Improving monoterpene geraniol production through geranyl diphosphate synthesis
regulation in Saccharomyces cerevisiae
Applied Microbiology and Biotechnology
Jianzhi Zhao1, Xiaoming Bao1, Chen Li, Yu Shen, Jin Hou*
State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, China
* Corresponding author: Dr. Jin Hou, State Key Laboratory of Microbial Technology, Shandong
University, Jinan 250100, China. Tel.: +86 531 8836 5827; Fax: +86 531 8836 5826; E-mail:
[email protected]
1
These authors contributed equally to the study.
Table S1 Primers used in this study
Primer name
Sequence (5’→ 3’)a, b
DNA fragments
LdGES-F
CGCGGATCCATGGCGTCTGCTAGATCAAC
LdGES and tLdGES
LdGES-R
AATCCTGCAGGTTATAATAATGGGGTGAAGAAC
tLdGES-F
CGCGGATCCATGTCATCTGTTTCTTTGCCTACAG
ObGES-F
CGCGGATCCATGTCTTGTGCAAGAATTACAGTT
ObGES-R
ACGCGTCGACTTACTGAGTAAAAAACAAAGCA
ObGES and tObGES
TCA
tObGES-F
CGCGGATCCATGCCATTGTCATCTACACCTT
VoGES-F
CGCGGATCCATGATTACTTCATCTTCATC
VoGES-R
ACGCGTCGACTTAAACAGAAACAGAAACTGGT
VoGES and tVoGES
GT
tVoGES-F
CGCGGATCCATGTCAACAGCTTCATCAATTGCT
G
IDI1-F
CGGGATCCATGACTGCCGACAACAATAGTA
IDI1-R
CCGCTCGAGTTATAGCATTCTATGAATTTGC
tHMG1-F
CGGGATCCATCTTAAGATGGCTGCAGACCAATT
IDI1
tHMG1
GGTGAAGACTGAAGTC
tHMG1-R
ATACCTGCAGGTTAGGATTTAATGCAGGTGACG
UPC2.1-F
CGCGGATCCATGAGCGAAGTCGGTATACAGA
UPC2.1-R1
CCGCCACCGAGGAAATCTAGCATCATATGCATA
UPC2-1
TCACCACCTCCACTGT
UPC2-1-R2
AATTTGTTGTTGTCATCGATGGTAATCCGCCACC
GAGGAAATCTAGCAT
UPC2-1-R3
ATACCTGCAGGTCATAACGAAAAATCAGAGAA
ATTTGTTGTTGTCATCG
tAgGPPS2-F
AGCTCTAGAATGGAATTCGATTTCAATAAATATA
tAgGPPS2
TGG
tAgGPPS2-R
CCGCTCGAGTTAATTTTGTCTGAAAGCGACGTA
G
tPaGPPS2-F
TGCTCATTAGAAAGAAAGCATAGCAATCTAATC
tPaGPPS2
TAAGTTTTCTAGAATGGAATTTGATTTTGATAAA
TACATGC
tPaGPPS2-R
GGAGGGCGTGAATGTAAGCGTGACATAACTAAT
TACATGACTCGAGTTAATTTTGTCTAGAAGCTAT
ATAA
tCrGPPS-F
TGCTCATTAGAAAGAAAGCATAGCAATCTAATC
tCrGPPS
TAAGTTTTCTAGAATGGTCGTCGCAGAAGTCCC
AAAGT
tCrGPPS-R
GGAGGGCGTGAATGTAAGCGTGACATAACTAAT
TACATGACTCGAGTTATTTCCTTCTAGTAATAAC
TCTC
ERG20-F
CGGACTAGTATGGCTTCAGAAAAAGAAATTA
ERG20-R
GGCGAGCTCTTATTTACTTCTCTTGTAAACC
ERG20
ERG20-197G-F
CTCCTTCATAGTTACTTTCGGTACTGCTTACTAT
ERG20K197G
TCTTTCT
ERG20-197G-R
AGAAAGAATAGTAAGCAGTACCGAAAGTAACT
ATGAAGGAG
ERG20-96W-F
TGAGTTGTTGCAGGCTTACTGGTTGGTCGCCGA
ERG20F96W
TGATAT
ERG20-96W-R
ATATCATCGGCGACCAACCAGTAAGCCTGCAAC
AACTCA
ERG20-96-127W-F
GTTGGGGAAATTGCCATCTGGGACGCATTCATG
ERG20F96W-N127W
TTAGAGG
ERG20-96-127W-R
CCTCTAACATGAATGCGTCCCAGATGGCAATTT
CCCCAAC
UAS3CITTEF1-F
CCAGTCACGACGTTGTAAAACGACGGCCAGTG
TEF1p-X-CYC1t
AATTCGAGCTCATTAACCCTCACTAAAGGGAAC
UAS3CITCYC1-R
CATTGTATTTTGAACGTACAAAGTATGCATTGTG
GTACCGAGCTCGAGCGTCCCAAAACCTTCTCA
A
GB-TEF1-UPC2-1-PG
ACCTTGCTTGAGAAGGTTTTGGGACGCTCGAA
TEF1p-UPC2-1-PGK
K1-KF
GATCCTTCCTTACATCACACCCAAT
1t
GB-TEF1-UPC2-1-PG
CATTAATGCAGCTGGATAAACGCCGGCGAAAC
K1-KR
GATCCGGAGAAACAGCTATGACCATGA
PCTR3-F
CGGGATCCGTATTCCAATGAGAATCGC
PCTR3-R
GCTCTAGACTTTGTATAGCCCTTAAAT
GBFusion(EW-GES)-
GAAAGCATAGCAATCTAATCTAAGTTTTAATTAC
ERG20WW-(GGGS)-
WF
AAAGGATCCATGGCTTCAGAAAAAGAAATTAG
tVoGES
CTR3 promoter
GAG
GBFusion(EW-GES)-
GCTGTTGACATAGAACCACCACCTTTACTTCTC
WR1
TTGTAAACCTTGTTC
GBFusion(EW-GES)-
ACAAGAGAAGTAAAGGTGGTGGTTCTATGTCA
GF1
ACAGCTTCATCAATTG
GBFusion(EW-GES)-
AAATTGATCTATCGATTTCAATTCAATTCAATCC
GR
TGCAGGTTAAACAGAAACAGAAACTGGTG
GBFusion(EW-GES)-
GCTGTTGACATAGAACCACCACCACCTTTACTT
ERG20WW-(GGGGS)-
WR2
CTCTTGTAAACCTTGTTC
tVoGES
GBFusion(EW-GES)-
AGAGAAGTAAAGGTGGTGGTGGTTCTATGTCA
GF2
ACAGCTTCATCAATTG
GBFusion(EW-GES)-
GCTGTTGACATACCAGAACCACCAGAACCTTTA
ERG20WW-(GSGGSG
WR3
CTTCTCTTGTAAACCTTGTTC
)-tVoGES
GBFusion(EW-GES)-
GAAGTAAAGGTTCTGGTGGTTCTGGTATGTCAA
GF3
CAGCTTCATCAATTG
GBFusion(GES-EW)-
AGAAAGCATAGCAATCTAATCTAAGTTTTAATTA
GF
CAAAGGATCCATGTCAACAGCTTCATCAATTGC
tVoGES-(GGGS)-
TG
ERG20WW
GBFusion(GES-EW)-
TCTTTTTCTGAAGCCATAGAACCACCACCAACA
GR1
GAAACAGAAACTGGTGTA
GBFusion(GES-EW)-
TGTTTCTGTTGGTGGTGGTTCTATGGCTTCAGA
WF1
AAAAGAAATTAGGAG
GBFusion(GES-EW)-
AAATTGATCTATCGATTTCAATTCAATTCAATCC
WR
TGCAGGTTATTTACTTCTCTTGTAAACCTTG
GBFusion(GES-EW)-
TCTTTTTCTGAAGCCATAGAACCACCACCACCA
tVoGES-(GGGGS)-
GR2
ACAGAAACAGAAACTGGTGTA
ERG20WW
GBFusion(GES-EW)-
TGTTTCTGTTGGTGGTGGTGGTTCTATGGCTTC
WF2
AGAAAAAGAAATTAGGAG
GBFusion(GES-EW)-
TTTTCTGAAGCCATACCAGAACCACCAGAACC
tVoGES-(GSGGSG)-
GR3
AACAGAAACAGAAACTGGTGTA
ERG20WW
GBFusion(GES-EW)-
TGTTTCTGTTGGTTCTGGTGGTTCTGGTATGGCT
WF3
TCAGAAAAAGAAATTAGGAG
a
b
The underlined characters indicate restriction sites.
The bold characters indicate mutant sites.
Table S2 Plasmids and strains used in this study
Plasmids/Strains
Characteristics /Genotype
Resource
p416mcstefcit3×
CEN6, URA3, UASCIT(3×)-PTEF1-yECitrine
(Blazeck et al. 2012)
pJFE3
2μm ori, URA3, PTEF1-TPGK1
(Shen et al. 2012)
pIYC04
2µm ori, HIS3, PTEF1-TADH1, PPGK1-TCYC1
(Chen et al. 2013)
pZGL(f)
pJFE3, 2μm ori, URA3, PTEF1-LdGES
This study
pZGL
pJFE3, 2μm ori, URA3, PTEF1-tLdGES
This study
pZGO(f)
pJFE3, 2μm ori, URA3, PTEF1-ObGES
This study
pZGO
pJFE3, 2μm ori, URA3, PTEF1-tObGES
This study
pZGV(f)
pJFE3, 2μm ori, URA3, PTEF1-VoGES
This study
pZGV
pJFE3, 2μm ori, URA3, PTEF1-tVoGES
This study
pZGV1
pJFE3, 2μm ori, URA3, PTEF1-tVoGES,
This study
Plasmids
UASCIT(3×)-PTEF1-ERG20
pJFE3, 2μm ori, URA3, PTEF1-tVoGES,
pZGV2
This study
UASCIT(3×)-PTEF1-tAgGPPS2
pJFE3, 2μm ori, URA3, PTEF1-tVoGES,
pZGV3
This study
UASCIT(3×)-PTEF1-tPaGPPS2
pJFE3, 2μm ori, URA3, PTEF1-tVoGES,
pZGV4
This study
UASCIT(3×)-PTEF1-tCrGPPS
pZGV5
a
pJFE3, 2μm ori, URA3, PTEF1-tVoGES,
UASCIT(3×)-PTEF1-ERG20
pZGV6
b
This study
G
pJFE3, 2μm ori, URA3, PTEF1-tVoGES,
This study
UASCIT(3×)-PTEF1-ERG20WW
pZMVA1
pIYC04, 2µm ori, HIS3, PTEF1, PPGK1-IDI1
This study
pZMVA2
pIYC04, 2µm ori, HIS3, PTEF1-tHMG1, PPGK1
This study
pZMVA3
pIYC04, 2µm ori, HIS3, PTEF1-tHMG1, PPGK1-IDI1
This study
pZMVA4
pIYC04, 2µm ori, HIS3, PTEF1-tHMG1,
This study
PPGK1-IDI1,PTEF1-UPC2.1
pZGV6-EG1
pZGV6-EG2
pZGV6-EG3
pJFE3, 2µ ori, URA3, PTEF1-ERG20WW-(GGGS)-tVoGES
This study
pJFE3, 2µ ori, URA3, PTEF1-ERG20
WW
-(GGGGS)-tVoGES
This study
pJFE3, 2µ ori, URA3, PTEF1-ERG20
WW
-(GSGGSG)-tVoGES
This study
WW
pZGV6-GE1
pJFE3, 2µ ori, URA3, PTEF1-tVoGES -(GGGS)-ERG20
pZGV6-GE2
pJFE3, 2µ ori, URA3, PTEF1-tVoGES-(GGGGS)-ERG20WW
pZGV6-GE3
pJFE3, 2µ ori, URA3, PTEF1-tVoGES-(GSGGSG)- ERG20
This study
WW
This study
This study
Strains
CEN.PK102-5B
MATa ura3-52 his3Δ1 leu2-3,112
Dr. P. Kötter,
Frankfurt, Germany
CEN.PK102-5B
CEN.PK102-5B, PERG20::PCTR3
This study
YZG00
pJFE3, pIYC04
This study
YZG01
pZGL, pIYC04
This study
YZG02
pZGO, pIYC04
This study
YZG03
pZGV, pIYC04
This study
-pCTR3
YZG04
pZGV, pZMVA1
This study
YZG05
pZGV, pZMVA2
This study
YZG06
pZGV, pZMVA3
This study
YZG07
pZGV1, pZMVA3
This study
YZG08
pZGV2, pZMVA3
This study
YZG09
pZGV3, pZMVA3
This study
YZG10
pZGV4, pZMVA3
This study
YZG11
pZGV5, pZMVA3
This study
YZG12
pZGV6, pZMVA3
This study
YZG12-pCTR3
pZGV6, pZMVA3,PERG20::PCTR3
This study
YZG13
pZGV6, pZMVA4
This study
YZG13-pCTR3
pZGV6, pZMVA4, PERG20::PCTR3
This study
YZG13-EG1
pZGV6-EG1, pZMVA4
This study
YZG13-EG2
pZGV6-EG2, pZMVA4
This study
YZG13-EG3
pZGV6-EG3, pZMVA4
This study
YZG13-GE1
pZGV6-GE1, pZMVA4
This study
YZG13-GE2
pZGV6-GE2, pZMVA4
This study
YZG13-GE3
pZGV6-GE3, pZMVA4
This study
a
ERG20G is abbreviation of ERG20K197G
b
ERG20WW is abbreviation of ERG20F96W-N127W
References
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