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,Email:huangluqi01@126com
Constructionofcelfactoriesforproductionoflupeolin
Saccharomycescerevisiae
LINTingting1,2,3,WANGDong2,DAIZhubo2,ZHANGXueli2,HUANGLuqi3
(1ShaanxiUniversityofChineseMedicine,Xianyang712046,China;
2TianjinInstituteofIndustrialBiotechnology,ChineseAcademyofSciences,Tianjin300308,China;
3StateKeyLaboratoryBreedingBaseofDaodiHerbs,NationalResourceCenterforChineseMateriaMedica,
ChinaAcademyofChineseMedicalSciences,Beijing100700,China)
[Abstract] Lupanetypetriterpenoids,suchasbetulinicacid,arederivedfromlupeolandhaveexcelentpropertiesinantiHIV,an
ticanceractivitiesandsoon.Forrealizingheterogenousproductionoflupanetypetriterpenoids,ourresearchfirstlyintegratedalthe
sevengenesintheMVApathwayinSaccharomycescerevisiaetoincreasethesupplyofsqualene(triterpenoidsuniversalprecursor)ina
singlestepusingtheDNAassemblermethodNext,celfactoriesforproductionoflupeolwasconstructedbyintegratingArabidopsis
thalianalupeolsyntheticgene(AtLUP)intochromosomeoftriterpenoidchassisstrainResultsshowedthattheMVApathway,about
20kbnucleotidelength,couldbeassembledinonepotprocessandthedoubledMVApathwaycouldsignificantlyimprovesqualeneby
500fold,reaching35400mg·L-1NK2LUPwasobtainedbyintroducingAtLUPgeneonchromosome,andcouldproduce823mg
·L-1lupeolThisstudysupportsthepossibilityoflargescalebiosyntheticpathwayassemblyinScerevisiaeandlaysthefoundationof
obtainingcelfactoriesforproductionoflupantypetriterpenoidsatthesametime
[Keywords] triterpenoids;lupeol;syntheticbiology;Saccharomycescerevisiae
doi:10.4268/cjcmm20160606
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boxylase; IDI1 S cerevisiaeisopentenyldiphosphateisomerase1;
ERG20ScerevisiaeFPPsynthase;ERG9Scerevisiaesqualenesyn
thase;ERG1Scerevisiaesqualeneepoxidase;AtLUPArabidopsis
thalianalupeolsynthase。
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Table1 Plasmidsandstrainsusedinthisstudy
d W¦ w$ ì>
´½
pEASYBluntsimple Cloningvectorforbluntligation,Amp,Km
w£×°
pGAL7URA3 CloningGAL7andURA3markerintopEASYBluntsimple
(¸ý
pHIS3TRP1 CloningHIS3andTRP1markerintopEASYBluntsimple
(¸ý
pM11ERG8 CloningPTDH3ERG8TTPI1casseteintopEASYBluntsimple (¸ý
pM3ERG10 CloningPTEF1ERG10TCYC1casseteintopEASYBluntsimple (¸ý
pM8ERG13 CloningPFBA1ERG13TTDH2casseteintopEASYBluntsimple (¸ý
pM9ERG12 CloningPPDC1ERG12TADH2casseteintopEASYBluntsimple (¸ý
pM5ERG19 CloningPPYK1ERG19TPGI1casseteintopEASYBluntsimple (¸ý
pM16IDI1 CloningPENO2IDI1TPDC1casseteintopEASYBluntsimple (¸ý
pδtHMG1 CloningPPGK1tHMG1TADH1casseteintopδUB (¸ý
pM2LUP CloningPPGK1LUPTADH1casseteintopEASYBluntsimple (¸ý
1°
NK2 CENPK21D (MATαura352;trp1289;leu23,112;his3Δ1;MAL28C;
SUC2)
\ÀÁÂø$S
NK2SQCK URA3markerwasintegratedintoGAL7siteofNK2
N
NK2SQ PPGK1tHMG1TADH1,PPDC1ERG12TADH2,PENO2IDI1TPDC1,PPYK1ERG19
TPGI1,PFBA1ERG13TTDH2,PTDH3ERG8TTPI1andPTEF1ERG10TCYC1cassetesto
getherwithURA3markerwereintegratedintoGAL7siteofNK2
N
NK2LUP PPGK1LUPTADH1cassetetogetherwithTRP1markerwasintegratedintoHIS3site
ofNK2SQ
N
Ü
2
N
³ir2
Table2 Primersusedinthisstudy
d W¦ ¹T
(5′3′)
¤>Ö2
1MpEASYPGK1F CTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCTTAATTAAACGCACAGATATTATAAC
S7G1MADHtPDC1R CCTTGCTTGTTTATCTTGCACATCACATCAGCGGAACATATGCTCACCCAGTCGCATGTCGGCATGCCGGTAGAGGTGTGGTCAATAAG
S7G1MADHtPDC1F GGTCAGGTTGCTTTCTCAGGTATAGCATGAGGTCGCTCTTATTGACCACACCTCTACCGGCATGCCGACATGCGACTGGGTGAGCATATGT
TCCG
3G1MADH2tENO2R TGTTAATCCACCTTCTAGCTCATTTTGGATGGCAACGGCAAGAGTAGGATTTAGAATTATATAACTTGATGAGATGAG
3G2MADH2tENO2F GGTAATTCTTCTGTCATTTACTCATCTCATCTCATCAAGTTATATAATTCTAAATCCTACTCTTGCCGTTGCCATCC
6G2MPDC1tFBA1R GTTATTTACAGAAGTTGGAAGGCTGGTATTGTTGTTCAAGCCAGCGGTGCCAGTTGGATCGGCAGTTTTGAATTGAGTAACCA
6G2MPDC1tFBA1F AGTGTAATAGAAGACTAGACACCTCGATACAAATAATGGTTACTCAATTCAAAACTGCCGATCCAACTGGCACCGCTGGCTTGA
S4G4MTDH2tTDH3R GGCCTCCGCGTCATTAAACTTCTTGTTGTTGACGCTAACATTCAACGCTAGTATGGCGAAAAGCCAATTAGTGTGATAC
S4G3MTDH2tTDH3F GGCATCACGGATTTTCGATAAAGCACTTAGTATCACACTAATTGGCTTTTCGCCATACTAGCGTTGAATGTTAGCGTCAAC
3G3MTPI1tTEF1R AGGAGTAGAAACATTTTGAAGCTATGGTGTGTGGGGGATCACTTTAATTAATCTATATAACAGTTGAAATTTGGA
3G2MTPI1tTEF1F GTCATTTTCGCGTTGAGAAGATGTTCTTATCCAAATTTCAACTGTTATATAGATTAATTAAAGTGATCCCCCACAC
MCYC1tpEASYR CGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGCGTTGGCCGATTCATTAATGC
X1MpEASYrtF CTTGCAAATGCCTATTGTGCAGATGTTATAATATCTGTGCGTTTAATTAAGGCTCGTATGTTGTGTGGAATTGT
GAL71 ATATTTGAAAGGCTTATGATTTTCTCTTGC
GAL72 GTCCAAATATTTGAAAACAAAGGTACAGC
X2MpEASYrtR CGAAGGCTTTAATTTGCAAGCTGCGGCCCTGCATTAATGAATCGGCCAACGCGCCAGGGTTTTCCCAGTCACGACGTTG
ZDHis3interg1 TTTATATAATGTATAATTCATT
ZDHis3interg2 CATTCTATACGTGTCATTCTGA
X1XppEASYM13RF CCGACTGGAAAGCGGGCAGTGAGCG
X2XppEASYM13FR CCATTCAGGCTGCGCAACTGTTGGGA
XpMpEASYM13RF GTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGTCACACAGGAAACAGCTATGACC
XpMpEASYM13FR CTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGACGACGTTGTAAAACGACGGCCAGT
Vv2
SacIPGK1 GCGCCGCGGACGCACAGATATTATAACATC
HMG1Asc TCGCGGCGCGCCTTAGGATTTAATGCAGGTGACGGAC
PacPDC1p GCGTTAATTAACATGCGACTGGGTGAGCATATGTTC
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IDI1Asc GCGGCGCGCCTTATAGCATTCTATGAATTTGCCT
PacPYK1p GCGTTAATTAAAATGCTACTATTTTGGAGATTAATC
ERG19Asc GCGGCGCGCCTTATTCCTTTGGTAGACCAGTCTTTGCG
PacpFBA GCGTTAATTAAGATCCAACTGGCACCGCTGGCTTGA
ERG13Asc GCGGCGCGCCTTATTTTTTAACATCGTAAGATCTTC
PacpTDH3 GCGTTAATTAAATACTAGCGTTGAATGTTAGCGTCA
ERG8Asc GCGGCGCGCCTTATTTATCAAGATAAGTTTCCGGATC
SacIpTEF1 GCGCCGCGGAGTGATCCCCCACACACCATAGCTT
ERG10Asc GCGGCGCGCCTCATATCTTTTCAATGACAATAGAG
SexAERG12 GCGACCWGGTATGTCATTACCGTTCTTAACTTCTGC
SexAERG19 GCGACCWGGTATGACCGTTTACACAGCATCCGTTACC
SexAERG13 GCGACCWGGTATGAAACTCTCAACTAAACTTTGTTGG
SexAIDI1 GCGACCWGGTATGACTGCCGACAACAATAG
SexAHMG1 TCGCGACCWGGTAAAACAATGGCTGCAGACCAATTGG
PacERG8 GCGTTAATTAAATGTCAGAGTTGAGAGCCTTCAGTGC
ENO2pSexA GCGACCWGGTTATTATTGTATGTTATAGTATTAGTTG
PYK1pSexA GCGACCWGGTTGTGATGATGTTTTATTTGTTTTGA
FBA1SexA GCGACCWGGTTTTGAATATGTATTACTTGGTTATG
PDC1pSexA GCGACCWGGTTTTGATTGATTTGACTGTGTTATTT
pTDH3SexA GCGACCWGGTTTTGTTTGTTTATGTGTGTTTATTC
TEF1SexA TGGCGACCWGGTTTTGTAATTAAAACTTAGATTAGA
LUPAsc GCGGCGCGCCTTAGTAGGAGTGAGCACATAAAACT
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PacPYK1p/ERG19Asc,PacpFBA/ERG13Asc,Pac
pTDH3/ERG8Asc,SacIpTEF1/ERG10Asc,SexA
HMG1/PDC1pSexA1,SexA1ERG12/ENO2pSexA1,
SexA1IDI1/PYK1pSexA1,SexA1ERG19/pFBASexA1,
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Fig2 StrainconstructionusingtheDNAassemblermethod:
al7genesofMVApathwaywereintegratedintoGAL7siteof
NK2
·1101·
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Vol41,No.6 March,2016
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Table3 InformationofDNAassembler
56 2W ¤>J
A
Ò5m ¤>J
B
A pδtHMG1 1MPEASYPGK1F LP1F PPGK1tHMG1TADH1 LP1R
S7G1MADHtPDC1R
pM9ERG12 S7G1MADHtPDC1F LP2F PPDC1ERG12TADH2 LP2R
3G1ADH2tENO2R
pM16IDI1 3G2ADH2tENO2F LP3F PENO2IDI1TPDC1 LP3R
3G2MPDC1tPYK1R
pM5ERG19 3G3MPDC1tPYK1F LP4F PPYK1ERG19TPGI1 LP4R
S7G1MPGI1tFBA1R
pM8ERG13 S7G1MPGI1tFBA1F LP5F PFBA1ERG13TTDH2 LP5R
S4G4MTDH2tTDH3R
pM11ERG8 S4G3MTDH2tTDH3F LP6F PTDH3ERG8TTPI1 LP6R
3G3MTPI1tTEF1R
pM3ERG10 3G2MTPI1tTEF1F LP7F PTEF1ERG10TCYC1 LP7R
2MCYC1tpEASYR
pGAL7URA3 X1MpEASYrtF GAL7Up HelperfragmentofGAL7sitewithURA3marker upR
GAL72
pGAL7URA3 X2MpEASYrtR downF HelperfragmentofGAL7site GAL7Down
GAL71
pGAL7URA3 GAL71 GAL7Up GAL7sitewithURA3marker GAL7Down
GAL72
B pM2LUP XpMpEASYM13RF LPF PPGK1LUPTADH1 LPR
XpMpEASYM13FR
pHIS3TRP1 X1XppEASYM13RF HIS3Up HelperfragmentofHIS3sitewithTRP1marker upR
ZDHis3interg1
pHIS3TRP1 ZDHis3interg2 downF HelperfragmentofHIS3site HIS3Down
X2XppEASYM13FR
22 NK2LUP
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32 MVA
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(35400±228)mg·L-1,
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gineeredNK2SQCKandNK2SQ
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q
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MTrans2Kplusmarker;
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Fig6 PCRverificationofengineeredNK2LUP
A
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q
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NK2LUP
mÂÆ$ÇÈÉÊ´
Lq
。
q
7 GCMS
ú
NK2LUP
1°mÂÆnVv
Fig7 IdentificationofNK2LUPfermentationproductsbyGC
MSmethod
4
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Fig8 Productionofergosterol,lanosterol,squaleneandlupeol
byengineeredNK2SQandNK2LUP
úÆ£àLkÖÀÁÂÃÄÅgj
NK2
SQ。
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1°
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NK2LUP。
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,
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yg?z
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