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Construction of cell factories for production of lupeol in Saccharomyces cerevisiae

创建酿酒酵母细胞工厂发酵生产羽扇豆醇



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Constructionofcelfactoriesforproductionoflupeolin
Saccharomycescerevisiae
LINTingting1,2,3,WANGDong2,DAIZhubo2,ZHANGXueli2,HUANGLuqi3
(1ShaanxiUniversityofChineseMedicine,Xianyang712046,China;
2TianjinInstituteofIndustrialBiotechnology,ChineseAcademyofSciences,Tianjin300308,China;
3StateKeyLaboratoryBreedingBaseofDaodiHerbs,NationalResourceCenterforChineseMateriaMedica,
ChinaAcademyofChineseMedicalSciences,Beijing100700,China)
[Abstract] Lupanetypetriterpenoids,suchasbetulinicacid,arederivedfromlupeolandhaveexcelentpropertiesinantiHIV,an
ticanceractivitiesandsoon.Forrealizingheterogenousproductionoflupanetypetriterpenoids,ourresearchfirstlyintegratedalthe
sevengenesintheMVApathwayinSaccharomycescerevisiaetoincreasethesupplyofsqualene(triterpenoidsuniversalprecursor)ina
singlestepusingtheDNAassemblermethodNext,celfactoriesforproductionoflupeolwasconstructedbyintegratingArabidopsis
thalianalupeolsyntheticgene(AtLUP)intochromosomeoftriterpenoidchassisstrainResultsshowedthattheMVApathway,about
20kbnucleotidelength,couldbeassembledinonepotprocessandthedoubledMVApathwaycouldsignificantlyimprovesqualeneby
500fold,reaching35400mg·L-1NK2LUPwasobtainedbyintroducingAtLUPgeneonchromosome,andcouldproduce823mg
·L-1lupeolThisstudysupportsthepossibilityoflargescalebiosyntheticpathwayassemblyinScerevisiaeandlaysthefoundationof
obtainingcelfactoriesforproductionoflupantypetriterpenoidsatthesametime
[Keywords] triterpenoids;lupeol;syntheticbiology;Saccharomycescerevisiae
doi:10.4268/cjcmm20160606
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HMGCoA3hydroxy3methylglutarylcoenzymeA;IPPisopentenyl
diphosphate;DMAPPdimethylalyldiphosphate;FPPfarnesyldiphos
phate;ERG10ScerevisiaeacetoacetylCoAthiolase;ERG13Scerevi
siaeHMGCoA synthase;ScerevisiaeHMG1HMGCoAreductase;
ERG12Scerevisiaemevalonatekinase;ERG8Scerevisiaephosphom
evalonatekinase;ERG19Scerevisiaemevalonatepyrophosphatedecar
boxylase; IDI1 S cerevisiaeisopentenyldiphosphateisomerase1;
ERG20ScerevisiaeFPPsynthase;ERG9Scerevisiaesqualenesyn
thase;ERG1Scerevisiaesqualeneepoxidase;AtLUPArabidopsis
thalianalupeolsynthase。
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Fig1 Lupeolbiosyntheticpathwayinyeast
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Table1 Plasmidsandstrainsusedinthisstudy
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pEASYBluntsimple Cloningvectorforbluntligation,Amp,Km
w£×°‹
pGAL7URA3 CloningGAL7andURA3markerintopEASYBluntsimple
(¸ý
pHIS3TRP1 CloningHIS3andTRP1markerintopEASYBluntsimple
(¸ý
pM11ERG8 CloningPTDH3ERG8TTPI1casseteintopEASYBluntsimple (¸ý
pM3ERG10 CloningPTEF1ERG10TCYC1casseteintopEASYBluntsimple (¸ý
pM8ERG13 CloningPFBA1ERG13TTDH2casseteintopEASYBluntsimple (¸ý
pM9ERG12 CloningPPDC1ERG12TADH2casseteintopEASYBluntsimple (¸ý
pM5ERG19 CloningPPYK1ERG19TPGI1casseteintopEASYBluntsimple (¸ý
pM16IDI1 CloningPENO2IDI1TPDC1casseteintopEASYBluntsimple (¸ý
pδtHMG1 CloningPPGK1tHMG1TADH1casseteintopδUB (¸ý
pM2LUP CloningPPGK1LUPTADH1casseteintopEASYBluntsimple (¸ý

NK2  CENPK21D (MATαura352;trp1289;leu23,112;his3Δ1;MAL28C;
SUC2)
…\ÀÁÂݸ$S
NK2SQCK URA3markerwasintegratedintoGAL7siteofNK2
N„…
NK2SQ   PPGK1tHMG1TADH1,PPDC1ERG12TADH2,PENO2IDI1TPDC1,PPYK1ERG19
TPGI1,PFBA1ERG13TTDH2,PTDH3ERG8TTPI1andPTEF1ERG10TCYC1cassetesto
getherwithURA3markerwereintegratedintoGAL7siteofNK2
N„…
NK2LUP  PPGK1LUPTADH1cassetetogetherwithTRP1markerwasintegratedintoHIS3site
ofNK2SQ
N„…
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2 
N„…³irƒ2‹
Table2 Primersusedinthisstudy
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(5′3′)
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1MpEASYPGK1F CTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCTTAATTAAACGCACAGATATTATAAC
S7G1MADHtPDC1R CCTTGCTTGTTTATCTTGCACATCACATCAGCGGAACATATGCTCACCCAGTCGCATGTCGGCATGCCGGTAGAGGTGTGGTCAATAAG
S7G1MADHtPDC1F  GGTCAGGTTGCTTTCTCAGGTATAGCATGAGGTCGCTCTTATTGACCACACCTCTACCGGCATGCCGACATGCGACTGGGTGAGCATATGT
TCCG
3G1MADH2tENO2R TGTTAATCCACCTTCTAGCTCATTTTGGATGGCAACGGCAAGAGTAGGATTTAGAATTATATAACTTGATGAGATGAG
3G2MADH2tENO2F GGTAATTCTTCTGTCATTTACTCATCTCATCTCATCAAGTTATATAATTCTAAATCCTACTCTTGCCGTTGCCATCC
6G2MPDC1tFBA1R GTTATTTACAGAAGTTGGAAGGCTGGTATTGTTGTTCAAGCCAGCGGTGCCAGTTGGATCGGCAGTTTTGAATTGAGTAACCA
6G2MPDC1tFBA1F AGTGTAATAGAAGACTAGACACCTCGATACAAATAATGGTTACTCAATTCAAAACTGCCGATCCAACTGGCACCGCTGGCTTGA
S4G4MTDH2tTDH3R GGCCTCCGCGTCATTAAACTTCTTGTTGTTGACGCTAACATTCAACGCTAGTATGGCGAAAAGCCAATTAGTGTGATAC
S4G3MTDH2tTDH3F GGCATCACGGATTTTCGATAAAGCACTTAGTATCACACTAATTGGCTTTTCGCCATACTAGCGTTGAATGTTAGCGTCAAC
3G3MTPI1tTEF1R AGGAGTAGAAACATTTTGAAGCTATGGTGTGTGGGGGATCACTTTAATTAATCTATATAACAGTTGAAATTTGGA
3G2MTPI1tTEF1F GTCATTTTCGCGTTGAGAAGATGTTCTTATCCAAATTTCAACTGTTATATAGATTAATTAAAGTGATCCCCCACAC
MCYC1tpEASYR CGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGCGTTGGCCGATTCATTAATGC
X1MpEASYrtF CTTGCAAATGCCTATTGTGCAGATGTTATAATATCTGTGCGTTTAATTAAGGCTCGTATGTTGTGTGGAATTGT
GAL71 ATATTTGAAAGGCTTATGATTTTCTCTTGC
GAL72 GTCCAAATATTTGAAAACAAAGGTACAGC
X2MpEASYrtR CGAAGGCTTTAATTTGCAAGCTGCGGCCCTGCATTAATGAATCGGCCAACGCGCCAGGGTTTTCCCAGTCACGACGTTG
ZDHis3interg1 TTTATATAATGTATAATTCATT
ZDHis3interg2 CATTCTATACGTGTCATTCTGA
X1XppEASYM13RF CCGACTGGAAAGCGGGCAGTGAGCG
X2XppEASYM13FR CCATTCAGGCTGCGCAACTGTTGGGA
XpMpEASYM13RF GTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGTCACACAGGAAACAGCTATGACC
XpMpEASYM13FR CTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGACGACGTTGTAAAACGACGGCCAGT
Vv2‹
SacIPGK1 GCGCCGCGGACGCACAGATATTATAACATC
HMG1Asc TCGCGGCGCGCCTTAGGATTTAATGCAGGTGACGGAC
PacPDC1p GCGTTAATTAACATGCGACTGGGTGAGCATATGTTC
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ERG12Asc GCGGCGCGCCTTATGAAGTCCATGGTAAATTCGTG
PacENO2p GCGTTAATTAAAATCCTACTCTTGCCGTTGCCATC
IDI1Asc GCGGCGCGCCTTATAGCATTCTATGAATTTGCCT
PacPYK1p GCGTTAATTAAAATGCTACTATTTTGGAGATTAATC
ERG19Asc GCGGCGCGCCTTATTCCTTTGGTAGACCAGTCTTTGCG
PacpFBA GCGTTAATTAAGATCCAACTGGCACCGCTGGCTTGA
ERG13Asc GCGGCGCGCCTTATTTTTTAACATCGTAAGATCTTC
PacpTDH3 GCGTTAATTAAATACTAGCGTTGAATGTTAGCGTCA
ERG8Asc GCGGCGCGCCTTATTTATCAAGATAAGTTTCCGGATC
SacIpTEF1 GCGCCGCGGAGTGATCCCCCACACACCATAGCTT
ERG10Asc GCGGCGCGCCTCATATCTTTTCAATGACAATAGAG
SexAERG12 GCGACCWGGTATGTCATTACCGTTCTTAACTTCTGC
SexAERG19 GCGACCWGGTATGACCGTTTACACAGCATCCGTTACC
SexAERG13 GCGACCWGGTATGAAACTCTCAACTAAACTTTGTTGG
SexAIDI1 GCGACCWGGTATGACTGCCGACAACAATAG
SexAHMG1 TCGCGACCWGGTAAAACAATGGCTGCAGACCAATTGG
PacERG8 GCGTTAATTAAATGTCAGAGTTGAGAGCCTTCAGTGC
ENO2pSexA GCGACCWGGTTATTATTGTATGTTATAGTATTAGTTG
PYK1pSexA GCGACCWGGTTGTGATGATGTTTTATTTGTTTTGA
FBA1SexA GCGACCWGGTTTTGAATATGTATTACTTGGTTATG
PDC1pSexA GCGACCWGGTTTTGATTGATTTGACTGTGTTATTT
pTDH3SexA GCGACCWGGTTTTGTTTGTTTATGTGTGTTTATTC
TEF1SexA TGGCGACCWGGTTTTGTAATTAAAACTTAGATTAGA
LUPAsc GCGGCGCGCCTTAGTAGGAGTGAGCACATAAAACT
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PacPYK1p/ERG19Asc,PacpFBA/ERG13Asc,Pac
pTDH3/ERG8Asc,SacIpTEF1/ERG10Asc,SexA
HMG1/PDC1pSexA1,SexA1ERG12/ENO2pSexA1,
SexA1IDI1/PYK1pSexA1,SexA1ERG19/pFBASexA1,
SexA1ERG13/pTDH3SexA1,SexA1ERG8/pTEF1
SexA1
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Fig2 StrainconstructionusingtheDNAassemblermethod:
al7genesofMVApathwaywereintegratedintoGAL7siteof
NK2
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Table3 InformationofDNAassembler
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A pδtHMG1 1MPEASYPGK1F LP1F PPGK1tHMG1TADH1 LP1R
S7G1MADHtPDC1R
pM9ERG12 S7G1MADHtPDC1F LP2F PPDC1ERG12TADH2 LP2R
3G1ADH2tENO2R
pM16IDI1 3G2ADH2tENO2F LP3F PENO2IDI1TPDC1 LP3R
3G2MPDC1tPYK1R
pM5ERG19 3G3MPDC1tPYK1F LP4F PPYK1ERG19TPGI1 LP4R
S7G1MPGI1tFBA1R
pM8ERG13 S7G1MPGI1tFBA1F LP5F PFBA1ERG13TTDH2 LP5R
S4G4MTDH2tTDH3R
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3G3MTPI1tTEF1R
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pGAL7URA3 X1MpEASYrtF GAL7Up HelperfragmentofGAL7sitewithURA3marker upR
GAL72
pGAL7URA3 X2MpEASYrtR downF HelperfragmentofGAL7site GAL7Down
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pGAL7URA3 GAL71 GAL7Up GAL7sitewithURA3marker GAL7Down
GAL72
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XpMpEASYM13FR
pHIS3TRP1 X1XppEASYM13RF HIS3Up HelperfragmentofHIS3sitewithTRP1marker upR
ZDHis3interg1
pHIS3TRP1 ZDHis3interg2 downF HelperfragmentofHIS3site HIS3Down
X2XppEASYM13FR
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AtLUPwasintegratedintoHIS3siteofNK2SQ
2ʲÇ
30min,13×103r·min-1
•S
3min,
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32 MVA
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p
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5。
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Õ

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PGK1¨
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ADH1
ðè~

+=
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HIS3

(TRP1
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),
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NK2LUP,
q
  
q
5 
ÀÁÂÃgû1°m°Ʈ™»Ê

œßŒÊ¨£à
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gineeredNK2SQCKandNK2SQ
3,6。NK2LUP
²
YPD
Æ¥Ô$mÂ
6d
~

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GCMS
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NK2LUP
Òm°ÆÇÈÉÊ

q
7。
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öÝr
(823±118)mg·L-1
q
8;
†$m1
NK2SQ
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8。
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Vol41,No.6 March,2016
MTrans2Kplusmarker;
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1~2PPGK1AtLUP5m²NK2LUP¨
NK2SQ
1°$ƒ(Ì

q
6 NK2LUP

PCR

Fig6 PCRverificationofengineeredNK2LUP
A
mÂƋ
GCMS
q(ƒu•3®KLq
;Lupeol
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³
;NK2LUP
gû1
NK2LUP
mÂƋ
;NK2SQ
gû1
NK2SQ
mÂƋ

q

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);B
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NK2LUP
mÂƋ$ÇÈÉʃ´
Lq

q
7 GCMS
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NK2LUP
1°mÂƋ—nVv
Fig7 IdentificationofNK2LUPfermentationproductsbyGC
MSmethod
4 
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nŸ™Ðmë¡ã]öÇÈɜ­Lk
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e
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9
ÁiÀÁÂðÆkd_=‹ˆw?Bú?>
ƒ½ó

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7
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gj

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£
à¨ÇÈÉÊ
Fig8 Productionofergosterol,lanosterol,squaleneandlupeol
byengineeredNK2SQandNK2LUP
úÆ£àƒLkÖÀÁÂÃÄÅgj
NK2
SQ。
Ÿ~

/x9¤ì>ƒ
AtLUP
ÔÕ+=lÖ

NK2SQ
ÔՕ$

c¿úœ°ÆÇÈɜƒ
ÀÁÂÃÄÅgj
NK2LUP。
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$ÇÈÉʃá_¬’Æö:?H

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(ERG1)
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CQÓ
[11],
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LUP
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Mt£à)¾£>ƒ

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yg?z

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·5101·