 of yarrowia lipolytica for producing β -carotene is obtained by knocking a carRP gene and a carB gene into a chromosome of uracil and leucine auxotrophic yarrowia lipolytica to construct and obtain the genetic engineering bacterium XK2 of yarrowia lipolytica and knocking in a GGS1 gene.
According to the invention, the yarrowia lipolytica genetically engineered bacterium XK3 is constructed by transforming yarrowia lipolytica genetically engineered bacterium XK2 with plasmids pHR _ A08_ GGS1 and pCRISPRIyl _ A08.
A gene engineering bacterium XK4 of yarrowia lipolytica for producing β -carotene is constructed by knocking the carRP gene and carB gene into the chromosome of uracil and leucine auxotrophic yarrowia lipolytica, and knocking the GGS1 gene and HMG gene into the chromosome.
According to the invention, the yarrowia lipolytica genetically engineered bacterium XK4 is constructed by transforming plasmids pHR _ AXP _ HMG and pCRISPRIyl _ AXP into yarrowia lipolytica genetically engineered bacterium XK 3.
A gene engineering bacterium XK5 of yarrowia lipolytica for producing β -carotene is constructed by knocking the carRP gene and carB gene into the chromosome of uracil and leucine auxotrophic yarrowia lipolytica, and knocking GGS1 gene, HMG gene and 1 copy of carB gene.
According to the invention, the yarrowia lipolytica genetically engineered bacterium XK5 is constructed by sequentially transforming plasmids pHR _ POX2_ carB and pCRISPRyl _ POX2 into yarrowia lipolytica genetically engineered bacterium XK 4.
A gene engineering bacterium XK6 of yarrowia lipolytica for producing β -carotene is constructed by knocking the carRP gene and carB gene into the chromosome of uracil and leucine auxotrophic yarrowia lipolytica, and knocking GGS1 gene, HMG gene, 1 copy of carRP gene and 1 copy of carB gene.
According to the invention, the yarrowia lipolytica genetically engineered bacterium XK6 is constructed by sequentially transferring plasmids pHR _ MFE1_ carRP and pCRISPRyl _ MFE1 into yarrowia lipolytica genetically engineered bacterium XK 5.
A gene engineering bacterium XK7 of yarrowia lipolytica for producing β -carotene is constructed by knocking the carRP gene and carB gene into the chromosome of uracil and leucine auxotrophic yarrowia lipolytica, and knocking GGS1 gene, HMG gene, two copies of carRP gene and 1 copy of carB gene.
According to the invention, the yarrowia lipolytica genetically engineered bacterium XK7 is constructed by sequentially transferring pHR _ POX3_ carRP and pCRISPRyl _ POX3 into yarrowia lipolytica genetically engineered bacterium XK 6.
A genetically engineered yarrowia lipolytica strain XK17 capable of producing β -carotene is constructed by knocking the carRP gene and carB gene into the chromosome of uracil and leucine auxotrophic yarrowia lipolytica, and knocking GGS1 gene, HMG gene, two copies of carRP gene, 1 copy of carB gene and ERG13 gene.
According to the invention, the yarrowia lipolytica genetically engineered bacterium XK17 is constructed by transferring plasmids pHR _ LIP1_ ERG13 and pCRISPRyl _ LIP1 into yarrowia lipolytica genetically engineered bacterium XK 7.
A genetically engineered yarrowia lipolytica strain XK19 capable of producing β -carotene is prepared by knocking a carRP gene and a carB gene into chromosomes of uracil and leucine auxotrophic yarrowia lipolytica, knocking GGS1 gene, HMG gene, two copies of the carRP gene, 1 copy of the carB gene and ERG13 gene into the chromosomes, and finally complementing two auxotrophic screening markers of uracil and leucine into the chromosomes.
According to the invention, the yarrowia lipolytica genetically engineered bacterium XK19 is characterized in that a linearized pINA1269 plasmid is transferred into a yarrowia lipolytica genetically engineered bacterium XK17 to supplement a leucine screening marker; then the linearized pINA1312 plasmid is transferred into yarrowia lipolytica genetic engineering bacteria XK17 supplemented with leucine selection markers for supplementing uracil selection markers, and the strain is constructed.
A genetically engineered yarrowia lipolytica strain XK10 capable of producing β -carotene is constructed by knocking the carRP gene and carB gene into the chromosome of uracil and leucine auxotrophic yarrowia lipolytica, and knocking GGS1 gene, HMG gene, 1 copy of carRP gene, 1 copy of carB gene and ERG13 gene.
According to the invention, the yarrowia lipolytica genetically engineered bacterium XK10 is constructed by transferring plasmids pHR _ LIP1_ ERG13 and pCRISPRyl _ LIP1 into yarrowia lipolytica genetically engineered bacterium XK 6.
A genetically engineered yarrowia lipolytica strain XK11 capable of producing β -carotene is constructed by knocking the carRP gene and carB gene into the chromosome of uracil and leucine auxotrophic yarrowia lipolytica, and knocking GGS1 gene, HMG gene, 1 copy of carRP gene, 1 copy of carB gene and ERG10-11099 gene into the chromosome.
According to the invention, the yarrowia lipolytica genetically engineered bacterium XK11 is constructed by transferring pHR _ E1_ ERG10-11099 and pCRISPRIyl _ E1 into yarrowia lipolytica genetically engineered bacterium XK 6.
A genetically engineered yarrowia lipolytica strain XK12 capable of producing β -carotene is constructed by knocking the carRP gene and carB gene into the chromosome of uracil and leucine auxotrophic yarrowia lipolytica, and knocking GGS1 gene, HMG gene, 1 copy of carRP gene, 1 copy of carB gene and ERG10-08536 gene into the chromosome.
According to the invention, the yarrowia lipolytica genetically engineered bacterium XK12 is constructed by transferring pHR _ A1_ ERG10-08536 and pCRISPRIyl _ A1 into yarrowia lipolytica genetically engineered bacterium XK 6.
A genetically engineered yarrowia lipolytica strain XK13 capable of producing β -carotene is constructed by knocking the carRP gene and carB gene into the chromosome of uracil and leucine auxotrophic yarrowia lipolytica, and knocking GGS1 gene, HMG gene, 1 copy of carRP gene, 1 copy of carB gene and ERG8 gene.
According to the invention, the yarrowia lipolytica genetically engineered bacterium XK13 is constructed by transferring pHR _ B1_ ERG8 and pCRISPRIyl _ B1 into yarrowia lipolytica genetically engineered bacterium XK 6.
A genetically engineered yarrowia lipolytica strain XK14 capable of producing β -carotene is constructed by knocking the carRP gene and carB gene into the chromosome of uracil and leucine auxotrophic yarrowia lipolytica, and knocking GGS1 gene, HMG gene, 1 copy of carRP gene, 1 copy of carB gene and ERG19 gene.
According to the invention, the yarrowia lipolytica genetically engineered bacterium XK14 is constructed by transferring pHR _ A2_ ERG19 and pCRISPRIyl _ A2 into yarrowia lipolytica genetically engineered bacterium XK 6.
The β -carotene-producing yarrowia lipolytica gene engineering bacterium XK15 is constructed by knocking the carRP gene and carB gene into the chromosome of uracil and leucine auxotrophic yarrowia lipolytica, and knocking GGS1 gene, HMG gene, 1 copy of carRP gene, 1 copy of carB gene and IDI gene into the chromosome.
According to the invention, the yarrowia lipolytica genetically engineered bacterium XK15 is constructed by transferring pHR _ POX6_ IDI and pCRISPRIyl _ POX6 into yarrowia lipolytica genetically engineered bacterium XK 6.
A genetically engineered yarrowia lipolytica strain XK16 capable of producing β -carotene is constructed by knocking the carRP gene and carB gene into the chromosome of uracil and leucine auxotrophic yarrowia lipolytica, and knocking GGS1 gene, HMG gene, 1 copy of carRP gene, 1 copy of carB gene and ERG12 gene.
According to the invention, the yarrowia lipolytica genetically engineered bacterium XK16 is constructed by transferring pHR _ E2_ ERG12 and pCRISPRIyl _ E2 into yarrowia lipolytica genetically engineered bacterium XK 6.
A gene engineering bacterium XK8 of yarrowia lipolytica for producing β -carotene is constructed by knocking the carRP gene and carB gene into the chromosome of uracil and leucine auxotrophic yarrowia lipolytica, and knocking GGS1 gene, HMG gene, 1 copy of carRP gene and 2 copies of carB gene.
According to the invention, the yarrowia lipolytica genetically engineered bacterium XK8 is constructed by transferring pHR _ POX4_ carB and pCRISPRyl _ POX4 into yarrowia lipolytica genetically engineered bacterium XK 6.
According to the invention, the accession number of the GGS1 gene in NCBI is GB: NC-006070, the accession number of the HMG gene in NCBI is GB: NC-006071, the accession number of the ERG13 gene in NCBI is GB: NC-006072.1; the accession numbers of the ERG10 gene in NCBI are NC _006071.1(ERG10-11099) and NC _006068.1(ERG10-08536) respectively; the ERG8 gene has the accession number GB: NC-006071.1 in NCBI; the ERG19 gene has the accession number GB: NC-006072.1 in NCBI; the accession number of the IDI gene in NCBI is GB: NC-006072.1; the ERG12 gene has the accession number GB: NC-006068.1 in NCBI.
According to the invention, the uracil and leucine auxotrophic yarrowia lipolytica is yarrowia lipolytica Po1 f; the carRP gene, the carB gene, the GGS1 gene, the HMG gene, the ERG13 gene, the ERG10-11099 gene, the ERG10-08536 gene, the ERG8 gene, the ERG19 gene, the IDI gene and the ERG12 gene are knocked into yarrowia lipolytica Po1f by adopting a CRISPR/Cas9 operating system.
As a second aspect of the present invention, a knock-in plasmid pair of genetically engineered yarrowia lipolytica for gene knock-in, said knock-in plasmid pair being a pair of CRISPR/Cas 9-based plasmids for gene knock-in yarrowia lipolytica, said pair of knock-in plasmids being pair of pcr _ POX2_ hrGFP and pcrisryl _ POX2, pcr _ POX3_ hrGFP and pcrisryl _ POX3, pcr _ LIP1_ hrGFP and pcrisryl _ LIP1, pcr _ POX4_ hrGFP and pcrisryl _ POX4, pcr _ E1_ hrGFP and pcrisryl _ E1, pcr _ a1_ hrryl gfp and pcrisryl _ a1, pair of pcr _ B6 _ phhrryl _ B1, pcr _ a2 and pcrisryl _ p 4684, pcrisryl _ gfp and pcrisryl _ p 2, pcrisryl _ gfp 4643;
(1) plasmid pair pHR _ POX2_ hrGFP and pCRISPRyl _ POX 2:
the plasmid pair pHR _ POX2_ hrGFP and pCRISPRIyl _ POX2 can be used for knocking-in of genes, such as carB gene;
the pCRISPRyl _ POX2 is obtained by linearizing a plasmid pCRISPRyl by using an AvrII restriction enzyme and seamlessly cloning and connecting the plasmid pCRISPRyl with a sgRNA fragment designed aiming at a POX2 site, wherein the nucleotide sequence of the sgRNA fragment designed aiming at a POX2 site is shown as SEQ ID NO:1 is shown in the specification;
the pHR _ POX2_ hrGFP is obtained by taking a genome of yarrowia lipolytica as a template, respectively amplifying 1200bp at both ends of a sgRNA sequence at a POX2 site by PCR (polymerase chain reaction) by using a primer pair POX2-LF/POX2-LR and POX2-RF/POX2-RR as a left homologous arm and a right homologous arm, taking a plasmid pHR _ AXP _ hrGFP as a template, and amplifying plas-F and plas-R by PCR by using a primer pair to obtain a plasmid basic skeleton, and seamlessly cloning and connecting the three fragments to obtain a plasmid pHR _ POX 2; connecting the hrGFP gene expression cassette obtained by double digestion of the plasmid pHR _ AXP _ hrGFP by using the restriction enzymes SpeI and AvrII with the plasmid pHR _ POX2 which is also subjected to double digestion by using the restriction enzymes SpeI and AvrII to obtain pHR _ POX2_ hrGFP; wherein the primer sequences of the primers POX2-LF, POX2-LR, POX2-RF and POX2-RR are respectively shown as SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13 and SEQ ID NO: 14 is shown in the figure; the sequences of the primers of the plas-F and plas-Rd are respectively shown as SEQ ID NO: 53 and SEQ ID NO: 54 is shown;
(2) plasmid pair pHR _ POX3_ hrGFP and pCRISPRyl _ POX 3:
the plasmid pair pHR _ POX3_ hrGFP and pCRISPRIyl _ POX3 can be used for knocking-in of genes, such as the carRP gene;
the pCRISPRyl _ POX3 is obtained by performing single-enzyme linearization on a plasmid pCRISPRyl by using an AvrII restriction enzyme and performing seamless cloning and connection with a sgRNA fragment designed aiming at a POX3 site, wherein the nucleotide sequence of the sgRNA fragment designed aiming at a POX3 site is shown as SEQ ID NO: 2 is shown in the specification;
the pHR _ POX3_ hrGFP is constructed by taking the genome of yarrowia lipolytica as a template, respectively amplifying 1200bp at both ends of a sgRNA sequence at a POX3 site by PCR (polymerase chain reaction) by using a primer pair POX3-LF/POX3-LR and POX3-RF/POX3-RR as left and right homologous arms, taking a plasmid pHR _ AXP _ hrGFP as a template, amplifying by PCR by using a primer pair plas-F and plas-R to obtain a plasmid basic skeleton, and seamlessly cloning and connecting the three fragments to obtain a plasmid pHR _ POX 3; connecting the hrGFP gene expression cassette obtained by double digestion of the plasmid pHR _ AXP _ hrGFP by using the restriction enzymes SpeI and AvrII with the plasmid pHR _ POX3 which is also subjected to double digestion by using the restriction enzymes SpeI and AvrII to obtain pHR _ POX3_ hrGFP; wherein the primer sequences of the primers POX3-LF, POX3-LR, POX3-RF and POX3-RR are respectively shown as SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17 and SEQ ID NO: 18 is shown in the figure;
(3) pHR _ LIP1_ hrGFP and pCRISPRIyl _ LIP 1:
the plasmid pair pHR _ LIP1_ hrGFP and pCRISRYL _ LIP1 can be used for knocking-in of genes, such as ERG 13;
the pCRISPRyl _ LIP1 is obtained by using AvrII restriction endonuclease to perform single-enzyme linearization on plasmid pCRISPRyl and seamlessly cloning and connecting the plasmid pCRISPRyl with sgRNA fragment designed aiming at LIP1 site, wherein the nucleotide sequence of the sgRNA fragment designed aiming at LIP1 site is shown as SEQ ID NO:5 is shown in the specification;
the pHR _ LIP1_ hrGFP is a plasmid pHR _ LIP1 obtained by using a genome of yarrowia lipolytica as a template, respectively amplifying 1200bp at both ends of a sgRNA sequence at a LIP1 site by PCR (polymerase chain reaction) by using primer pairs LIP1-LF/LIP1-LR and LIP1-RF/LIP1-RR, and obtaining a plasmid basic skeleton by using a plasmid pHR _ AXP _ hrGFP as a template and performing PCR (polymerase chain reaction) amplification by using primer pairs plas-F and plas-R; the hrGFP gene expression cassette obtained by double digestion of the plasmid pHR _ AXP _ hrGFP with the restriction enzymes SpeI and AvrII was ligated to the plasmid pHR _ LIP1 similarly double digested with the restriction enzymes SpeI and AvrII to obtain pHR _ LIP1_ hrGFP; wherein the primer sequences of the primers LIP1-LF, LIP1-LR, LIP1-RF and LIP1-RR are respectively shown as SEQ ID NO: 27. SEQ ID NO: 28. SEQ ID NO: 29 and SEQ ID NO: 30 is shown in the figure;
(4) pHR _ POX4_ hrGFP and pCRISPRIyl _ POX 4:
the plasmid pair pHR _ POX4_ hrGFP and pCRISPRIyl _ POX4 can be used for knocking-in of genes, such as carB gene;
the pCRISPRyl _ POX4 is obtained by using AvrII restriction endonuclease to perform single-enzyme linearization on a plasmid pCRISPRyl, and seamlessly cloning and connecting the plasmid pCRISPRyl with a sgRNA fragment designed aiming at a POX4 site, wherein the nucleotide sequence of the sgRNA fragment designed aiming at a POX4 site is shown as SEQ ID NO: 3 is shown in the specification;
the pHR _ POX4_ hrGFP is obtained by taking a genome of yarrowia lipolytica as a template, respectively amplifying 1200bp at both ends of a sgRNA sequence at a POX4 site by PCR (polymerase chain reaction) by using a primer pair POX4-LF/POX4-LR and POX4-RF/POX4-RR as a left homologous arm and a right homologous arm, taking a plasmid pHR _ AXP _ hrGFP as a template, and amplifying plas-F and plas-R by PCR by using a primer pair to obtain a plasmid basic skeleton, and seamlessly cloning and connecting the three fragments to obtain a plasmid pHR _ POX 4; connecting the hrGFP gene expression cassette obtained by double digestion of the plasmid pHR _ AXP _ hrGFP by using the restriction enzymes SpeI and AvrII with the plasmid pHR _ POX4 which is also subjected to double digestion by using the restriction enzymes SpeI and AvrII to obtain pHR _ POX4_ hrGFP; wherein the primer sequences of the primers POX4-LF, POX4-LR, POX4-RF and POX4-RR are respectively shown as SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21 and SEQ ID NO: 22;
(5) pHR _ E1_ hrGFP and pCRISPRIyl _ E1:
the plasmid pair pHR _ E1_ hrGFP and pCRISPRIyl _ E1 can be used for knocking-in of genes, such as ERG 10-11099;
the pCRISPRyl _ E1 is constructed by using AvrII restriction endonuclease to perform single-enzyme linearization on plasmid pCRISPRyl and seamlessly cloning and connecting the plasmid pCRISPRyl with a sgRNA fragment designed aiming at the E1 site, wherein the nucleotide sequence of the sgRNA fragment designed aiming at the E1 site is shown as SEQ ID NO: 6 is shown in the specification;
the pHR _ E1_ hrGFP is obtained by taking the genome of yarrowia lipolytica as a template, respectively amplifying 1200bp at both ends of an sgRNA sequence of an E1 site by PCR (polymerase chain reaction) by using primer pairs E1-LF/E1-LR and E1-RF/E1-RR as a left homologous arm and a right homologous arm, taking a plasmid pHR _ AXP _ hrGFP as a template, amplifying plas-F and plas-R by PCR by using the primer pairs to obtain a basic plasmid skeleton, and seamlessly cloning and connecting the three fragments to obtain a plasmid pHR _ E1; the hrGFP gene expression cassette obtained by double digestion of the plasmid pHR _ AXP _ hrGFP with the restriction enzymes SpeI and AvrII was ligated to the plasmid pHR _ E1 similarly subjected to double digestion with the restriction enzymes SpeI and AvrII to obtain pHR _ E1_ hrGFP; wherein the primer sequences of the primers E1-LF, E1-LR, E1-RF and E1-RR are respectively shown in SEQ ID NO: 31. SEQ ID NO: 32. SEQ ID NO: 33 and SEQ ID NO: 34;
(6) pHR _ A1_ hrGFP and pCRISPRIyl _ A1:
the plasmid pair pHR _ A1_ hrGFP and pCRISPRIyl _ A1 can be used for knocking-in of genes, such as ERG10-08536 gene;
the pCRISPRyl _ A1 is obtained by using AvrII restriction endonuclease to perform single-enzyme linearization on plasmid pCRISPRyl, and seamlessly cloning and connecting the plasmid pCRISPRyl with a sgRNA fragment designed aiming at A1 site, wherein the nucleotide sequence of the sgRNA fragment designed aiming at A1 site is shown in SEQ ID NO: 7 is shown in the specification;
pHR _ A1_ hrGFP is a plasmid pHR _ A1 obtained by taking the genome of yarrowia lipolytica as a template, respectively amplifying 1200bp at both ends of an sgRNA sequence at an A1 site by PCR (polymerase chain reaction) by using primer pairs A1-LF/A1-LR and A1-RF/A1-RR, and taking a plasmid pHR _ AXP _ hrGFP as a template, amplifying by PCR by using the primer pairs plas-F and plas-R to obtain a plasmid basic skeleton, and seamlessly cloning and connecting the three fragments; the hrGFP gene expression cassette obtained by double digestion of the plasmid pHR _ AXP _ hrGFP with the restriction enzymes SpeI and AvrII was ligated to the plasmid pHR _ A1 similarly subjected to double digestion with the restriction enzymes SpeI and AvrII to obtain pHR _ A1_ hrGFP; wherein the primer sequences of the primers A1-LF, A1-LR, A1-RF and A1-RR are respectively shown in SEQ ID NO: 35. SEQ ID NO: 36. SEQ ID NO: 37 and SEQ ID NO: 38;
(7) pHR _ B1_ hrGFP and pCRISPRIyl _ B1:
the plasmid pair pHR _ B1_ hrGFP and pCRISPRIyl _ B1 can be used for knocking-in of genes, such as ERG 8;
the pCRISPRyl _ B1 is obtained by using AvrII restriction endonuclease to perform single-enzyme linearization on plasmid pCRISPRyl, and seamlessly cloning and connecting the plasmid pCRISPRyl with a sgRNA fragment designed aiming at the B1 site, wherein the nucleotide sequence of the sgRNA fragment designed aiming at the B1 site is shown as SEQ ID NO: 8 is shown in the specification;
the pHR _ B1_ hrGFP is a plasmid pHR _ B1 obtained by using a genome of yarrowia lipolytica as a template, respectively amplifying 1200bp at both ends of a sgRNA sequence at a site B1 as left and right homologous arms by using a primer pair B1-LF/B1-LR, B1-RF1/B1-RR1 and B1-RF2/B1-RR2 through PCR, using a plasmid pHR _ AXP _ hrGFP as a template, and obtaining a plasmid basic skeleton by using a primer pair plas-F and plas-R through PCR amplification, wherein the four fragments are subjected to seamless cloning connection to obtain the plasmid pHR _ B1; the hrGFP gene expression cassette obtained by double digestion of the plasmid pHR _ AXP _ hrGFP with the restriction enzymes SpeI and AvrII was ligated to the plasmid pHR _ B1 similarly subjected to double digestion with the restriction enzymes SpeI and AvrII to obtain pHR _ B1_ hrGFP; wherein, the primer sequences of the primers B1-LF, B1-LR, B1-RF1, B1-RR1, B1-RF2 and B1-RR2 are respectively shown in SEQ ID NO: 39. SEQ ID NO: 40. SEQ ID NO: 41. SEQ ID NO: 42. SEQ ID NO: 43 and SEQ ID NO: 44 is shown;
(8) pHR _ A2_ hrGFP and pCRISPRIyl _ A2:
the plasmid pair pHR _ A2_ hrGFP and pCRISPRIyl _ A2 can be used for knocking-in of genes, such as ERG 19;
the pCRISPRyl _ A2 is obtained by using AvrII restriction endonuclease to perform single-enzyme linearization on plasmid pCRISPRyl, and seamlessly cloning and connecting the plasmid pCRISPRyl with a sgRNA fragment designed aiming at A2 site, wherein the nucleotide sequence of the sgRNA fragment designed aiming at A2 site is shown in SEQ ID NO: 9 is shown in the figure;
the pHR _ A2_ hrGFP is obtained by taking the genome of yarrowia lipolytica as a template, respectively amplifying 1200bp at both ends of an A2 site sgRNA sequence by using primer pairs A2-LF/A2-LR and A2-RF/A2-RR through PCR as left and right homologous arms, taking plasmid pHR _ AXP _ hrGFP as a template, obtaining a plasmid basic skeleton by using primer pairs plas-F and plas-R through PCR amplification, and obtaining a plasmid pHR _ A2 by seamlessly cloning and connecting the three fragments; the hrGFP gene expression cassette obtained by double digestion of the plasmid pHR _ AXP _ hrGFP with the restriction enzymes SpeI and AvrII was ligated to the plasmid pHR _ A2 similarly subjected to double digestion with the restriction enzymes SpeI and AvrII to obtain pHR _ A2_ hrGFP; wherein the primer sequences of the primers A2-LF, A2-LR, A2-RF and A2-RR are respectively shown in SEQ ID NO: 45. SEQ ID NO: 46. SEQ ID NO: 47 and SEQ ID NO: 48 is shown;
(9) pHR _ POX6_ hrGFP and pCRISPRIyl _ POX 6:
the plasmid pair pHR _ POX6_ hrGFP and pCRISPRIyl _ POX6 can be used for knocking-in of genes, such as IDI gene;
the pCRISPRyl _ POX6 is obtained by using AvrII restriction endonuclease to perform single-enzyme linearization on a plasmid pCRISPRyl, and seamlessly cloning and connecting the plasmid pCRISPRyl with a sgRNA fragment designed aiming at a POX6 site, wherein the nucleotide sequence of the sgRNA fragment designed aiming at a POX6 site is shown as SEQ ID NO: 4 is shown in the specification;
the pHR _ POX6_ hrGFP is obtained by taking a genome of yarrowia lipolytica as a template, respectively amplifying 1200bp at both ends of a sgRNA sequence at a POX6 site by PCR (polymerase chain reaction) by using a primer pair POX6-LF/POX6-LR and POX6-RF/POX6-RR as a left homologous arm and a right homologous arm, taking a plasmid pHR _ AXP _ hrGFP as a template, and amplifying plas-F and plas-R by PCR by using a primer pair to obtain a plasmid basic skeleton, and seamlessly cloning and connecting the three fragments to obtain a plasmid pHR _ POX 6; connecting the hrGFP gene expression cassette obtained by double digestion of the plasmid pHR _ AXP _ hrGFP by using the restriction enzymes SpeI and AvrII with the plasmid pHR _ POX6 which is also subjected to double digestion by using the restriction enzymes SpeI and AvrII to obtain pHR _ POX6_ hrGFP; wherein the primer sequences of the primers POX6-LF, POX6-LR, POX6-RF and POX6-RR are respectively shown as SEQ ID NO: 23. SEQ ID NO: 24. SEQ ID NO: 25 and SEQ ID NO: 26 is shown;
(10) pHR _ E2_ hrGFP and pCRISPRIyl _ E2:
the plasmid pair pHR _ E2_ hrGFP and pCRISPRIyl _ E2 can be used for knocking-in of genes, such as ERG 12;
the pCRISPRyl _ E2 is obtained by using AvrII restriction endonuclease to perform single-enzyme linearization on plasmid pCRISPRyl, and seamlessly cloning and connecting the plasmid pCRISPRyl with a sgRNA fragment designed aiming at the E2 site, wherein the nucleotide sequence of the sgRNA fragment designed aiming at the E2 site is shown as SEQ ID NO:10 is shown in the figure;
the pHR _ E2_ hrGFP is obtained by taking the genome of yarrowia lipolytica as a template, respectively amplifying 1200bp at both ends of an sgRNA sequence of an E2 site by PCR (polymerase chain reaction) by using primer pairs E2-LF/E2-LR and E2-RF/E2-RR as a left homologous arm and a right homologous arm, taking a plasmid pHR _ AXP _ hrGFP as a template, amplifying plas-F and plas-R by PCR by using the primer pairs to obtain a basic plasmid skeleton, and seamlessly cloning and connecting the three fragments to obtain a plasmid pHR _ E2; the hrGFP gene expression cassette obtained by double digestion of the plasmid pHR _ AXP _ hrGFP with the restriction enzymes SpeI and AvrII was ligated to the plasmid pHR _ E2 similarly subjected to double digestion with the restriction enzymes SpeI and AvrII to obtain pHR _ E2_ hrGFP; wherein the primer sequences of the primers E2-LF, E2-LR, E2-RF and E2-RR are respectively shown in SEQ ID NO: 49. SEQ ID NO: 50. SEQ ID NO: 51 and SEQ ID NO: shown at 52.
According to the invention, the knock-in plasmid pair is constructed based on CRISPR/Cas9 operating system, and plasmids pHR _ POX2_ hrGFP and pCRISPRyl _ POX2 are subjected to gene knock-in at POX2 site to construct the knock-in plasmid pair; plasmids pHR _ POX3_ hrGFP and pCRISPRIyl _ POX3 are knock-in plasmid pairs constructed by gene knock-in at the POX3 site; plasmids pHR _ POX4_ hrGFP and pCRISPRIyl _ POX4 are knock-in plasmid pairs constructed by gene knock-in at the POX4 site; plasmids pHR _ POX6_ hrGFP and pCRISPRIyl _ POX6 are knock-in plasmid pairs constructed by gene knock-in at the POX6 site; plasmids pHR _ LIP1_ hrGFP and pCRISRyl _ LIP1 are knock-in plasmid pairs constructed by gene knock-in at the LIP1 site; plasmids pHR _ E1_ hrGFP and pCRISPRyl _ E1 are knock-in plasmid pairs constructed by gene knock-in at the E1 site; plasmids pHR _ a1_ hrGFP and pCRISPRyl _ a1 are knock-in plasmid pairs constructed by gene knock-in at the a1 site; plasmids pHR _ B1_ hrGFP and pCRISPRyl _ B1 are knock-in plasmid pairs constructed by gene knock-in at the B1 site; plasmids pHR _ a2_ hrGFP and pCRISPRyl _ a2 are knock-in plasmid pairs constructed by gene knock-in at the a2 site; plasmids pHR _ E2_ hrGFP and pCRISPRIyl _ E2 are knock-in plasmid pairs constructed by gene knock-in at the E2 site.
As a third aspect of the invention, a construction method of β -carotene-producing yarrowia lipolytica gene engineering bacteria XK19 comprises the following steps:
(1) based on the existing CRISPR/Cas9 operating system, constructing a knock-in plasmid pair containing a carRP gene and constructing a knock-in plasmid pair containing a carB gene, wherein the knock-in plasmid pair of the carRP gene is pHR _ XPR2_ carRP and pCRISPRyl _ XPR2, and the knock-in plasmid pair of the carB gene is pHR _ D17_ carB and pCRISPRyl _ D17;
(2) transforming the plasmid pHR _ XPR2_ carRP and the plasmid pCRISPRIyl _ XPR2 obtained in the step (1) into uracil and leucine auxotrophic yarrowia lipolytica to obtain yarrowia lipolytica XK1, and then simultaneously transforming the plasmid pHR _ D17_ carB and the plasmid pCRISPRIyl _ D17 obtained in the step (1) into yarrowia lipolytica XK1 to obtain β -carotene producing yarrowia lipolytica XK 2;
(3) constructing plasmid pair pHR _ A08_ GGS1 and pCRISPRyl _ A08 and pHR _ AXP _ HMG and pCRISPRyl _ AXP, simultaneously transforming the plasmid pair pHR _ A08_ GGS1 and pCRISPRyl _ A08 into the yarrowia lipolytica XK2 obtained in step (2) to obtain XK3 of yarrowia lipolytica producing β -carotene, simultaneously transforming the plasmid pair pHR _ AXP _ HMG and pCRISPRyl _ AXP into XK3 to obtain XK4 of yarrowia lipolytica producing β -carotene;
(4) respectively constructing plasmid pair pHR _ POX2_ carB and pCRISPRIyl _ POX2, pHR _ MFE1_ carRP and pCRISPRIyl _ MFE1, and transforming the plasmid pair pHR _ POX2_ carB and pCRISPRIyl _ POX2 into the yarrowia lipolytica XK4 obtained in the step (3) to obtain XK5 of the yarrowia lipolytica producing β -carotene, transforming the plasmid pair pHR _ MFE1_ carRP and pCRISPRIyl _ MFE1 into XK5 of the yarrowia lipolytica to obtain XK6 of the yarrowia lipolytica producing β -carotene;
(5) respectively constructing plasmid pairs pHR _ POX3_ carRP and pCRISPRyl _ POX3, and sequentially transforming into the yarrowia lipolytica XK6 obtained in the step (4) to obtain XK7 of the yarrowia lipolytica producing β -carotene;
(6) constructing a plasmid pair pHR _ LIP1_ ERG13 and pCRISPRIyl _ LIP1, and transferring into the yarrowia lipolytica XK7 of the step (5), so as to obtain the yarrowia lipolytica XK17 producing β -carotene;
(7) the plasmid pINA1269 is linearized by double digestion using the PmlI and BamHI enzyme cleavage sites, the linearized pINA1269 plasmid is transferred to strain XK17 to complement the leucine selection marker, and then the linearized pINA1312 plasmid is linearized by double digestion using the PmlI and BamHI enzyme cleavage sites, and the linearized pINA1312 plasmid is transferred to XK17 strain supplemented with the leucine selection marker to complement the uracil selection marker, obtaining the β -carotene producing yarrowia lipolytica XK 19.
According to the invention, the plasmid pHR _ XPR2_ carRP in the step (1) is constructed by the following steps: the carRP gene is connected to the plasmid pHR _ XPR2_ hrGFP through the restriction enzyme cutting sites BssHII and NheI to obtain a plasmid pHR _ XPR2_ carRP;
the plasmid pHR _ D17_ carB in the step (1) is constructed by the following steps: the carB gene is connected to a plasmid pHR _ D17_ hrGFP through a restriction enzyme site BssHII and NheI to obtain a plasmid pHR _ D17_ carB;
the plasmid pHR _ AXP _ HMG obtained in the step (3) is constructed by the following steps: the HMG gene is connected to a plasmid pHR _ AXP _ hrGFP through enzyme cutting sites BssHII and NheI to obtain a plasmid pHR _ AXP _ HMG;
the plasmid pHR _ A08_ GGS1 in the step (3) is constructed by the following steps: the GGS1 gene is connected to a plasmid pHR _ A08_ hrGFP through enzyme cutting sites BssHII and NheI to obtain a plasmid pHR _ A08_ GGS 1;
the construction of the plasmid pHR _ MFE1_ carRP of step (4) comprises the following steps: the carRP gene is connected to a plasmid pHR _ MFE1_ hrGFP through enzyme cutting sites BssHII and NheI to obtain a plasmid pHR _ MFE1_ carRP;
the plasmid pHR _ POX2_ carB in the step (4) is constructed by the following steps: the carB gene is connected to a plasmid pHR _ POX2_ hrGFP through a restriction enzyme site BssHII and NheI to obtain a plasmid pHR _ POX2_ carB;
the construction steps of the plasmid pHR _ POX3_ carRP in the step (5) are as follows: the carRP gene is connected to the plasmid pHR _ POX3_ hrGFP through the restriction enzyme cutting sites BssHII and NheI to obtain a plasmid pHR _ POX3_ carRP;
the plasmid pHR _ LIP1_ ERG13 of the step (6) is constructed by the following steps: the ERG13 gene from yarrowia lipolytica was ligated to plasmid pHR _ LIP1_ hrGFP via cleavage sites BssHII and NheI to give plasmid pHR _ LIP1_ ERG 13.
As a fourth aspect of the invention, a construction method of a yarrowia lipolytica gene engineering bacterium capable of producing β -carotene comprises the following steps:
(1) based on the existing CRISPR/Cas9 operating system, constructing a knock-in plasmid pair containing a carRP gene and constructing a knock-in plasmid pair containing a carB gene, wherein the knock-in plasmid pair of the carRP gene is pHR _ XPR2_ carRP and pCRISPRyl _ XPR2, and the knock-in plasmid pair of the carB gene is pHR _ D17_ carB and pCRISPRyl _ D17;
(2) transforming the plasmid pHR _ XPR2_ carRP and the plasmid pCRISPRIyl _ XPR2 obtained in the step (1) into uracil and leucine auxotrophic yarrowia lipolytica to obtain yarrowia lipolytica XK1, and then simultaneously transforming the plasmid pHR _ D17_ carB and the plasmid pCRISPRIyl _ D17 obtained in the step (1) into yarrowia lipolytica XK1 to obtain β -carotene producing yarrowia lipolytica XK 2;
(3) constructing plasmid pair pHR _ A08_ GGS1 and pCRISPRyl _ A08 and pHR _ AXP _ HMG and pCRISPRyl _ AXP, simultaneously transforming the plasmid pair pHR _ A08_ GGS1 and pCRISPRyl _ A08 into the yarrowia lipolytica XK2 obtained in step (2) to obtain XK3 of yarrowia lipolytica producing β -carotene, simultaneously transforming the plasmid pair pHR _ AXP _ HMG and pCRISPRyl _ AXP into XK3 to obtain XK4 of yarrowia lipolytica producing β -carotene;
(4) respectively constructing plasmid pair pHR _ POX2_ carB and pCRISPRIyl _ POX2, pHR _ MFE1_ carRP and pCRISPRIyl _ MFE1, and transforming the plasmid pair pHR _ POX2_ carB and pCRISPRIyl _ POX2 into the yarrowia lipolytica XK4 obtained in the step (3) to obtain XK5 of the yarrowia lipolytica producing β -carotene, transforming the plasmid pair pHR _ MFE1_ carRP and pCRISPRIyl _ MFE1 into XK5 of the yarrowia lipolytica to obtain XK6 of the yarrowia lipolytica producing β -carotene;
(5) plasmid pairs pHR _ LIP1_ ERG13 and pCRISPRIyl _ LIP1, pHR _ E1_ ERG10-11099 and pCRISPRIyl _ E1, pHR _ A1_ ERG 1-1 and pCRISPRIyl _ A1, pHR _ B1_ ERG1 and pCRISPRIyl _ B1, pHR _ A1_ ERG1 and pCRISPRIyl _ A1, pHR _ POX 1_ IDI and pCRISPRIyl _ POX 1, pHR _ E1_ ERG1 and pCRISPRIyl _ E1 were constructed respectively, and strain XK1 was transformed to obtain lipolytics XK1, XK1, XK1, XK1 and XK 1.
According to the invention, the plasmid pHR _ XPR2_ carRP in the step (1) is constructed by the following steps: the carRP gene is connected to the plasmid pHR _ XPR2_ hrGFP through the restriction enzyme cutting sites BssHII and NheI to obtain a plasmid pHR _ XPR2_ carRP;
the plasmid pHR _ D17_ carB in the step (1) is constructed by the following steps: the carB gene is connected to a plasmid pHR _ D17_ hrGFP through a restriction enzyme site BssHII and NheI to obtain a plasmid pHR _ D17_ carB;
the plasmid pHR _ AXP _ HMG obtained in the step (3) is constructed by the following steps: the HMG gene is connected to a plasmid pHR _ AXP _ hrGFP through enzyme cutting sites BssHII and NheI to obtain a plasmid pHR _ AXP _ HMG;
the plasmid pHR _ A08_ GGS1 in the step (3) is constructed by the following steps: the GGS1 gene is connected to a plasmid pHR _ A08_ hrGFP through enzyme cutting sites BssHII and NheI to obtain a plasmid pHR _ A08_ GGS 1;
the construction of the plasmid pHR _ MFE1_ carRP of step (4) comprises the following steps: the carRP gene is connected to a plasmid pHR _ MFE1_ hrGFP through enzyme cutting sites BssHII and NheI to obtain a plasmid pHR _ MFE1_ carRP;
the plasmid pHR _ POX2_ carB in the step (4) is constructed by the following steps: the carB gene is connected to a plasmid pHR _ POX2_ hrGFP through a restriction enzyme site BssHII and NheI to obtain a plasmid pHR _ POX2_ carB;
the construction steps of the plasmids pHR _ LIP1_ ERG13, pHR _ E1_ ERG10-11099, pHR _ A1_ ERG10-08536, pHR _ B1_ ERG8, pHR _ A2_ ERG19, pHR _ POX6_ IDI and pHR _ E2_ ERG12 in the step (5) are as follows: ERG13, ERG10-11099, ERG10-08536, ERG8, ERG19, IDI and ERG12 genes derived from yarrowia lipolytica were ligated to plasmids pHR _ LIP1_ hrGFP, pHR _ E1_ hrGFP, pHR _ A1_ hrGFP, pHR _ B1_ hrGFP, pHR _ A2_ hrGFP, pHR _ POX6_ hrGFP and pHR _ E2_ hrGFP, respectively, and constructed.
As a fifth aspect of the invention, the application of the yarrowia lipolytica gene engineering bacterium capable of producing β -carotene in the preparation of β -carotene is provided.
As a sixth aspect of the invention, the use of the β -carotene-producing yarrowia lipolytica genetically engineered bacterium in the preparation of carotenoids and derivatives thereof is provided.
According to the invention, the carotenoids are zeaxanthin, astaxanthin, crocin, canthaxanthin, echinenone, and the like.
As a seventh aspect of the invention, a method for producing β -carotene, β -carotene is obtained by fermentation production of the above-mentioned genetically engineered yarrowia lipolytica producing β -carotene.
The yarrowia lipolytica genetic engineering bacterium capable of producing β -carotene has the advantages that the construction method is simple and convenient, only 7 genes are introduced in the yarrowia lipolytica genetic engineering bacterium XK7-XK8 and XK10-XK16 without a anaplerosis screening marker mode, β -carotene can be produced at high yield through fermentation, and the yarrowia lipolytica genetic engineering bacterium has a good application prospect.
The yarrowia lipolytica genetic engineering bacterium XK19 capable of producing β -carotene has the beneficial effects that (1) the yield of β -carotene is high, the yarrowia lipolytica genetic engineering bacterium capable of producing β -carotene can enable the yield of β -carotene to reach 4.5g/L in a 5L fermentation tank, (2) the construction method is simple and convenient, only 8 genes are introduced in a CRISPR/Cas9 and integrated plasmid anaplerosis screening and marking mode, the obtained strain is good in stability after continuous subculture, and can be applied to large-scale commercial production, and the obtained β -carotene can be used safely and has a good prospect.
Drawings
FIG. 1 is a diagram showing the metabolic pathway of yarrowia lipolytica for β -carotene production after the introduction of β -carotene synthesis gene.
FIG. 2 is a graph showing the results of shake flask fermentation for production of β -carotene by strains XK2, XK3, XK4, XK5, XK6, XK7, XK8, XK10, XK11, XK12, XK13, XK14, XK15, XK16, XK17 and XK 19.
FIG. 3 is a graph showing the results of fed batch fermentation of strain XK19 in a 5L fermentor for β -carotene production.
FIG. 4 is a schematic diagram of the construction of the β -carotene-producing yarrowia lipolytica genetically engineered bacterium of the present invention.
Detailed Description
The present invention will be further described with reference to the following specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. For example, the CRISPR/Cas9 operating system in the embodiments of the present invention is prepared according to the preparation method described in Schwartz, c., Shabbir-Hussain, m., food, k., Blenner, M., Wheeldon, I.2017.Standard marked wrerless gene integration for path engineering in acs Synth Biol,6(3), 402-.
The yarrowia lipolytica used in the application can synthesize GGPP by itself, therefore, two β -carotene synthesis genes-carRP and carB after codon optimization from mucor circinelloides are transformed into yarrowia lipolytica, β -carotene can be produced, on the basis, two copies of carRP gene and one copy of carB gene are added, GGS1 gene, HMG gene and ERG13 gene are over-expressed, the yield of β -carotene can be greatly improved, and finally two auxotrophic screening markers of uracil and leucine are complemented back to successfully obtain a high-yield strain of β -carotene (see figure 1).
The reagents and starting materials used in the present invention are commercially available.
The strains and plasmid sources related to the invention are as follows:
1. yarrowia lipolytica Po1f and plasmid pINA 1269: prepared according to the preparation method described in Madzak, C.A., Treton, B.A., blanch-Roland, S.2000.Strong hybrid precursors and integral expressions/relational expressions for the use of a quantitative expression of heterologous proteins in the layer Yarrowia lipolytica. J.mol Microbiol Biotechnology, 2(2), 207-.
2. Plasmid pINA 1312: see Nicaud, J.M., Madzak, C., van den Broek, P., Gysler, C., Duboc, P., Niederberger, P., Garlardin, C.2002.protein expression and secretion in the layer Yarrowia lipolytica. FEMS Yeast Res,2(3), 371-.
3. Plasmid pCRISPRIyl, plasmid pHR _ XPR2_ hrGFP, plasmid pHR _ D17_ hrGFP, plasmid pCRISPRIyl _ XPR2, plasmid pCRISPRIyl _ D17, plasmid pHR _ AXP _ hrGFP, plasmid pHR _ A08_ hrGFP, plasmid pCRISPRIyl _ AXP, plasmid pCRISPRIyl _ A08, plasmid pHR _ MFE1_ hrGFP, plasmid pCRISPRIyl _ MFE1 are known plasmids and are all purchased from addgene.
4. Plasmid pHR _ XPR2_ carRP: contains the gene carRP derived from Mucor circinelloides; plasmid pHR _ D17_ carB: contains a gene carB derived from Mucor circinelloides; plasmid pHR _ a08_ GGS 1: comprising gene GGS1 derived from yarrowia lipolytica; plasmid pHR _ AXP _ HMG: contains gene HMG derived from yarrowia lipolytica; plasmid pHR _ POX2_ carB: contains a gene carB derived from Mucor circinelloides; plasmid pHR _ MFE1_ carRP: contains the gene carRP derived from Mucor circinelloides; plasmid pHR _ POX3_ carRP: contains the gene carRP derived from Mucor circinelloides; plasmid pHR _ POX4_ carB: contains a gene carB derived from Mucor circinelloides; plasmid pHR _ LIP1_ ERG 13: contains gene ERG13 from yarrowia lipolytica; plasmid pHR _ E1_ ERG 10-11099: contains gene ERG10-11099 derived from yarrowia lipolytica; plasmid pHR _ A1_ ERG 10-08536: contains gene ERG10-08536 derived from yarrowia lipolytica; plasmid pHR _ B1_ ERG 8: contains gene ERG8 from yarrowia lipolytica; plasmid pHR _ a2_ ERG 19: contains gene ERG19 from yarrowia lipolytica; plasmid pHR _ POX6_ IDI: contains gene IDI derived from yarrowia lipolytica; plasmid pHR _ E2_ ERG 12: contains gene ERG12 originated from yarrowia lipolytica.
5. The invention constructs ten pairs of knock-in plasmid pairs according to the existing CRISPR/Cas9 operating system, wherein ten available sites are developed for gene knock-in on the basis of the CRISPR/Cas9 operating system published on Addgene, POX2 (the locustag on NCBI is YALI0F10857g), POX3 (the locustag on NCBI is YALI0D24750g), POX4 (the locustag on NCBI is YALI0E27654g), POX6 (the locustag on NCBI is YALI0E06567g), LIP1 (the locustag on NCBI is YALI0E 1063559), E1 (the locustag on NCBI is YALI0E27555g), YAA 1 (the locustag on NCBI is YALI0A11286g), B1 (the locustag on NCBI is YACUS 3500E 3503527), and the locustag on NCBI is YALI 24A g), and the NCBI 584645 is NCBI 4930A 4935 (the NCBI). The gene knock-in was performed at these ten sites, and plasmid pairs, pHR _ POX2_ hrGFP and pCRISRyl _ POX2, pHR _ POX3_ hrGFP and pCRISRyl _ POX3, pHR _ LIP1_ hrGFP and pCRISRyl _ LIP1, pHR _ POX4_ hrGFP and pCRISRyl _ POX4, pHR _ E1_ hrGFP and pCRISRyl _ E1, pHR _ A1_ hrGFP and pCRISPRyl _ A1, pHR _ B1_ hrGFP and pCRISRyl _ B1, pHR _ A2_ hrGFP and pCRISyl _ A2, pHR _ POX6_ hrGFP and pCRISRyl _ POX6, pHR _ E2_ hrGFP and pCRISRyl _ 539E 2, were constructed for each site. Wherein, the sequences of the sgRNA fragments with 10 designed sites are shown in Table 1; the primer sequences for constructing the above ten pairs of plasmid pairs are shown in Table 2.
Table 1 sequence of sgrnas of knock-in sites
Figure BDA0002388807230000101
Figure BDA0002388807230000111
TABLE 2 primer sequences of plasmid pairs
Figure BDA0002388807230000112
Figure BDA0002388807230000121
The chemical names and gene names related to the present invention are explained as follows:
1. GGPP: geranylgeranyl pyrophosphate
2. FPP (field programmable Gate array): farnesyl diphosphate
3. IPP: isopentenyl pyrophosphate
4. DMAPP: dimethylallyl pyrophosphoric acid
5. HMG-CoA: 3-hydroxy-3-methylglutaryl coenzyme A
6. carRP is a bifunctional enzyme gene which can catalyze two molecules of GGPP to generate phytoene and catalyze the lycopene to generate β -carotene, and the accession number in NCBI is GB: AJ 250827.1;
7. a carB: the gene which can catalyze phytoene to generate lycopene has the accession number GB: AJ238028.1 in NCBI;
8. GGS 1: the gene which can catalyze FPP to generate GGPP has the accession number GB: NC-006070 in NCBI;
9. HMG: a gene encoding HMG-CoA reductase which can reduce HMG-CoA to mevalonate, having accession number GB: NC-006071 in NCBI;
10. ERG 13: a gene which codes HMG-CoA synthetase and can catalyze acetoacetyl-CoA and acetyl-CoA to generate HMG-CoA, and the accession number of the gene in NCBI is GB: NC-006072.1;
11. ERG 10: the gene codes acetoacetyl-CoA thiolase, can catalyze two molecules of acetyl-CoA to generate acetoacetyl-CoA, has two isozymes, and has the accession numbers of NC-006071.1 (ERG10-11099) and NC-006068.1 (ERG10-08536) in NCBI;
12. ERG 8: a gene for coding phospho MVA kinase which can catalyze mevalonate-5-phosphate to generate mevalonate-5-diphosphate, and the accession number of the gene is GB: NC-006071.1 in NCBI;
13. ERG 19: a gene for coding MVA pyrophosphate decarboxylase and catalyzing mevalonate-5-diphosphate to generate IPP, and the accession number of the gene in NCBI is GB: NC-006072.1;
14. IDI: a gene which codes IPP isomerase and can catalyze IPP to generate DMAPP, and the accession number of the gene in NCBI is GB: NC-006072.1;
15. ERG 12: a gene encoding MVA kinase, which catalyzes the production of mevalonate-5-phosphate from mevalonate, under the accession number GB: NC-006068.1 at NCBI.
16. The carRP gene and carB gene referred to in the following examples refer to the optimized carRP gene and carB gene, the nucleotide sequences of which are shown in SEQ ID NO:55 and SEQ ID NO:56, respectively.
The following examples employ yarrowia lipolytica Po1f as the starting strain. It should be noted that conventional uracil and leucine auxotrophic Yarrowia lipolytica (Yarrowia lipolytica) can be used as the starting strain for transformation for the experiments described in the examples below.
Example 1 construction of CRISPR/Cas9 Gene knock-in plasmid pairs
Plasmid construction for different sgrnas is based on the purchased plasmid pCRISPRyl, which has an enzyme cleavage site avrli, upstream of which contains the promoter SCR 1' -tRNAGly for promoting expression of the sgRNA, and the sgRNA plasmid is obtained by inserting the 20bp sequence of the sgRNA into this site. The plasmid pCRISPRyl was purified after single cleavage with AvrII restriction enzyme to obtain linearized plasmid pCRISPRyl. The linearized plasmids were seamlessly cloned with sgRNA fragments (SEQ ID NO:1-SEQ ID NO:10 in Table 1) designed for POX2, POX3, POX4, LIP1, E1, A1, B1, A2, POX6, and E2, respectively, to obtain the vectors pCRISPRIyl _ POX2, pCRISPRIyl _ POX3, pCRISPRIyl _ POX4, pCRISPRIyl _ LIP1, pCRISPRIyl _ E1, pCRISPRIyl _ A1, pCRISPRIyl _ B1, pCRISPRIyl _ A2, pCRISPRIyl _ POX6, and pCRISPRIyl _ E2.
(1) Construction of plasmid pHR _ POX2_ hrGFP: using yarrowia lipolytica genome as a template, respectively using primer pairs POX2-LF/POX2-LR and POX2-RF/POX2-RR to amplify 1200bp at each end of a sgRNA sequence at a POX2 site by PCR as a left homologous arm and a right homologous arm, using a plasmid pHR _ AXP _ hrGFP as a template, using primer pairs plas-F and plas-R to amplify by PCR to obtain a plasmid basic skeleton, and seamlessly cloning and connecting the three fragments to obtain a plasmid pHR _ POX 2; the hrGFP gene expression cassette obtained by double-digesting the plasmid pHR _ AXP _ hrGFP with the restriction enzymes SpeI and AvrII was ligated to the plasmid pHR _ POX2 similarly double-digested with the restriction enzymes SpeI and AvrII, and pHR _ POX2_ hrGFP was obtained after the verification.
(2) Construction of plasmid pHR _ POX3_ hrGFP: using yarrowia lipolytica genome as a template, respectively using primer pairs POX3-LF/POX3-LR and POX3-RF/POX3-RR to amplify 1200bp at each end of a sgRNA sequence at a POX3 site by PCR as a left homologous arm and a right homologous arm, using a plasmid pHR _ AXP _ hrGFP as a template, using primer pairs plas-F and plas-R to amplify by PCR to obtain a plasmid basic skeleton, and seamlessly cloning and connecting the three fragments to obtain a plasmid pHR _ POX 3; the hrGFP gene expression cassette obtained by double-digesting the plasmid pHR _ AXP _ hrGFP with the restriction enzymes SpeI and AvrII was ligated to the plasmid pHR _ POX3 similarly double-digested with the restriction enzymes SpeI and AvrII, and pHR _ POX3_ hrGFP was obtained after the verification.
(3) Construction of plasmid pHR _ LIP1_ hrGFP: using yarrowia lipolytica genome as a template, respectively using primer pairs LIP1-LF/LIP1-LR and LIP1-RF/LIP1-RR to amplify 1200bp at each end of sgRNA sequence at LIP1 site by PCR as a left homologous arm and a right homologous arm, using plasmid pHR _ AXP _ hrGFP as a template, using primer pairs plas-F and plas-R to amplify by PCR to obtain a plasmid basic skeleton, and seamlessly cloning and connecting the three fragments to obtain a plasmid pHR _ LIP 1; the hrGFP gene expression cassette obtained by double-digesting the plasmid pHR _ AXP _ hrGFP with the restriction enzymes SpeI and AvrII was ligated to the plasmid pHR _ LIP1 similarly double-digested with the restriction enzymes SpeI and AvrII to obtain pHR _ LIP1_ hrGFP.
(4) Construction of plasmid pHR _ POX4_ hrGFP: using yarrowia lipolytica genome as a template, respectively using primer pairs POX4-LF/POX4-LR and POX4-RF/POX4-RR to amplify 1200bp at each end of a sgRNA sequence at a POX4 site by PCR as a left homologous arm and a right homologous arm, using a plasmid pHR _ AXP _ hrGFP as a template, using primer pairs plas-F and plas-R to amplify by PCR to obtain a plasmid basic skeleton, and seamlessly cloning and connecting the three fragments to obtain a plasmid pHR _ POX 4; the hrGFP gene expression cassette obtained by double-digesting the plasmid pHR _ AXP _ hrGFP with the restriction enzymes SpeI and AvrII was ligated to the plasmid pHR _ POX4 similarly double-digested with the restriction enzymes SpeI and AvrII to obtain pHR _ POX4_ hrGFP.
(5) Construction of plasmid pHR _ E1_ hrGFP: using yarrowia lipolytica genome as a template, respectively using primer pairs E1-LF/E1-LR and E1-RF/E1-RR to amplify 1200bp at each end of an E1 site sgRNA sequence by PCR as a left homologous arm and a right homologous arm, using a plasmid pHR _ AXP _ hrGFP as a template, using primer pairs plas-F and plas-R to amplify by PCR to obtain a plasmid basic skeleton, and seamlessly cloning and connecting the three fragments to obtain a plasmid pHR _ E1; the hrGFP gene expression cassette obtained by double-digesting the plasmid pHR _ AXP _ hrGFP with the restriction enzymes SpeI and AvrII was ligated to the plasmid pHR _ E1 similarly double-digested with the restriction enzymes SpeI and AvrII to obtain pHR _ E1_ hrGFP.
(6) Construction of plasmid pHR _ a1_ hrGFP: using yarrowia lipolytica genome as a template, respectively using primer pairs A1-LF/A1-LR and A1-RF/A1-RR to amplify 1200bp at both ends of an A1 site sgRNA sequence by PCR as a left homologous arm and a right homologous arm, using a plasmid pHR _ AXP _ hrGFP as a template, using primer pairs plas-F and plas-R to amplify by PCR to obtain a plasmid basic skeleton, and seamlessly cloning and connecting the three fragments to obtain a plasmid pHR _ A1; the hrGFP gene expression cassette obtained by double-digesting the plasmid pHR _ AXP _ hrGFP with the restriction enzymes SpeI and AvrII was ligated to the plasmid pHR _ A1 similarly double-digested with the restriction enzymes SpeI and AvrII to obtain pHR _ A1_ hrGFP.
(7) Plasmid pHR _ B1_ hrGFP was constructed in parts: using yarrowia lipolytica genome as a template, respectively using primer pairs B1-LF/B1-LR, B1-RF1/B1-RR1 and B1-RF2/B1-RR2 to amplify 1200bp at both ends of a sgRNA sequence of a B1 site by PCR as left and right homologous arms, using a plasmid pHR _ AXP _ hrGFP as a template, using primer pairs plas-F and plas-R to amplify by PCR to obtain a plasmid basic skeleton, and seamlessly cloning and connecting the four fragments to obtain a plasmid pHR _ B1; the hrGFP gene expression cassette obtained by double-digesting the plasmid pHR _ AXP _ hrGFP with the restriction enzymes SpeI and AvrII was ligated to the plasmid pHR _ B1 similarly double-digested with the restriction enzymes SpeI and AvrII to obtain pHR _ B1_ hrGFP.
(8) Construction of plasmid pHR _ a2_ hrGFP: using yarrowia lipolytica genome as a template, respectively using primer pairs A2-LF/A2-LR and A2-RF/A2-RR to amplify 1200bp at both ends of an A2 site sgRNA sequence by PCR as a left homologous arm and a right homologous arm, using a plasmid pHR _ AXP _ hrGFP as a template, using primer pairs plas-F and plas-R to amplify by PCR to obtain a plasmid basic skeleton, and seamlessly cloning and connecting the three fragments to obtain a plasmid pHR _ A2; the hrGFP gene expression cassette obtained by double-digesting the plasmid pHR _ AXP _ hrGFP with the restriction enzymes SpeI and AvrII was ligated to the plasmid pHR _ A2 similarly double-digested with the restriction enzymes SpeI and AvrII to obtain pHR _ A2_ hrGFP.
(9) Construction of plasmid pHR _ POX6_ hrGFP: using yarrowia lipolytica genome as a template, respectively using primer pairs POX6-LF/POX6-LR and POX6-RF/POX6-RR to amplify 1200bp at each end of a sgRNA sequence at a POX6 site by PCR as a left homologous arm and a right homologous arm, using a plasmid pHR _ AXP _ hrGFP as a template, using primer pairs plas-F and plas-R to amplify by PCR to obtain a plasmid basic skeleton, and seamlessly cloning and connecting the three fragments to obtain a plasmid pHR _ POX 6; the hrGFP gene expression cassette obtained by double-digesting the plasmid pHR _ AXP _ hrGFP with the restriction enzymes SpeI and AvrII was ligated to the plasmid pHR _ POX6 similarly double-digested with the restriction enzymes SpeI and AvrII to obtain pHR _ POX6_ hrGFP.
(10) Construction of plasmid pHR _ E2_ hrGFP: using yarrowia lipolytica genome as a template, respectively using primer pairs E2-LF/E2-LR and E2-RF/E2-RR to amplify 1200bp at each end of an E2 site sgRNA sequence by PCR as a left homologous arm and a right homologous arm, using a plasmid pHR _ AXP _ hrGFP as a template, using primer pairs plas-F and plas-R to amplify by PCR to obtain a plasmid basic skeleton, and seamlessly cloning and connecting the three fragments to obtain a plasmid pHR _ E2; the hrGFP gene expression cassette obtained by double-digesting the plasmid pHR _ AXP _ hrGFP with the restriction enzymes SpeI and AvrII was ligated to the plasmid pHR _ E2 similarly double-digested with the restriction enzymes SpeI and AvrII to obtain pHR _ E2_ hrGFP.
Example 2 construction of genetically engineered yarrowia lipolytica producing β -Carotene
FIG. 4 shows the construction scheme of the β -carotene-producing yarrowia lipolytica genetically engineered bacterium of the present invention.
(1) Based on the existing CRISPR/Cas9 operating system, two pairs of knock-in plasmid pairs were constructed, respectively for the construction of knock-in plasmid pairs containing the optimized carRP gene and for the construction of knock-in plasmid pairs containing the optimized carB gene.
Both knock-in plasmid pairs contained cleavage sites for NheI and BssHII on the donor plasmid.
The donor plasmid also contains promoter and/or terminator sequences. More preferably, the donor plasmid further comprises the promoter UAS1B8-TEF (136).
The knock-in plasmid pair in this example is a recombinant vector conventional in the art, wherein the sgRNA plasmid contains a leucine selection marker, the donor plasmid contains a uracil selection marker, which is capable of transforming uracil and leucine auxotrophic yarrowia lipolytica, and the two knock-in plasmid pairs are capable of knocking in the optimized carRP gene and carB gene, respectively.
The method comprises the following specific steps:
the optimized sequence of carRP (the nucleotide sequence of which is shown in SEQ ID No. 1) and the optimized sequence of carB (the nucleotide sequence of which is shown in SEQ ID No. 2) derived from the gene of Mucor circinelloides (Mucor circinelloides) were constructed into plasmid pHR _ XPR2_ hrGFP and plasmid pHR _ D17_ hrGFP by means of restriction enzyme sites NheI and BssHII, respectively, to obtain plasmids pHR _ XPR2_ carRP and pHR _ D17_ carB. Wherein the codon-optimized carRP and carB genes are obtained by optimizing the nucleotide sequences of carRP and carB derived from Mucor circinelloides.
(2) The plasmid pHR _ XPR2_ carRP and the plasmid pCRISPRyl _ XPR2 obtained in step (1) were simultaneously transformed into yarrowia lipolytica Po1f to obtain strain XK1, and it was verified that the carRP gene was knocked in strain XK 1. Wherein the Transformation kit Frozen EZ Yeast Transformation II is used for TransformationTM(purchased from Zymo Research) according to the protocol described in the kit instructions. After recovering the selection marker from strain XK1, plasmid pHR _ D17_ carB and plasmid pCRISPRyl _ D17 obtained in step (1) were simultaneously transferred into yarrowia lipolytica XK1 to obtain strain XK2, and it was verified that the carB gene was knocked into strain XK 2.
(3) HMG gene and GGS1 gene derived from yarrowia lipolytica were ligated to plasmids pHR _ AXP _ hrGFP and pHR _ A08_ hrGFP via restriction sites BssHII and NheI, respectively, to obtain plasmids pHR _ AXP _ HMG and pHR _ A08_ GGS 1. The resulting plasmids pHR _ A08_ GGS1 and pCRISPRIyl _ A08 were sequentially transformed into strain XK2 to obtain strain XK3, the resulting plasmids pHR _ AXP _ HMG and pCRISPRIyl _ AXP were transformed into strain XK3 to obtain strain XK4, and it was confirmed that HMG gene and GGS1 gene were knocked into strain XK 4.
(4) The carRP gene was ligated into plasmid pHR _ MFE1_ hrGFP via the cleavage sites BssHII and NheI, resulting in plasmid pHR _ MFE1_ carRP. The carB gene was ligated into plasmid pHR _ POX2_ hrGFP via the cleavage sites BssHII and NheI, resulting in plasmid pHR _ POX2_ carB. The resulting plasmids, pHR _ POX2_ carB and pCRISPRyl _ POX2, were sequentially transformed into strain XK4 to obtain strain XK5, and the resulting plasmids, pHR _ MFE1_ carRP and pCRISPRyl _ MFE1, were sequentially transformed into strain XK5 to obtain strain XK6, and it was confirmed that the carRP gene and the carB gene were knocked into strain XK 6.
(5) The carRP gene and the carB gene were ligated to plasmids pHR _ POX3_ hrGFP and pHR _ POX4_ hrGFP, respectively, to obtain plasmids pHR _ POX3_ carRP and pHR _ POX4_ carB. The resulting plasmids, pHR _ POX3_ carRP and pCRISPRyl _ POX3, pHR _ POX4_ carB and pCRISPRyl _ POX4, were transformed into strain XK6, respectively, to obtain strains XK7 and XK8, and it was confirmed that the carRP gene was knocked in strain XK7 and the carB gene was knocked in strain XK 8.
ERG13, ERG10-11099, ERG10-08536, ERG8, ERG19, IDI, ERG12 genes were ligated to plasmids pHR _ LIP1_ hrGFP, pHR _ E1_ hrGFP, pHR _ A1_ hrGFP, pHR _ B1_ hrGFP, pHR _ A2_ hrGFP, pHR _ POX6_ hrGFP and pHR _ E2_ hrGFP, respectively, to obtain plasmids pHR _ LIP1_ ERG13, pHR _ E1_ ERG10-11099, pHR _ A1_ ERG10-08536, pHR _ B1_ ERG8, pHR _ A2_ ERG19, pHR _ POX6_ IDI and pHR _ E2_ ERG 12. The resulting plasmids pHR _ LIP1_ ERG13 and pCRISPRIyl _ LIP1, pHR _ E1_ ERG10-11099 and pCRISPRIyl _ E1, pHR _ A1_ ERG10-08536 and pCRISPRIyl _ A1, pHR _ B1_ ERG8 and pCRISPRIyl _ B1, pHR _ A2_ ERG19 and pCRISPRIyl _ A2, pHR _ POX6_ IDI and pCRISPRIyl _ POX6, pHR _ E2_ ERG12 and pCRISPRIyl _ E2 were transformed into strain XK6, respectively, to obtain strains XK10, XK11, XK12, XK13, XK14, XK15 and XK 387 16. According to verification, ERG13, ERG10-11099, ERG10-08536, ERG8, ERG19, IDI and ERG12 genes are knocked in the seven strains respectively.
(6) The above-constructed plasmids pHR _ LIP1_ ERG13 and pCRISPRIyl _ LIP1 were transformed into strain XK7 to obtain strain XK 17. The ERG13 gene was knocked in by strain XK 17.
(7) Carrying out double enzyme digestion linearization on the plasmid pINA1269 by using PmlI and BamHI enzyme cleavage sites, and transferring the linearized pINA1269 plasmid into a strain XK17 to supplement leucine screening marker; then, the plasmid pINA1312 is subjected to double enzyme digestion linearization by using PmlI and BamHI enzyme cleavage sites, and the linearized pINA1312 plasmid is transferred into an XK17 strain supplemented with the leucine selection marker to supplement the uracil selection marker, so that a strain XK19 is obtained. Wherein the transformation method is the same as the transformation method in the step (2). The strain XK19 was verified to be successful in complementing uracil and leucine selection markers. It should be noted that the plasmids pINA1269 and pINA1312 are recombinant vectors conventional in the art, capable of transforming uracil and leucine auxotrophic yarrowia lipolytica, said recombinant vectors containing cleavage sites for PmlI and BamHI.
EXAMPLE 3 determination of the β -carotene production by strains
Yarrowia lipolytica Po1f, strain XK2, XK3, XK4, XK5, XK6, XK7, XK8, XK10, XK11, XK12, XK13, XK14, XK15, XK16, XK17 and XK19 were inoculated into 2mL YPD medium (composed of 2% glucose, 2% peptone and 1% yeast extract, balance water, said percentages being mass percentages), respectively, cultured for 24 hours, and then cultured as the initial OD600The culture was carried out by inoculating the culture in a new 50mL YPD medium at an inoculum size of 0.01, and after 4 days of fermentation culture, β -carotene extraction was carried out using DMSO and acetone.
The results of the test are shown in table 3 and fig. 2.
TABLE 3 production of β -Carotene by different strains
Figure BDA0002388807230000161
The results in table 3 and fig. 2 demonstrate that the ability of strain XK19 to produce β -carotene is greatly improved, and the overexpression of ERG13 gene significantly improves the yield of β -carotene by overexpression of each gene in the MVA pathway, which is another rate-limiting gene in the pathway besides HMG gene.
Example 4 continuous feed fermentation culture of XK19 Strain
After the fermentation of the screened yarrowia lipolytica strain XK19, which produces high yields of β -carotene, in a 5L fermenter, the culture medium selected for the experiment was 2 × YPD medium (4% peptone, 2% yeast extract, 0.5% glucose), the initial tank volume was 2L, the inoculum was streaked on YPD solid plates from the glycerol seed-holding tubes, the grown single clones were inoculated into two flasks each containing 50mL YPD medium (ampicillin resistance and kanamycin resistance were added to the flasks to prevent contamination), after overnight culture of the flasks in a 30 ℃ constant temperature shaker at 220rpm, the cells in exponential growth phase were inoculated into the fermenter after incubation in 30 ℃ constant temperature shaker at 30 ℃ with aeration rate of 2vvm and dissolved oxygen at 20%, the stirrer speed (300 + 1000rpm) was coupled with dissolved oxygen, pH was adjusted to 5.5 using 3M sodium hydroxide or 3M hydrochloric acid to control pH, after 6 hours, 500g/L glucose was added to the fermenter at 12h rate, and the results of fermentation were obtained by fermentation in a 12.5L fermenter, and the results were obtained in a12 h fermenter using 3M sodium hydroxide or 3M hydrochloric acid solution, and the results of the fermenter were obtained in a12 h fermentation process, as shown in FIGS. 36H.
FIG. 3 shows that the biomass of the cells was 78.9g DCW/L on day 6.5 of the fermentation, and that the yields of β -carotene were 4.5g/L and 57.5mg/g DCW.
The foregoing is merely a preferred embodiment of this invention and it will be appreciated by those skilled in the art that numerous modifications and adaptations can be made without departing from the principles of the invention. Such modifications and refinements are also to be considered within the scope of the present invention.
Sequence listing
<110> university of east China's college of science
<120> yarrowia lipolytica gene engineering bacterium for producing β -carotene and application thereof
<141>2020-02-21
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<213> Artificial Sequence (Artificial Sequence)
<400>20
cctagggatc gttgtcagaa gtaagactag ttggaaaatt cgcttcacag atcgct 56
<210>21
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>21
actagtctta cttctgacaa cgatccctag gaccccgagc ccatgtcttt ccgaaa 56
<210>22
<211>55
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>22
aacagctatg accatgatta cgccaagctt tggcgttggc aagctccttg gaccg 55
<210>23
<211>55
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>23
gttcgaaggt accaaggaag catgcggtac cttgtctctt ggatacattg cactt 55
<210>24
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>24
cctagggatc gttgtcagaa gtaagactag tcggtaaacg tgttgaggga ctgttg 56
<210>25
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>25
actagtctta cttctgacaa cgatccctag ggagcgaaac cagacttcct tcaacc 56
<210>26
<211>55
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>26
aacagctatg accatgatta cgccaagctt cgcctgtctg cactggtcaa tggtg 55
<210>27
<211>55
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>27
gttcgaaggt accaaggaag catgcggtac cttccttgtg tttgtcaccg ccttt 55
<210>28
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>28
cctagggatc gttgtcagaa gtaagactag tcggaacagc aagataccca aaggca 56
<210>29
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>29
actagtctta cttctgacaa cgatccctag gcaattgggg tgcccgagac caatac 56
<210>30
<211>55
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>30
aacagctatg accatgatta cgccaagctt agtgcctgaa atcctggctg ccccc 55
<210>31
<211>55
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>31
gttcgaaggt accaaggaag catgcggtac gttacggaaa accatgaatc cggag 55
<210>32
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>32
cctagggatc gttgtcagaa gtaagactag tattatgaga cacgatccac aggcat 56
<210>33
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>33
actagtctta cttctgacaa cgatccctag gatggactca tggaccagga cgtgta 56
<210>34
<211>55
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>34
aacagctatg accatgatta cgccaagctt agagacgaaa ccaatcgccc gtggt 55
<210>35
<211>55
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>35
gttcgaaggt accaaggaag catgcggtac tggagtggaa cttggacagt attaa 55
<210>36
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>36
cctagggatc gttgtcagaa gtaagactag tcacagcttg tcactttgcg ttgaca 56
<210>37
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>37
actagtctta cttctgacaa cgatccctag gcatgtccag tgaagcctcc gagttc 56
<210>38
<211>55
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>38
aacagctatg accatgatta cgccaagctt agttcacttt ggacaccttc tatgg 55
<210>39
<211>54
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>39
ttcgaaggta ccaaggaagc atgcggtacc gaatcacaac ttcctcttga cata 54
<210>40
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>40
cctagggatc gttgtcagaa gtaagactag ttccgggaag aggagctcca gccgat 56
<210>41
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>41
actagtctta cttctgacaa cgatccctag gaatgaagaa cagaaggtcg gctgga 56
<210>42
<211>38
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>42
tatccatgcg atactcttct tcatacgcgc acgttgct 38
<210>43
<211>38
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>43
gcgcgtatga agaagagtat cgcatggata cttgtaga 38
<210>44
<211>55
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>44
aacagctatg accatgatta cgccaagctt gaagggaaga gaggcaaagt aaatg 55
<210>45
<211>55
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>45
gttcgaaggt accaaggaag catgcggtac tgtggtgtca cctcttcaag agctt 55
<210>46
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>46
cctagggatc gttgtcagaa gtaagactag taaactcttg cacgctgcgg ccggat 56
<210>47
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>47
actagtctta cttctgacaa cgatccctag gatttcatga atggcaagaa gtttcc 56
<210>48
<211>55
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>48
aacagctatg accatgatta cgccaagctt tgtaccaagg taaacggctc gccag 55
<210>49
<211>55
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>49
gttcgaaggt accaaggaag catgcggtac gagaggttgg aggacaggtt gagat 55
<210>50
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>50
cctagggatc gttgtcagaa gtaagactag tgcatgcaca aagggacgat cgatca 56
<210>51
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>51
actagtctta cttctgacaa cgatccctag gtatcggaaa ctttggtaca gatact 56
<210>52
<211>55
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>52
aacagctatg accatgatta cgccaagctt agataatata taatatattt gaaga 55
<210>53
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>53
aagcttggcg taatcatggt c 21
<210>54
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>54
gtaccgcatg cttccttgg 19
<210>55
<211>1845
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>55
atgctgctga cctacatgga ggtgcacctg tactacaccc tgcccgtgct gggagtcctg 60
tcttggctgt cccgacccta ctacaccgcc accgacgctc tgaagttcaa gttcctgacc 120
ctggtggcct tcaccaccgc ctctgcttgg gacaactaca tcgtctacca caaggcctgg 180
tcctactgtc ccacctgcgt gaccgctgtc attggttacg tccccctgga ggagtacatg 240
ttcttcatca ttatgaccct gctgaccgtg gccttcacca acctggtcat gcgatggcac 300
ctgcactctt tcttcatccg acccgagacc cccgtgatgc agtccgtgct ggtccgactg 360
gtccccatca ccgccctgct gattaccgcc tacaaggctt ggcacctggc tgtgcctggc 420
aagcctctgt tctacggctc ttgtatcctg tggtacgcct gccccgtcct ggctctgctg 480
tggttcggag ccggagagta catgatgcga cgacccctgg ccgtgctggt ctccattgct 540
ctgcccaccc tgttcctgtg ttgggtggac gtggtcgcca tcggcgctgg aacctgggac 600
atttctctgg ccacctccac cggcaagttc gtggtccctc acctgcctgt cgaggagttc 660
atgttcttcg ctctgattaa caccgtgctg gtcttcggaa cctgtgccat cgaccgaact 720
atggctattc tgcacctgtt caagaacaag tctccctacc agcgacccta ccagcactct 780
aagtccttcc tgcaccagat cctggagatg acctgggctt tctgcctgcc tgaccaggtg 840
ctgcactctg acaccttcca cgacctgtct gtctcctggg acattctgcg aaaggcttct 900
aagtccttct acaccgcctc cgctgtgttc cctggagacg tccgacagga gctgggtgtg 960
ctgtacgcct tctgtcgagc taccgacgac ctgtgcgaca acgagcaggt gcccgtccag 1020
acccgaaagg agcagctgat cctgacccac cagttcgtct ctgacctgtt cggtcagaag 1080
acctccgccc ccaccgctat cgactgggac ttctacaacg accagctgcc cgcctcttgt 1140
atttccgctt tcaagtcctt cacccgactg cgacacgtgc tggaggctgg tgctatcaag 1200
gagctgctgg acggatacaa gtgggacctg gagcgacgat ctattcgaga ccaggaagac 1260
ctgcgatact actccgcctg tgtggcttct tccgtcggag agatgtgcac ccgaatcatt 1320
ctggcccacg ctgacaagcc cgcctctcga cagcagaccc agtggatcat tcagcgagct 1380
cgagagatgg gactggtgct gcagtacacc aacatcgccc gagacattgt caccgactcc 1440
gaggagctgg gtcgatgcta cctgccccag gactggctga ccgagaagga agtggccctg 1500
atccagggcg gactggctcg agagattgga gaggagcgac tgctgtctct gtcccaccga 1560
ctgatctacc aggccgacga gctgatggtg gtcgctaaca agggcattga caagctgccc 1620
tctcactgtc agggtggcgt gcgagccgct tgcaacgtct acgcctctat cggtaccaag 1680
ctgaagtcct acaagcacca ctacccctct cgagcccacg tgggcaactc caagcgagtc 1740
gagatcgctc tgctgtccgt gtacaacctg tacaccgccc ccattgctac ctcttccacc 1800
acccactgcc gacagggcaa gatgcgaaac ctgaacacca tctag 1845
<210>56
<211>1740
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>56
atgtccaaga agcacatcgt catcattggt gctggtgtgg gcggaaccgc taccgctgct 60
cgactggccc gagagggctt caaggtcacc gtggtcgaga agaacgactt cggtggcgga 120
cgatgttccc tgattcacca ccagggccac cgattcgacc agggaccctc tctgtacctg 180
atgcccaagt acttcgagga cgccttcgct gacctggacg agcgaatcca ggaccacctg 240
gagctgctgc gatgcgacaa caactacaag gtccacttcg acgacggaga gtccattcag 300
ctgtcttccg acctgacccg aatgaaggct gagctggacc gagtggaggg tcctctgggt 360
ttcggacgat tcctggactt catgaaggag acccacatcc actacgagtc cggcaccctg 420
attgccctga agaagaactt cgagtctatc tgggacctga tccgaattaa gtacgctccc 480
gagatcttcc gactgcacct gttcggaaag atctacgacc gagcttctaa gtacttcaag 540
accaagaaga tgcgaatggc cttcaccttc cagaccatgt acatgggaat gtccccctac 600
gacgcccccg ctgtgtactc tctgctgcag tacaccgagt tcgccgaggg tatctggtac 660
ccccgaggtg gcttcaacat ggtggtccag aagctggagg ccattgctaa gcagaagtac 720
gacgccgagt tcatctacaa cgcccccgtc gctaagatta acaccgacga cgctaccaag 780
caggtcaccg gtgtgaccct ggagaacggc cacatcattg acgccgacgc tgtggtctgt 840
aacgccgacc tggtgtacgc ttaccacaac ctgctgcctc cctgccgatg gacccagaac 900
accctggcct ccaagaagct gacctcttcc tctatctctt tctactggtc catgtctacc 960
aaggtccccc agctggacgt gcacaacatc ttcctggccg aggcttacca ggagtccttc 1020
gacgagattt tcaaggactt cggcctgccc tccgaggcct ctttctacgt gaacgtcccc 1080
tcccgaattg acccctctgc cgctcccgac ggaaaggact ctgtgatcgt cctggtgccc 1140
attggacaca tgaagtccaa gaccggtgac gcttctaccg agaactaccc cgccatggtc 1200
gacaaggctc gaaagatggt cctggctgtg atcgagcgac gactgggaat gtccaacttc 1260
gccgacctga ttgagcacga gcaggtcaac gaccccgctg tgtggcagtc taagttcaac 1320
ctgtggcgag gatccattct gggtctgtct cacgacgtcc tgcaggtgct gtggttccga 1380
ccctccacca aggactctac cggccgatac gacaacctgt tcttcgtggg agcctccacc 1440
caccctggta ccggagtccc tatcgtgctg gctggttcca agctgacctc tgaccaggtg 1500
gtcaagtctt tcggcaagac ccccaagccc cgaaagattg agatggagaa cacccaggct 1560
cccctggagg agcctgacgc tgagtccacc ttccctgtct ggttctggct gcgagccgct 1620
ttctgggtca tgttcatgtt cttctacttc ttccctcagt ccaacggaca gacccctgcc 1680
tctttcatca acaacctgct gcccgaggtc ttccgagtgc acaactctaa cgtgatctag 1740

Claims (7)

1. A genetically engineered yarrowia lipolytica producing β -carotene is characterized in that,
the yarrowia lipolytica gene engineering bacterium producing β -carotene is constructed by knocking carRP gene and carB gene into chromosome of uracil and leucine auxotrophic yarrowia lipolytica, or,
the β -carotene-producing yarrowia lipolytica gene engineering bacterium is constructed by knocking the carRP gene and carB gene into the chromosome of uracil and leucine auxotrophic yarrowia lipolytica and knocking GGS1 gene, or,
the β -carotene-producing yarrowia lipolytica gene engineering bacterium is constructed by knocking the carRP gene and carB gene into the chromosome of uracil and leucine auxotrophic yarrowia lipolytica and knocking GGS1 gene and HMG gene, or,
the β -carotene-producing yarrowia lipolytica gene engineering bacterium is constructed by knocking the carRP gene and carB gene into the chromosome of uracil and leucine auxotrophic yarrowia lipolytica, then knocking GGS1 gene, HMG gene and 1 copy of carB gene, or,
the yarrowia lipolytica gene engineering bacterium producing β -carotene is constructed by knocking the carRP gene and carB gene into the chromosome of uracil and leucine auxotrophic yarrowia lipolytica, then knocking GGS1 gene, HMG gene, 1 copy of carRP gene and 1 copy of carB gene, or,
the β -carotene-producing yarrowia lipolytica gene engineering bacterium is constructed by knocking the carRP gene and carB gene into the chromosome of uracil and leucine auxotrophic yarrowia lipolytica, then knocking GGS1 gene, HMG gene, two copies of carRP gene and 1 copy of carB gene, or,
the yarrowia lipolytica gene engineering bacterium producing β -carotene is constructed by knocking the carRP gene and carB gene into the chromosome of uracil and leucine auxotrophic yarrowia lipolytica, then knocking GGS1 gene, HMG gene, two copies of carRP gene and 1 copy of carB gene, ERG13 gene, or,
the yarrowia lipolytica genetic engineering bacterium capable of producing β -carotene is constructed by knocking a carRP gene and a carB gene into chromosomes of uracil and leucine auxotrophic yarrowia lipolytica, knocking GGS1 gene, HMG gene, two copies of carRP gene, 1 copy of carB gene and ERG13 gene, and finally complementing two auxotrophic screening markers of uracil and leucine, or,
the yarrowia lipolytica gene engineering bacterium producing β -carotene is constructed by knocking the carRP gene and carB gene into the chromosome of uracil and leucine auxotrophic yarrowia lipolytica, then knocking GGS1 gene, HMG gene, 1 copy of carRP gene, 1 copy of carB gene and ERG13 gene, or,
the yarrowia lipolytica genetic engineering bacterium capable of producing β -carotene is constructed by knocking the carRP gene and carB gene into the chromosome of uracil and leucine auxotrophic yarrowia lipolytica, then knocking GGS1 gene, HMG gene, 1 copy of carRP gene, 1 copy of carB gene and ERG10-11099 gene, or,
the yarrowia lipolytica genetic engineering bacterium capable of producing β -carotene is constructed by knocking the carRP gene and carB gene into the chromosome of uracil and leucine auxotrophic yarrowia lipolytica, then knocking GGS1 gene, HMG gene, 1 copy of carRP gene, 1 copy of carB gene and ERG10-08536 gene, or,
the β -carotene-producing yarrowia lipolytica gene engineering bacterium is constructed by knocking the carRP gene and carB gene into the chromosome of uracil and leucine auxotrophic yarrowia lipolytica, then knocking GGS1 gene, HMG gene, 1 copy of carRP gene, 1 copy of carB gene and ERG8 gene, or,
the β -carotene-producing yarrowia lipolytica gene engineering bacterium is constructed by knocking the carRP gene and carB gene into the chromosome of uracil and leucine auxotrophic yarrowia lipolytica, then knocking GGS1 gene, HMG gene, 1 copy of carRP gene, 1 copy of carB gene and ERG19 gene, or,
the β -carotene-producing yarrowia lipolytica gene engineering bacterium is constructed by knocking the carRP gene and the carB gene into the chromosome of uracil and leucine auxotrophic yarrowia lipolytica, and knocking GGS1 gene, HMG gene, 1 copy of carRP gene, 1 copy of carB gene and IDI gene, or,
the β -carotene-producing yarrowia lipolytica genetic engineering bacterium is constructed by knocking a carRP gene and a carB gene into a chromosome of uracil-leucine auxotrophic yarrowia lipolytica, and knocking GGS1 gene, HMG gene, 1 copy of carRP gene, 1 copy of carB gene and ERG12 gene;
the β -carotene-producing yarrowia lipolytica gene engineering bacterium is constructed by knocking a carRP gene and a carB gene into a chromosome of uracil-leucine auxotrophic yarrowia lipolytica, and knocking GGS1 gene, HMG gene, 1 copy of carRP gene and 2 copies of carB gene;
the nucleotide sequence of the carRP gene is shown as SEQ ID NO:55 is shown; the nucleotide sequence of the carB gene is shown as SEQ ID NO. 56.
2. The β -carotene producing genetically engineered yarrowia lipolytica of claim 1, wherein said uracil and leucine auxotrophic yarrowia lipolytica is yarrowia lipolytica Po1f, said carRP gene, carB gene, GGS1 gene, HMG gene, ERG13 gene, ERG10-11099 gene, ERG10-08536 gene, ERG8 gene, ERG19 gene, IDI gene, ERG12 gene are knocked into yarrowia lipolytica Po1f using CRISPR/9 operating system.
3. A knock-in plasmid pair of genetically engineered yarrowia lipolytica for gene knock-in, characterized in that said knock-in plasmid pair is a CRISPR/Cas9 system-based plasmid pair for knock-in yarrowia lipolytica, said knock-in plasmid pair being a pair of phrrp _ POX2_ hrGFP and pcrispry _ POX2, phrrp _ POX3_ hrGFP and pcrispry _ POX3, phrrp _ LIP1_ hrGFP and pcrispryryl _ LIP1, phrrp _ POX4_ hrGFP and pcrispry _ POX4, phrrp _ E _ 1_ hrGFP and pcrispry _ E1, phrrp _ a1_ hrysyl _ a gfp and pcrispry _ a 8, phrrp _ B6 _ hrGFP and pcrisyl _ B1, phrrp _ a2_ hrhrysyl _ a and pcrisryl _ p _ a 4684, at least one pair of phrrp _ p and pcrisryl _ p 3, phrase _ p 2 and pcrisyl _ p 3;
(1) plasmid pair pHR _ POX2_ hrGFP and pCRISPRyl _ POX 2:
the pCRISPRyl _ POX2 is obtained by linearizing a plasmid pCRISPRyl by using an AvrII restriction enzyme and seamlessly cloning and connecting the plasmid pCRISPRyl with a sgRNA fragment designed aiming at a POX2 site, wherein the nucleotide sequence of the sgRNA fragment designed aiming at a POX2 site is shown as SEQ ID NO:1 is shown in the specification;
the pHR _ POX2_ hrGFP is obtained by taking a genome of yarrowia lipolytica as a template, respectively amplifying 1200bp at both ends of a sgRNA sequence at a POX2 site by PCR (polymerase chain reaction) by using a primer pair POX2-LF/POX2-LR and POX2-RF/POX2-RR as a left homologous arm and a right homologous arm, taking a plasmid pHR _ AXP _ hrGFP as a template, and amplifying plas-F and plas-R by PCR by using a primer pair to obtain a plasmid basic skeleton, and seamlessly cloning and connecting the three fragments to obtain a plasmid pHR _ POX 2; connecting the hrGFP gene expression cassette obtained by double digestion of the plasmid pHR _ AXP _ hrGFP by using the restriction enzymes SpeI and AvrII with the plasmid pHR _ POX2 which is also subjected to double digestion by using the restriction enzymes SpeI and AvrII to obtain pHR _ POX2_ hrGFP; wherein the primer sequences of the primers POX2-LF, POX2-LR, POX2-RF and POX2-RR are respectively shown as SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13 and SEQ ID NO: 14 is shown in the figure; the sequences of the primers of the plas-F and plas-Rd are respectively shown as SEQ ID NO: 53 and SEQ ID NO: 54 is shown;
(2) plasmid pair pHR _ POX3_ hrGFP and pCRISPRyl _ POX 3:
the pCRISPRyl _ POX3 is obtained by performing single-enzyme linearization on a plasmid pCRISPRyl by using an AvrII restriction enzyme and performing seamless cloning and connection with a sgRNA fragment designed aiming at a POX3 site, wherein the nucleotide sequence of the sgRNA fragment designed aiming at a POX3 site is shown as SEQ ID NO: 2 is shown in the specification;
the pHR _ POX3_ hrGFP is constructed by taking the genome of yarrowia lipolytica as a template, respectively amplifying 1200bp at both ends of a sgRNA sequence at a POX3 site by PCR (polymerase chain reaction) by using a primer pair POX3-LF/POX3-LR and POX3-RF/POX3-RR as left and right homologous arms, taking a plasmid pHR _ AXP _ hrGFP as a template, amplifying by PCR by using a primer pair plas-F and plas-R to obtain a plasmid basic skeleton, and seamlessly cloning and connecting the three fragments to obtain a plasmid pHR _ POX 3; connecting the hrGFP gene expression cassette obtained by double digestion of the plasmid pHR _ AXP _ hrGFP by using the restriction enzymes SpeI and AvrII with the plasmid pHR _ POX3 which is also subjected to double digestion by using the restriction enzymes SpeI and AvrII to obtain pHR _ POX3_ hrGFP; wherein the primer sequences of the primers POX3-LF, POX3-LR, POX3-RF and POX3-RR are respectively shown as SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17 and SEQ ID NO: 18 is shown in the figure;
(3) pHR _ LIP1_ hrGFP and pCRISPRIyl _ LIP 1:
the pCRISPRyl _ LIP1 is obtained by using AvrII restriction endonuclease to perform single-enzyme linearization on plasmid pCRISPRyl and seamlessly cloning and connecting the plasmid pCRISPRyl with sgRNA fragment designed aiming at LIP1 site, wherein the nucleotide sequence of the sgRNA fragment designed aiming at LIP1 site is shown as SEQ ID NO:5 is shown in the specification;
the pHR _ LIP1_ hrGFP is a plasmid pHR _ LIP1 obtained by using a genome of yarrowia lipolytica as a template, respectively amplifying 1200bp at both ends of a sgRNA sequence at a LIP1 site by PCR (polymerase chain reaction) by using primer pairs LIP1-LF/LIP1-LR and LIP1-RF/LIP1-RR, and obtaining a plasmid basic skeleton by using a plasmid pHR _ AXP _ hrGFP as a template and performing PCR (polymerase chain reaction) amplification by using primer pairs plas-F and plas-R; the hrGFP gene expression cassette obtained by double digestion of the plasmid pHR _ AXP _ hrGFP with the restriction enzymes SpeI and AvrII was ligated to the plasmid pHR _ LIP1 similarly double digested with the restriction enzymes SpeI and AvrII to obtain pHR _ LIP1_ hrGFP; wherein the primer sequences of the primers LIP1-LF, LIP1-LR, LIP1-RF and LIP1-RR are respectively shown as SEQ ID NO: 27. SEQ ID NO: 28. SEQ ID NO: 29 and SEQ ID NO: 30 is shown in the figure;
(4) pHR _ POX4_ hrGFP and pCRISPRIyl _ POX 4:
the pCRISPRyl _ POX4 is obtained by using AvrII restriction endonuclease to perform single-enzyme linearization on a plasmid pCRISPRyl, and seamlessly cloning and connecting the plasmid pCRISPRyl with a sgRNA fragment designed aiming at a POX4 site, wherein the nucleotide sequence of the sgRNA fragment designed aiming at a POX4 site is shown as SEQ ID NO: 3 is shown in the specification;
the pHR _ POX4_ hrGFP is obtained by taking a genome of yarrowia lipolytica as a template, respectively amplifying 1200bp at both ends of a sgRNA sequence at a POX4 site by PCR (polymerase chain reaction) by using a primer pair POX4-LF/POX4-LR and POX4-RF/POX4-RR as a left homologous arm and a right homologous arm, taking a plasmid pHR _ AXP _ hrGFP as a template, and amplifying plas-F and plas-R by PCR by using a primer pair to obtain a plasmid basic skeleton, and seamlessly cloning and connecting the three fragments to obtain a plasmid pHR _ POX 4; connecting the hrGFP gene expression cassette obtained by double digestion of the plasmid pHR _ AXP _ hrGFP by using the restriction enzymes SpeI and AvrII with the plasmid pHR _ POX4 which is also subjected to double digestion by using the restriction enzymes SpeI and AvrII to obtain pHR _ POX4_ hrGFP; wherein the primer sequences of the primers POX4-LF, POX4-LR, POX4-RF and POX4-RR are respectively shown as SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21 and SEQ ID NO: 22;
(5) pHR _ E1_ hrGFP and pCRISPRIyl _ E1:
the pCRISPRyl _ E1 is constructed by using AvrII restriction endonuclease to perform single-enzyme linearization on plasmid pCRISPRyl and seamlessly cloning and connecting the plasmid pCRISPRyl with a sgRNA fragment designed aiming at the E1 site, wherein the nucleotide sequence of the sgRNA fragment designed aiming at the E1 site is shown as SEQ ID NO: 6 is shown in the specification;
the pHR _ E1_ hrGFP is obtained by taking the genome of yarrowia lipolytica as a template, respectively amplifying 1200bp at both ends of an sgRNA sequence of an E1 site by PCR (polymerase chain reaction) by using primer pairs E1-LF/E1-LR and E1-RF/E1-RR as a left homologous arm and a right homologous arm, taking a plasmid pHR _ AXP _ hrGFP as a template, amplifying plas-F and plas-R by PCR by using the primer pairs to obtain a basic plasmid skeleton, and seamlessly cloning and connecting the three fragments to obtain a plasmid pHR _ E1; the hrGFP gene expression cassette obtained by double digestion of the plasmid pHR _ AXP _ hrGFP with the restriction enzymes SpeI and AvrII was ligated to the plasmid pHR _ E1 similarly subjected to double digestion with the restriction enzymes SpeI and AvrII to obtain pHR _ E1_ hrGFP; wherein the primer sequences of the primers E1-LF, E1-LR, E1-RF and E1-RR are respectively shown in SEQ ID NO: 31. SEQ ID NO: 32. SEQ ID NO: 33 and SEQ ID NO: 34;
(6) pHR _ A1_ hrGFP and pCRISPRIyl _ A1:
the pCRISPRyl _ A1 is obtained by using AvrII restriction endonuclease to perform single-enzyme linearization on plasmid pCRISPRyl, and seamlessly cloning and connecting the plasmid pCRISPRyl with a sgRNA fragment designed aiming at A1 site, wherein the nucleotide sequence of the sgRNA fragment designed aiming at A1 site is shown in SEQ ID NO: 7 is shown in the specification;
pHR _ A1_ hrGFP is a plasmid pHR _ A1 obtained by taking the genome of yarrowia lipolytica as a template, respectively amplifying 1200bp at both ends of an sgRNA sequence at an A1 site by PCR (polymerase chain reaction) by using primer pairs A1-LF/A1-LR and A1-RF/A1-RR, and taking a plasmid pHR _ AXP _ hrGFP as a template, amplifying by PCR by using the primer pairs plas-F and plas-R to obtain a plasmid basic skeleton, and seamlessly cloning and connecting the three fragments; the hrGFP gene expression cassette obtained by double digestion of the plasmid pHR _ AXP _ hrGFP with the restriction enzymes SpeI and AvrII was ligated to the plasmid pHR _ A1 similarly subjected to double digestion with the restriction enzymes SpeI and AvrII to obtain pHR _ A1_ hrGFP; wherein the primer sequences of the primers A1-LF, A1-LR, A1-RF and A1-RR are respectively shown as SEQ ID NO: 35. SEQ ID NO: 36. SEQ ID NO: 37 and SEQ ID NO: 38;
(7) pHR _ B1_ hrGFP and pCRISPRIyl _ B1:
the pCRISPRyl _ B1 is obtained by using AvrII restriction endonuclease to perform single-enzyme linearization on plasmid pCRISPRyl, and seamlessly cloning and connecting the plasmid pCRISPRyl with a sgRNA fragment designed aiming at the B1 site, wherein the nucleotide sequence of the sgRNA fragment designed aiming at the B1 site is shown as SEQ ID NO: 8 is shown in the specification;
the pHR _ B1_ hrGFP is a plasmid pHR _ B1 obtained by using a genome of yarrowia lipolytica as a template, respectively amplifying 1200bp at both ends of a sgRNA sequence at a site B1 as left and right homologous arms by using a primer pair B1-LF/B1-LR, B1-RF1/B1-RR1 and B1-RF2/B1-RR2 through PCR, using a plasmid pHR _ AXP _ hrGFP as a template, and obtaining a plasmid basic skeleton by using a primer pair plas-F and plas-R through PCR amplification, wherein the four fragments are subjected to seamless cloning connection to obtain the plasmid pHR _ B1; the hrGFP gene expression cassette obtained by double digestion of the plasmid pHR _ AXP _ hrGFP with the restriction enzymes SpeI and AvrII was ligated to the plasmid pHR _ B1 similarly subjected to double digestion with the restriction enzymes SpeI and AvrII to obtain pHR _ B1_ hrGFP; wherein, the primer sequences of the primers B1-LF, B1-LR, B1-RF1, B1-RR1, B1-RF2 and B1-RR2 are respectively shown in SEQ ID NO: 39. SEQ ID NO: 40. SEQ ID NO: 41. SEQ ID NO: 42. SEQ ID NO: 43 and SEQ ID NO: 44 is shown;
(8) pHR _ A2_ hrGFP and pCRISPRIyl _ A2:
the pCRISPRyl _ A2 is obtained by using AvrII restriction endonuclease to perform single-enzyme linearization on plasmid pCRISPRyl, and seamlessly cloning and connecting the plasmid pCRISPRyl with a sgRNA fragment designed aiming at A2 site, wherein the nucleotide sequence of the sgRNA fragment designed aiming at A2 site is shown in SEQ ID NO: 9 is shown in the figure;
the pHR _ A2_ hrGFP is obtained by taking the genome of yarrowia lipolytica as a template, respectively amplifying 1200bp at both ends of an A2 site sgRNA sequence by using primer pairs A2-LF/A2-LR and A2-RF/A2-RR through PCR as left and right homologous arms, taking plasmid pHR _ AXP _ hrGFP as a template, obtaining a plasmid basic skeleton by using primer pairs plas-F and plas-R through PCR amplification, and obtaining a plasmid pHR _ A2 by seamlessly cloning and connecting the three fragments; the hrGFP gene expression cassette obtained by double digestion of the plasmid pHR _ AXP _ hrGFP with the restriction enzymes SpeI and AvrII was ligated to the plasmid pHR _ A2 similarly subjected to double digestion with the restriction enzymes SpeI and AvrII to obtain pHR _ A2_ hrGFP; wherein the primer sequences of the primers A2-LF, A2-LR, A2-RF and A2-RR are respectively shown in SEQ ID NO: 45. SEQ ID NO: 46. SEQ ID NO: 47 and SEQ ID NO: 48 is shown;
(9) pHR _ POX6_ hrGFP and pCRISPRIyl _ POX 6:
the pCRISPRyl _ POX6 is obtained by using AvrII restriction endonuclease to perform single-enzyme linearization on a plasmid pCRISPRyl, and seamlessly cloning and connecting the plasmid pCRISPRyl with a sgRNA fragment designed aiming at a POX6 site, wherein the nucleotide sequence of the sgRNA fragment designed aiming at a POX6 site is shown as SEQ ID NO: 4 is shown in the specification;
the pHR _ POX6_ hrGFP is obtained by taking a genome of yarrowia lipolytica as a template, respectively amplifying 1200bp at both ends of a sgRNA sequence at a POX6 site by PCR (polymerase chain reaction) by using a primer pair POX6-LF/POX6-LR and POX6-RF/POX6-RR as a left homologous arm and a right homologous arm, taking a plasmid pHR _ AXP _ hrGFP as a template, and amplifying plas-F and plas-R by PCR by using a primer pair to obtain a plasmid basic skeleton, and seamlessly cloning and connecting the three fragments to obtain a plasmid pHR _ POX 6; connecting the hrGFP gene expression cassette obtained by double digestion of the plasmid pHR _ AXP _ hrGFP by using the restriction enzymes SpeI and AvrII with the plasmid pHR _ POX6 which is also subjected to double digestion by using the restriction enzymes SpeI and AvrII to obtain pHR _ POX6_ hrGFP; wherein the primer sequences of the primers POX6-LF, POX6-LR, POX6-RF and POX6-RR are respectively shown as SEQ ID NO: 23. SEQ ID NO: 24. SEQ ID NO: 25 and SEQ ID NO: 26 is shown;
(10) pHR _ E2_ hrGFP and pCRISPRIyl _ E2:
the pCRISPRyl _ E2 is obtained by using AvrII restriction endonuclease to perform single-enzyme linearization on plasmid pCRISPRyl, and seamlessly cloning and connecting the plasmid pCRISPRyl with a sgRNA fragment designed aiming at the E2 site, wherein the nucleotide sequence of the sgRNA fragment designed aiming at the E2 site is shown as SEQ ID NO:10 is shown in the figure;
the pHR _ E2_ hrGFP is obtained by taking the genome of yarrowia lipolytica as a template, respectively amplifying 1200bp at both ends of an sgRNA sequence of an E2 site by PCR (polymerase chain reaction) by using primer pairs E2-LF/E2-LR and E2-RF/E2-RR as a left homologous arm and a right homologous arm, taking a plasmid pHR _ AXP _ hrGFP as a template, amplifying plas-F and plas-R by PCR by using the primer pairs to obtain a basic plasmid skeleton, and seamlessly cloning and connecting the three fragments to obtain a plasmid pHR _ E2; the hrGFP gene expression cassette obtained by double digestion of the plasmid pHR _ AXP _ hrGFP with the restriction enzymes SpeI and AvrII was ligated to the plasmid pHR _ E2 similarly subjected to double digestion with the restriction enzymes SpeI and AvrII to obtain pHR _ E2_ hrGFP; wherein the primer sequences of the primers E2-LF, E2-LR, E2-RF and E2-RR are respectively shown in SEQ ID NO: 49. SEQ ID NO: 50. SEQ ID NO: 51 and SEQ ID NO: shown at 52.
4. Use of the β -carotene producing yarrowia lipolytica genetically engineered bacterium of claim 1 or 2 in the preparation of β -carotene.
5. Use of the β -carotene producing yarrowia lipolytica genetically engineered bacterium of claim 1 or 2 in the preparation of carotenoids and derivatives thereof.
6. The use according to claim 5, wherein the carotenoid is zeaxanthin, astaxanthin, crocin, canthaxanthin or echinenone.
7. A method for producing β -carotene, characterized in that β -carotene is obtained by fermentation production of the β -carotene-producing yarrowia lipolytica genetically engineered bacterium of claim 1 or 2.
CN202010107322.1A 2020-02-21 2020-02-21 Yarrowia lipolytica gene engineering bacterium for producing β -carotene and application thereof Pending CN111321087A (en)

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CN114317307A (en) * 2021-12-30 2022-04-12 广州智特奇生物科技股份有限公司 Genetically engineered bacterium capable of improving astaxanthin biosynthesis yield and construction method and application thereof
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