CN117736895A - Recombinant yarrowia lipolytica capable of producing palmitoleic acid in high yield, and construction method and application thereof - Google Patents
Recombinant yarrowia lipolytica capable of producing palmitoleic acid in high yield, and construction method and application thereof Download PDFInfo
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- CN117736895A CN117736895A CN202311757991.3A CN202311757991A CN117736895A CN 117736895 A CN117736895 A CN 117736895A CN 202311757991 A CN202311757991 A CN 202311757991A CN 117736895 A CN117736895 A CN 117736895A
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- yarrowia lipolytica
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- promoter
- acyltransferase
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- 235000021319 Palmitoleic acid Nutrition 0.000 title claims abstract description 35
- SECPZKHBENQXJG-UHFFFAOYSA-N cis-palmitoleic acid Natural products CCCCCCC=CCCCCCCCC(O)=O SECPZKHBENQXJG-UHFFFAOYSA-N 0.000 title claims abstract description 35
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides recombinant yarrowia lipolytica capable of producing palmitoleic acid in high yield, and a construction method and application thereof, and belongs to the technical field of bioengineering. The recombinant yarrowia lipolytica is prepared by fermenting yarrowia lipolyticaYarrowia lipolytica) The peroxisome biogenesis factor 10 and triacylglycerol lipase 4 were knocked out in the genome and acetyl-coa carboxylase, Δ9 desaturase, acetyl-coa diacylglycerol acyltransferase, glycerol-3-phosphate acyltransferase, lysophosphatidylcholine acyltransferase, phosphatidylcholine were inserted: the diacylglycerol acyltransferase gene expression cassette. Experiments prove that the recombinant yarrowia lipolytica can be used for producing the palmitoleic acid by high-efficiency fermentation, and the high-efficiency synthesis of the natural product palmitoleic acid of plant sources in the yarrowia lipolytica is realized.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to recombinant yarrowia lipolytica capable of producing palmitoleic acid in high yield, and a construction method and application thereof.
Background
Palmitoleic acid (C16:1Δ9) is a monounsaturated fatty acid with a carbon chain length of 16, and is classified as an omega-7 fatty acid according to its double bond position. Palmitoleic acid, a specific fatty acid, has a variety of functional applications. Since the 20 th century, the increase in chronic diseases (e.g. obesity, cardiovascular diseases) has posed a major threat to human health. Many studies have linked these diseases to unhealthy diets, primarily by overintake of macronutrients such as sugar, protein and fat. Recent studies have demonstrated that palmitoleic acid is a key indicator of human fatty acids and may be closely associated with the development of a variety of diseases, including obesity, cardiovascular disease, and fatty liver. It is also widely used in the pharmaceutical field as an antithrombotic agent for preventing stroke. Because of its ability to inhibit melanin production, it is also commonly used in skin cleansers and has potential as an effective skin lightening agent.
At present, the synthesis of palmitoleic acid mainly depends on plant synthesis and microalgae production. However, there are a number of disadvantages to the use of plants to produce palmitoleic acid. First, these plants require relatively special climatic conditions, while the enormous site costs and lengthy growth periods introduce uncontrollable factors to the planting, such as climate change, artifacts, etc. The microalgae which is another production means also faces a plurality of problems, the gene operation of the microalgae is difficult, the metabolic transformation lifting space is not large, and meanwhile, the extraction of microalgae grease and the cost are also dilemma of industrial production. Compared with a plant extraction method, the microbial fermentation method has short growth cycle and can produce the palmitoleic acid all-weather, and simultaneously the oleaginous microorganism has high oil content and large biomass, so that the oleaginous microorganism is an economic and efficient way for producing palmitoleic acid. However, in the prior art, there is a lack of microorganisms capable of producing palmitoleic acid with high efficiency.
Disclosure of Invention
The invention aims to provide a recombinant yarrowia lipolytica capable of efficiently producing palmitoleic acid.
The invention also aims to provide a construction method of the recombinant yarrowia lipolytica, which is efficient and simple to operate.
It is a further object of the present invention to provide the use of said recombinant yarrowia lipolytica in the production of palmitoleic acid.
Recombinant yarrowia lipolytica with high palmitoleic acid yield is obtained by knocking out peroxisome biogenesis factor 10 and triacylglycerol lipase 4 in the genome of yarrowia lipolytica (Yarrowia lipolytica), and inserting acetyl-CoA carboxylase, delta 9 desaturase, acetyl-CoA diacylglycerol acyltransferase, glycerol-3-phosphate acyltransferase, lysophosphatidylcholine acyltransferase, phosphatidylcholine: the diacylglycerol acyltransferase gene expression cassette.
In the present invention, the acetyl-coa carboxylase gene is derived from yarrowia lipolytica (Yarrowia lipolytica); the delta 9 desaturase is obtained by optimizing a gene from caenorhabditis elegans by codons; glycerol-3-phosphoryl transferase, lysophosphatidylcholine acyltransferase, acetyl-coa diacylglycerol acyltransferase, and phosphatidylcholine: the diacylglycerol acyltransferase is obtained by codon-optimizing a gene derived from Hippophae rhamnoides (Sea buckthorn).
In the present invention, the Δ9 desaturase, glycerol-3-phosphoryl transferase, lysophosphatidylcholine acyltransferase, acetyl-coa diacylglycerol acyltransferase, phosphatidylcholine: the gene sequence of the diacylglycerol acyltransferase is shown in SEQ ID No.1-5 in sequence.
In the present invention, the Δ9 desaturase, acetyl-coa carboxylase, acetyl-coa diacylglycerol acyltransferase, glycerol-3-phosphate acyltransferase, lysophosphatidylcholine acyltransferase, phosphatidylcholine: the sites of integration of the diacylglycerol acyltransferase gene expression cassette in the yarrowia lipolytica (Yarrowia lipolytica) genome are in order: a08 site, intA, DGAT2, GPAT, LPAAT, PDAT.
In the present invention, the promoter of the expression cassette is any one of a TEF promoter, an hp4d promoter, a TEFin promoter, a POX2 promoter, or a GPDin promoter of yarrowia lipolytica; the terminator is any one of xpr2t terminator, mig1t terminator and lip2t terminator of yarrowia lipolytica.
In the present invention, P in the genome of the recombinant yarrowia lipolytica ELO1 Promoter site and/or P SCD The promoter site is also inserted with an endogenous copper ion inhibition promoter P CTR1 。
In the present invention, each gene expression cassette is inserted into the genome by a homologous recombination method, and the gene to be knocked out is knocked out by a homologous recombination method.
The invention also provides application of the recombinant yarrowia lipolytica in preparing palmitoleic acid.
In the present invention, the recombinant yarrowia lipolytica was fermented to produce palmitoleic acid using the following fermentation medium: 130-170g/L of glucose, 10-12g/L of ammonium sulfate, 2.5-3.5g/L of yeast extract, 0.085-0.105g/L of corn peptone, 3.5-4.5g/L of monopotassium phosphate, 1.8-2.2g/L of magnesium sulfate, 0.78-0.82g/L of calcium sulfate, 0.35-0.45g/L of sodium chloride, 10-14mg/L of ammonium sulfate hydrochloride, 0.85-1.15mg/L of biotin, 140-180mg/L of sodium molybdate, 0.18-0.22mg/L of copper sulfate, 38-42mg/L of boric acid, 160-200mg/L of manganese sulfate and 70-80mg/L of ferrous chloride.
In the present invention, glucose is fed during fermentation.
The beneficial effects are that: the recombinant yarrowia lipolytica (Yarrowia lipolytica) is based on knocking out the yarrowia lipolytica responsible for encoding the non-homologous recombinant gene ku70, so that the homologous recombination capacity of the yarrowia lipolytica is enhanced, and the genetic stability of the introduced gene can be greatly improved by realizing the integration of the genes through the homologous recombination function of the yarrowia lipolytica. The construction method of the recombinant yarrowia lipolytica is efficient and simple to operate. The recombinant yarrowia lipolytica (Yarrowia lipolytica) knocks out peroxisome biogenesis factor 10 (PEX 10 locus) and triacylglycerol lipase 4, overexpresses caenorhabditis elegans delta 9 desaturase, acetyl-CoA carboxylase, heterologous acetyl-CoA diacylglycerol acyltransferase, heterologous glycerol-3-phosphate acyltransferase, heterologous lysophosphatidylcholine acyltransferase, heterologous phosphatidylcholine: the diacylglycerol acyltransferase, experiments prove that the recombinant yarrowia lipolytica can be used for producing the palmitoleic acid by high-efficiency fermentation, and the high-efficiency synthesis of the natural product palmitoleic acid of plant sources in the yarrowia lipolytica is realized.
Drawings
Fig. 1 is a schematic diagram of palmitoleic acid synthesis strategy according to an embodiment of the present invention. ACC1: acetyl-coa carboxylase; DGAT2: diacylglycerol acyltransferase; PDAT: phosphatidylcholine: diacylglycerol acyltransferase; ELO1: fatty acid elongase 1; ELO2: fatty acid elongase 2; FAD2: Δ12 oleic acid desaturase; FAS: fatty acid elongase; TGL4: triacylglycerol lipase 4; GPAT: 3-phosphoglycerol acyltransferase; LPAAT: lysophosphatidic acid acyltransferase; LPCAT: lysophosphatidylcholine acyltransferase; CPT: choline phosphotransferase.
FIG. 2 is a block diagram of recombinant plasmid pUC-Leu-A08-FAT5, wherein A08-up represents homology arm A08-up upstream of the start codon of A08 site, A08-dm represents homology arm A08-dm downstream of the stop codon of A08 site, and TEFin represents promoter P TEFin Xpr2T represents terminator T xpr2t LEU represents a 3 (beta) -isopropyl malate dehydrogenase encoding gene expression cassette, and FAT5 is a Δ9 desaturase from caenorhabditis elegans.
FIG. 3 is a recombinant plasmid pUC-HUH-P ELO1 -P CTR1 Wherein P is as follows ELO1 -up represents P ELO1 Site upstream homology arm, P ELO1 -dm represents P ELO1 Downstream homology arm of the site, URA represents orotidine-5' -phosphate decarboxylase encoding gene expression cassette (comprising Yarrowia lipolytica endogenous promoter P TEFin MilkNucleotide-5' -phosphate decarboxylase and terminator T xpr2t ) CTR1 is P CTR1 Genes (yarrowia lipolytica endogenous copper ion inhibition promoter).
FIG. 4 is a structural diagram of a recombinant plasmid pUC-HUH-GPAT-SbGPAT, wherein GPAT-up represents a homology arm upstream of the start codon of the GPAT site, GPAT-dm represents a homology arm downstream of the stop codon of the SbGPAT site, and GPDin represents a promoter P GPDin Mig1T represents terminator T mig1t URA represents the expression cassette of the gene encoding orotidine-5' -phosphate decarboxylase (comprising promoter P endogenous to Yarrowia lipolytica) TEFin Orotidine-5' -phosphate decarboxylase and terminator T xpr2t ) SbGPAT is Hippophae rhamnoides glycerol-3-phosphate acyltransferase.
FIG. 5 is a block diagram of recombinant plasmid pUC-HUH-TGL4, wherein TGL4-up represents the homology arm upstream of the start codon of TGL4 site, TGL4-down represents the homology arm downstream of the stop codon of TGL4 site, URA represents the orotidine-5' -phosphate decarboxylase encoding gene expression cassette (comprising Yarrowia lipolytica endogenous promoter P TEFin Orotidine-5' -phosphate decarboxylase and terminator T xpr2t )。
FIG. 6 is a chart showing the analysis of fatty acid composition in example 3 of the present invention.
Detailed Description
The invention is further illustrated by the following examples.
The experimental methods used in the examples below are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Yarrowia lipolytica (Yarrowia lipolytica) Po1f, available from American type culture Collection under the accession number ATCC MYA-2613.
Yarrowia lipolytica (Yarrowia lipolytica) Po1fΔku70 (MatA, Δku70:: hisG, leu2-270, ura3-302, xpr2-322, axp 1-2), abbreviated as Yarrowia lipolytica Po fΔku70.Yarrowia lipolytica Po1 f.DELTA.ku70 was constructed from yarrowia lipolytica (Yarrowia lipolytica) Po1f with the coding gene ku70 responsible for non-homologous recombination knocked out (disclosed in KretzschmarA, et al, current Genetics,2013,59 (1-2): 63-72).
P SCD The site-integrating plasmid was obtained by introducing P on chromosome C in the Yarrowia lipolytica Po f.DELTA.ku70 genome SCD The sequence of size 2059bp upstream of the start codon of the promoter (GenBank accession number: XP_ 501496) (upstream homology arm) was inserted into the EcoRI cleavage site of the pUC57-hisG-Ura-hisG vector (construction method see example 1), and P on chromosome C in the Yarrowia lipolyticaPo fΔku70 genome was digested with EcoRI SCD The sequence (downstream homology arm) with the size of 2019bp downstream of the stop codon of the site promoter is inserted into the HindIII enzyme cutting site of the pUC57-hisG-Ura-hisG vector, and two hisG tag coding genes are positioned between the upstream homology arm and the downstream homology arm of the promoter site.
P ELO1 The site-integrating plasmid was obtained by introducing P on chromosome F in the Yarrowia lipolytica Po fΔku70 genome ELO1 The sequence (upstream homology arm) of 1501bp upstream of the start codon of the site (GenBank accession number: XM_ 505094.1) was inserted into the EcoRI cleavage site of the pUC57-hisG-Ura-hisG vector, and P on chromosome F in the Yarrowia lipolytica Po fΔku70 genome was introduced ELO1 The sequence (downstream homology arm) with the size 1501bp downstream of the site termination codon is inserted into the HindIII enzyme cutting site in the pUC57-hisG-Ura-hisG vector, and two hisG tag coding genes are positioned between the upstream homology arm and the downstream homology arm of the promoter site.
The GPAT site integration plasmid was obtained by inserting a sequence of 1500bp upstream of the start codon of the GPAT site (GenBank accession number: XM_ 501275.1) in the genome of Yarrowia lipolytica Po fΔku70 (upstream homology arm GPAT-up) into the HindIII cleavage site of the pUC57-hisG-Ura-hisG vector, inserting a sequence of 1500bp downstream of the stop codon of the GPAT site in the genome of Yarrowia lipolytica Po fΔku70 (downstream homology arm GPAT-dm) into the EcoRI cleavage site of the pUC57-hisG-Ura-hisG vector, and inserting two hisG tag-encoding genes between the upstream and downstream homology arms.
The LPAAT site integrating plasmid was prepared by inserting a sequence of 1500bp upstream of the start codon of the LPAAT site (GenBank accession number: XM_ 504127.1) in the genome of Yarrowia lipolytica Po fΔku70 (upstream homology arm LPAAT-up) into the KpnI cleavage site in the pUC57-hisG-Ura-hisG vector, inserting a sequence of 1500bp downstream of the stop codon of the LPAAT site in the genome of Yarrowia lipolytica Po fΔku70 (downstream homology arm LPAAT-dm) into the HindIII cleavage site in the pUC57-hisG-Ura-hisG vector, and inserting two hisG tag encoding genes between the downstream homology arms.
The PDAT site integrating plasmid was obtained by inserting a 1666bp sequence (upstream homology arm) upstream of the start codon of the PDAT site (GenBank accession No.: XM_ 504038.1) on chromosome E in the Yarrowia lipolytica Po fΔku70 genome into the EcoRI cleavage site of the pUC57-hisG-Ura-hisG vector, and inserting a 1666bp sequence (downstream homology arm) downstream of the stop codon of the PDAT site on chromosome E in the Yarrowia lipolytica Po fΔku70 genome into the PacI cleavage site of the pUC57-hisG-Ura-hisG vector, with two hisG tag encoding genes between the upstream homology arms.
The DGAT2 site integration plasmid was obtained by inserting a sequence of 1500bp upstream of the start codon of the DGAT2 site (GenBank accession number: XM_ 504700.1) in the genome of Yarrowia lipolytica Po fΔku70 at the KpnI cleavage site in the pUC57-hisG-Ura-hisG vector, and inserting a sequence of 1486bp downstream of the stop codon of the DGAT2 site in the genome of Yarrowia lipolytica Po fΔku70 (downstream homology arm) in the genome of pUC57-hisG-Ura-hisG vector between the two hisG tag encoding genes at the sites.
The PEX10 site-integrating plasmid was obtained by inserting a sequence (upstream homology arm) of 2250bp upstream of the initiation codon of the PEX10 site (GenBank accession number: XM_ 501311.1) on chromosome C in the Yarrowia lipolytica Po fΔku70 genome into the HindIII cleavage site in the pUC57-hisG-Ura-hisG vector, and inserting a sequence (downstream homology arm) of 1500bp downstream of the termination codon of the PEX10 site on chromosome C in the Yarrowia lipolytica Po fΔku70 genome into the EcoRI cleavage site in the pUC57-hisG-Ura-hisG vector, with the two hisG tag-encoding genes between the downstream homology arms on the PEX10 site.
The TGL4 site-integrating plasmid was obtained by inserting a sequence (upstream homology arm) of 1500bp upstream of the initiation codon of the TGL4 site (GenBank accession number: XM_ 505230.1) on chromosome F in the Yarrowia lipolytica Po fΔku70 genome into the pUC57-hisG-Ura-hisG vector at the PacI cleavage site, and inserting a sequence (downstream homology arm) of 1500bp downstream of the termination codon of the TGL4 site on chromosome F in the Yarrowia lipolytica Po fΔku70 genome into the pUC57-hisG-Ura-hisG vector at the EcoRI cleavage site, and the two hisG tag-encoding genes were located between the upstream and downstream homology arms of the TGL4 site.
The IntA site integration plasmid was obtained by inserting a 1633bp sequence upstream of the start codon (upstream homology arm) in the IntA site (GenBank accession number: CR 382127.1) on chromosome A in the Yarrowia lipolytica Po fΔku70 genome into the EcoRI cleavage site in the pUC57-hisG-Ura-hisG vector, and inserting a 1621bp sequence downstream of the stop codon (downstream homology arm) in the IntA site on chromosome A in the Yarrowia lipolytica Po fΔku70 genome into the HindIII cleavage site in the pUC57-hisG-Ura-hisG vector, with the two hisG tag-encoding genes between the IntA site and the downstream homology arm.
The A08 site integrating plasmid was obtained by inserting a sequence (upstream homology arm) of 2521bp upstream of the start codon of the A08 site (GenBank accession number: CP 096871.1) in the chromosome of Yarrowia lipolytica Po fΔku70 genome into the EcoRI cleavage site of pUC57-Leu vector (construction method see example 1), and inserting a sequence (downstream homology arm) of 2031bp downstream of the stop codon of the A08 site in the chromosome of Yarrowia lipolytica Po fΔku70 genome into the HindIII cleavage site of pUC57-Leu vector (construction method see example 1), the Leu coding gene being located between the upstream and downstream homology arms of the A08 site.
Example 1 amplification of Gene elements and preparation of plasmid of interest
Preparation of the target Gene
The optimized Δ9 desaturase gene FAT5 (SEQ ID No: 1) was synthesized by the Toxose Biotechnology Co., ltd. After codon optimization based on the nucleotide sequence of Δ9 desaturase (FAT 5, genBank No: AAF 97548.1) from caenorhabditis elegans (Caenorhabditis elegans) provided on NCBI, and inserted between the HindI and EcoRI cleavage sites of plasmid pUC57 (purchased from GenScript Co.) to obtain plasmid pUC57-FAT5.
The optimized glycerol-3-phosphate acyltransferase gene SbGPAT (SEQ ID No: 2) was synthesized by the Kirschner Biotechnology Co., ltd. After codon optimization based on the nucleotide sequence of the glycerol-3-phosphate acyltransferase gene (Ding J, et al BMC plant biology,2019,19 (1): 1-18) from Hippophae rhamnoides (Sea buckthrorn) and inserted between HindI and EcoRI cleavage sites of plasmid pUC57 to obtain plasmid pUC57-SbGPAT.
The nucleotide sequence of the lysophosphatidylcholine acyltransferase gene (Ding J, et al BMC plant biology,2019,19 (1): 1-18) from Sea buckthorn fruit (Sea buckthorn) provided in the literature was subjected to codon optimization, and the optimized lysophosphatidylcholine acyltransferase encoding gene SbLPAAT (SEQ ID No: 3) was synthesized by the company of Style Biotechnology, and inserted between HindI and EcoRI cleavage sites of the plasmid pUC57 to obtain the plasmid pUC57-SbLPAAT.
The nucleotide sequence of acetyl CoA diacylglycerol acyltransferase (Ding J, et al BMC plant biology,2019,19 (1): 1-18) from Sea buckthorn fruit (Sea buckthorn) provided in the literature is subjected to codon optimization, and the optimized coding gene SbDGAT2 (SEQ ID No: 4) of acetyl CoA diacylglycerol acyltransferase is synthesized by the company of Kirschner Biotech Co., ltd.) and inserted between HindIII and PacI cleavage sites of the plasmid pUC57, thereby obtaining the plasmid pUC57-SbDGAT2.
Phosphatidylcholine from seabuckthorn fruit (Sea buckthorm) as provided in the literature: nucleotide sequences of diacylglycerol acyltransferases (Ding J, et al BMC plant biology,2019,19 (1): 1-18) were codon optimized and the optimized phosphatidylcholine was synthesized by the company Style Biotechnology Co., ltd: the diacylglycerol acyltransferase encoding gene SbPDAT (SEQ ID No: 5) was inserted between the HindIII and PacI cleavage sites of plasmid pUC57 to obtain plasmid pUC57-SbPDAT.
Orotidine-5' -phosphate decarboxylase according to Yarrowia lipolytica provided on NCBINucleotide sequences encoding Gene Ura (GenBank accession number: AJ 306421.1) and a hisG tag (GenBank accession number: AF 324729.1), were synthesized by Suzhou Jinweizhi biotechnology Co., ltd, two hisG tag-encoding gene sequences were inserted between HindIII and PacI cleavage sites of plasmid pUC57 (from Jinweizhi Co., ltd.), and a orotidine-5' -phosphate decarboxylase-encoding gene expression cassette (consisting of Yarrowia lipolytica endogenous promoter P) was inserted between the two hisG tag-encoding gene sequences TEFin Orotidine-5' -phosphate decarboxylase encoding gene Ura and terminator T xpr2t Composition, abbreviated as P TEFin -URA-T xpr2t ) So as to achieve the recovery of the Ura marker, the plasmid pUC57-hisG-Ura-hisG was obtained.
The synthesis was delegated from the Biotechnology Co.Ltd. Of Suzhou gold, based on the nucleotide sequence of the gene Leu encoding 3 (. Beta. -isopropyl malate dehydrogenase gene Yarrowia lipolytica provided at NCBI (GenBank accession number: CP 061014.1). The 3 (. Beta. -isopropyl malate dehydrogenase-encoding gene expression cassette (from promoter P endogenous to Yarrowia lipolytica) was inserted between KpnI and PacI cleavage sites of plasmid pUC57 TEFin 3 (beta) -isopropyl malate dehydrogenase encoding gene Leu and terminator T xpr2t Composition), plasmid pUC57-Leu was obtained.
Acc1 (GenBank accession number: YALI0C11407 g) was amplified using Yarrowia lipolytica Po fDeltaku 70 genomic DNA as a template and the primers IntA:: ACC1-F and IntA:: ACC 1-R.
Using Yarrowia lipolytica Po f delta ku70 genome DNA as a template and P ELO1 ::P CTR1 -F and P ELO1 ::P CTR1 R is a primer, and a copper ion inhibition promoter CTR1 (GenBank accession number: YALI0B10153 g) is amplified.
Yarrowia lipolytica endogenous promoter P hp4d ，P TEFin ，P GPDin And endogenous terminator T xpr2t The nucleotide sequence of (a) is described in "Wang, kaifeng, et al, ACSSpotential biology 11.4 (2022): 1542-1554".
Construction of recombinant plasmid
The structure of the recombinant plasmid is shown in Table 1 and FIGS. 2-5; the primers used to construct the recombinant plasmids are shown in Table 2.
1. Construction of recombinant plasmid pUC-leu-A08-FAT5
The recombinant plasmid pUC-Leu-A08-FAT5 takes pUC57-Leu as a framework, and is inserted with an A08 site initiation codon upstream homology arm A08-up and a termination codon downstream homology arm A08-dm in Yarrowia lipolytica Po fDeltaku 70, and a FAT5 expression cassette (P TEFin -FAT5-T xpr2t ) LEU expression cassette (comprising promoter P endogenous to Yarrowia lipolytica TEFin 3 (beta) -isopropyl malate dehydrogenase encoding gene Leu and terminator T xpr2t ) Also between the upstream and downstream homology arms, see FIG. 2 for specific structure. Wherein FAT5 is a Δ9 desaturase from caenorhabditis elegans. In the present invention, the expression cassette is expressed as a promoter-gene of interest-terminator, for example, expression cassette P TEFin -FAT5-T xpr2t ，P TEFin Is a promoter, FAT5 is a target gene, T xpr2t Is a terminator.
P is shown in A08: TEFin f and A08:: P TEFin -R is a primer, yarrowia lipolytica Po f delta ku70 genome DNA is used as a template, and FAT5 expression cassette promoter P is amplified TEFin . A08 is T xpr2t F and A08:: T xpr2t R is a primer, yarrowia lipolytica Po f delta ku70 genome DNA is used as a template, and FAT5 expression cassette terminator T is amplified xpr2t 。
Plasmid pUC57-FAT5 is used as a template, A08:: FAT5-F and A08:: FAT5-R are used as primers, and the two amplified ends are respectively provided with a promoter P TEFin And terminator T xpr2t FAT5 gene of homology arm.
The PCR amplification system is as follows:
| component (A) | Volume of |
| PrimerSTAR Max Premix | 25μl |
| Template | 1μl |
| Primer 1 | 2μl |
| Primer 2 | 2μl |
| Distilled water | 20μl |
PrimerSTAR Max Premix is available from Bao Ri doctor Material technology (Beijing) Inc.
The procedure for the PCR was as follows: denaturation at 98℃for 10s, annealing at 55℃for 5s, extension at 72℃ (extension time=target fragment length/1 kb, unit min), 30 cycles were repeated.
Each fragment was recovered by purification with TaKaRa MiniBEST DNA Fragment Purification Kit (available from Shanghai Baisai Biotechnology Co., ltd.).
After cleavage of the A08 site integrating plasmid with NEB restriction enzyme SnaBI, the linearized A08 site integrating plasmid was recovered by agarose gel electrophoresis.
Each element (promoter P) in the FAT5 gene expression cassette constructed by linearization of the A08 site integration plasmid and title 1 of this example TEFin Gene FAT5 and terminator T xpr2t ) One-step cloning was achieved using ClonExpress MultiS One Step Cloning Kit from the biological technology limited company of nanking nuozhen, inserting the FAT5 gene expression cassette between the upstream and downstream homology arms of the a08 locus integrating plasmid. The reaction system is shown in the following table. After the reaction system was incubated at 37℃for 30min, a circular recombinant vector was obtained.
The system of the one-step clone is shown in the following table:
wherein the usage of linearizing vector (x), insert (y=sum of the optimal usage of all inserts) can be calculated by the following formula: optimal amount of used per fragment or linearized vector= [0.02 x base pair number of fragment or linearized vector ] ng.
The circular recombinant vector is transformed into escherichia coli DH5 alpha competent cells, and positive recombinant plasmid pUC-leu-A08-FAT5 is obtained through screening of ampicillin resistance plates and colony PCR and sequencing verification.
The plasmid pUC-leu-A08-FAT5 was digested with NEB restriction enzyme NotI, and the linearized pUC-leu-A08-FAT5 plasmid was recovered by agarose gel electrophoresis.
2. Recombinant plasmid pUC-HUH-P ELO1 -P CTR1 Construction of (3)
Recombinant plasmid pUC-HUH-P ELO1 -P CTR1 pUC57-hisG-Ura-hisG is used as a skeleton, and P in Yarrowia lipolytica Po fDeltaku 70 is inserted ELO1 Site upstream homology arm P ELO1 -up and downstream homology arm P ELO1 Dm, P is also inserted between the downstream homology arm and the hisG tag encoding gene CTR1 Gene, two hisG tag coding gene sequences and orotidine-5' -phosphate decarboxylase coding gene expression cassette are also located in the upstream homology arm P ELO1 -up and downstream homology arm P ELO1 Between dm, the specific structure is shown in FIG. 3.
Using Yarrowia lipolytica Po f delta ku70 genome DNA as a template and P ELO1 ::P CTR1 -F and P ELO1 ::P CTR1 R is a primer, and the amplified ends are respectively provided with P ELO1 P of homology arm of genes encoding dm and hisG tag CTR1 And (3) a gene.
Will P ELO1 Site integration plasmid was restricted by NEB companyAfter cleavage with the endoprotease HindIII, the linearized pUC-HUH integrative plasmid was recovered by agarose gel electrophoresis.
Will linearize P ELO1 Site integration plasmid and the title 2 construct of this example have P at each end ELO1 P of homology arm of genes encoding dm and hisG tag CTR1 Gene was cloned in one step using ClonExpress One Step Cloning Kit from Nanjinouzan Biotechnology Co., ltd, and P was isolated CTR1 Insertion of Gene into P ELO1 And (3) integrating the sites between the upstream and downstream homologous arms of the plasmid to obtain a circular recombinant vector.
Transforming the circular recombinant vector into escherichia coli DH5 alpha competent cells, screening by an ampicillin resistance flat plate, and obtaining a positive recombinant plasmid pUC-HUH-P through colony PCR and sequencing verification ELO1 -P CTR1 。
Plasmid pUC-HUH-P was subjected to restriction enzyme BstAPI from NEB company ELO1 -P CTR1 After enzyme digestion, the agarose gel electrophoresis gel is used for recovering linearized pUC-HUH-P ELO1 -P CTR1 A plasmid.
3. Recombinant plasmid pUC-HUH-P SCD -P CTR1 Construction of (3)
Recombinant plasmid pUC-HUH-P SCD -P CTR1 pUC57-hisG-Ura-hisG is used as a skeleton, and P in Yarrowia lipolytica Po fDeltaku 70 is inserted SCD Site upstream homology arm P SCD -up and downstream homology arm P SCD Dm, P is inserted between the downstream homology arm and the hisG tag encoding gene sequence CTR1 The gene, two hisG tag encoding gene sequences and the orotidine-5' -phosphate decarboxylase encoding gene expression cassette are also between the upstream homology arm and the downstream homology arm. Recombinant plasmid pUC-HUH-P SCD -P CTR1 The structure of (a) is the same as that of FIG. 3, but is different from that of P SCD Site homology arm substitution P ELO1 Site homology arms.
Using Yarrowia lipolytica Po f delta ku70 genome DNA as a template and P SCD ::P CTR1 -F and P SCD ::P CTR1 R is a primer, and the amplified ends are respectively provided with P ELO1 P of homology arm of genes encoding dm and hisG tag CTR1 And (3) a gene.
Will P SCD After the site-integrating plasmid was digested with NEB restriction enzyme HindIII, the linearized P was recovered by agarose gel electrophoresis SCD Site integration plasmid.
P to be linearized SCD Site integration plasmid and P constructed in the title 3 of this example with homology arms at both ends CTR1 Gene was cloned in one step using ClonExpress One Step Cloning Kit from Nanjinouzan Biotechnology Co., ltd, and P was isolated CTR1 Insertion of Gene into P SCD And (3) integrating a downstream homology arm of the plasmid with the hisG tag coding gene to obtain a circular recombinant vector.
Transforming the circular recombinant vector into E.coli DH5 alpha competent cells, screening by an ampicillin resistance plate, and obtaining a positive recombinant plasmid pUC-HUH-P through colony PCR and sequencing verification SCD -P CTR1 。
Plasmid pUC-HUH-P was subjected to restriction enzyme BstAPI from NEB company SCD -P CTR1 After enzyme digestion, the agarose gel electrophoresis gel is used for recovering linearized pUC-HUH-P SCD -P CTR1 A plasmid.
4. Construction of recombinant plasmid pUC-HUH-GPAT-SbGPAT
pUC-HUH-GPAT-SbGPAT uses pUC57-hisG-Ura-hisG as skeleton, and inserts GPAT-up of GPAT site initiation codon upstream homology arm GPAT-up and GPAT-dm of termination codon downstream homology arm in Yarrowia lipolytica Po fΔku70, and also inserts SbGPAT gene expression cassette (P) GPDin -SbGPAT-T mig1t ) URA expression cassette (comprising promoter P endogenous to Yarrowia lipolytica TEFin Orotidine-5' -phosphate decarboxylase and terminator T xpr2t ) Also between the upstream and downstream homology arms. The structure of the recombinant plasmid pUC-HUH-GPAT-SbGPAT is shown in FIG. 4.
With GPAT:: P GPDin F and GPAT:: P GPDin R is a primer, yarrowia lipolytica Po fΔku70 genome DNA is used as a template, and SbGPAT expression cassette promoter P is amplified GPDin . By GPAT:: T mig1t F and GPAT:: T mig1t R is a primer, and R is a primer,amplifying the terminator T of the SbGPAT expression cassette by taking Yarrowia lipolytica Po fDeltaku 70 genome DNA as a template mig1t 。
The plasmid pUC57-SbGPAT is used as a template, GPDin:: sbGPAT-F and GPDin::: sbGPAT-R are used as primers, and the two amplified ends are respectively provided with a promoter P GPDin And terminator T mig1t The SbGPAT gene of the homology arm.
The linearized GPAT plasmid was recovered by agarose gel electrophoresis after cleavage of the GPAT site-integrating plasmid with NEB restriction enzyme PacI.
The linearized GPAT site integrating plasmid and the elements (P) in the SbGPAT gene expression cassette constructed in title 4 of this example GPDin Gene SbGPAT and terminator T mig1t ) By Nanjinouzan biotechnology Co.LtdIIOne Step Cloning Kit one-step cloning was performed to obtain a circular recombinant vector.
The circular recombinant vector is transformed into escherichia coli DH5 alpha competent cells, and positive recombinant plasmid pUC-HUH-GPAT-SbGPAT is obtained through screening of ampicillin resistance plates and colony PCR and sequencing verification.
The linearized pUC-HUH-GPAT-SbGPAT plasmid was recovered by agarose gel electrophoresis after cleavage of the plasmid pUC-HUH-GPAT-SbGPAT with the restriction enzyme NotI from NEB.
5. Construction of recombinant plasmid pUC-HUH-LPAAT-SbLPAAT
The recombinant plasmid pUC-HUH-LPAAT-SbLPAAT uses pUC57-hisG-Ura-hisG as skeleton, and inserts the upstream homology arm LPAAT-up of LPAAT site initiation codon and downstream homology arm LPAAT-dm of termination codon in Yarrowia lipolytica Po fΔku70, and also inserts SbLPAAT gene expression cassette (P) TEFin -SbLPAAT-T xpr2t ) Orotidine-5' -phosphate decarboxylase encoding gene expression cassette (comprising Yarrowia lipolytica endogenous promoter P TEFin Orotidine-5' -phosphate decarboxylase and terminator T xpr2t ) Also between the upstream and downstream homology arms. Recombinant plasmid pUCThe block diagram of HUH-LPAAT-Sb LPAAT is identical to FIG. 4, except that GPAT-up and GPAT-dm are replaced with LPAAT-up and LPAAT-dm, respectively, and the SbGPAT gene expression cassette is replaced with the SbLPAAT gene expression cassette.
P is shown in LPAAT-up: TEFin f and LPAAT-up:: P TEFin R is a primer, yarrowia lipolytica Po f delta ku70 genome DNA is used as a template, and SbLPAAT gene expression cassette promoter P is amplified TEFin . The LPAAT-dm is:: T xpr2t F and LPAAT-dm::: T xpr2t R is a primer, yarrowia lipolytica Po fDeltaku 70 genome DNA is used as a template, and SbLPAAT expression cassette terminator T is amplified xpr2t 。
Using Yarrowia lipolytica Po f delta ku70 genome DNA as a template and P TEFin SbLPAAT-F and T xpr2t SbLPAAT-R as primer, and amplified two ends with promoter P TEFin And terminator T xpr2t The SbLPAAT gene of the homology arm.
After cleavage of the LPAAT site-integrating plasmid with NEB restriction enzyme PacI, the linearized LPAAT plasmid was recovered by agarose gel electrophoresis.
The linearized LPAAT site integrating plasmid and the elements (P) in the SbLPAAT gene expression cassette constructed in title 5 of this example TEFin SbLPAAT gene and terminator T xpr2t ) By Nanjinouzan biotechnology Co.LtdIIOne Step Cloning Kit one-step cloning was performed to obtain a circular recombinant vector.
Transforming the circular recombinant vector into escherichia coli DH5 alpha competent cells, screening by an ampicillin resistance flat plate, and obtaining positive recombinant plasmid by colony PCR and sequencing verification
pUC-HUH-LPAAT-SbLPAAT。
The linearized pUC-HUH-LPAAT-SbLPAAT plasmid was recovered by agarose gel electrophoresis after cleavage of the plasmid pUC-HUH-LPAAT-SbLPAAT with the restriction enzyme NotI from NEB.
6. Construction of recombinant plasmid pUC-HUH-DGAT2-SbDGAT2
The recombinant plasmid pUC-HUH-DGAT2-SbDGAT2 uses pUC57-hisG-Ura-hisG as skeleton, and the upstream homology arm DGAT2-up of DGAT2 site initiation codon and downstream homology arm DGAT2-dm of termination codon in Yarrowia lipolytica Po fΔku70 are inserted, and between said downstream homology arm and hisG tag coding gene the SbDGAT2 gene expression cassette (P TEFin -SbDGAT2-T xpr2t ) Orotidine-5' -phosphate decarboxylase encoding gene expression cassette (comprising Yarrowia lipolytica endogenous promoter P TEFin Orotidine-5' -phosphate decarboxylase and terminator T xpr2t ) Also between the upstream and downstream homology arms. The construction of the recombinant plasmid pUC-HUH-DGAT2-SbDGAT2 is the same as that of FIG. 4, except that DGAT2-up and DGAT2-dm are used to replace GPAT-up and GPAT-dm, respectively, and the SbDGAT2 gene expression cassette is used to replace the SbGPAT gene expression cassette.
With DGAT2:: P TEFin F and DGAT2:: P TEFin R is a primer, yarrowia lipolytica Po f delta ku70 genome DNA is used as a template, and SbDGAT2 gene expression cassette promoter P is amplified TEFin . With DGAT2:: T xpr2t F and DGAT 2::: T xpr2t R is a primer, yarrowia lipolytica Po f delta ku70 genome DNA is used as a template, and SbDGAT2 gene expression cassette terminator T is amplified xpr2t 。
Using Yarrowia lipolytica Po f delta ku70 genome DNA as a template and P TEFin SbDGAT2-F and T xpr2t SbDGAT2-R as primer, and the amplified ends are respectively provided with a promoter P TEFin And terminator T xpr2t The SbDGAT2 gene of the homology arm.
The linearized DGAT2 site integrating plasmid was recovered by digestion of the DGAT2 site integrating plasmid with NEB restriction enzyme HindIII, agarose gel electrophoresis gel.
Each element (promoter P) in the SbDGAT2 gene expression cassette constructed by linearizing the DGAT2 site integration plasmid and title 6 of this example TEFin SbDGAT2 gene and terminator T xpr2t ) One-step cloning was achieved using ClonExpress MultiS One Step Cloning Kit from the biological technology limited company of nanking nuozhen to obtain a circular recombinant vector.
Transforming the circular recombinant vector into escherichia coli DH5 alpha competent cells, screening by an ampicillin resistance flat plate, and obtaining positive recombinant plasmid by colony PCR and sequencing verification
pUC-HUH-DGAT2-SbDGAT2。
The linearized pUC-HUH-DGAT2-SbDGAT2 plasmid was recovered by agarose gel electrophoresis after cleavage of the plasmid pUC-HUH-DGAT2-SbDGAT2 with NEB restriction enzyme EcoRI.
7. Construction of recombinant plasmid pUC-HUH-IntA-ACC1
The recombinant plasmid pUC-HUH-IntA-ACC takes pUC57-hisG-Ura-hisG as a framework, and is inserted with an IntA-up upstream homology arm of an IntA site initiation codon and an IntA-dm downstream homology arm of a termination codon in Yarrowia lipolytica Po fDeltaku 70, and an ACC1 gene expression cassette (P hp4d -ACC1-T mig1t ) Orotidine-5' -phosphate decarboxylase encoding gene expression cassette (comprising Yarrowia lipolytica endogenous promoter P TEFin Orotidine-5' -phosphate decarboxylase and terminator T xpr2t ) Also between the upstream and downstream homology arms. The construction of the recombinant plasmid pUC-HUH-IntA-ACC1 is the same as that of FIG. 4, except that the GPAT-up and GPAT-dm are replaced with IntA-up and IntA-dm, respectively, and the GPAT gene expression cassette is replaced with the ACC1 gene expression cassette.
P is shown in IntA:: hp4d f and IntA:: P hp4d R is a primer, yarrowia lipolytica Po f delta ku70 genome DNA is used as a template, and ACC1 gene expression cassette promoter P is amplified hp4d . Let IntA be T mig1t F and IntA:: T mig1t R is a primer, and Yarrowia lipolytica Po f delta ku70 genome DNA is used as a template to amplify ACC1 expression cassette terminator T mig1t 。
The Yarrowia lipolytica Po F delta ku70 genome DNA is used as a template, intA:: ACC1-F and IntA::: ACC1-R are used as primers, and the two ends of the amplified DNA are respectively provided with a promoter P hp4d And terminator T mig1t ACC1 gene of homology arm.
The linearized IntA site-integrating plasmid was recovered by agarose gel electrophoresis after cleavage of the IntA site-integrating plasmid with NEB restriction enzyme PacI.
Integration of linearized IntA site into plasmidAnd each element (promoter P) in the ACC1 gene expression cassette constructed in title 7 of this example hp4d ACC1 Gene and terminator T mig1t ) One-step cloning was achieved using ClonExpress MultiS One Step Cloning Kit from the biological technology limited company of nanking nuozhen, and the ACC1 gene expression cassette was inserted between the upstream and downstream homology arms of the IntA site integrating plasmid to obtain a circular recombinant vector.
The circular recombinant vector is transformed into escherichia coli DH5 alpha competent cells, and positive recombinant plasmid pUC-HUH-IntA-ACC1 is obtained through screening of ampicillin resistance plates and colony PCR and sequencing verification.
The linearized pUC-HUH-IntA-ACC plasmid was recovered by agarose gel electrophoresis after cleavage of the plasmid pUC-HUH-IntA-ACC1 with NEB restriction enzyme EcoRI.
8. Construction of recombinant plasmid pUC-HUH-PDAT-SbPDAT
The recombinant plasmid pUC-HUH-PDAT-SbPDAT takes pUC57-hisG-Ura-hisG as a framework, a PDAT-up homologous arm on the upstream side of a PDAT site initiation codon and a PDAT-dm homologous arm on the downstream side of a termination codon in Yarrowia lipolytica Po fDeltaku 70 are inserted, and a SbPDAT gene expression cassette (P TEFin -SbPDAT-T xpr2t ) Orotidine-5' -phosphate decarboxylase encoding gene expression cassette (comprising Yarrowia lipolytica endogenous promoter P TEFin Orotidine-5' -phosphate decarboxylase and terminator T xpr2t ) Also between the upstream and downstream homology arms. The construction of the recombinant plasmid pUC-HUH-PDAT-SbPDAT is the same as that of FIG. 4, except that GPAT-up and GPAT-dm are replaced with PDAT-up and PDAT-dm, respectively, and the SbPDAT gene expression cassette is replaced with SbGPAT gene expression cassette.
P is defined as PDAT:: TEFin f and PDAT:: P TEFin R is a primer, yarrowia lipolytica Po fDeltaku 70 genome DNA is used as a template, and SbPDAT expression cassette promoter P is amplified TEFin . With PDAT:: T xpr2t F and PDAT:: T xpr2t R is a primer, yarrowia lipolytica Po fDeltaku 70 genome DNA is used as a template, and SbPDAT expression cassette terminator T is amplified xpr2t 。
pUC57-SbPDAT as templateIn P TEFin SbPDAT-F and T xpr2t SbPDAT-R as primer, and the amplified two ends are respectively provided with promoter P TEFin And terminator T xpr2t SbPDAT gene of homology arm.
After cleavage of the PDAT site-integrating plasmid with NEB restriction enzyme PacI, the linearized PDAT site-integrating plasmid was recovered by agarose gel electrophoresis.
The linearized PDAT site integrating plasmid and the elements (promoter P) in the SbPDAT gene expression cassette constructed in title 8 of this example TEFin SbPDAT gene and terminator T xpr2t ) The one-step cloning is realized by utilizing ClonExpress MultiS One Step Cloning Kit of Nanjinouzan biotechnology limited company, and the SbPDAT gene expression cassette is inserted between a downstream homology arm of a PDAT site integrating plasmid and a hisG tag coding gene, and a circular recombinant vector is obtained between the upstream homology arm and the downstream homology arm.
The circular recombinant vector is transformed into escherichia coli DH5 alpha competent cells, and positive recombinant plasmid pUC-HUH-PDAT-SbPDAT is obtained through screening of ampicillin resistance plates and colony PCR and sequencing verification.
The linearized pUC-HUH-PDAT-SbPDAT plasmid was recovered by agarose gel electrophoresis after cleavage of the plasmid pUC-HUH-PDAT-SbPDAT with the restriction enzyme NotI from NEB.
9. Construction of recombinant knockout plasmid pUC-HUH-PEX10
Recombinant plasmid pUC-HUH-PEX10 takes pUC57-hisG-Ura-hisG as a framework, and is inserted with a homologous arm PEX10-up with 1500bp upstream of initiation codon and a homologous arm PEX10-dm with 1500bp downstream of termination codon of PEX10 site in Yarrowia lipolytica Po fDeltaku 70, and a orotidine-5' -phosphate decarboxylase encoding gene expression cassette (comprising Yarrowia lipolytica endogenous promoter P TEFin Orotidine-5' -phosphate decarboxylase and terminator T xpr2t ) Also between the upstream and downstream homology arms, the specific structure is shown in FIG. 5, except that TGL4-up and TGL4-down are replaced with PEX10-up and PEX10-dm, respectively.
The homology arm PEX10-up upstream of the initiation codon of TGL4 site was amplified using PEX10-up-F and PEX10-up-R as primers and Yarrowia lipolytica Po fΔku70 genomic DNA as template.
The linearized pUC57-hisG-Ura-hisG plasmid was recovered by agarose gel electrophoresis after cleavage of the plasmid pUC57-hisG-Ura-hisG with NEB restriction enzyme EcoRI.
The linearized pUC57-hisG-Ura-hisG plasmid and the PEX 10-homology arm PEX10-up (with pUC57-hisG-Ura-hisG homology arm sequences at both ends) constructed in title 9 of this example were used by Nannofurovone Biotech Co., ltdIIOne Step Cloning Kit one-step cloning was performed to obtain a circular recombinant vector.
The circular recombinant vector is transformed into escherichia coli DH5 alpha competent cells, and positive recombinant plasmid pUC-HUH-PEX10-up is obtained through screening of ampicillin resistance plates and colony PCR and sequencing verification.
And amplifying the homology arm PEX10-dm downstream of the termination codon of the PEX10 site by using PEX10-dm-F and PEX10-dm-R as primers and Yarrowia lipolytica Po F delta ku70 genomic DNA as a template.
The linearized pUC-HUH-PEX10-up plasmid was recovered by agarose gel electrophoresis after cleavage of the plasmid pUC-HUH-PEX10-up with NEB restriction enzyme HindIII.
The linearized pUC-HUH-PEX10-up plasmid and the homology arm PEX10-dm (with pUC-HUH-PEX10-up homology arm sequences at both ends) downstream of the PEX10 site stop codon constructed in title 9 of this example were used by Nanjinouzan Biotech Co., ltd IIOne Step Cloning Kit one-step cloning was carried out to obtain recombinant plasmid pUC-HUH-PEX10.
The linearized pUC-HUH-PEX10 plasmid was recovered by agarose gel electrophoresis after cleavage of the plasmid pUC-HUH-PEX10 with NEB restriction enzyme HindIII.
10. Construction of recombinant knockout plasmid pUC-HUH-TGL4
Recombinant plasmidpUC-HUH-TGL4 was constructed by taking pUC57-hisG-Ura-hisG as a backbone, inserting a homology arm TGL4-up of 1500bp upstream of the initiation codon and a homology arm (TGL 4-down) of 1500bp downstream of the termination codon in the TGL4 site in Yarrowia lipolytica Po fΔku70, and a nucleotide whey nucleotide-5' -phosphate decarboxylase encoding gene expression cassette (comprising Yarrowia lipolytica endogenous promoter P TEFin Orotidine-5' -phosphate decarboxylase and terminator T xpr2t ) The specific structure between the upstream and downstream homology arms is shown in FIG. 5.
The homology arm TGL4-up upstream of the initiation codon of TGL4 site was amplified using TGL4-up-F and TGL4-up-R as primers and Yarrowia lipolytica Po fΔku70 genomic DNA as template.
The linearized pUC57-hisG-Ura-hisG plasmid was recovered by agarose gel electrophoresis after cleavage of the plasmid pUC57-hisG-Ura-hisG with NEB restriction enzyme EcoRI.
The linearized pUC57-hisG-Ura-hisG plasmid and the TGL4 site initiation codon upstream homology arm TGL4-up constructed in title 9 of this example (with pUC57-hisG-Ura-hisG homology arm sequences at both ends) were used by Nannofurovone Biotechnology Co.Ltd IIOne Step Cloning Kit one-step cloning was performed to obtain a circular recombinant vector.
The circular recombinant vector is transformed into escherichia coli DH5 alpha competent cells, and positive recombinant plasmid pUC-HUH-TGL4-up is obtained through screening of ampicillin resistance plates and colony PCR and sequencing verification.
The homology arm TGL4-Down downstream of the TGL4 site stop codon was amplified using TGL4-dm-F and TGL4-dm-R as primers and Yarrowia lipolytica Po fΔku70 genomic DNA as template.
The linearized pUC-HUH-TGL4-up plasmid was recovered by agarose gel electrophoresis after cleavage of the plasmid pUC-HUH-TGL4-up with NEB restriction enzyme HindIII.
The linearized pUC-HUH-TGL4-up plasmid and the homology arm TGL4-Down (with pUC-HUH-TGL4-up homology at both ends) downstream of the TGL4 site stop codon constructed in this example title 9Arm sequence) using Nanjinouzan biotechnology limitedIIOne Step Cloning Kit one-step cloning was performed to obtain recombinant plasmid pUC-HUH-TGL4./>
The linearized pUC-HUH-TGL4 plasmid was recovered by agarose gel electrophoresis after cleavage of the plasmid pUC-HUH-TGL4 with NEB restriction enzyme HindIII.
TABLE 2 primer sequences
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EXAMPLE 2 construction of recombinant yarrowia lipolytica (one) construction of recombinant bacterium 1
Linearizing recombinant plasmid pUC-HUH-P ELO1 -P CTR1 Introduced into Yarrowia lipolytica Po fΔku70, P CTR1 Integration of genes into genome P ELO1 At the locus, and then losing a hisG tag and Ura selection marker under 5-fluoroorotic acid selection pressure to obtainRecombinant bacterium 1.
The specific method comprises the following steps:
(1) linearized recombinant plasmid pUC-HUH-P ELO1 -P CTR1 Is introduced and positive recombinant bacteria are screened and identified: yarrowia lipolytica Po1 f.DELTA.ku70 competent cells were prepared by overnight culture in YPD liquid medium (containing 20g/L peptone, 10g/L yeast extract and 20g/L glucose) and linearized recombinant plasmid pUC-HUH-P using Zymogen Frozen EZYeast Transformation Kit II of Zymo Research Corporation ELO1 -P CTR1 Transformation into Yarrowia lipolytica Po fΔku70, homologous recombination, screening positive clones using SD-Ura plates, and PCR identification. Wherein the SD-Ura plate contained 20g/L glucose, 6.7g/L YNB (no amino yeast nitrogen source, available from BBI Life Sciences), 0.67g/L CSM-Ura (complete supplementation of mixture to remove uracil, available from MP Biomedicals), 23g/L agar powder, and water as solvent.
(2) One hisG tag and Ura selection marker were lost: the PCR was performed to identify the correct positive clones by plating on YPD plates containing 5-fluoroorotic acid (containing 20g/L peptone, 10g/L yeast extract, 20g/L glucose, 5-fluoroorotic acid (2.5 g/L), agar powder 23 g/L), and then streaking single colonies simultaneously on YPD plates containing 5-fluoroorotic acid and SD-Ura plates. Single colonies which were able to grow on YPD plates containing 5-fluoroorotic acid but not on SD-Ura plates were selected to give recombinant bacterium 1.
Construction of recombinant bacterium 2
Linearizing recombinant plasmid pUC-HUH-P SCD -P CTR1 Introduction into recombinant bacterium 1, P CTR1 Integration of genes into genome P SCD At the locus, one hisG tag and Ura selection marker were then lost under 5-fluoroorotic acid selection pressure to give recombinant 2.
The specific method comprises the following steps:
(1) linearized recombinant plasmid pUC-HUH-P SCD -P CTR1 Is introduced and positive recombinant bacteria are screened and identified: recombinant strain 1 was cultured overnight in YPD liquid medium to prepare competent cells, and the linearized recombinant plasmid was used as Zymogen Frozen EZYeast Transformation Kit II of Zymo Research CorporationpUC-HUH-P SCD -P CTR1 And (3) transforming into recombinant bacterium 1, carrying out homologous recombination, screening positive clones by using an SD-Ura flat plate, and then carrying out PCR identification.
(2) One hisG tag and Ura selection marker were lost: the PCR was used to identify the correct positive clones by plating onto YPD plates containing 5-fluoroorotic acid and then streaking single colonies onto both YPD plates and SD-Ura plates containing 5-fluoroorotic acid. Single colonies were selected which were able to grow on YPD plates containing 5-fluoroorotic acid but not on SD-Ura plates to give recombinant 2.
Construction of recombinant bacterium 3
The linearized recombinant plasmid pUC-leu-A08-FAT5 is introduced into recombinant bacterium 2, and the FAT5 gene expression cassette is integrated at the site of genome A08 to obtain recombinant bacterium 3. The specific method comprises the following steps:
(1) Introduction of linearization recombinant plasmid pUC-leu-A08-FAT5 and screening and identification of positive recombinant bacteria: the recombinant bacterium 2 is cultured overnight in YPD liquid culture medium to prepare competent cells, the Zymogen Frozen EZYeast Transformation Kit II of Zymo Research Corporation is utilized to transform the linearization recombinant plasmid pUC-Leu-A08-FAT5 into the recombinant bacterium 2 for homologous recombination, the screening medium SD-Leu is adopted to screen positive clones, and then PCR identification is carried out. Wherein the screening culture medium SD-Leu contains 20g/L of glucose, 6.7g/L of YNB, 0.67g/L of CSM-Ura, 23g/L of agar powder and water as solvent.
(2) One hisG tag and Ura selection marker were lost: the PCR identified the correct positive clones were plated on YPD plates containing 5-fluoroorotic acid and single colonies were then streaked simultaneously on YPD plates containing 5-fluoroorotic acid and SD-Leu plates. Single colonies were selected which were able to grow on YPD plates containing 5-fluoroorotic acid but not on SD-Leu plates to give recombinant bacterium 3.
Construction of recombinant bacterium 4
The linearized plasmids pUC-HUH-TGL4 and pUC-HUH-Pex10 and the linearized plasmid pUC-HUH-IntA-ACC1 carrying the ACC1 gene expression cassette are sequentially introduced into the recombinant bacterium 3, the Pex10 and TGL4 genes are knocked out through a homologous recombination method, and the ACC1 expression cassette is integrated into an IntA site to obtain the recombinant bacterium 4.
The specific method comprises the following steps: the recombinant bacterium 3 is cultured overnight in YPD liquid medium to prepare competent cells, the Zymogen Frozen EZ Yeast Transformation Kit II of Zymo Research Corporation is utilized to introduce the linearization recombinant plasmid pUC-HUH-TGL4 into the recombinant bacterium 3, homologous recombination is carried out, the TGL4 gene is knocked out, the positive recombinant bacterium from which the TGL4 gene is knocked out is screened and identified, and the specific method is the same as that of (1) linearization recombinant plasmid pUC-HUH-P in the title (one) of the embodiment ELO1 -P CTR1 Is introduced and positive recombinant bacteria are screened and identified. Then, linearized plasmids pUC-HUH-Pex10 and pUC-HUH-IntA-ACC1 were introduced into the positive recombinant bacteria obtained in the previous step in this order by the same method, and after each plasmid was introduced, positive recombinant bacteria were selected and identified. After the last linearized plasmid pUC-HUH-IntA-ACC1 is introduced, the obtained positive recombinant bacteria are screened and identified, and a hisG tag and Ura screening marker are lost: the PCR was used to identify the correct positive clones by plating onto YPD plates containing 5-fluoroorotic acid and then streaking single colonies onto both YPD plates and SD-Ura plates containing 5-fluoroorotic acid. Single colonies were selected which were able to grow on YPD plates containing 5-fluoroorotic acid but not on SD-Ura plates to give recombinant bacterium 4.
(fifth) construction of recombinant bacterium 5
The linearized recombinant plasmids pUC-HUH-GPAT-SbGPAT, pUC-HUH-LPAAT-SbLPAAT, pUC-HUH-DGAT2-SbDGAT2 and pUC-HUH-PDAT-SbPDAT are sequentially introduced into recombinant bacteria 4, sbGPAT, sbLPAAT, sbDGAT and SbPDAT are respectively integrated into GPAT, LPAAT, DGAT and PDAT sites by a homologous recombination method, so as to obtain recombinant bacteria 5. After each plasmid was introduced, positive recombinant bacteria were selected and used as host bacteria for the next plasmid introduction. The method for introducing and screening each plasmid was the same as (1) the linearized recombinant plasmid pUC-HUH-P in the title (one) of this example ELO1 -P CTR1 Is introduced and positive recombinant bacteria are screened and identified. After the last linearization plasmid pUC-HUH-PDAT-SbPDAT is introduced, the obtained positive recombinant bacterium is screened and identified, and after a hisG label and Ura screening mark are lost, recombinant bacterium 5 is obtained. The specific method for losing a hisG tag and Ura selection marker is the same as in example heading (one).
EXAMPLE 3 cultivation of recombinant yarrowia lipolytica and extraction of the products of engineering bacteria
Palmitoleic acid was produced using yarrowia lipolytica (Yarrowia lipolytica) Po1fΔku70, recombinant bacteria 1-5 of example 2, respectively. The specific method comprises the following steps: each strain was streaked on YPD plates (containing 20g/L peptone, 10g/L yeast extract, 20g/L glucose and 10g/L agar) and cultured overnight to activate each strain, and then the activated strain was inoculated into YPD liquid medium (containing 20g/L peptone, 10g/L yeast extract, 20g/L glucose, water as a solvent) and cultured at 30℃and 220rpm for 16 hours to obtain a seed solution. The seed solution was inoculated into 50ml of a fermentation medium at an inoculum size of 1%, shake-cultured at 30℃and 220rpm for 5 days, and 4g/L of an aqueous solution of copper sulfate was added at 48 hours of the shake culture to give a final concentration of copper sulfate in the fermentation broth of 40. Mu.M. After the fermentation, transferring the fermentation broth to a 50ml centrifuge tube, centrifuging at 5000rpm for 15min, discarding the supernatant, and drying the bacterial precipitate in an oven (75 ℃) until the weight is constant to obtain dry bacterial.
Wherein the formula of the fermentation medium is as follows: glucose 60g/L, yeast nitrogen source without amino group (YNB) 1.7g/L, ammonium sulfate 1.3g/L, and water as solvent.
Qualitative and quantitative analysis of palmitoleic acid
1. Methyl esterification of fatty acids
About 0.05g of dry cells was weighed, 100. Mu.L of a methyl tridecanoate n-hexane solution at a concentration of 0.2408g/L was added as an internal standard, and 500. Mu.L of a 1M NaOH methanol solution was added; the sample was vortexed at 1200rpm for 1 hour at room temperature (25 ℃) and then neutralized with 40 μl of 98% strength by mass sulfuric acid; 400. Mu.L of n-hexane was added and vortexed at 1200rpm for 10 minutes at room temperature (25 ℃ C.) at which time fatty acid methyl esters were extracted into n-hexane; centrifugation was carried out at 8000rpm for 2 minutes, and the supernatant was filtered with organic Ni Long Lvmo (0.22 μm) and transferred to a glass bottle for GC detection.
2. Palmitoleic acid detection
GC detection conditions: FID detector, sample inlet temperature 250 ℃, sample volume 1 μl, split ratio: 50:1, column: DB-23 chromatographic column (60 m 0.25 m 0.15 m). Chromatographic conditions: the initial temperature was 100deg.C, raised to 196℃at a rate of 25deg.C/min, then raised to 220℃at 2℃/min, and maintained for 2min. Qualitative and quantitative analysis was performed using a fatty acid mixture standard from Sigma-Aldrich.
Under the above conditions, the peak time of methyl palmitoleate detected by GC was about 6.2min (fig. 6).
After 5 days of fermentation, accumulation of palmitoleic acid was detected in all strains. The mass percentage of palmitoleic acid in the total fatty acid in the recombinant bacteria 1, 2, 3, 4 and 5 is 7.62%, 15.67%, 20.14%, 24.15% and 41.57%, respectively. The highest palmitoleic acid yield of recombinant bacterium 5 (Table 3) was achieved at 2.84g/L, i.e., 2.84g palmitoleic acid per liter of broth, significantly higher than the initial and other recombinant strains.
TABLE 3 palmitoleic acid production by initial, recombinant 3 and recombinant 5 bacteria
Recombinant bacterium 5 was inoculated into 50ml of a seed culture (YPD medium) and cultured for 24 hours, and then the seed solution was inoculated into a 5L fermenter at an inoculum size of 5%. Adding 2.5L of fermentation medium into a 5L fermentation tank, fermenting for 0-48h, and controlling dissolved oxygen to be 20%; the fermentation time is more than 48 hours, and the dissolved oxygen is controlled to be 5 percent. During the fermentation, the pH value is constantly controlled at 5.5 until the fermentation is finished. The feed was fed once at 40h and 96h, 800ml of 800g/L glucose aqueous solution each time. The fermentation temperature is 28 ℃, the stirring rotation speed is regulated according to dissolved oxygen, and the culture time is 6 days. Wherein the fermentation medium comprises: glucose 150g/L, ammonium sulfate 11g/L, yeast extract 3g/L, corn peptone 0.1g/L, potassium dihydrogen phosphate 4g/L, magnesium sulfate 2g/L, calcium sulfate 0.8g/L, sodium chloride 0.4g/L, ammonium sulfate hydrochloride 12mg/L, biotin 1mg/L, sodium molybdate 160mg/L, copper sulfate 0.2mg/L, boric acid 40mg/L, manganese sulfate 180mg/L, and ferrous chloride 75mg/L. After 5 days of fermentation, the yield of palmitoleic acid from recombinant bacterium 5 was highest, reaching 10.56g/L, i.e., 10.56g palmitoleic acid per liter of fermentation broth (Table 3), significantly higher than that from the initial and other recombinant bacteria.
Claims (10)
1. Recombinant yarrowia lipolytica with high palmitoleic acid production, characterized in that peroxisome biogenesis factor 10 and triacylglycerol lipase 4 are knocked out in the genome of yarrowia lipolytica (Yarrowia lipolytica), and acetyl-coa carboxylase, Δ9 desaturase, acetyl-coa diacylglycerol acyltransferase, glycerol-3-phosphate acyltransferase, lysophosphatidylcholine acyltransferase, phosphatidylcholine are inserted: the diacylglycerol acyltransferase gene expression cassette.
2. The recombinant yarrowia lipolytica of claim 1, wherein the acetyl-coa carboxylase gene is derived from yarrowia lipolytica (Yarrowia lipolytica); the delta 9 desaturase is obtained by optimizing a gene from caenorhabditis elegans by codons; glycerol-3-phosphoryl transferase, lysophosphatidylcholine acyltransferase, acetyl-coa diacylglycerol acyltransferase, and phosphatidylcholine: the diacylglycerol acyltransferase is obtained by codon-optimizing a gene derived from Hippophae rhamnoides (Sea buckthorn).
3. Recombinant yarrowia lipolytica according to claim 1 or 2, characterized in that the Δ9 desaturase, glycerol-3-phosphate acyltransferase, lysophosphatidylcholine acyltransferase, acetyl-coa diacylglycerol acyltransferase, phosphatidylcholine: the gene sequence of the diacylglycerol acyltransferase is shown in SEQ ID No: 1-5.
4. The recombinant yarrowia lipolytica of claim 3, wherein said Δ9 desaturase, acetyl-coa carboxylase, acetyl-coa diacylglycerol acyltransferase, glycerol-3-phosphate acyltransferase, lysophosphatidylcholine acyltransferase, phosphatidylcholine: the sites of integration of the diacylglycerol acyltransferase gene expression cassette in the yarrowia lipolytica (Yarrowia lipolytica) genome are in order: a08 site, intA, DGAT2, GPAT, LPAAT, PDAT.
5. The recombinant yarrowia lipolytica according to claim 4, wherein the promoter of the expression cassette is any one of the TEF promoter, hp4d promoter, TEFin promoter, POX2 promoter or GPDin promoter of yarrowia lipolytica; the terminator is any one of xpr2t terminator, mig1t terminator and lip2t terminator of yarrowia lipolytica.
6. The recombinant yarrowia lipolytica of claim 5, wherein P in the genome of said recombinant yarrowia lipolytica ELO1 Promoter site and/or P SCD The promoter site is also inserted with an endogenous copper ion inhibition promoter P CTR1 。
7. The method for preparing the recombinant yarrowia lipolytica as claimed in claim 6, wherein the gene expression cassettes are inserted into the genome by homologous recombination and the gene to be knocked out is knocked out by homologous recombination.
8. Use of the recombinant yarrowia lipolytica of claim 1 for the preparation of palmitoleic acid.
9. Use according to claim 8, characterized in that the recombinant yarrowia lipolytica is fermented to palmitoleic acid using the following fermentation medium: 130-170g/L of glucose, 10-12g/L of ammonium sulfate, 2.5-3.5g/L of yeast extract, 0.085-0.105g/L of corn peptone, 3.5-4.5g/L of monopotassium phosphate, 1.8-2.2g/L of magnesium sulfate, 0.78-0.82g/L of calcium sulfate, 0.35-0.45g/L of sodium chloride, 10-14mg/L of ammonium sulfate hydrochloride, 0.85-1.15mg/L of biotin, 140-180mg/L of sodium molybdate, 0.18-0.22mg/L of copper sulfate, 38-42mg/L of boric acid, 160-200mg/L of manganese sulfate and 70-80mg/L of ferrous chloride.
10. Use according to claim 9, characterized in that glucose is fed in during fermentation.
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