CN114958636A - Recombinant yarrowia lipolytica with high punicic acid yield as well as construction method and application thereof - Google Patents
Recombinant yarrowia lipolytica with high punicic acid yield as well as construction method and application thereof Download PDFInfo
- Publication number
- CN114958636A CN114958636A CN202210520028.2A CN202210520028A CN114958636A CN 114958636 A CN114958636 A CN 114958636A CN 202210520028 A CN202210520028 A CN 202210520028A CN 114958636 A CN114958636 A CN 114958636A
- Authority
- CN
- China
- Prior art keywords
- yarrowia lipolytica
- expression cassette
- site
- recombinant
- plasmid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000235015 Yarrowia lipolytica Species 0.000 title claims abstract description 92
- CUXYLFPMQMFGPL-BGDVVUGTSA-N (9Z,11E,13Z)-octadecatrienoic acid Chemical compound CCCC\C=C/C=C/C=C\CCCCCCCC(O)=O CUXYLFPMQMFGPL-BGDVVUGTSA-N 0.000 title claims abstract description 89
- CUXYLFPMQMFGPL-UYWAGRGNSA-N trichosanic acid Natural products CCCCC=C/C=C/C=CCCCCCCCC(=O)O CUXYLFPMQMFGPL-UYWAGRGNSA-N 0.000 title claims abstract description 45
- 238000010276 construction Methods 0.000 title abstract description 33
- 239000013612 plasmid Substances 0.000 claims abstract description 133
- 230000014509 gene expression Effects 0.000 claims abstract description 109
- 230000010354 integration Effects 0.000 claims abstract description 36
- 102100030554 Peroxisome biogenesis factor 10 Human genes 0.000 claims abstract description 27
- 102100038805 Lysophospholipid acyltransferase 2 Human genes 0.000 claims abstract description 15
- 108010052187 1-Acylglycerophosphocholine O-Acyltransferase Proteins 0.000 claims abstract description 13
- 238000002744 homologous recombination Methods 0.000 claims abstract description 10
- 230000006801 homologous recombination Effects 0.000 claims abstract description 10
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 claims abstract description 10
- 102000000452 Acetyl-CoA carboxylase Human genes 0.000 claims abstract description 9
- 108010016219 Acetyl-CoA carboxylase Proteins 0.000 claims abstract description 9
- 102000013444 Diacylglycerol Cholinephosphotransferase Human genes 0.000 claims abstract description 9
- 102000002148 Diacylglycerol O-acyltransferase Human genes 0.000 claims abstract description 9
- 108010001348 Diacylglycerol O-acyltransferase Proteins 0.000 claims abstract description 9
- 108010051225 Diacylglycerol cholinephosphotransferase Proteins 0.000 claims abstract description 9
- 101710119209 Triacylglycerol lipase 4 Proteins 0.000 claims abstract description 7
- 101710196890 Peroxisome biogenesis factor 10 Proteins 0.000 claims abstract description 6
- 101001065073 Punica granatum Bifunctional fatty acid conjugase/Delta(12)-oleate desaturase Proteins 0.000 claims abstract description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 57
- 101150085005 ku70 gene Proteins 0.000 claims description 55
- 241000894006 Bacteria Species 0.000 claims description 46
- 238000000855 fermentation Methods 0.000 claims description 23
- 230000004151 fermentation Effects 0.000 claims description 23
- 101001126498 Homo sapiens Peroxisome biogenesis factor 10 Proteins 0.000 claims description 21
- 235000014113 dietary fatty acids Nutrition 0.000 claims description 19
- 229930195729 fatty acid Natural products 0.000 claims description 19
- 239000000194 fatty acid Substances 0.000 claims description 19
- 150000004665 fatty acids Chemical class 0.000 claims description 18
- BSMKSTAEJFRBHA-UHFFFAOYSA-N tgl-4 Chemical compound O1C(C2=3)=C(O4)C(O)=CC=3CCNC2CC(C=C2)=CC=C2OC(=C2)C(OC)=CC=C2CC2N(C)CCC3=CC(OC)=C1C4=C23 BSMKSTAEJFRBHA-UHFFFAOYSA-N 0.000 claims description 16
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 14
- 101100099200 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) TGL4 gene Proteins 0.000 claims description 14
- 239000008103 glucose Substances 0.000 claims description 14
- 108020004705 Codon Proteins 0.000 claims description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- 102000004190 Enzymes Human genes 0.000 claims description 10
- 108090000790 Enzymes Proteins 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 9
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 7
- 235000014360 Punica granatum Nutrition 0.000 claims description 6
- 238000005457 optimization Methods 0.000 claims description 6
- 108010018763 Biotin carboxylase Proteins 0.000 claims description 5
- 235000004443 Ricinus communis Nutrition 0.000 claims description 5
- 240000000528 Ricinus communis Species 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 4
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 4
- 241001083505 Punica Species 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 101100084403 Homo sapiens PRODH gene Proteins 0.000 claims description 2
- 101150059359 POX2 gene Proteins 0.000 claims description 2
- 102100028772 Proline dehydrogenase 1, mitochondrial Human genes 0.000 claims description 2
- 101100256199 Starmerella bombicola sble gene Proteins 0.000 claims description 2
- 101100029251 Zea mays PER2 gene Proteins 0.000 claims description 2
- 101150077696 lip-1 gene Proteins 0.000 claims description 2
- YAFQFNOUYXZVPZ-UHFFFAOYSA-N liproxstatin-1 Chemical compound ClC1=CC=CC(CNC=2C3(CCNCC3)NC3=CC=CC=C3N=2)=C1 YAFQFNOUYXZVPZ-UHFFFAOYSA-N 0.000 claims description 2
- 244000294611 Punica granatum Species 0.000 claims 1
- 230000015572 biosynthetic process Effects 0.000 abstract description 4
- 238000003786 synthesis reaction Methods 0.000 abstract description 4
- 229930014626 natural product Natural products 0.000 abstract description 2
- 238000011144 upstream manufacturing Methods 0.000 description 55
- 101100342585 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) mus-51 gene Proteins 0.000 description 53
- 101100342589 Schizosaccharomyces pombe (strain 972 / ATCC 24843) pku70 gene Proteins 0.000 description 53
- 241000201640 Yarrowia lipolytica PO1f Species 0.000 description 50
- 108020004414 DNA Proteins 0.000 description 35
- 238000010367 cloning Methods 0.000 description 21
- SEHFUALWMUWDKS-UHFFFAOYSA-N 5-fluoroorotic acid Chemical compound OC(=O)C=1NC(=O)NC(=O)C=1F SEHFUALWMUWDKS-UHFFFAOYSA-N 0.000 description 20
- 108091081024 Start codon Proteins 0.000 description 19
- 238000000246 agarose gel electrophoresis Methods 0.000 description 19
- 108091008146 restriction endonucleases Proteins 0.000 description 19
- 238000012216 screening Methods 0.000 description 18
- 102100037214 Orotidine 5'-phosphate decarboxylase Human genes 0.000 description 17
- 108010055012 Orotidine-5'-phosphate decarboxylase Proteins 0.000 description 17
- 101150032598 hisG gene Proteins 0.000 description 17
- 210000004027 cell Anatomy 0.000 description 15
- 239000002609 medium Substances 0.000 description 12
- 101000848262 Punica granatum Delta(12)-acyl-lipid-desaturase Proteins 0.000 description 11
- 108020005038 Terminator Codon Proteins 0.000 description 11
- 101000848260 Trichosanthes kirilowii Delta(12)-oleate desaturase Proteins 0.000 description 11
- 239000002773 nucleotide Substances 0.000 description 10
- 125000003729 nucleotide group Chemical group 0.000 description 10
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 9
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 9
- 229960000723 ampicillin Drugs 0.000 description 9
- 238000010586 diagram Methods 0.000 description 9
- 238000012163 sequencing technique Methods 0.000 description 9
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 8
- 210000000349 chromosome Anatomy 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 101150058703 ACC1 gene Proteins 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 241000196324 Embryophyta Species 0.000 description 6
- 239000001888 Peptone Substances 0.000 description 6
- 108010080698 Peptones Proteins 0.000 description 6
- 230000003321 amplification Effects 0.000 description 6
- 229940041514 candida albicans extract Drugs 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 239000003550 marker Substances 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 238000003199 nucleic acid amplification method Methods 0.000 description 6
- 235000019319 peptone Nutrition 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 239000002904 solvent Substances 0.000 description 6
- 239000012138 yeast extract Substances 0.000 description 6
- 101710190443 Acetyl-CoA carboxylase 1 Proteins 0.000 description 5
- 241000219991 Lythraceae Species 0.000 description 5
- 238000000605 extraction Methods 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 5
- 239000000843 powder Substances 0.000 description 5
- 230000001131 transforming effect Effects 0.000 description 5
- 238000012795 verification Methods 0.000 description 5
- 229920001817 Agar Polymers 0.000 description 4
- 102100021334 Bcl-2-related protein A1 Human genes 0.000 description 4
- 101710088194 Dehydrogenase Proteins 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 108010062466 Enzyme Precursors Proteins 0.000 description 4
- 102000010911 Enzyme Precursors Human genes 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- KYOBSHFOBAOFBF-XVFCMESISA-N orotidine 5'-phosphate Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1C(O)=O KYOBSHFOBAOFBF-XVFCMESISA-N 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 101710097496 Lysophospholipid acyltransferase Proteins 0.000 description 2
- 101710163746 Lysophospholipid acyltransferase 2 Proteins 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- JNDDPBOKWCBQSM-UHFFFAOYSA-N methyl tridecanoate Chemical compound CCCCCCCCCCCCC(=O)OC JNDDPBOKWCBQSM-UHFFFAOYSA-N 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000011218 seed culture Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- 239000007222 ypd medium Substances 0.000 description 2
- RYCNUMLMNKHWPZ-SNVBAGLBSA-N 1-acetyl-sn-glycero-3-phosphocholine Chemical compound CC(=O)OC[C@@H](O)COP([O-])(=O)OCC[N+](C)(C)C RYCNUMLMNKHWPZ-SNVBAGLBSA-N 0.000 description 1
- 101710124165 1-acyl-sn-glycerol-3-phosphate acyltransferase Proteins 0.000 description 1
- 108010054662 2-acylglycerophosphate acyltransferase Proteins 0.000 description 1
- OSJPPGNTCRNQQC-UWTATZPHSA-N 3-phospho-D-glyceric acid Chemical compound OC(=O)[C@H](O)COP(O)(O)=O OSJPPGNTCRNQQC-UWTATZPHSA-N 0.000 description 1
- 102000057234 Acyl transferases Human genes 0.000 description 1
- 108700016155 Acyl transferases Proteins 0.000 description 1
- 102100039239 Amidophosphoribosyltransferase Human genes 0.000 description 1
- 108010039224 Amidophosphoribosyltransferase Proteins 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 241000284466 Antarctothoa delta Species 0.000 description 1
- 101000963440 Bacillus subtilis (strain 168) Biotin carboxylase 1 Proteins 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 101100118654 Caenorhabditis elegans elo-1 gene Proteins 0.000 description 1
- 102100036869 Diacylglycerol O-acyltransferase 1 Human genes 0.000 description 1
- 108050004099 Diacylglycerol O-acyltransferase 1 Proteins 0.000 description 1
- 102100032052 Elongation of very long chain fatty acids protein 5 Human genes 0.000 description 1
- 108050007807 Elongation of very long chain fatty acids protein 5 Proteins 0.000 description 1
- 101150058769 FAD2 gene Proteins 0.000 description 1
- 101150013497 FADX gene Proteins 0.000 description 1
- 102000036181 Fatty Acid Elongases Human genes 0.000 description 1
- 108010058732 Fatty Acid Elongases Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 101710163717 Lysophospholipid acyltransferase 5 Proteins 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108010079590 Phosphatidylcholine desaturase Proteins 0.000 description 1
- 101710172946 Probable 1-acyl-sn-glycerol-3-phosphate acyltransferase Proteins 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 241000218989 Trichosanthes Species 0.000 description 1
- 101900063294 Yarrowia lipolytica Orotidine 5'-phosphate decarboxylase Proteins 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- DTOSIQBPPRVQHS-PDBXOOCHSA-N alpha-linolenic acid Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC(O)=O DTOSIQBPPRVQHS-PDBXOOCHSA-N 0.000 description 1
- 235000020661 alpha-linolenic acid Nutrition 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 241000684086 bacterium 1-2 Species 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 150000001982 diacylglycerols Chemical class 0.000 description 1
- ADHFGLVXSIGCIG-UHFFFAOYSA-N diazanium sulfate hydrochloride Chemical compound [NH4+].[NH4+].Cl.[O-]S([O-])(=O)=O ADHFGLVXSIGCIG-UHFFFAOYSA-N 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 235000019197 fats Nutrition 0.000 description 1
- 125000005313 fatty acid group Chemical group 0.000 description 1
- 235000019387 fatty acid methyl ester Nutrition 0.000 description 1
- 229960002089 ferrous chloride Drugs 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- KQQKGWQCNNTQJW-UHFFFAOYSA-N linolenic acid Natural products CC=CCCC=CCC=CCCCCCCCC(O)=O KQQKGWQCNNTQJW-UHFFFAOYSA-N 0.000 description 1
- 229960004488 linolenic acid Drugs 0.000 description 1
- 230000004132 lipogenesis Effects 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 229940099596 manganese sulfate Drugs 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- ZUVVLBGWTRIOFH-UHFFFAOYSA-N methyl 4-methyl-2-[(4-methylphenyl)sulfonylamino]pentanoate Chemical compound COC(=O)C(CC(C)C)NS(=O)(=O)C1=CC=C(C)C=C1 ZUVVLBGWTRIOFH-UHFFFAOYSA-N 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- -1 polyethylene terephthalate Polymers 0.000 description 1
- 229920000139 polyethylene terephthalate Polymers 0.000 description 1
- 239000005020 polyethylene terephthalate Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000011684 sodium molybdate Substances 0.000 description 1
- 235000015393 sodium molybdate Nutrition 0.000 description 1
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
- C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1288—Transferases for other substituted phosphate groups (2.7.8)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/93—Ligases (6)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6409—Fatty acids
- C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- C12Y114/19—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with oxidation of a pair of donors resulting in the reduction of molecular oxygen to two molecules of water (1.14.19)
- C12Y114/19006—DELTA12-fatty-acid desaturase (1.14.19.6), i.e. oleoyl-CoA DELTA12 desaturase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y203/00—Acyltransferases (2.3)
- C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
- C12Y203/01158—Phospholipid:diacylglycerol acyltransferase (2.3.1.158)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y604/00—Ligases forming carbon-carbon bonds (6.4)
- C12Y604/01—Ligases forming carbon-carbon bonds (6.4.1)
- C12Y604/01002—Acetyl-CoA carboxylase (6.4.1.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Abstract
The invention provides a recombinant yarrowia lipolytica strain for high yield of punicic acid, a construction method and application thereof, belonging to the field of bioengineering. The recombinant yarrowia lipolytica is yarrowia lipolytica XJ-11 strain. The construction method of the recombinant yarrowia lipolytica with high punicic acid yield comprises the following steps of preparing a peroxisome biogenesis factor 10 knockout box, a triacylglycerol lipase 4 knockout box, an acetyl coenzyme A carboxylase expression box, a delta 12 desaturase expression box and phosphatidylcholine: a step of introducing a diacylglycerol acyltransferase expression cassette, a choline phosphotransferase expression cassette, a heterologous fatty acid conjugase expression cassette, a heterologous lysophosphatidylcholine acyltransferase expression cassette into said yarrowia lipolytica in the form of a plasmid, followed by integration into the genome of yarrowia lipolytica by homologous recombination. The recombinant yarrowia lipolytica can efficiently ferment and produce punicic acid, and realizes the efficient synthesis of the plant-derived natural product punicic acid in yarrowia lipolytica.
Description
Technical Field
The invention belongs to the field of bioengineering, and particularly relates to recombinant yarrowia lipolytica with high punicic acid yield, a construction method and application thereof.
Background
Punicacid (Punic acid, C18: 3) Δ9cis,11trans,13cis ) The learning name is as follows: cis-9, trans-11, cis-13-octadecatrienoic acid, which is an omega-5 type conjugated linolenic acid with 3 conjugated double bonds, is named because it is mainly from pomegranate seeds. Punicate has wide physiological activity, and the research shows that punicic acid can inhibit the amplification of prostate cancer and breast cancer cells. Related researches on a series of animal models also show that the punicic acid has the physiological activities of obesity prevention, inflammation resistance, oxidation resistance and the like. In addition, punicic acid has certain activity on the metabolic burden of the heart, which can reduce fat accumulation and inhibit lipogenesis.
The punicic acid is conventionally obtained by extracting plant seeds (such as pomegranate seed and trichosanthes seed). However, the plant extraction method has many disadvantages, such as large plant growing area, long growth period, and environmental susceptibility, and the extraction of punicic acid from plants consumes a lot of energy because punicic acid is usually covered by cell walls and seed hulls, and has low extraction efficiency and difficult separation and purification. Compared with a plant extraction method, the microbial fermentation method has a short growth period and can produce all weather, and the oil-producing microorganisms have high oil content and large biomass, so that the method for producing the punicic acid by using the oil-producing microorganisms is an economic and efficient mode. In the prior art, microorganisms capable of efficiently producing punicic acid are lacked.
Disclosure of Invention
The invention aims to provide a recombinant yarrowia lipolytica capable of efficiently synthesizing punicic acid.
The invention further aims to provide a construction method of the recombinant yarrowia lipolytica, which is efficient and simple to operate.
Still another object of the present invention is to provide the use of said recombinant yarrowia lipolytica for the production of punicic acid.
The purpose of the invention is realized by adopting the following technical scheme:
the invention provides a recombinant Yarrowia lipolytica strain for high yield of punicic acid, which is a Yarrowia lipolytica (Yarrowia lipolytica) XJ-11 strain with the preservation number of CCTCC NO: m2022320.
In the present invention, Yarrowia lipolytica (Yarrowia lipolytica) XJ-11 strain is a Yarrowia lipolytica genome in which peroxisome biogenesis factor 10 and triacylglycerol lipase 4 are deleted, and a.DELTA.12 desaturase expression cassette, phosphatidylcholine: diacylglycerol acyltransferase expression cassette, acetyl coenzyme A carboxylase expression cassette, choline phosphotransferase expression cassette, heterologous fatty acid conjugase expression cassette, and heterologous lysophosphatidylcholine acyltransferase.
In the present invention, the acetyl-coa carboxylase, Δ 12 desaturase, phosphatidylcholine: diacylglycerol acyltransferase, choline phosphotransferase encoding genes are derived from Yarrowia lipolytica; the fatty acid conjugated enzyme encoding gene is obtained by codon optimization of a pomegranate (Punica grantum) derived fatty acid conjugated enzyme encoding gene; the heterologous lysophosphatidylcholine acyltransferase is obtained by codon optimization of a gene encoding lysophosphatidylcholine acyltransferase from Ricinus communis.
In the present invention, the promoter of each expression cassette is the promoter P of yarrowia lipolytica TEF 、P hp4d 、P TEFin 、P POX2 Or P GPDin Any one of the above; the terminator is terminator T of yarrowia lipolytica xpr2t 、T mig1t 、T lip2t 、T pex10t Or T pex20t Any one of them.
In the present invention, the integration site of the expression cassette is any one of A08 site, 26s rDNA site, PEX10 site, TGL4 site, IntA site, IntB site, IntC site, IntD site, IntE site, IntF site, lip1 site of yarrowia lipolytica.
In the invention, the sequence of the encoding gene of the fatty acid conjugated enzyme is shown as SEQ ID No. 1; the sequence of the encoding gene of the ricinus communis lysophosphatidylcholine acyltransferase is shown as SEQ ID No. 3.
The invention also provides a construction method of the recombinant yarrowia lipolytica with high punicic acid yield, which comprises the following steps of preparing a peroxisome biogenesis factor 10 knockout box, a triacylglycerol lipase 4 knockout box, an acetyl coenzyme A carboxylase expression box, a delta 12 desaturase expression box and phosphatidylcholine: a step of introducing a diacylglycerol acyltransferase expression cassette, a choline phosphotransferase expression cassette, a heterologous fatty acid conjugase expression cassette, a heterologous lysophosphatidylcholine acyltransferase expression cassette into said yarrowia lipolytica in the form of a plasmid, followed by integration into the genome of yarrowia lipolytica by homologous recombination.
In the present invention, the yarrowia lipolytica knocks out the ku70 gene.
The invention also provides application of the recombinant bacterium in producing punicic acid, which is characterized by comprising the step of culturing the recombinant bacterium in any one of claims 1 to 6 by adopting a fermentation culture medium to obtain a fermentation product.
In the invention, the fermentation medium contains 50-70g/L of glucose, 1.6-1.8g/L of nitrogen source of aminoyeast and 1.2-1.4g/L of ammonium sulfate.
Has the advantages that: the Yarrowia lipolytica (Yarrowia lipolytica) XJ-11 strain of the invention is based on the deletion of Yarrowia lipolytica responsible for coding non-homologous recombination gene ku70, so that the homologous recombination capability is enhanced, the gene integration is realized through the homologous recombination function of Yarrowia lipolytica, and the genetic stability of the introduced gene can be greatly improved. The construction method of the recombinant yarrowia lipolytica is efficient and simple to operate. The Yarrowia lipolytica (Yarrowia lipolytica) XJ-11 strain of the invention knocks out peroxisome biogenesis factor 10(PEX10 site) and triacylglycerol lipase 4(TGL4 site), and over-expresses endogenous delta 12 desaturase, phosphatidylcholine: experiments prove that the recombinant yarrowia lipolytica can efficiently ferment and produce the punicic acid, and the efficient synthesis of the plant-derived natural product punicic acid in the yarrowia lipolytica is realized. The synthetic scheme of the punicic acid provided by the embodiment of the invention is shown in figure 1.
Drawings
Fig. 1 is a schematic diagram of a synthesis strategy of punicic acid according to an embodiment of the present invention. ACC 1: acetyl-coa carboxylase; DGAT: diacylglycerol acyltransferase; LRO 1: phosphatidylcholine: diacylglycerol acyltransferase; ELO 1: fatty acid elongase 1; FAD 2: Δ 12 oleate desaturase; FADX: a fatty acid conjugated enzyme; FAS: a fatty acid elongase; TGL 4: triacylglycerol lipase 4; GPAT: 3-phosphoglycerate acyltransferase; and (3) LPAAT: lysophosphatidic acid acyltransferase; LPCAT: lysophosphatidylcholine acyltransferase; CPT: choline phosphotransferase.
FIG. 2 is a diagram showing the structure of plasmid pUC-HisG-Ura-HisG, wherein HisG represents a DNA fragment derived from Salmonella, and URA represents an orotidine-5' -phosphate decarboxylase-encoding gene expression cassette (containing the endogenous promoter P of Yarrowia lipolytica) TEFin Orotidine-5' -phosphate decarboxylase and terminator T xpr2t )。
FIG. 3 is a diagram of the structure of recombinant plasmid pUC-A08-PgFADX, in which A08-up represents the upstream homology arm at A08 site, A08-dm represents the downstream homology arm at A08 site, and TEFin represents promoter P TEFin And xpr2T denotes a terminator T xpr2t LEU denotes the expression cassette for the gene encoding 3 (. beta. -isopropylmalate dehydrogenase-containing the endogenous promoter P of Yarrowia lipolytica TEFin 3 (beta) -isopropylmalate dehydrogenase and terminator T xpr2t ) PgFADX is fatty acid conjugated enzyme coding gene.
FIG. 4 is a diagram showing the structure of recombinant plasmid pUC-HUH-IntA-YLFAD2, wherein IntA-up represents the upstream homology arm of IntA site, IntA-down represents the downstream homology arm of IntA site, and TEFin represents promoter P TEFin And xpr2T denotes a terminator T xpr2t URA denotes an orotidine-5' -phosphate decarboxylase-encoding gene expression cassette (containing the Yarrowia lipolytica endogenous promoter P) TEFin Orotidine-5' -phosphate decarboxylase and terminator T xpr2t ) YLFAD2 is a Δ 12 desaturase encoding gene.
FIG. 5 is a diagram showing the structure of a recombinant plasmid pUC-HUH-TGL4, wherein TGL4-up represents the upstream homology arm at TGL4 site, TGL4-down represents the downstream homology arm at TGL4 site, and URA represents the orotidine-5' -phosphate decarboxylase-encoding gene expression cassette (containing promoter P endogenous to Yarrowia lipolytica) TEFin Orotidine-5' -phosphate decarboxylase and terminator T xpr2t )。
Fig. 6 is a fatty acid composition analysis chart provided by an embodiment of the present invention.
Detailed Description
The present invention will be further illustrated by the following specific examples.
The experimental procedures used in the following examples are all conventional ones unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Yarrowia lipolytica (Yarrowia lipolytica) Po1f, available from American type culture Collection, accession number ATCC MYA-2613.
Yarrowia lipolytica (Yarrowia lipolytica) Po1 f. delta. ku70 (MatA. delta. ku70:: hisG, leu2-270, Ura3-302, xpr2-322, axp1-2), abbreviated as Yarrowia lipolytica Po1 f. delta. ku 70. Yarrowia lipolytica Po1 f. delta. ku70 was constructed from Yarrowia lipolytica Po1f after knocking out the coding gene ku70 responsible for non-homologous recombination (disclosed in Kretzschmar A, et al, Current Genetics,2013,59(1-2): 63-72).
The IntC site integration plasmid was obtained by inserting a 1402bp upstream sequence (upstream homology arm) of the start codon and a 1396bp downstream sequence (downstream homology arm) of the stop codon of the IntC site on chromosome C of Yarrowia lipolytica Po1 f. delta. ku70 genome into pUC57-hisG-Ura-hisG vector (construction method is shown in example 1), and two hisG tag-encoding genes were present between the upstream and downstream homology arms of the IntC site.
The PEX10 site integration plasmid is obtained by inserting a sequence with the upstream size of 2250bp of the initiation codon (upstream homology arm) of PEX10 site and a sequence with the downstream size of 1500bp of the termination codon (downstream homology arm) of the PEX10 site on chromosome C of Yarrowia lipolytica Po1f delta ku70 genome into pUC57-hisG-Ura-hisG vector (the construction method is shown in example 1), and two hisG tag coding genes are positioned on the PEX10 site and between the downstream homology arms.
The TGL4 site integration plasmid was obtained by inserting a sequence (upstream homology arm) with an upstream size of 1500bp from the start codon of TGL4 site and a sequence (downstream homology arm) with a downstream size of 1500bp from the stop codon into pUC57-hisG-Ura-hisG vector (the construction method is shown in example 1) on chromosome F of Yarrowia lipolytica Po 1F. delta. ku70 genome, and two hisG tag-encoding genes were located between the upstream and downstream homology arms at TGL4 site.
The IntF site integration plasmid was obtained by inserting a sequence (upstream homology arm) with a size of 1610bp upstream of the initiation codon and a sequence (downstream homology arm) with a size of 1852bp downstream of the termination codon into pUC57-hisG-Ura-hisG vector (the construction method is described in example 1) on chromosome F of Yarrowia lipolytica Po 1F. delta. ku70 genome, and two hisG tag-encoding genes were located between the upstream and downstream homology arms of the IntF site.
The IntA site integration plasmid was obtained by inserting a 1633bp upstream sequence of the start codon (upstream homology arm) and a 1621bp downstream sequence of the stop codon (downstream homology arm) of the IntA site on chromosome A of Yarrowia lipolytica Po1 f. delta. ku70 genome into pUC57-hisG-Ura-hisG vector (construction method is shown in example 1), and two hisG tag-encoding genes were present between the upstream and downstream homology arms of the IntA site.
The A08 site integration plasmid is obtained by inserting a sequence with the upstream size of 2521bp of the initiation codon (upstream homology arm) and a sequence with the downstream size of 2031bp of the termination codon (downstream homology arm) of an A08 site on a chromosome of Yarrowia lipolytica Po1f delta ku70 genome into a pUC57-Leu vector (the construction method is shown in example 1), and a Leu encoding gene is arranged between the upstream and downstream homology arms of the A08 site.
The IntE site integration plasmid was obtained by inserting a sequence 1533bp upstream of the start codon (upstream homology arm) and a sequence 1521bp downstream of the stop codon (downstream homology arm) of the IntE site on chromosome E of Yarrowia lipolytica Po1 f. delta. ku70 genome into pUC57-hisG-Ura-hisG vector (construction method is shown in example 1), and two hisG tag-encoding genes were present between the upstream and downstream homology arms of the IntE site.
The IntD site integration plasmid was obtained by inserting a sequence (upstream homology arm) with an upstream region of the start codon of the IntD site of 1443bp and a sequence (downstream homology arm) with a downstream region of 1189bp into a pUC57-hisG-Ura-hisG vector (the construction method is shown in example 1) on chromosome D of Yarrowia lipolytica Po1 f. delta. ku70 genome, and two hisG tag-encoding genes were present between the upstream and downstream homology arms of the IntD site.
Example 1 amplification of Gene elements and preparation of target plasmids
(first) preparation of target Gene
The nucleotide sequence of the fatty acid-conjugated enzyme encoding gene (GenBank accession No.: AY178446.1) from pomegranate (Punicandratam) provided at NCBI was codon-optimized, and then, the optimized fatty acid-conjugated enzyme encoding gene PgFADX (SEQ ID No: 1) was synthesized by Jinzhi Biotechnology Ltd, Suzhou, and inserted into plasmid pUC57 (available from GenScript) to obtain plasmid pUC 57-PgFADX.
Plasmid pUC57-PgFAD2 was obtained by synthesizing an optimized Δ 12 fatty acid desaturase-encoding gene PgFAD2(SEQ ID No: 2) by codon optimization based on the nucleotide sequence of a Δ 12 fatty acid desaturase-encoding gene (GenBank accession No: AY178447) from pomegranate (Punica grandium) provided at NCBI and inserting the gene into plasmid pUC 57.
Based on the nucleotide sequence of lysophosphatidylcholine acyltransferase gene (GenBank accession No: KC540908) from Ricinus communis provided at NCBI, after codon optimization, Suzhou Jinwei Biotechnology Ltd was entrusted to synthesize an optimized lysophosphatidylcholine acyltransferase-encoding gene RcLPCAT (SEQ ID No: 3), and inserted into plasmid pUC57 to obtain plasmid pUC 57-RcLPCAT.
Based on the nucleotide sequence of Yarrowia lipolytica orotidine-5 '-phosphate decarboxylase encoding gene Ura (GenBank accession No.: AJ306421.1) and hisG tag (GenBank accession No.: AF324729.1) provided at NCBI, the synthesis of Suzhou Jinzhi Biotechnology Limited was entrusted, two hisG tag encoding gene sequences were inserted into plasmid pUC57, and an orotidine-5' -phosphate decarboxylase encoding gene expression cassette (endogenous promoter P of Yarrowia lipolytica) was inserted between the two hisG tag encoding gene sequences TEFin Orotidine-5' -phosphate decarboxylase-encoding gene Ura and terminator T xpr2t Composition) to achieve Ura marker recovery, resulting in plasmid pUC57-hisG-Ura-hisG (see FIG. 2 for specific structure).
The nucleotide sequence of Leu, a gene encoding 3 (. beta. -isopropylmalate dehydrogenase gene of Yarrowia lipolytica provided at NCBI (GenBank accession No.: CP061014.1), was assigned to the national science and technology Ltd. The expression cassette for the gene encoding 3 (. beta. -isopropylmalate dehydrogenase) (promoter P endogenous to Yarrowia lipolytica) was inserted into plasmid pUC57 TEFin 3 (beta) -isopropylmalate dehydrogenase-encoding gene Leu and terminator T xpr2t Composition), a plasmid pUC57-Leu was obtained, which was identical in structure to FIG. 2 except that the hisG-Ura-hisG sequence was replaced with the LEU expression cassette.
acetyl-CoA carboxylase encoding gene ACC1(GenBank accession number: YALI0C11407g) was amplified using Yarrowia lipolytica Po 1F. delta. ku70 genomic DNA as a template and IntE:: ACC1-F and IntE:: ACC1-R as primers.
Delta 12 desaturase-encoding gene YLFAD2(GenBank accession: YALI0B10153g) was amplified using Yarrowia lipolytica Po1F delta ku70 genomic DNA as a template and IntA:: YLFAD2-F and IntA:: YLFAD2-R as primers.
The phosphatidylcholine was amplified using Yarrowia lipolytica Po 1F. delta. ku70 genomic DNA as template and PEX10:: YLLRO1-F and PEX10:: YLLRO1-R as primers: diacylglycerol acyltransferase-encoding gene YLLRO1(GenBank accession: YALI0E16797 g).
The choline phosphotransferase encoding gene YLCPT (GenBank accession number: YALI0C10989g) was amplified using Yarrowia lipolytica Po 1F. delta. ku70 genomic DNA as a template and IntC:: YLCPT-F and IntC:: YLCPT-R as primers.
(II) Yarrowia lipolytica endogenous promoter P hp4d The nucleotide sequence of (A) is shown as SEQ ID No. 4; endogenous promoter P of Yarrowia lipolytica TEFin The nucleotide sequence of (A) is shown as SEQ ID No. 5; endogenous promoter P of Yarrowia lipolytica GPDin The nucleotide sequence of (A) is shown as SEQ ID No. 6; endogenous terminator T of Yarrowia lipolytica mig1t The nucleotide sequence of (A) is shown as SEQ ID No. 7; endogenous terminator T of Yarrowia lipolytica xpr2t The nucleotide sequence is shown as SEQ ID No. 8.
(III) 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-A08-PgFADX
The recombinant plasmid pUC-A08-PgFADX takes pUC57-Leu as a framework, an upstream homology arm A08-up of an A08 site initiation codon and a downstream homology arm A08-dm of a termination codon in Yarrowia lipolytica Po1f delta ku70 are inserted, and a PgFADX expression cassette (P gFADX) is also inserted between the upstream homology arm and the downstream homology arm TEFin -PgFADX-T xpr2t ) LEU expression cassette (containing the endogenous promoter P of Yarrowia lipolytica) TEFin 3 (beta) -isopropylmalate dehydrogenase-encoding gene Leu and terminator T xpr2t ) Also between the upstream and downstream homology arms, the specific structure is shown in FIG. 3.
Using A08 as the reference P TEFin F and A08:: P TEFin -R is a primer, and genomic DNA of Yarrowia lipolytica Po1f delta ku70 is used as a template to amplify PgFADX expression cassette promoter P TEFin . T is defined as A08 xpr2t F and A08:: T xpr2t -R is a primer, and genomic DNA of Yarrowia lipolytica Po1f delta ku70 is used as a template to amplify a terminator T of a PgFADX expression cassette xpr2t 。
Plasmid pUC57-PgFADX is used as template, A08, PgFADX-F and A08, PgFADX-R are used as primer, and two amplified ends have promoter P TEFin And a terminator T xpr2t PgFADX gene of homologous arm.
The PCR amplification system is as follows:
| components | Volume of |
| PrimerSTARMaxPremix | 25ul |
| Form | 1ul |
| Primer | |
| 1 | 2ul |
| Primer 2 | 2ul |
| Distilled water | 20ul |
Of these, PrimerSTAR Max Premix was purchased from Baozi physician technology (Beijing) Inc.
The procedure for the above PCR was as follows: denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 5s, extension at 72 ℃ (extension time ═ length of target fragment/1 kb in min), and 30 cycles of repetition.
Each fragment was purified and recovered by using TaKaRaMiniBEST DNAsragment Purification Kit (purchased from Shanghai Baisai Biotechnology Ltd.).
The A08 site integration plasmid was digested with the NEB restriction enzyme SnaB I, and the linearized A08 site integration plasmid was recovered by agarose gel electrophoresis.
The linearized A08 site integration plasmid and each element (promoter P) in the PgFADX gene expression cassette constructed in title 1 of this example TEFin Gene PgFADX and terminator T xpr2t ) One-Step Cloning was achieved using the Clonexpress MultiS One Step Cloning Kit from Nanjing Novowed Biotechnology Ltd, and the PgFADD gene expression cassette was inserted between the upstream and downstream homology arms of the A08 site integration plasmid. The reaction system is shown in the following table. After incubating the reaction system at 37 ℃ for 30min, the circular recombinant vector is obtained.
The system for one-step cloning is as follows:
| components | Volume of |
| 5×CEMultiSBuffer | 4ul |
| ExnaseMultiS | 2ul |
| Linearized vector | xng |
| Insert fragment | yng |
| Distilled water | Make up the volume to 10ul |
The usage of linearized vector (x) and insert (y ═ the sum of the optimal usage of all inserts) can be calculated from the following formula: the optimum amount of the vector used per fragment or linearized vector was ═ 0.02 × base pair of fragments or linearized vector ] ng.
And transforming the circular recombinant vector into escherichia coli DH5 alpha competent cells, and obtaining a positive recombinant plasmid pUC-A08-PgFADX by screening ampicillin resistant plates and verifying colony PCR and sequencing.
After the plasmid pUC-A08-PgFADX was digested with the restriction enzyme Not I of NEB, the plasmid pUC-A08-PgFADX was linearized by agarose gel electrophoresis.
2. Construction of recombinant plasmid pUC-HUH-IntA-YLFAD2
The recombinant plasmid pUC-HUH-IntA-YLFAD2 is characterized in that pUC57-hisG-Ura-hisG is used as a framework, an upstream homology arm IntA-up of an IntA site initiation codon and a downstream homology arm IntA-dm of a termination codon in Yarrowia lipolytica Po1f delta ku70 are inserted, and a YLFAD2 gene expression cassette (P FAD2 gene) is inserted between the upstream and downstream homology arms TEFin -YlFAD2-T xpr2t ) And URA expression cassette (containing the endogenous promoter of Yarrowia lipolytica)
P TEFin Orotidine-5' -phosphate decarboxylase and terminator T xpr2t ) The specific structure is shown in figure 4.
Using IntA as P TEFin F and IntA:: P TEFin -R is a primer, and the genomic DNA of Yarrowia lipolytica Po1f delta ku70 is used as a template to amplify the promoter P of the YLFAD2 expression cassette TEFin . Using IntA as the base xpr2t -F and IntA:: T xpr2t -R is a primer, genomic DNA of Yarrowia lipolytica Po1f delta ku70 is taken as a template, and the terminator T of the YLFAD2 expression cassette is amplified xpr2t 。
The Yarrowia lipolytica Po1F delta ku70 genome DNA is used as a template, the IntA:: YLFAD2-F and IntA:: YLFAD2-R are used as primers, and the two ends of the amplification are respectively provided with a promoter P TEFin And a terminator T xpr2t The YLFAD2 gene of the homologous arm.
The IntA site integration plasmid was digested with the restriction enzyme PacI from NEB, and the linearized IntA site integration plasmid was recovered by agarose gel electrophoresis.
The linearized IntA plasmid and the elements (promoter P) in the expression cassette of the YLFAD2 gene constructed in title 2 of this example TEFin Gene YLFAD2 and terminator T xpr2t ) One-Step Cloning was achieved by using the Clonexpress MultiS One Step Cloning Kit of Nanjing Novowed Biotechnology Ltd, and the YLFAD2 gene expression cassette was inserted between the upstream and downstream homology arms of the IntA site integration plasmid.
And transforming the circular recombinant vector into escherichia coli DH5 alpha competent cells, and obtaining a positive recombinant plasmid pUC-HUH-IntA-YLFAD2 by screening ampicillin resistant plates and verifying colony PCR and sequencing.
After the plasmid pUC-HUH-IntA-YLFAD2 was digested with the restriction enzyme Ssp I from NEB, the linearized pUC-HUH-IntA-YLFAD2 plasmid was recovered by agarose gel electrophoresis.
3. Construction of recombinant plasmid pUC-HUH-IntA-PgFAD2
The recombinant plasmid pUC-HUH-IntA-PgFAD2 takes pUC57-hisG-Ura-hisG as a framework, an IntA upstream homology arm IntA-up of an IntA site start codon and an IntA-dm of a termination codon downstream homology arm in Yarrowia lipolytica Po1f delta ku70 are inserted, and a PgFAD2 gene expression cassette (P gFAD2 gene expression cassette) is also inserted between the IntA upstream homology arm and the PgFAD downstream homology arm (P gFAD-Dm) TEFin -PgFAD2-T xpr2t ) URA expression cassette (containing the endogenous promoter P of Yarrowia lipolytica) TEFin Orotidine-5' -phosphate decarboxylase and terminator T xpr2t ) Also between the upstream and downstream homology arms. The structure diagram of the recombinant plasmid pUC-HUH-IntA-PgFAD2 is the same as that in FIG. 3, but the difference is that the PgFAD2 gene expression cassette is used for replacing the YLFAD2 gene expression cassette.
Using IntA as P TEFin F and IntA:: P TEFin -R is a primer, and genomic DNA of Yarrowia lipolytica Po1f delta ku70 is used as a template to amplify a PgFAD2 expression cassette promoter P TEFin . Using IntA as the base xpr2t -F and IntA:: T xpr2t -R is a primer, genomic DNA of Yarrowia lipolytica Po1f delta ku70 is taken as a template, and the PgFAD2 expression cassette terminator T is amplified xpr2t 。
Plasmid pUC57-PgFAD2 as template, and IntA PgFAD2-F as templatePgFAD2-R as primer, with promoter P on two ends TEFin And a terminator T xpr2t PgFAD2 gene of homologous arm.
The IntA site integration plasmid was digested with the restriction enzyme PacI from NEB, and the linearized IntA site integration plasmid was recovered by agarose gel electrophoresis.
The linearized IntA site integration plasmid and each element (promoter P) in the PgFAD2 gene expression cassette constructed in title 3 of this example TEFin Gene PgFAD2 and terminator T xpr2t ) One-Step Cloning was achieved using the Clonexpress MultiS One Step Cloning Kit from Nanjing Novowed Biotechnology Ltd, and the PgFAD2 gene expression cassette was inserted between the upstream and downstream homology arms of the IntA site integration plasmid.
And transforming the circular recombinant vector into escherichia coli DH5 alpha competent cells, and obtaining a positive recombinant plasmid pUC-HUH-IntA-PgFAD2 by screening ampicillin resistant plates and verifying colony PCR and sequencing.
After the plasmid pUC-HUH-IntA-PgFAD2 was digested with the restriction enzyme Ssp I of NEB, the linearized pUC-HUH-IntA-PgFAD2 plasmid was recovered from the gel by agarose gel electrophoresis.
4. Construction of recombinant plasmid pUC-HUH-IntF-PgFADX
The recombinant plasmid pUC-HUH-IntF-PgFADX takes pUC57-hisG-Ura-hisG as a framework, an upstream homology arm IntF-up of an IntF site initiation codon and a downstream homology arm IntF-dm of a termination codon in Yarrowia lipolytica Po1f delta ku70 are inserted, and a PgFADX gene expression cassette (P gFADX gene) is also inserted between the upstream and downstream homology arms GPDin -PgFADX-T mig1t ) URA expression cassette (containing the endogenous promoter P of Yarrowia lipolytica) TEFin Orotidine-5' -phosphate decarboxylase and terminator T xpr2t ) Also between the upstream and downstream homology arms. The structure diagram of the recombinant plasmid pUC-HUH-IntF-PgFADX is the same as that in FIG. 3, the difference is only that IntF-up and IntF-dm are respectively used for replacing IntA-up and IntA-dm, and PgFADX gene expression box is used for replacing YLFAD2 gene expression box. Using IntF as follows GPDin P and IntF GPDin -R is a primer, and the genomic DNA of Yarrowia lipolytica Po1f delta ku70 is used as a template to amplify the PgFADX tableCassette promoter P GPDin . Using IntF as base station mig1t T and IntF mig1t -R is a primer, and genomic DNA of Yarrowia lipolytica Po1f delta ku70 is used as a template to amplify a terminator T of a PgFADX expression cassette mig1t 。
Plasmid pUC57-PgFADX is used as template, IntF:: PgFADX-F and IntF:: PgFADX-R are used as primers, and promoter P is respectively arranged at two amplification ends GPDin And a terminator T mig1t PgFADX gene of homologous arm.
After the IntF site-integrated plasmid was digested with the restriction enzyme PacI from NEB, the linearized IntF plasmid was recovered from the gel by agarose gel electrophoresis.
The linearized IntF plasmid and the elements (P) of the PgFADX gene expression cassette constructed in title 4 of this example were ligated GPDin Gene PgFADX and terminator T mig1t ) Using Nanjing NuoZan Biotechnology Ltd
II One Step Cloning was carried out by One Step Cloning Kit to obtain circular recombinant vector.
And transforming the circular recombinant vector into escherichia coli DH5 alpha competent cells, and obtaining a positive recombinant plasmid pUC-HUH-IntF-PgFADX by screening ampicillin resistant plates and verifying colony PCR and sequencing.
After the plasmid pUC-HUH-IntF-PgFADX was digested with the restriction enzyme Not I from NEB, the linearized pUC-HUH-IntF-PgFADX plasmid was recovered by agarose gel electrophoresis.
5. Construction of recombinant plasmid pUC-HUH-PEX10-YLLRO1
The recombinant plasmid pUC-HUH-PEX10-YLLRO1 is characterized in that pUC57-hisG-Ura-hisG is used as a framework, a homology arm PEX10-up with the upstream size of 2250bp of a PEX10 site start codon in Yarrowia lipolytica Po1f delta ku70 and a homology arm PEX10-dm with the downstream size of 1500bp of a stop codon are inserted, and a YLLRO1 expression cassette (P-HUH-PEX 10-dm) is also inserted between the upstream and downstream homology arms TEFin -YlLRO1-T xpr2t ) Orotidine-5' -phosphate decarboxylase encoding gene expression cassettes (containing the Yarrowia lipolytica endogenous promoter P) TEFin Orotidine-5' -phosphateDecarboxylase and terminator T xpr2t ) Also between the upstream and downstream homology arms. The structure of recombinant plasmid pUC-HUH-PEX10-YLLRO1 is the same as that shown in FIG. 3, except that PEX10-up and PEX10-dm are respectively used to replace IntA-up and IntA-dm, and YLLRO1 gene expression cassette is used to replace YLFAD2 gene expression cassette. P is a polyethylene terephthalate (PEX) 10 TEFin F and PEX10:: P TEFin -R is a primer, and the genomic DNA of Yarrowia lipolytica Po1f delta ku70 is used as a template to amplify the YLLRO1 expression cassette promoter P TEFin . Using PEX10: (T) xpr2t F and PEX10:: T xpr2t -R is a primer, and genomic DNA of Yarrowia lipolytica Po1f delta ku70 is used as a template to amplify the terminator T of the YLLRO1 expression cassette xpr2t 。
The genomic DNA of Yarrowia lipolytica Po1F delta ku70 is taken as a template, PEX10, YLLRO1-F and PEX10, YLLRO1-R are taken as primers, and promoters P are respectively arranged at two ends of amplification TEFin And a terminator T xpr2t The YLLRO1 gene of the homology arm (GenBank accession: YALI0E16797 g).
The plasmid integrated at the PEX10 site was digested with Pac I, a restriction enzyme from NEB, and the linearized PEX10 plasmid was recovered from the gel by agarose gel electrophoresis.
The linearized PEX10 plasmid and each element (P) in the YLLRO1 gene expression cassette constructed in title 5 of this example were ligated TEFin Gene YLLRO1 and terminator T xpr2t ) Clonexpress using Nanjing Novozam Biotechnology Ltd
II One Step Cloning was carried out by One Step Cloning Kit to obtain circular recombinant vector.
The circular recombinant vector is transformed into escherichia coli DH5 alpha competent cells, and a positive recombinant plasmid pUC-HUH-PEX10-YLLRO1 is obtained through ampicillin resistance plate screening, colony PCR and sequencing verification.
After the plasmid pUC-HUH-PEX10-YLLRO1 was digested with the restriction enzyme Not I from NEB, the linearized pUC-HUH-PEX10-YLLRO1 plasmid was recovered from the gel by agarose gel electrophoresis.
6. Construction of recombinant plasmid pUC-HUH-IntC-YLCPT
Recombinant plasmidpUC-HUH-IntC-YLCPT is pUC57-hisG-Ura-hisG as framework, IntC-up homology arm IntC-up of IntC site initiation codon and IntC-dm of termination codon downstream homology arm in Yarrowia lipolytica Po1f delta ku70 are inserted, and YLCPT gene expression cassette (P-HUH-IntC-YLCPT) is inserted between the upstream and downstream homology arms TEFin -YlCPT-T xpr2t ) Orotidine-5' -phosphate decarboxylase encoding gene expression cassettes (containing the Yarrowia lipolytica endogenous promoter P) TEFin Orotidine-5' -phosphate decarboxylase and terminator T xpr2t ) Also between the upstream and downstream homology arms. The structure diagram of the recombinant plasmid pUC-HUH-IntC-YLCPT is the same as that in FIG. 3, except that IntA-up and IntA-dm are respectively replaced by IntC-up and IntC-dm, and the YLCPT gene expression cassette is used to replace the YLFAD2 gene expression cassette.
From IntC:: P TEFin F and IntC:: P TEFin -R is a primer, and the genomic DNA of Yarrowia lipolytica Po1f delta ku70 is used as a template to amplify the YLCPT expression cassette promoter P TEFin . Using IntC as the base xpr2t -F and IntC:: T xpr2t -R is a primer, and the genomic DNA of Yarrowia lipolytica Po1f delta ku70 is used as a template to amplify the terminator T of the YLCPT expression cassette xpr2t 。
The Yarrowia lipolytica Po1F delta ku70 genome DNA is taken as a template, the IntC:: YLCPT-F and IntC:: YLCPT-R are taken as primers, and the two ends of the amplification are respectively provided with a promoter P TEFin And a terminator T xpr2t The YLCPT gene of the homology arm.
The IntC site integration plasmid was digested with HindIII, a restriction enzyme from NEB, and the linearized IntC site integration plasmid was recovered from the gel by agarose gel electrophoresis.
The linearized IntC site integration plasmid and each element (promoter P) in the YLCPT gene expression cassette constructed in title 6 of this example TEFin Gene YLCPT and terminator T xpr2t ) One-Step Cloning is realized by using Clonexpress Multi S One Step Cloning Kit of Nanjing Novowed Biotechnology Co., Ltd to obtain a recombinant plasmid pUC-HUH-intC-YLCPT.
The circular recombinant vector is transformed into escherichia coli DH5 alpha competent cells, and positive recombinant plasmid pUC-HUH-intC-YLCPT is obtained through ampicillin resistance plate screening and colony PCR and sequencing verification.
After the plasmid pUC-HUH-IntC-YLCPT was digested with EcoRI, a restriction enzyme from NEB, the linearized pUC-HUH-IntC-YLCPT plasmid was recovered by agarose gel electrophoresis.
7. Construction of recombinant plasmid pUC-HUH-IntE-ACC1
The recombinant plasmid pUC-HUH-IntE-ACC1 takes pUC57-hisG-Ura-hisG as a framework, an IntE-up homology arm IntE-up of an IntE site initiation codon and an IntE-dm of a downstream homology arm of a termination codon in Yarrowia lipolytica Po1f delta ku70 are inserted, and an ACC1 gene expression cassette (P1 gene expression cassette) is also inserted between the IntE-up homology arm and the downstream homology arm hp4d -ACC1-T mig1t ) Orotidine-5' -phosphate decarboxylase encoding gene expression cassettes (containing the Yarrowia lipolytica endogenous promoter P) TEFin Orotidine-5' -phosphate decarboxylase and terminator T xpr2t ) Also between the upstream and downstream homology arms. The structure diagram of the recombinant plasmid pUC-HUH-IntE-ACC1 is the same as that in FIG. 3, except that IntA-up and IntA-dm are respectively replaced by IntE-up and IntE-dm, and an ACC1 gene expression cassette is replaced by YLFAD2 gene expression cassette. P is expressed by IntE hp4d P and IntE hp4d -R is a primer, and the ACC1 expression cassette promoter P is amplified by using Yarrowia lipolytica Po1f delta ku70 genome DNA as a template hp4d . Using IntE as the base mig1t -F and IntE:: T mig1t -R is a primer, and uses Yarrowia lipolytica Po1f delta ku70 genome DNA as a template to amplify ACC1 expression cassette terminator T mig1t 。
Uses Yarrowia lipolytica Po1F delta ku70 genome DNA as a template, uses IntE (ACC 1-F) and IntE (ACC 1-R) as primers, and amplifies two ends of the primer with a promoter P hp4d And a terminator T mig1t ACC1 gene of homologous arm.
After the IntE site integration plasmid was digested with the restriction enzyme PacI of NEB, the linearized IntE site integration plasmid was recovered by agarose gel electrophoresis.
The linearized IntE site integration plasmid and each element (promoter P) in the ACC1 gene expression cassette constructed in title 7 of this example hp4d ACC1 gene and terminator T mig1t ) Clonexpress MultiS One Step using Nanjing NuoZan Biotechnology LtdThe Cloning Kit enabled one-step Cloning, inserting the ACC1 gene expression cassette between the upstream and downstream homology arms of the IntE site integration plasmid.
The circular recombinant vector is transformed into escherichia coli DH5 alpha competent cells, and a positive recombinant plasmid pUC-HUH-IntE-ACC1 is obtained through ampicillin resistance plate screening and colony PCR and sequencing verification.
After the plasmid pUC-HUH-IntE-ACC1 was digested with EcoRI, a restriction enzyme from NEB, the linearized pUC-HUH-IntE-ACC1 plasmid was recovered by agarose gel electrophoresis.
8. Construction of recombinant plasmid pUC-HUH-IntD-RcLPCAT
The recombinant plasmid pUC-HUH-IntD-RcLPCAT takes pUC57-hisG-Ura-hisG as a framework, an upstream homology arm IntD-up of an IntD site initiation codon and an downstream homology arm IntD-dm of a termination codon in Yarrowia lipolytica Po1f delta ku70 are inserted, and a RcLPCAT gene expression cassette (P) is also inserted between the upstream and downstream homology arms TEFin -RcLPCAT-T xpr2t ) Orotidine-5' -phosphate decarboxylase encoding gene expression cassettes (containing the Yarrowia lipolytica endogenous promoter P) 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-IntD-RcLPCAT is the same as that shown in FIG. 3, except that IntA-up and IntA-dm are replaced with IntD-up and IntD-dm, respectively, and the YLFAD2 gene expression cassette is replaced with RcLPCAT gene expression cassette.
Using IntD as the reference TEFin F and IntD:: P TEFin -R is a primer, and the genomic DNA of Yarrowia lipolytica Po1f delta ku70 is used as a template to amplify the promoter P of the RcLPCAT expression cassette TEFin . From IntD as follows: (T) xpr2t T and IntD xpr2t -R is a primer, and RcLPCAT expression cassette terminator T is amplified by using Yarrowia lipolytica Po1f delta ku70 genome DNA as a template xpr2t 。
Using pUC57-RcLPCAT as template and RcLPCAT-F and RcLPCAT-R as primers to amplify two ends with promoter P TEFin And a terminator T xpr2t The RcLPCAT gene of the homologous arm.
After the IntD site integration plasmid was digested with the restriction enzyme PacI of NEB, the linearized IntD site integration plasmid was recovered by agarose gel electrophoresis.
The linearized IntD site integration plasmid and each element (promoter P) in the RcLPCAT gene expression cassette constructed in title 8 of this example TEFin Gene RcLPCAT and terminator T xpr2t ) One-Step Cloning is realized by using Clon express Multi S One Step Cloning Kit of Nanjing Novowed Biotechnology Limited, and the RcLPCAT gene expression cassette is inserted between the upstream and downstream homology arms of the IntD locus integration 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-IntD-RcLPCAT is obtained through ampicillin resistance plate screening and colony PCR and sequencing verification.
After the plasmid pUC-HUH-IntD-RcLPCAT was digested with the restriction enzyme Not I from NEB, the linearized pUC-HUH-IntD-RcLPCAT plasmid was recovered by agarose gel electrophoresis.
9. Construction of recombinant knockout plasmid pUC-HUH-TGL4
The recombinant plasmid pUC-HUH-TGL4 takes pUC57-hisG-Ura-hisG as a framework, a homologous arm (TGL4-up) with the upstream size of the initiation codon of TGL4 site being 1500bp and a homologous arm (TGL4-dm) with the downstream size of the termination codon being 1500bp in Yarrowia lipolytica Po1f delta ku70 are inserted, and an orotidine-5' -phosphate decarboxylase encoding gene expression cassette (containing a promoter P endogenous to Yarrowia lipolytica) TEFin Orotidine-5' -phosphate decarboxylase and terminator T xpr2t ) Also between the upstream and downstream homology arms, the detailed structure is shown in FIG. 5.
TGL4-up-F and TGL4-up-R are used as primers, Yarrowia lipolytica Po1F delta ku70 genome DNA is used as a template, and upstream homology arm TGL4-up of an initiation codon of TGL4 site is amplified.
After digesting plasmid pUC57-hisG-Ura-hisG with EcoRI, a restriction enzyme of NEB, the linearized pUC57-hisG-Ura-hisG plasmid was recovered by agarose gel electrophoresis.
The linearized pUC57-hisG-Ura-hisG plasmid and TGL4 site initiation codon upstream homology arm TGL4-up (with pUC at both ends) constructed in title 9 of this example were used57-hisG-Ura-hisG homology arm sequences) Using Nanjing Novozam Biotech Ltd
II One Step Cloning was carried out by One Step Cloning Kit to obtain 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 ampicillin resistance plate screening and colony PCR and sequencing verification.
TGL4-dm-F and TGL4-dm-R are used as primers, Yarrowia lipolytica Po1F delta ku70 genome DNA is used as a template, and a downstream homology arm TGL4-dm of a stop codon at the TGL4 site is amplified.
After the plasmid pUC-HUH-TGL4-up was digested with the restriction enzyme Hind III from NEB, the linearized pUC-HUH-TGL4-up plasmid was recovered by agarose gel electrophoresis.
The linearized pUC-HUH-TGL4-up plasmid and TGL4 site stop codon downstream homology arm TGL4-dm (with pUC-HUH-TGL4-up homology arm sequence at both ends) constructed in title 9 of this example were used with Nanjing Nodezam Biotech Co., Ltd
II One Step Cloning was performed with the One Step Cloning Kit to obtain recombinant plasmid pUC-HUH-TGL 4.
After the plasmid pUC-HUH-TGL4 was digested with the restriction enzyme Hind III from NEB, the linearized pUC-HUH-TGL4 plasmid was recovered by agarose gel electrophoresis.
TABLE 1 insertion sequence in each recombinant plasmid
TABLE 2 primer sequences
Example 2 construction of recombinant yarrowia lipolytica
The plasmids introduced in this example were all linearized plasmids.
(I) construction of recombinant bacterium 1-2
Recombinant plasmid pUC-HUH-IntA-YLFAD2 is introduced into Yarrowia lipolytica Po1f delta ku70, an expression cassette of YLFAD2 is integrated to the IntA locus of a genome, and then a hisG tag and a Ura selection tag are lost under 5-fluoroorotic acid selection pressure, so that recombinant bacterium 1 is obtained. pUC-HUH-IntA-PgFAD2 was introduced into Yarrowia lipolytica Po1 f. delta. ku70, the PgFAD2 expression cassette was integrated into the genomic IntA site, and recombinant strain 2 was obtained after losing one hisG tag and Ura selection marker under 5-fluoroorotic acid selection pressure.
The specific method for constructing the recombinant bacterium 1 is as follows:
yarrowia lipolytica Po1 f. delta. ku70 competent cells were prepared after overnight culture in YPD liquid medium (containing 2% peptone, 1% Yeast extract and 2% glucose), the recombinant plasmid pUC-HUH-IntA-YLFAD2 was transformed into Yarrowia lipolytica Po1 f. delta. ku70 using Zymogen FROZEN EZ Yeast Transformation Kit II of Zymo Research Corporation for homologous recombination, positive clones were selected using selection medium SD-Ura, and then PCR identification was performed. Wherein the selection medium SD-Ura contains 20g/L glucose, 6.7g/L YNB (without amino yeast nitrogen source, purchased from BBI Life Sciences), 0.67g/L CSM-Ura (complete supplement mixture for removing uracil, purchased from MPBiomedia), 23g/L agar powder, and water as solvent.
Secondly, coating positive clones which are correctly identified by PCR on YPD plates containing 5-fluoroorotic acid, and then taking a single colony to streak on the YPD plates containing 5-fluoroorotic acid and SD-Ura plates at the same time. A single colony that grew on YPD plates containing 5-fluoroorotic acid but failed to grow on SD-Ura plates was selected to obtain recombinant bacterium 1.
YPD plates containing 5-fluoroorotic acid: 1g of 5-fluoroorotic acid powder was dissolved in 10mL of dimethyl sulfoxide (DMSO) to obtain a 5-fluoroorotic acid solution. To 99 parts by volume of YPD medium, 1 part by volume of 5-fluoroorotic acid solution was added to obtain YPD plates containing 5-fluoroorotic acid.
The construction method of the recombinant bacterium 2 is the same as that of the recombinant bacterium 1, and only the difference is that pUC-HUH-IntA-PgFAD2 is used for replacing pUC-HUH-IntA-YLFAD 2.
(II) construction of recombinant bacterium 3
Plasmids pUC-A08-PgFADX and pUC-HUH-IntF-PgFADX containing PgFADX gene expression cassettes are sequentially introduced into the recombinant bacterium 1, and the PgFADX expression cassettes are integrated to a genome A08 locus and an IntF locus to obtain a recombinant bacterium 3.
The specific method comprises the following steps:
the recombinant bacterium 1 was cultured overnight in YPD liquid medium (containing 2% peptone, 1% yeast extract and 2% glucose) to prepare competent cells.
Secondly, pUC-A08-PgFADX was transformed into competent cells of recombinant bacterium 1 using Zymogen FROzen EZ Yeast Transformation Kit II from Zymo Research Corporation, positive clones were selected using selection medium SD-Leu, and PCR was performed. Wherein the screening medium SD-Leu contains: 20g/L glucose, 6.7g/L YNB (Aminoyeast nitrogen source without amino group, from BBI Life Sciences), 0.67g/L CSM-Leu (leucine removal from complete make-up mixture, from MPBiomedia), 23g/L agar powder, and water as solvent.
And thirdly, converting pUC-HUH-IntF-PgFADX into the positive clone obtained in the second step, and screening the positive clone by SD-Ura and carrying out PCR identification. The positive clone which is correctly identified by PCR is named as recombinant bacterium 3.
(III) construction of recombinant bacterium 4
Losing one hisG label and Ura selection marker for the recombinant bacterium 3, then sequentially introducing plasmids pUC-HUH-PEX10-YLLRO1 for knocking out plasmid pUC-HUH-TGL4 and YLLRO1 gene expression cassettes, plasmids pUC-HUH-IntC-YLCPT for YLCPT gene expression cassettes and plasmid pUC-HUH-IntE-ACC1 for ACC1 gene expression cassettes, integrating YLLRO1 expression cassettes to PEX10 sites, integrating YLCPT expression cassettes to IntC sites, integrating ACC1 expression cassettes to IntE sites, and knocking out PEX10 and TGL4 sites to obtain the recombinant bacterium 4.
The specific method comprises the following steps:
coating the recombinant bacterium 3 on an YPD plate containing 5-fluoroorotic acid, and then taking a single colony to simultaneously streak the YPD plate containing 5-fluoroorotic acid and an SD-Ura plate. Single colonies that grew on YPD plates containing 5-fluoroorotic acid but failed to grow on SD-Ura plates were selected, losing one hisG tag and Ura selection marker.
② recombinant bacterium 3 which has lost one hisG tag and Ura selection marker is cultured overnight in YPD liquid medium (containing 2% peptone, 1% Yeast extract, 2% glucose) to prepare competent cells, and recombinant plasmids pUC-HUH-TGL4, pUC-HUH-PEX10-YLLRO1, pUC-HUH-intC-YLCPT and pUC-HUH-IntE-ACC1 are transformed into recombinant bacterium 3 in sequence by using Zymogen FROZEN EZ Yeast Transformation Kit II of Zymo Research Corporation for homologous recombination. After each plasmid is introduced, positive clones are screened by adopting a screening culture medium SD-Ura, and then PCR identification is carried out. Wherein the screening culture medium SD-Ura contains 20g/L glucose, 6.7g/L YNB, 0.67g/L CSM-Ura, 23g/L agar powder and water as solvent.
Thirdly, positive clones which are correctly identified by PCR are coated on YPD plates containing 5-fluoroorotic acid, and single colonies are taken and streaked on YPD plates containing 5-fluoroorotic acid and SD-Ura plates at the same time. A single colony that grew on YPD plates containing 5-fluoroorotic acid but failed to grow on SD-Ura plates was selected and designated as recombinant bacterium 4.
(IV) construction of recombinant bacterium 5 (overexpression RcLPCAT based on over-expression PgFADX, YLFAD2, ACC1, YLLRO1, YLCPT knockout PEX10 and TGL 4)
And (3) introducing a recombinant plasmid pUC-HUH-IntD-RcLPCAT into the recombinant bacterium 4, integrating an RcLPCAT expression cassette into a genome IntD locus, and then losing a hisG label and a Ura screening marker under the 5-fluoroorotic acid screening pressure to obtain a recombinant bacterium 5.
The specific method comprises the following steps:
culturing recombinant bacterium 4 in YPD liquid culture medium (containing 2% peptone, 1% Yeast extract and 2% glucose) overnight to prepare competent cells, transforming recombinant plasmid pUC-HUH-intD-RcLPCAT into recombinant bacterium 4 by using Zymogen FROzen EZ Yeast Transformation Kit II of Zymo Research Corporation for homologous recombination, screening positive clones by using screening culture medium SD-Ura, and carrying out PCR identification. Wherein the screening culture medium SD-Ura contains 20g/L glucose, 6.7g/L YNB, 0.67g/L CSM-Ura, 23g/L agar powder and water as solvent.
Secondly, coating positive clones which are correctly identified by PCR on YPD plates containing 5-fluoroorotic acid, and taking single colonies to streak on the YPD plates containing 5-fluoroorotic acid and SD-Ura plates at the same time. A single colony that grew on YPD plates containing 5-fluoroorotic acid but failed to grow on SD-Ura plates was selected and designated as recombinant bacterium 5.
The above 5 recombinant bacteria were constructed, wherein recombinant bacteria 5 was named yarrowia lipolytica XJ-11, and was deposited in China Center for Type Culture Collection (CCTCC) at 2022, 3, 25 d, address: wuhan, Wuhan university; the preservation number is CCTCC NO: m2022320; the classification is named as: yarrowia lipolytica XJ-11, Yarrowia lipolytica XJ-11.
Example 3 use of recombinant yarrowia lipolytica in the production of punicic acid
Engineering bacteria culture and product extraction
Punicate was produced using Yarrowia lipolytica (Yarrowia lipolytica) Po1 f. delta. ku70 as the starting bacterium and the recombinant bacteria 1-5 of example 2, respectively. The specific method comprises the following steps: respectively activating the initial strain and the recombinant strain, and culturing in YPD liquid culture medium (containing 2% peptone, 1% yeast extract, 2% glucose, and water as solvent) at 30 deg.C and 220rpm for 16h to obtain seed liquid. The seed solution was inoculated in 50ml of a fermentation medium at an inoculum size of 1%, and shake-cultured at 220rpm at 30 ℃ for 5 days. After fermentation, transferring the fermentation liquor to a 50ml centrifuge tube, centrifuging for 15min at 5000rpm, removing supernatant, taking the precipitate, and drying in an oven (75 ℃) to constant weight to obtain dry thalli.
Wherein the formula of the fermentation medium is as follows: 60g/L glucose, 1.7g/L nitrogen source (YNB) without amino yeast, 1.3g/L ammonium sulfate and water as solvent.
Qualitative and quantitative analysis of punicic acid
1. Methyl esterification of fatty acids
Weighing about 0.05g of dry thallus, adding 100 mu L of normal hexane solution of methyl tridecanoate with the concentration of 0.2408g/L as an internal standard, then adding 500 mu L of methanol solution of NaOH with the concentration of 1M, whirling at 1200rpm for 1 hour at room temperature (25 ℃), and neutralizing with 40 mu L of 98% sulfuric acid aqueous solution; add 400. mu.L n-hexane and vortex at 1200rpm for 10 minutes at room temperature (25 ℃) where the fatty acid methyl esters are extracted into the n-hexane; after centrifugation at 8000rpm for 2 minutes, the supernatant was filtered through an organic nylon filter (0.22 μm) and transferred to a glass bottle for GC detection.
2. Detection of total fatty acids and punicic acid
GC detection conditions are as follows: FID detector, injection port temperature 250 ℃, injection volume 1 μ l, split ratio: 50:1, chromatographic column: DB-23(60m 0.25 m 0.15 m). Chromatographic conditions are as follows: the initial temperature is 100 deg.C, and the temperature is increased to 196 deg.C at a rate of 25 deg.C/min, then increased to 220 deg.C at a rate of 2 deg.C/min, and maintained for about 2 min. The total fatty acids and punicic acid were quantified qualitatively and quantitatively using a fatty acid mix standard from Sigma-Aldrich.
Under the above conditions, the peak time of methyl puniciate detected by GC was about 18.9min (FIG. 6).
After 5 days of fermentation, no punicic acid accumulation was detected in recombinant bacteria 1 and 2. The mass percentages of the punicic acid of the recombinant bacteria 3, the recombinant bacteria 4 and the recombinant bacteria 5 in the total fatty acid (the sum of all detected fatty acids) are 3.68%, 16.40% and 21.64%, respectively. The yield of the punicic acid of the recombinant bacterium 5 is the highest (table 3), and reaches 0.92g/L, namely, 0.92g of the punicic acid is produced in each liter of fermentation liquor, which is obviously higher than that of the initial bacterium and the recombinant bacterium 3.
TABLE 3 punicic acid yields of initial, recombinant 3 and recombinant 5 bacteria
The recombinant bacterium 5 was inoculated into 50ml of a seed culture (YPD medium) and cultured for 24 hours, and then the seed culture was inoculated into a fermenter at an inoculum size of 5%. Adding 2.5L fermentation medium into 5L fermentation tank, fermenting for 0-48 hr, and controlling dissolved oxygen to 20%; the fermentation time is more than 48h, and the dissolved oxygen is controlled to be 0-5%. During the fermentation, the pH value was constantly controlled at 5.5 until the end of the fermentation. The fermentation time was 40h and 96h, and 800ml of 800g/L aqueous glucose solution was added each time. The fermentation temperature was 28 ℃ and the cultivation time was six days. Wherein the fermentation medium comprises 150g/L glucose, 11g/L ammonium sulfate, 3g/L yeast extract, 0.1g/L corn peptone, 4g/L potassium dihydrogen phosphate, 2g/L magnesium sulfate, 0.8g/L calcium sulfate, 0.4g/L sodium chloride, 12mg/L ammonium sulfate hydrochloride, 1mg/L biotin, 160mg/L sodium molybdate, 0.2mg/L copper sulfate, 40mg/L boric acid, 180mg/L manganese sulfate and 75mg/L ferrous chloride. After 5 days of fermentation, the yield of the punicic acid of the recombinant bacteria 5 reaches 9.08g/L to the maximum, namely 9.08g of punicic acid is produced per liter of fermentation liquor (shown in table 3), which is obviously higher than that of the initial bacteria and the recombinant bacteria 3.
SEQUENCE LISTING
<110> Nanjing university of industry
<120> recombinant yarrowia lipolytica strain with high punicic acid yield as well as construction method and application thereof
<130> 20220513
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 1188
<212> DNA
<213> artificial
<220>
<223> PgFADX
<400> 1
atgggcgcgg acgggactat gtctcccgtg ctgaccaagc gacgacccga ccaagagatc 60
aacaagctgg acatcaagcc caaccacgag gtggacatcg cccgacgagc cccccactct 120
aagcccccct tcaccctgtc tgacctgcga tctgccatcc ccccccactg tttccaccga 180
tctctgctga tgtcttcttc ttacctgatc cgagacttcg ccctggcctt cctgttctac 240
cactctgccg tgacctacat ccccctgctg cccaagcccc tggcctgtat ggcctggccc 300
gtgtactggt tcctgcaagg ctctaacatg ctgggcatct gggtgatcgc ccacgagtgt 360
ggccaccaag ccttctctaa ctacggctgg gtgaacgacg ccgtgggctt cttcctgcac 420
acctctctgc tggtgcccta cttccccttc aagtactctc accgacgaca ccactctaac 480
accaactctg tggagcacga cgaggtgttc gtgccccgac acaaggacgg cgtgcagtgg 540
tactaccgat tcttcaacaa cacccccggc cgagtgctga ccctgaccct gaccctgctg 600
gtgggctggc cctcttacct ggccttcaac gcctctggcc gaccctacga cggcttcgcc 660
tctcactaca accccaacgc tcagatcttc aacctgcgag agcgattctg ggtgcacgtg 720
tctaacatcg gcatcctggc catctactac atcctgtacc gactggccac caccaagggc 780
ctgccctggc tgctgtctat ctacggcgtg cccgtgctga tcctgaacgc cttcgtggtg 840
ctgatcacct tcctgcagca ctctcacccc gccctccccc attacaattc cgatgagtgg 900
gactggctgc gaggcgccct ggccaccgtg gaccgagact acggcttcct gaacgaggtg 960
ttccacgaca tcaccgacac ccacgtgatc caccacctgt tccccaccat gccccactac 1020
aacgccaaag aggccaccgt gtctatccga cccatcctga aggactacta caagttcgac 1080
cgaaccccca tctggcgagc cctgtggcga gaggccaagg agtgtctgta cgtggaggcc 1140
gacggaacgg gttctaaggg cgtgctgtgg ttcaagtcta agttctaa 1188
<210> 2
<211> 1161
<212> DNA
<213> artificial
<220>
<223> PgFAD2
<400> 2
atgggcgccg gcggccgaat gaccgtgccc aacaagtggg agggcgaggg cgacgagaag 60
tctcagaagc ccgtgcagcg agtgccctct gccaagcccc ccttcaccct gtctgagatc 120
aagaaggcca tcccccccca ctgtttcaag cgatctctgc tgaagtcttt ctcttacgtg 180
ctgtacgacc tgaccctggt ggccatcttc tactacgtgg ccaccaccta catcgacgcc 240
ctgcccggcc ccctgcgata cgccgcctgg cccgtgtact gggccctgca aggctgtgtg 300
ctgaccggcg tgtgggtgat cgcccacgag tgtggccacc acgccttctc tgactatcag 360
tgggtggacg actgtgtggg cctggtgctg cactctgccc tgctggtgcc ctacttctct 420
tggaagtact ctcaccgacg acaccactct aacaccggct ctctggagcg agacgaggtg 480
ttcgtgccca agcccaagtc taagatgccc tggttctcta agtacctgaa caaccccccc 540
ggccgagtga tgaccctgat cgtgaccctg accctgggct ggcccctgta cctggccctg 600
aacgtgtctg gccgacccta cgaccgattc gcctgtcact tcgaccccta cggccccatc 660
tacaccgacc gagagcgact gcagatctac atctctgacg tgggcatcat ggccgccacc 720
tacaccctgt acaagatcgc cgctgcccga ggcctggcct ggctggtgtg tgtgtacggc 780
gtgcccctgc tgatcgtgaa cgccttcctg gtgaccatca cctacctgca gcacacccac 840
cccgccctgc cccactacga ctcttctgag tgggactggc tgcgaggcgc cctggccacc 900
gccgaccgag actacggcat cctgaacaag gtgttccaca acatcaccga cacccacgtg 960
gcccaccacc tgttctctac catgccccac taccacgcca tggaggccac caaggccatc 1020
aagcccatcc tgggcgacta ctatcagttc gacggcaccc ccgtgtacaa ggccatgtgg 1080
cgagaggccc gagagtgtct gtacgtggag cccgacgacg gcgccaactc taagggcgtg 1140
ttctggtaca agaagaacct g 1161
<210> 3
<211> 1398
<212> DNA
<213> artificial
<220>
<223> RcLPCAT
<400> 3
atggatttag acttggaatc aatggcatcg gcgatcgggg tatcaattcc agttttacga 60
tttttactct gctttgtagc gacgattcca gtgagtttta tgcaccgcct cgcgcctggt 120
agtctcggta aacatttgta tgctgctttg actggggctt tcttgtcata cctatcattt 180
gggttctctt caaatcttca ctttttggtg cctatgttat tgggttacgc ttccatggtt 240
ctgtttcgct ctcactgtgg aatcttggtt ttccttttgg gtttcggtta tctcattggc 300
tgccatgttt attacatgag tggagatgca tggaaggaag gaggcattga tgctactggg 360
gccttaatgg tgttaacact gaaagtcata tcatgcgcga taaattacaa agatggatta 420
ctaaaagagg aagagttaca gggatcacag aagaaaaacc gtctgattaa actgccgtct 480
ttgattgagt actttggtta ttgcctctgc tgtggtagcc actttgccgg tcctgtttat 540
gaaatgaagg actatcttga atggactgaa aggaagggga tatgggctgg cacagagaaa 600
ggaccctcac catcaccttt tggggcaaca attcgagcta tccttcaagc tgctatttgc 660
atggtcattc atttatactt ggtgccccac tatcctttat cccggttcac tgatcctgtg 720
taccaagaat ggggcttctg gaaacgatta acttatcagt atatgtcagg tttaacagca 780
cgttggaaat actacttcat ctggtcaatt tccgaggcct ccattattat ctctggattg 840
ggtttcagtg gttggacaga tacttctcca ccaaagccac agtgggatcg cgctagaaac 900
gttgacattc taggtgttga gtttgcaaag agtgcagctg agttgccact tgtgtggaac 960
atacaagtca gcacatggct tcgccactat gtttatgatc gacttgttcc aaagggaaag 1020
aaagctggtt tccttcagtt gttggccact cagactacca gtgctgtttg gcatggatta 1080
tatcctggat acattatatt ctttgtccag tcagcattaa tgattgcagg ttcgaaagtc 1140
atatacagat ggcaacaagc tataccttca aataaggctc ttgaaaagaa gatactagtg 1200
tttatgaact ttgcttacac agttttggtt ctaaattact cctgtgttgg tttcatggtt 1260
ttaagcttgc atgaaacgat tgcagcatat ggaagtgtat attttattgg caccatagtg 1320
cccgttgtat ttttcctcct tggcttcatt attaaaccag caaggccttc caggtctaaa 1380
acacggaagg atgagtga 1398
<210> 4
<211> 545
<212> DNA
<213> yarrowia lipolytica
<400> 4
gtgcatgctg aggtgtctca caagtgccgt gcagtcccgc ccccacttgc ttctctttgt 60
gtgtagtgta cgtacattat cgagaccgtt gttcccgccc acctcgatcc ggcatgctga 120
ggtgtctcac aagtgccgtg cagtcccgcc cccacttgct tctctttgtg tgtagtgtac 180
gtacattatc gagaccgttg ttcccgccca cctcgatccg gcatgctgag gtgtctcaca 240
agtgccgtgc agtcccgccc ccacttgctt ctctttgtgt gtagtgtacg tacattatcg 300
agaccgttgt tcccgcccac ctcgatccgg catgctgagg tgtctcacaa gtgccgtgca 360
gtcccgcccc cacttgcttc tctttgtgtg tagtgtacgt acattatcga gaccgttgtt 420
cccgcccacc tcgatccggc atgcactgat cacgggcaaa agtgcgtata tatacaagag 480
cgtttgccag ccacagattt tcactccaca caccacatca cacatacaac cacacacatc 540
cacgt 545
<210> 5
<211> 531
<212> DNA
<213> yarrowia lipolytica
<400> 5
agagaccggg ttggcggcgc atttgtgtcc caaaaaacag ccccaattgc cccaattgac 60
cccaaattga cccagtagcg ggcccaaccc cggcgagagc ccccttcacc ccacatatca 120
aacctccccc ggttcccaca cttgccgtta agggcgtagg gtactgcagt ctggaatcta 180
cgcttgttca gactttgtac tagtttcttt gtctggccat ccgggtaacc catgccggac 240
gcaaaataga ctactgaaaa tttttttgct ttgtggttgg gactttagcc aagggtataa 300
aagaccaccg tccccgaatt acctttcctc ttcttttctc tctctccttg tcaactcaca 360
cccgaaatcg ttaagcattt ccttctgagt ataagaatca ttcaaaatgg tgagtttcag 420
aggcagcagc aattgccacg ggctttgagc acacggccgg gtgtggtccc attcccatcg 480
acacaagacg ccacgtcatc cgaccagcac tttttgcagt actaaccgca g 531
<210> 6
<211> 1127
<212> DNA
<213> yarrowia lipolytica
<400> 6
gacgcagtag gatgtcctgc acgggtcttt ttgtggggtg tggagaaagg ggtgcttgga 60
gatggaagcc ggtagaaccg ggctgcttgg ggggatttgg ggccgctggg ctccaaagag 120
gggtaggcat ttcgttgggg ttacgtaatt gcggcatttg ggtcctgcgc gcatgtccca 180
ttggtcagaa ttagtccgga taggagactt atcagccaat cacagcgccg gatccacctg 240
taggttgggt tgggtgggag cacccctcca cagagtagag tcaaacagca gcagcaacat 300
gatagttggg ggtgtgcgtg ttaaaggaaa aaaaaagaag cttgggttat attcccgctc 360
tatttagagg ttgcgggata gacgccgacg gagggcaatg gcgccatgga accttgcgga 420
tatcgatacg ccgcggcgga ctgcgtccga accagctcca gcagcgtttt ttccgggcca 480
ttgagccgac tgcgaccccg ccaacgtgtc ttggcccacg cactcatgtc atgttggtgt 540
tgggaggcca ctttttaagt agcacaaggc acctagctcg cagcaaggtg tccgaaccaa 600
agaagcggct gcagtggtgc aaacggggcg gaaacggcgg gaaaaagcca cgggggcacg 660
aattgaggca cgccctcgaa tttgagacga gtcacggccc cattcgcccg cgcaatggct 720
cgccaacgcc cggtcttttg caccacatca ggttacccca agccaaacct ttgtgttaaa 780
aagcttaaca tattataccg aacgtaggtt tgggcgggct tgctccgtct gtccaaggca 840
acatttatat aagggtctgc atcgccggct caattgaatc ttttttcttc ttctcttctc 900
tatattcatt cttgaattaa acacacatca acaatggcca tcaaagtcgg tattaacgga 960
ttcggacgaa tcggacgaat tgtgagtacc atagaaggtg atggaaacat gacccaacag 1020
aaacagatga caagtgtcgt cgacccacca gagcccaatt gagctcatac taacagtcga 1080
caacctgtcg aaccaattga tgactccccg acaatgtact aacacag 1127
<210> 7
<211> 502
<212> DNA
<213> yarrowia lipolytica
<400> 7
cactggccgg tcgataattt aacgtgctga gctcagcaca cgcattgccc attggctgta 60
tatagatgaa tgtaatgata ccgtaagaga atgagagcac ggtattgtat tacaggggat 120
taagtacaca tttacttgga gttctgtacc agaagacact actatacatg gtattactta 180
cattagagtc ggtgaccgta ttcgtctcgt atagacataa tattttccta ccccacattg 240
ttcctgggcc ttcggagcac atctacagtg agtgactgtt tcagttgagc ttgaggggtt 300
aagtaagtgg gggaagggtt tgcgattctg aaaaagagca tgactaatct ctctgtggag 360
gagcaatgaa gtcacgtgat gcaatcatac cggtgtatcg gatctgcctg ggtgtctgat 420
tactaatcat ttactcacct gttttcccca gctatctcat ccatctcaga gcctcggccc 480
agccttcggc ccttttgggt tt 502
<210> 8
<211> 519
<212> DNA
<213> yarrowia lipolytica
<400> 8
gatccaacta cggaacttgt gttgatgtct ttgcccccgg ctccgatatc atctctgcct 60
cttaccagtc cgactctggt actttggtct actccggtac ctccatggcc tgtccccacg 120
ttgccggtct tgcctcctac tacctgtcca tcaatgacga ggttctcacc cctgcccagg 180
tcgaggctct tattactgag tccaacaccg gtgttcttcc caccaccaac ctcaagggct 240
ctcccaacgc tgttgcctac aacggtgttg gcatttaggc aattaacaga tagtttgccg 300
gtgataattc tcttaacctc ccacactcct ttgacataac gatttatgta acgaaactga 360
aatttgacca gatattgttg taaatagaaa atctggcttg taggtggcaa aatgcggcgt 420
ctttgttcat caattccctc tgtgactact cgtcatccct ttatgttcga ctgtcgtatt 480
tcttattttc catacatatg caagtgagat gcccgtgtc 519
Claims (10)
1. A recombinant Yarrowia lipolytica strain for high yield of punicic acid is a Yarrowia lipolytica (Yarrowia lipolytica) XJ-11 strain, the preservation number is CCTCC NO: m2022320.
2. The recombinant Yarrowia lipolytica with high punicic acid yield of claim 1, wherein said Yarrowia lipolytica strain XJ-11 is a Yarrowia lipolytica genome in which peroxisome biogenesis factor 10, triacylglycerol lipase 4, Δ 12 desaturase expression cassette, phosphatidylcholine: diacylglycerol acyltransferase expression cassette, acetyl coenzyme A carboxylase expression cassette, choline phosphotransferase expression cassette, heterologous fatty acid conjugase expression cassette, and heterologous lysophosphatidylcholine acyltransferase.
3. The punicic acid-producing recombinant yarrowia lipolytica of claim 2, wherein said acetyl-coa carboxylase, Δ 12 desaturase, phosphatidylcholine: diacylglycerol acyltransferase, choline phosphotransferase encoding genes are derived from Yarrowia lipolytica; the fatty acid conjugated enzyme encoding gene is obtained by codon optimization of a pomegranate (Punica grantum) derived fatty acid conjugated enzyme encoding gene; the heterologous lysophosphatidylcholine acyltransferase is obtained by codon optimization of a gene encoding lysophosphatidylcholine acyltransferase from Ricinus communis.
4. The recombinant yarrowia lipolytica with high punicic acid yield of claim 3, wherein said promoter of each expression cassette is the promoter P of yarrowia lipolytica TEF 、P hp4d 、P TEFin 、P POX2 Or P GPDin Any one of the above; the terminator is terminator T of yarrowia lipolytica xpr2t 、T mig1t 、T lip2t 、T pex10t Or T pex20t Any one of them.
5. The recombinant yarrowia lipolytica with high punicic acid yield of claim 4, wherein said integration site of said expression cassette is any one of A08 site, 26s rDNA site, PEX10 site, TGL4 site, IntA site, IntB site, IntC site, IntD site, IntE site, IntF site, lip1 site of yarrowia lipolytica.
6. The recombinant yarrowia lipolytica with high punicic acid yield of claim 5, wherein said fatty acid-conjugated enzyme encoding gene sequence is set forth in SEQ ID No. 1; the sequence of the encoding gene of the ricinus communis lysophosphatidylcholine acyltransferase is shown as SEQ ID No. 3.
7. The method of claim 1, comprising the steps of combining a peroxisome biogenesis factor 10 knockout cassette, a triacylglycerol lipase 4 knockout cassette, an acetyl-coa carboxylase expression cassette, a Δ 12 desaturase expression cassette, and phosphatidylcholine: a step of introducing a diacylglycerol acyltransferase expression cassette, a choline phosphotransferase expression cassette, a heterologous fatty acid conjugase expression cassette, a heterologous lysophosphatidylcholine acyltransferase expression cassette into said yarrowia lipolytica in the form of a plasmid, followed by integration into the genome of yarrowia lipolytica by homologous recombination.
8. The method of claim 7, wherein said yarrowia lipolytica knocks out the ku70 gene.
9. The application of the recombinant bacterium in any one of claims 1 to 7 in the production of punicic acid is characterized by comprising the step of culturing the recombinant bacterium in any one of claims 1 to 6 by using a fermentation medium to obtain a fermentation product.
10. The application of the recombinant bacterium in the production of punicic acid according to claim 9, wherein the fermentation medium contains 50-70g/L of glucose, 1.6-1.8g/L of amino-free yeast nitrogen source and 1.2-1.4g/L of ammonium sulfate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210520028.2A CN114958636B (en) | 2022-05-13 | 2022-05-13 | Recombinant yarrowia lipolytica capable of producing punica granatum at high yield as well as construction method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210520028.2A CN114958636B (en) | 2022-05-13 | 2022-05-13 | Recombinant yarrowia lipolytica capable of producing punica granatum at high yield as well as construction method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114958636A true CN114958636A (en) | 2022-08-30 |
| CN114958636B CN114958636B (en) | 2023-07-14 |
Family
ID=82982576
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210520028.2A Active CN114958636B (en) | 2022-05-13 | 2022-05-13 | Recombinant yarrowia lipolytica capable of producing punica granatum at high yield as well as construction method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114958636B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111235045A (en) * | 2020-01-19 | 2020-06-05 | 天津大学 | Recombinant yarrowia lipolytica for heterologous synthesis of β -balsam stem and oleanolic acid and construction method thereof |
| CN111235047A (en) * | 2020-02-12 | 2020-06-05 | 天津大学 | Recombinant yarrowia lipolytica for heterogeneously synthesizing α -coumarol and ursolic acid and construction method |
| US20210032665A1 (en) * | 2018-04-09 | 2021-02-04 | Qingdao Institute Of Bioenergy And Bioprocess Technology, Chinese Academy Of Sciences | Recombinant yeast strain for producing nervonic acids and application thereof |
-
2022
- 2022-05-13 CN CN202210520028.2A patent/CN114958636B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20210032665A1 (en) * | 2018-04-09 | 2021-02-04 | Qingdao Institute Of Bioenergy And Bioprocess Technology, Chinese Academy Of Sciences | Recombinant yeast strain for producing nervonic acids and application thereof |
| CN111235045A (en) * | 2020-01-19 | 2020-06-05 | 天津大学 | Recombinant yarrowia lipolytica for heterologous synthesis of β -balsam stem and oleanolic acid and construction method thereof |
| CN111235047A (en) * | 2020-02-12 | 2020-06-05 | 天津大学 | Recombinant yarrowia lipolytica for heterogeneously synthesizing α -coumarol and ursolic acid and construction method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114958636B (en) | 2023-07-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11603545B2 (en) | Recombinant yeast strain for producing nervonic acids and application thereof | |
| US8597931B2 (en) | Mutant yeast strains capable of accumulating a large quantity of lipids | |
| CN105400796A (en) | Gene for adjusting and controlling production of long-chain diacid and application of gene | |
| EP2582798A1 (en) | Production of biodiesel by yeast from lignocellulose and glycerol | |
| CN112831427A (en) | Yarrowia lipolytica for high yield of beta-carotene and application thereof | |
| CN112538438B (en) | Recombinant yarrowia lipolytica with high oleic acid yield as well as construction method and application thereof | |
| CN112941119B (en) | Method for increasing yield of fatty acid ethyl ester of saccharomyces cerevisiae engineering bacteria | |
| CN112608936B (en) | Promoter for regulating and controlling expression of exogenous gene of yeast, regulating and controlling method and application thereof | |
| CN114891653B (en) | Recombinant yarrowia lipolytica strain capable of producing nervonic acid in high yield, construction method and application thereof | |
| CN114958636B (en) | Recombinant yarrowia lipolytica capable of producing punica granatum at high yield as well as construction method and application thereof | |
| EP4249583A1 (en) | Use of fatty acid elongase gene and esterase gene in synthesis of nervonic acid and grease in yeast | |
| CN113122461A (en) | Single cell protein producing strain and its application | |
| CN113403213A (en) | Yarrowia lipolytica engineering bacterium for producing triacetic acid lactone by using xylose and application | |
| CN111304104A (en) | Recombinant yarrowia lipolytica for heterologous synthesis of betulinic acid and construction method thereof | |
| CN116769622A (en) | Recombinant yarrowia lipolytica capable of producing double high-gamma-linolenic acid with high yield, construction method and application thereof | |
| CN117736895A (en) | Recombinant yarrowia lipolytica capable of producing palmitoleic acid in high yield, and construction method and application thereof | |
| CN114806914B (en) | Yarrowia lipolytica capable of producing beta-carotene at high yield and application thereof | |
| CN116179381A (en) | Genetically engineered bacterium for producing OPO structural ester, construction method and application thereof | |
| CN114958637B (en) | Engineering bacterium for producing beta-eucalyptol as well as construction method and application thereof | |
| CN114891822B (en) | Construction method of high-yield gamma-linolenic acid mucor circinelloides recombinant bacterium, recombinant bacterium constructed by method and application | |
| CN117778217A (en) | Yarrowia lipolytica capable of producing nervonic acid and construction method and application thereof | |
| CN107475269B (en) | acyl-CoA thioesterase gene of candida tropicalis and application thereof | |
| CN117625654A (en) | Marine nannochloropsis fatty acid elongase and gene and application thereof | |
| CN117070385A (en) | Recombinant strain, method and application of yarrowia lipolytica for constructing cell wall formation related gene deletion | |
| CN116445311A (en) | Method for constructing high-yield fatty acid saccharomyces cerevisiae engineering bacteria |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |