药学学报

Name	Description	Source
Plasmids
pEASY-Blunt simple	Cloning vector for blunt ligation, Amp, Km	TransGen Biotech
p-P_GAL7	Cloning P_GAL7 and T_SPG5 into pEASY-Blunt simple	Lab collection
p-P_GAL10	Cloning P_GAL10 and T_CWP2 into pEASY-Blunt simple	Lab collection
p-P_GAL1	Cloning P_GAL1 and T_PRM9 into pEASY-Blunt simple	Lab collection
pM3-tHMG1	Cloning P_TEF1-tHMG1-T_CYC1cassette into pEASY-Blunt simple	Lab collection
P_GAL7-cHMG2-T_SPG5	Cloning truncated HMG2 into p-P_GAL7	Lab collection
P_GAL10-HMGR-N-T_CWP2	Cloning HMGR-N into p-P_GAL10	Lab collection
P_GAL1-tHMG1-T_PRM9	Cloning tHMG1 into p-P_GAL1	Lab collection
P_GAL10-CnVS-T_CWP2	Cloning CnVS into p-P_GAL10	This study
P_GAL10-SmFPS-CsVal-T_CWP2	Cloning SmFPS-CsVal into p-P_GAL10	This study
P_GAL10-SmFPS-EgVal-T_CWP2	Cloning SmFPS-EgVal into p-P_GAL10	This study
P_GAL10-SmFPS-CnVS-T_CWP2	Cloning SmFPS-CnVS into p-P_GAL10	This study
pRS425-LEU2-TRP1-URA3-TEF1p-ObGES	Cloning LEU2, TRP1, URA3 makersand P_TEF1-ObGES-T_CYC1 cassette into pRS425	Lab collection
NDT80-HIS3	Cloning NDT80 and HIS3 marker into pEASY-Blunt simple	Lab collection
P_ACT3-PDR3m-T_PDR3	Cloning P_ACT3-PDR3^Q763L-T_PDR3 cassette into pEASY-Blunt simple	Lab collection
P_GAL1-INO2-T_PRM9	Cloning INO2 into p-P_GAL1	Lab collection
NDT80-gRNA	NDT80 disruption gRNA cassette, 2 micron, URA3 marker	Lab collection
LEU2-gRNA	LEU2 disruption gRNA cassette, 2 micron, URA3 marker	Lab collection
HIS3-gRNA	HIS3 disruption gRNA cassette, 2 micron, URA3 marker	Lab collection
HO-gRNA	HO disruption gRNA cassette, 2 micron, URA3 marker	Lab collection
GAL80-gRNA	GAL80 disruption gRNA cassette, 2 micron, URA3 marker	Lab collection
ROX1-gRNA	ROX1 disruption gRNA cassette, 2 micron, URA3 marker	Lab collection
HXK2-gRNA	HXK2 disruption gRNA cassette, 2 micron, URA3 marker	Lab collection
Strains
YTT-T5	A platform yeast for mono- and sesquiterpenoid production	Lab collection
VAL-01	P_GAL10-CnVS-T_CWP2 and P_GAL1-tHMG1-T_PRM9 cassettes were integrated into NDT80 site of YTT-T5	This study
VAL-02	P_GAL10-SmFPS-CnVS-T_CWP2 and P_GAL1-tHMG1-T_PRM9 cassettes were integrated into NDT80 site of YTT-T5	This study
VAL-03	P_GAL10-SmFPS-CsVal-T_CWP2 and P_GAL1-tHMG1-T_PRM9 cassettes were integrated into NDT80 site of YTT-T5	This study
VAL-04	P_GAL10-SmFPS-EgVal-T_CWP2 and P_GAL1-tHMG1-T_PRM9 cassettes were integrated into NDT80 site of YTT-T5	This study
VAL-05	P_GAL10-SmFPS-CnVS-T_CWP2 and P_GAL1-tHMG1-T_PRM9 cassettes were integrated into LEU2 site of VAL-02	This study
VAL-06	P_GAL10-SmFPS-CnVS-T_CWP2 and P_GAL1-tHMG1-T_PRM9 cassettes were integrated into HIS3 site of VAL-05	This study
VAL-07	P_GAL10-SmFPS-CnVS-T_CWP2 and P_GAL1-tHMG1-T_PRM9 cassettes were integrated into HO site of VAL-06	This study
VAL-08	ROX1 was deleted inVAL-05	This study
VAL-09	HXK2 was deleted in VAL-08 and subsequently P_GAL4-GAL4-T_GAL4 cassette was integrated into GAL80 site	This study
VAL-10	LEU2, TRP1, URA3, HIS3 makers, P_ACT3-PDR3m-T_PDR3, P_TEF1-tHMG1-T_CYC1 and P_GAL1-INO2-T_PRM9 were integrated into YPRCdelta15 site of VAL-09	This study

Name	Description	Source
Plasmids
pEASY-Blunt simple	Cloning vector for blunt ligation, Amp, Km	TransGen Biotech
p-P_GAL7	Cloning P_GAL7 and T_SPG5 into pEASY-Blunt simple	Lab collection
p-P_GAL10	Cloning P_GAL10 and T_CWP2 into pEASY-Blunt simple	Lab collection
p-P_GAL1	Cloning P_GAL1 and T_PRM9 into pEASY-Blunt simple	Lab collection
pM3-tHMG1	Cloning P_TEF1-tHMG1-T_CYC1cassette into pEASY-Blunt simple	Lab collection
P_GAL7-cHMG2-T_SPG5	Cloning truncated HMG2 into p-P_GAL7	Lab collection
P_GAL10-HMGR-N-T_CWP2	Cloning HMGR-N into p-P_GAL10	Lab collection
P_GAL1-tHMG1-T_PRM9	Cloning tHMG1 into p-P_GAL1	Lab collection
P_GAL10-CnVS-T_CWP2	Cloning CnVS into p-P_GAL10	This study
P_GAL10-SmFPS-CsVal-T_CWP2	Cloning SmFPS-CsVal into p-P_GAL10	This study
P_GAL10-SmFPS-EgVal-T_CWP2	Cloning SmFPS-EgVal into p-P_GAL10	This study
P_GAL10-SmFPS-CnVS-T_CWP2	Cloning SmFPS-CnVS into p-P_GAL10	This study
pRS425-LEU2-TRP1-URA3-TEF1p-ObGES	Cloning LEU2, TRP1, URA3 makersand P_TEF1-ObGES-T_CYC1 cassette into pRS425	Lab collection
NDT80-HIS3	Cloning NDT80 and HIS3 marker into pEASY-Blunt simple	Lab collection
P_ACT3-PDR3m-T_PDR3	Cloning P_ACT3-PDR3^Q763L-T_PDR3 cassette into pEASY-Blunt simple	Lab collection
P_GAL1-INO2-T_PRM9	Cloning INO2 into p-P_GAL1	Lab collection
NDT80-gRNA	NDT80 disruption gRNA cassette, 2 micron, URA3 marker	Lab collection
LEU2-gRNA	LEU2 disruption gRNA cassette, 2 micron, URA3 marker	Lab collection
HIS3-gRNA	HIS3 disruption gRNA cassette, 2 micron, URA3 marker	Lab collection
HO-gRNA	HO disruption gRNA cassette, 2 micron, URA3 marker	Lab collection
GAL80-gRNA	GAL80 disruption gRNA cassette, 2 micron, URA3 marker	Lab collection
ROX1-gRNA	ROX1 disruption gRNA cassette, 2 micron, URA3 marker	Lab collection
HXK2-gRNA	HXK2 disruption gRNA cassette, 2 micron, URA3 marker	Lab collection
Strains
YTT-T5	A platform yeast for mono- and sesquiterpenoid production	Lab collection
VAL-01	P_GAL10-CnVS-T_CWP2 and P_GAL1-tHMG1-T_PRM9 cassettes were integrated into NDT80 site of YTT-T5	This study
VAL-02	P_GAL10-SmFPS-CnVS-T_CWP2 and P_GAL1-tHMG1-T_PRM9 cassettes were integrated into NDT80 site of YTT-T5	This study
VAL-03	P_GAL10-SmFPS-CsVal-T_CWP2 and P_GAL1-tHMG1-T_PRM9 cassettes were integrated into NDT80 site of YTT-T5	This study
VAL-04	P_GAL10-SmFPS-EgVal-T_CWP2 and P_GAL1-tHMG1-T_PRM9 cassettes were integrated into NDT80 site of YTT-T5	This study
VAL-05	P_GAL10-SmFPS-CnVS-T_CWP2 and P_GAL1-tHMG1-T_PRM9 cassettes were integrated into LEU2 site of VAL-02	This study
VAL-06	P_GAL10-SmFPS-CnVS-T_CWP2 and P_GAL1-tHMG1-T_PRM9 cassettes were integrated into HIS3 site of VAL-05	This study
VAL-07	P_GAL10-SmFPS-CnVS-T_CWP2 and P_GAL1-tHMG1-T_PRM9 cassettes were integrated into HO site of VAL-06	This study
VAL-08	ROX1 was deleted inVAL-05	This study
VAL-09	HXK2 was deleted in VAL-08 and subsequently P_GAL4-GAL4-T_GAL4 cassette was integrated into GAL80 site	This study
VAL-10	LEU2, TRP1, URA3, HIS3 makers, P_ACT3-PDR3m-T_PDR3, P_TEF1-tHMG1-T_CYC1 and P_GAL1-INO2-T_PRM9 were integrated into YPRCdelta15 site of VAL-09	This study

Primer name	Sequence (5′ to 3′)
3G-1-M-GAL7p-CWP2t-R	CCCTTAGCTGCGATTAAAGAGTCAAGGCGTGAAAAACATAGGCGAAAGTGGTGTCACAGCGAATTTCCTCAC
3G-2-M-GAL7p-CWP2t-F	GAACTTGTAAACAATTCGGTCCCTACATGTGAGGAAATTCGCTGTGACACCACTTTCGCCTATGTTTTTCACGC
GAL1p-R	TATAGTTTTTTCTCCTTGACGTTAAAGTATAGAGGTAT
GAL1p-F	TTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGGATGG
NDT80-50-CWP2t-F	TACTAACCTTTCATTAAAGAGAAATAACAATATTATAAAAAGCGCTTAAACACTTTCGCCTATGTTTTTCACGC
NDT80-50-PRM9t-down	GATCCACAGAATTCGCATATTTTTTTAACGATTTAAAATCATTAGTTTATTCAACATCGTATTTTCCGAAGCG
LEU2-50-CWP2t-F	TTTACATTTCAGCAATATATATATATATATTTCAAGGATATACCATTCTACACTTTCGCCTATGTTTTTCACGC
LEU2-50-PRM9t-down	CGGTGGTACTGTTGGAACCACCTAAATCACCAGTTCTGATACCTGCATCCTCAACATCGTATTTTCCGAAGCG
HIS3-50-CWP2t-F	CTTCGAAGAATATACTAAAAAATGAGCAGGCAAGATAAACGAAGGCAAAGCACTTTCGCCTATGTTTTTCACGC
HIS3-50-PRM9t-down	TATACACATGTATATATATCGTATGCTGCAGCTTTAAATAATCGGTGTCATCAACATCGTATTTTCCGAAGCG
HO-50-CWP2t-F	TCTAAATCCATATCCTCATAAGCAGCAATCAATTCTATCTATACTTTAAACACTTTCGCCTATGTTTTTCACGC
HO-50-PRM9t-down	ATTAAATTTTACTTTTATTACATACAACTTTTTAAACTAATATACACATTTCAACATCGTATTTTCCGAAGCG
GAL80-GAL4p-F	CCAGCGTATACAATCTCGATAGTTGGTTTCCCGTTCTTTCCACTCCCGTCGACAGCATTCGCCCAGTATTTTTTTTATTC
GAL4t-GAL80-R	GTTTTTATAACGTTCGCTGCACTGGGGGCCAAGCACAGGGCAAGATGCTTTTAACGTTCTACTTTACACTGGTGGTAGG
delta15-LEU2t-F	GTAGTTTTAAAATTTCAAATCCGAACAACAGAGCATAGGGTTTCGCAAACCGTTGAGCCATTAGTATCAATTTGCTTAC
URA3t-HIS3p-R	CGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAACTCTTCCTTTTTCAATGGGTAATAACTG
URA3t-HIS3p-F	AGAGCTTCAATTTAATTATATCAGTTATTACCCATTGAAAAAGGAAGAGTTCGCGCGTTTCGGTGATGA
HIS3t-ACT3p-R	ATGTTAGAACCACCTGGGCCAACAATCATATTGAATCTTCTGGTATCAGCGTGTCACTACATAAGAACACCTTTGGTG
HIS3t-ACT3p-F	ATGGTACCAACGATGTTCCCTCCACCAAAGGTGTTCTTATGTAGTGACACGCTGATACCAGAAGATTCAATATGATTGTTG
PDR3t-TEF1p-R	GTAAAAAAGGAGTAGAAACATTTTGAAGCTATGGTGTGTGGGGGATCACTCTTTTTGAACAGCGCGCCCA
PDR3t-TEF1p-F	ACATAAGATCAACGATTTCTTCATTAATTTTGGGCGCGCTGTTCAAAAAGAGTGATCCCCCACACACCAT
CYC1t-GAL1p-R	CTTTGCGTCCATCCAAAAAAAAAGTAAGAATTTTTGAAAATTCAATATAAGCGCGTTGGCCGATTCATTA
CYC1t-GAL1p-F	GGCTTTAATTTGCAAGCTGCGGCCCTGCATTAATGAATCGGCCAACGCGCTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGGA
Delta15-50-PRM9t-R	TCTGTATTGTTCTTCTTAGTGCTTGTATATGCTCATCCCGACCTTCCATTTCAACATCGTATTTTCCGAAGCGT
ROX1-KO-up-F	CCGACAAATCCCCTGGATATCATTG
ROX1-KO-up-R	ACTTTTATTAAACAGGAGCCTGTTGATTGTCTAACTGCGTTCTTTTGT
ROX1-KO-down-F	ACGCAGTTAGACAATCAACAGGCTCCTGTTTAATAAAAGTTTAAATCGCC
ROX1-KO-down-R	CGATTGTGTCTATATCTACAATATGAACCTG
HXK2-KO-UP-F	ATGTTACTACGAGTTTTCTGAACCTCCTC
HXK2-KO-UP-R	GTAGAAAAAGGGCACCTTCTTGTTGTTCAAACTTAATTTACAAATTAAGTTTTATTTAATTAGCGTACTTATTATGTGTGGAGAATT
HXK2-KO-DOWN-F	GTTGTAGGAATATAATTCTCCACACATAATAAGTACGCTAATTAAATAAAACTTAATTTGTAAATTAAGTTTGAACAACAAGAAGGT
HXK2-KO-DOWN-R	CGCGTAAAAATCGCGGGTAT
GAL10p-F(251bp)	CGGAGGGCTGTCGCC
AscI-CnVS-R	GCGGCGCGCCTTAAGGGATGATTGGTTCAACGAAAAACT
CsVal-YZ-R	CTATATCGATTCTAACAGAGCAGATTTGC
EgVal-YZ-R(486)	CAAACCTCTCGCGTCGTTAAC
GAL1p-F(349)	GCCGCACTGCTCCGA
ASC1-tHMG1-R	GCGGCGCGCCTTAGGATTTAATGCAGGTGACGGACC
NDT80-OUT-F	TGTTAATTCCTTTGTGCCAGATAGTATTG
NDT80-OUT-R	CACCCAATCTTCATTATTCACCAAATATTCCC
LEU2-OUT-F	GCTTGCATCACAATACTTGAAGTTGAC
ASC1-CWP2t-F	GGCGCGCCGAAATCTCTGATTTTTTATAATATCTATATGGCTTTTTTC
ASC1-PRM9t-F	GGCGCGCCACAGAAGACGGGAGACACTAGC
LEU2-OUT-R	GTGCCCTCCTCCTTGTCAATATTAAT
HIS3-OUT-F	GCCTCGGTAATGATTTTCATTTTTTTTTTTCC
HIS3-OUT-R	GCGCCTCGTTCAGAATGACAC
HO-OUT-F	CTGGGCGTTATTAGGTGTGAAACC
HO-OUT-R	CGCGTAAATATTTAGCTTGATAACTGTTACTG
ROX1-YZ-F	CAGGTGCAGGCGTACTTTAAAG
ROX1-YZ-R	GCCACTTGAGCTTTCGGTTTG
HXK2-OUT-F	AACAAAGAAAAGGCAAGAGTAAATAGCG
HXK2-OUT-R	CGGTGTTCATCCAGGTGGTAATATC
GAL80-OUT-F	GTTGGCCTCTACTTACTCCATCGAC
pGAL4-SexAI	GCGACCTGGTCTTTCAGGAGGCTTGCTTCTCT
GAL4t-F	AATGAATCGTAGATACTGAAAAACCCC
GAL80-OUT-R	GGCATTATATGGCCGATTTGTATTAGTATCC
PDR3-YZ-F(207)	GGAGAGGAATCGTACACTCAAGAC
TEF1-PAC1-R	GCGTTAATTAATTTGTAATTAAAACTTAGATTAGATTGCTATGCTTTCTTTC
TEF1p-YZ-F(350bp)	GCCGTACCACTTCAAAACACC
INO2-R	TCAGGAATCATCCAGTATGTGCTGT

Primer name	Sequence (5′ to 3′)
3G-1-M-GAL7p-CWP2t-R	CCCTTAGCTGCGATTAAAGAGTCAAGGCGTGAAAAACATAGGCGAAAGTGGTGTCACAGCGAATTTCCTCAC
3G-2-M-GAL7p-CWP2t-F	GAACTTGTAAACAATTCGGTCCCTACATGTGAGGAAATTCGCTGTGACACCACTTTCGCCTATGTTTTTCACGC
GAL1p-R	TATAGTTTTTTCTCCTTGACGTTAAAGTATAGAGGTAT
GAL1p-F	TTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGGATGG
NDT80-50-CWP2t-F	TACTAACCTTTCATTAAAGAGAAATAACAATATTATAAAAAGCGCTTAAACACTTTCGCCTATGTTTTTCACGC
NDT80-50-PRM9t-down	GATCCACAGAATTCGCATATTTTTTTAACGATTTAAAATCATTAGTTTATTCAACATCGTATTTTCCGAAGCG
LEU2-50-CWP2t-F	TTTACATTTCAGCAATATATATATATATATTTCAAGGATATACCATTCTACACTTTCGCCTATGTTTTTCACGC
LEU2-50-PRM9t-down	CGGTGGTACTGTTGGAACCACCTAAATCACCAGTTCTGATACCTGCATCCTCAACATCGTATTTTCCGAAGCG
HIS3-50-CWP2t-F	CTTCGAAGAATATACTAAAAAATGAGCAGGCAAGATAAACGAAGGCAAAGCACTTTCGCCTATGTTTTTCACGC
HIS3-50-PRM9t-down	TATACACATGTATATATATCGTATGCTGCAGCTTTAAATAATCGGTGTCATCAACATCGTATTTTCCGAAGCG
HO-50-CWP2t-F	TCTAAATCCATATCCTCATAAGCAGCAATCAATTCTATCTATACTTTAAACACTTTCGCCTATGTTTTTCACGC
HO-50-PRM9t-down	ATTAAATTTTACTTTTATTACATACAACTTTTTAAACTAATATACACATTTCAACATCGTATTTTCCGAAGCG
GAL80-GAL4p-F	CCAGCGTATACAATCTCGATAGTTGGTTTCCCGTTCTTTCCACTCCCGTCGACAGCATTCGCCCAGTATTTTTTTTATTC
GAL4t-GAL80-R	GTTTTTATAACGTTCGCTGCACTGGGGGCCAAGCACAGGGCAAGATGCTTTTAACGTTCTACTTTACACTGGTGGTAGG
delta15-LEU2t-F	GTAGTTTTAAAATTTCAAATCCGAACAACAGAGCATAGGGTTTCGCAAACCGTTGAGCCATTAGTATCAATTTGCTTAC
URA3t-HIS3p-R	CGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAACTCTTCCTTTTTCAATGGGTAATAACTG
URA3t-HIS3p-F	AGAGCTTCAATTTAATTATATCAGTTATTACCCATTGAAAAAGGAAGAGTTCGCGCGTTTCGGTGATGA
HIS3t-ACT3p-R	ATGTTAGAACCACCTGGGCCAACAATCATATTGAATCTTCTGGTATCAGCGTGTCACTACATAAGAACACCTTTGGTG
HIS3t-ACT3p-F	ATGGTACCAACGATGTTCCCTCCACCAAAGGTGTTCTTATGTAGTGACACGCTGATACCAGAAGATTCAATATGATTGTTG
PDR3t-TEF1p-R	GTAAAAAAGGAGTAGAAACATTTTGAAGCTATGGTGTGTGGGGGATCACTCTTTTTGAACAGCGCGCCCA
PDR3t-TEF1p-F	ACATAAGATCAACGATTTCTTCATTAATTTTGGGCGCGCTGTTCAAAAAGAGTGATCCCCCACACACCAT
CYC1t-GAL1p-R	CTTTGCGTCCATCCAAAAAAAAAGTAAGAATTTTTGAAAATTCAATATAAGCGCGTTGGCCGATTCATTA
CYC1t-GAL1p-F	GGCTTTAATTTGCAAGCTGCGGCCCTGCATTAATGAATCGGCCAACGCGCTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGGA
Delta15-50-PRM9t-R	TCTGTATTGTTCTTCTTAGTGCTTGTATATGCTCATCCCGACCTTCCATTTCAACATCGTATTTTCCGAAGCGT
ROX1-KO-up-F	CCGACAAATCCCCTGGATATCATTG
ROX1-KO-up-R	ACTTTTATTAAACAGGAGCCTGTTGATTGTCTAACTGCGTTCTTTTGT
ROX1-KO-down-F	ACGCAGTTAGACAATCAACAGGCTCCTGTTTAATAAAAGTTTAAATCGCC
ROX1-KO-down-R	CGATTGTGTCTATATCTACAATATGAACCTG
HXK2-KO-UP-F	ATGTTACTACGAGTTTTCTGAACCTCCTC
HXK2-KO-UP-R	GTAGAAAAAGGGCACCTTCTTGTTGTTCAAACTTAATTTACAAATTAAGTTTTATTTAATTAGCGTACTTATTATGTGTGGAGAATT
HXK2-KO-DOWN-F	GTTGTAGGAATATAATTCTCCACACATAATAAGTACGCTAATTAAATAAAACTTAATTTGTAAATTAAGTTTGAACAACAAGAAGGT
HXK2-KO-DOWN-R	CGCGTAAAAATCGCGGGTAT
GAL10p-F(251bp)	CGGAGGGCTGTCGCC
AscI-CnVS-R	GCGGCGCGCCTTAAGGGATGATTGGTTCAACGAAAAACT
CsVal-YZ-R	CTATATCGATTCTAACAGAGCAGATTTGC
EgVal-YZ-R(486)	CAAACCTCTCGCGTCGTTAAC
GAL1p-F(349)	GCCGCACTGCTCCGA
ASC1-tHMG1-R	GCGGCGCGCCTTAGGATTTAATGCAGGTGACGGACC
NDT80-OUT-F	TGTTAATTCCTTTGTGCCAGATAGTATTG
NDT80-OUT-R	CACCCAATCTTCATTATTCACCAAATATTCCC
LEU2-OUT-F	GCTTGCATCACAATACTTGAAGTTGAC
ASC1-CWP2t-F	GGCGCGCCGAAATCTCTGATTTTTTATAATATCTATATGGCTTTTTTC
ASC1-PRM9t-F	GGCGCGCCACAGAAGACGGGAGACACTAGC
LEU2-OUT-R	GTGCCCTCCTCCTTGTCAATATTAAT
HIS3-OUT-F	GCCTCGGTAATGATTTTCATTTTTTTTTTTCC
HIS3-OUT-R	GCGCCTCGTTCAGAATGACAC
HO-OUT-F	CTGGGCGTTATTAGGTGTGAAACC
HO-OUT-R	CGCGTAAATATTTAGCTTGATAACTGTTACTG
ROX1-YZ-F	CAGGTGCAGGCGTACTTTAAAG
ROX1-YZ-R	GCCACTTGAGCTTTCGGTTTG
HXK2-OUT-F	AACAAAGAAAAGGCAAGAGTAAATAGCG
HXK2-OUT-R	CGGTGTTCATCCAGGTGGTAATATC
GAL80-OUT-F	GTTGGCCTCTACTTACTCCATCGAC
pGAL4-SexAI	GCGACCTGGTCTTTCAGGAGGCTTGCTTCTCT
GAL4t-F	AATGAATCGTAGATACTGAAAAACCCC
GAL80-OUT-R	GGCATTATATGGCCGATTTGTATTAGTATCC
PDR3-YZ-F(207)	GGAGAGGAATCGTACACTCAAGAC
TEF1-PAC1-R	GCGTTAATTAATTTGTAATTAAAACTTAGATTAGATTGCTATGCTTTCTTTC
TEF1p-YZ-F(350bp)	GCCGTACCACTTCAAAACACC
INO2-R	TCAGGAATCATCCAGTATGTGCTGT

Template	Primer name	Module
P_GAL10-CnVS-T_CWP2	NDT80-50-CWP2t-F	P_GAL10-CnVS-T_CWP2
GAL1p-R
P_GAL1-tHMG1-T_PRM9	GAL1p-F NDT80-50-PRM9t-R	P_GAL1-tHMG1-T_CWP2

Template	Primer name	Module
P_GAL10-CnVS-T_CWP2	NDT80-50-CWP2t-F	P_GAL10-CnVS-T_CWP2
GAL1p-R
P_GAL1-tHMG1-T_PRM9	GAL1p-F NDT80-50-PRM9t-R	P_GAL1-tHMG1-T_CWP2

Strain	Template	Primer name	Module
VAL-02	P_GAL10-SmFPS-CnVS-T_CWP2	NDT80-50-CWP2t-F GAL1p-R	P_GAL10-SmFPS-CnVS-T_CWP2
P_GAL1-tHMG1-T_PRM9	GAL1p-F NDT80-50-PRM9t-R	P_GAL1-tHMG1-T_PRM9
VAL-03	P_GAL10-SmFPS-CsVal-T_CWP2	NDT80-50-CWP2t-F GAL1p-R	P_GAL10-SmFPS-CsVal-T_CWP2
P_GAL1-tHMG1-T_PRM9	GAL1p-F NDT80-50-PRM9t-R	P_GAL1-tHMG1-T_PRM9
VAL-04	P_GAL10-SmFPS-EgVal-T_CWP2	NDT80-50-CWP2t-F GAL1p-R	P_GAL10-SmFPS-EgVal-T_CWP2
P_GAL1-tHMG1-T_PRM9	GAL1p-F NDT80-50-PRM9t-R	P_GAL1-tHMG1-T_PRM9

Strain	Template	Primer name	Module
VAL-02	P_GAL10-SmFPS-CnVS-T_CWP2	NDT80-50-CWP2t-F GAL1p-R	P_GAL10-SmFPS-CnVS-T_CWP2
P_GAL1-tHMG1-T_PRM9	GAL1p-F NDT80-50-PRM9t-R	P_GAL1-tHMG1-T_PRM9
VAL-03	P_GAL10-SmFPS-CsVal-T_CWP2	NDT80-50-CWP2t-F GAL1p-R	P_GAL10-SmFPS-CsVal-T_CWP2
P_GAL1-tHMG1-T_PRM9	GAL1p-F NDT80-50-PRM9t-R	P_GAL1-tHMG1-T_PRM9
VAL-04	P_GAL10-SmFPS-EgVal-T_CWP2	NDT80-50-CWP2t-F GAL1p-R	P_GAL10-SmFPS-EgVal-T_CWP2
P_GAL1-tHMG1-T_PRM9	GAL1p-F NDT80-50-PRM9t-R	P_GAL1-tHMG1-T_PRM9

Strain	Template	Primer name	Module
VAL-05	P_GAL10-SmFPS-CnVS-T_CWP2	LEU2-50-CWP2t-F GAL1p-R	P_GAL10-SmFPS-CnVS-T_CWP2
P_GAL1-tHMG1-T_PRM9	GAL1p-F LEU2-50-PRM9t-R	P_GAL1-tHMG1-T_PRM9
VAL-06	P_GAL10-SmFPS-CnVS-T_CWP2	HIS3-50-CWP2t-F GAL1p-R	P_GAL10-SmFPS-CnVS-T_CWP2
P_GAL1-tHMG1-T_PRM9	GAL1p-F HIS3-50-PRM9t-R	P_GAL1-tHMG1-T_PRM9
VAL-07	P_GAL10-SmFPS-CnVS-T_CWP2	HO-50-CWP2t-F GAL1p-R	P_GAL10-SmFPS-CnVS-T_CWP2
P_GAL1-tHMG1-T_PRM9	GAL1p-F HO-50-PRM9t-R	P_GAL1-tHMG1-T_PRM9

Strain	Template	Primer name	Module
VAL-05	P_GAL10-SmFPS-CnVS-T_CWP2	LEU2-50-CWP2t-F GAL1p-R	P_GAL10-SmFPS-CnVS-T_CWP2
P_GAL1-tHMG1-T_PRM9	GAL1p-F LEU2-50-PRM9t-R	P_GAL1-tHMG1-T_PRM9
VAL-06	P_GAL10-SmFPS-CnVS-T_CWP2	HIS3-50-CWP2t-F GAL1p-R	P_GAL10-SmFPS-CnVS-T_CWP2
P_GAL1-tHMG1-T_PRM9	GAL1p-F HIS3-50-PRM9t-R	P_GAL1-tHMG1-T_PRM9
VAL-07	P_GAL10-SmFPS-CnVS-T_CWP2	HO-50-CWP2t-F GAL1p-R	P_GAL10-SmFPS-CnVS-T_CWP2
P_GAL1-tHMG1-T_PRM9	GAL1p-F HO-50-PRM9t-R	P_GAL1-tHMG1-T_PRM9

Template	Primer name	Module
Yeast genome	ROX1-KO-up-F ROX1-KO-up-R	ROX1-up
Yeast genome	ROX1-KO-down-F ROX1-KO-down-R	ROX1-down
ROX1-upand ROX1-down	ROX1-KO-up-F ROX1-KO-down-R	ROX1-KO
Yeast genome	HXK2-KO-UP-F HXK2-KO-UP-R	HXK2-up
Yeast genome	HXK2-KO-DOWN-F HXK2-KO-DOWN-R	HXK2-down
HXK2-upand HXK2-down	HXK2-KO-UP-F HXK2-KO-DOWN-R	HXK2-KO
Yeast genome	GAL80-GAL4p-F GAL4t-GAL80-R	P_GAL4-GAL4-T_GAL4

Template	Primer name	Module
Yeast genome	ROX1-KO-up-F ROX1-KO-up-R	ROX1-up
Yeast genome	ROX1-KO-down-F ROX1-KO-down-R	ROX1-down
ROX1-upand ROX1-down	ROX1-KO-up-F ROX1-KO-down-R	ROX1-KO
Yeast genome	HXK2-KO-UP-F HXK2-KO-UP-R	HXK2-up
Yeast genome	HXK2-KO-DOWN-F HXK2-KO-DOWN-R	HXK2-down
HXK2-upand HXK2-down	HXK2-KO-UP-F HXK2-KO-DOWN-R	HXK2-KO
Yeast genome	GAL80-GAL4p-F GAL4t-GAL80-R	P_GAL4-GAL4-T_GAL4

Template	Primer name	Module
pRS425-LEU2-TRP1-URA3-TEF1p-ObGES	Delta15-LEU2t-F URA3t-HIS3p-R	LEU2-TRP1-URA3 Maker
NDT80-HIS3	URA3t-HIS3p-F HIS3t-ACT3p-R	HIS3 Maker
P_ACT3-PDR3m-T_PDR3	HIS3t-ACT3p-F PDR3t-TEF1p-R	P_ACT3-PDR3m-T_PDR3
pM3-tHMG1	PDR3t-TEF1p-F CYC1t-GAL1p-R	P_TEF1-tHMG1-T_CYC1
P_GAL1-INO2-T_PRM9	CYC1t-GAL1p-F Delta15-50-PRM9t-R	P_GAL1-INO2-T_PRM9

Template	Primer name	Module
pRS425-LEU2-TRP1-URA3-TEF1p-ObGES	Delta15-LEU2t-F URA3t-HIS3p-R	LEU2-TRP1-URA3 Maker
NDT80-HIS3	URA3t-HIS3p-F HIS3t-ACT3p-R	HIS3 Maker
P_ACT3-PDR3m-T_PDR3	HIS3t-ACT3p-F PDR3t-TEF1p-R	P_ACT3-PDR3m-T_PDR3
pM3-tHMG1	PDR3t-TEF1p-F CYC1t-GAL1p-R	P_TEF1-tHMG1-T_CYC1
P_GAL1-INO2-T_PRM9	CYC1t-GAL1p-F Delta15-50-PRM9t-R	P_GAL1-INO2-T_PRM9

创建酿酒酵母细胞工厂发酵生产芳香精油瓦伦烯

PDF下载

杨婷婷 ¹^,²^,³ , 王冬 ²^,³ , 李文豪 ¹^,²^,³ , 石玉松 ²^,³ , 李荣生 ²^,³ , 马文建 ¹ , 戴住波 ²^,³^,^* , 张学礼 ²^,³

药学学报 | 研究论文 2023,58(6): 1619-1628

收起

药学学报 |研究论文 2023 , 58 (6) : 1619 -1628

创建酿酒酵母细胞工厂发酵生产芳香精油瓦伦烯

全屏

杨婷婷¹^,²^,³, 王冬²^,³, 李文豪¹^,²^,³, 石玉松²^,³, 李荣生²^,³, 马文建¹, 戴住波²^,³^,^*, 张学礼²^,³

作者信息

1.天津科技大学生物工程学院, 天津 300457

2.中国科学院天津工业生物技术研究所, 天津 300308

3.中国科学院系统微生物工程重点实验室, 天津 300308

通讯作者:

*戴住波, E-mail: dai_zb@tib.cas.cn

Construction of cell factories for production of valencene in Saccharomyces cerevisiae

Ting-ting YANG¹^,²^,³, Dong WANG²^,³, Wen-hao LI¹^,²^,³, Yu-song SHI²^,³, Rong-sheng LI²^,³, Wen-jian MA¹, Zhu-bo DAI²^,³^,^*, Xue-li ZHANG²^,³

Affiliations

1. College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China

2. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China

3. Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China

出版时间: 2023-06-12 doi: 10.16438/j.0513-4870.2022-1382

文章导航

摘要

收起

瓦伦烯是一种具有柑橘气味的倍半萜类化合物, 常被用于化妆品和食品添加剂, 以及工业合成诺卡酮。本研究拟用合成生物学技术创建酿酒酵母细胞工厂发酵生产瓦伦烯。首先, 在底盘细胞YTT-T5中引入北美金柏来源的瓦伦烯合酶(CnVS), 成功获得可生产1.1 mg·L^-1瓦伦烯的工程菌VAL-01; 采用蛋白质融合技术, 不同植物来源的瓦伦烯合酶筛选, 并调整了关键基因拷贝数后, 瓦伦烯产量提升至436.4 mg·L^-1。进一步工作中, 删除转录因子ROX1, 瓦伦烯的产量提高了17.4%。最后, 对半乳糖诱导系统进行调控, 并过表达多药耐药性调控因子PDR3和调控内质网大小转录因子INO2, 所得工程菌株VAL-10经高密度发酵能生产2 798.6 mg·L^-1瓦伦烯(产量是初始菌株的2 500倍), 为瓦伦烯绿色生产提供了基础。

关键词

合成生物学 / 酿酒酵母 / 倍半萜 / 瓦伦烯

Abstract

收起

Valencene, a kind of sesquiterpenoid with a citrus flavor, is mainly found in Valencia orange and is commonly used in cosmetics and food additives, as well as industrial synthetic nootkatone. In this study, synthetic biology was used to create a Saccharomyces cerevisiae cell factory to produce valencene. Fistly, valencene synthase gene (CnVS) from Callitropsis nootkatensis was inserted into the chromosome of the chassis strain YTT-T5. The resulting strain VAL-01 could produce 1.1 mg·L^-1 valencene. Protein fusion technique was used, different valencene synthases were compared and the copy number of key genes was adjusted, yielding valencene to 436.4 mg·L^-1. Then, knocking-out the transcription factor ROX1 resulted in valencene improvement by 17.4%. Moreover, the induction system of galactose was regulated, transcription factor PDR3 and INO2 were overexpressed. The engineered strain VAL-10 could produce 2 798.6 mg·L^-1 valencene by high cell density fermentation method (nearly 2 500 times higher than VAL-01). This study provides a basis for green production of valencene.

Key words

synthetic biology / Saccharomyces cerevisiae / sesquiterpenoid / valencene

引用本文

杨婷婷, 王冬, 李文豪, 石玉松, 李荣生, 马文建, 戴住波, 张学礼. 创建酿酒酵母细胞工厂发酵生产芳香精油瓦伦烯. 药学学报, 2023 , 58 (6) : 1619 -1628 . DOI: 10.16438/j.0513-4870.2022-1382

Ting-ting YANG, Dong WANG, Wen-hao LI, Yu-song SHI, Rong-sheng LI, Wen-jian MA, Zhu-bo DAI, Xue-li ZHANG. Construction of cell factories for production of valencene in Saccharomyces cerevisiae[J]. Acta Pharmaceutica Sinica, 2023 , 58 (6) : 1619 -1628 . DOI: 10.16438/j.0513-4870.2022-1382

正文

收起

瓦伦烯(valencene) 是一种具有柑橘气味的倍半萜类化合物, 主要存在于柑橘属植物中, 常被用作化妆品和食品的添加剂^{[1, 2]}, 以及工业上合成诺卡酮(nootkatone)。诺卡酮具有约1 μg·L^-1的低气味阈值, 被广泛用于化妆品、食品、芳香治疗、驱蚊杀虫等领域^[3]。植物提取是瓦伦烯的主要来源, 但是植物来源的供给不稳定。同时, 经济高速增长带来物质需求增长, 仅从原植物中提取天然产物的方法无法满足市场需求^[4]。随着合成生物学的发展, 构建微生物细胞工厂实现天然产物的生物合成一直是合成生物学的研究热点^[5]。目前许多科学家已经在微生物中实现了多种高价值药效成分的生产, 例如青蒿素^[6]、大麻素^[7]、人参皂苷^[8-10]、山莨菪碱和东莨菪碱等^[4]。

酿酒酵母作为一种真核模式生物, 因其被普遍认为是安全的(GRAS) 微生物, 是合成生物学技术产业中的一个重要平台微生物, 已经被广泛应用于玫瑰精油、榄香烯、番茄红素和β-胡萝卜素等天然萜类产物的发酵生产^[4-11]。酿酒酵母生产萜类化合物主要是通过甲羟戊酸途径(MVA pathway), 即乙酰辅酶A经过7步酶促反应合成萜类化合物基本单元异戊烯基焦磷酸(IPP) 和二甲基烯丙基焦磷酸(DMAPP)。在倍半萜化合物生物合成途径中, IPP和DMAPP可在法尼基焦磷酸合成酶(FPS, ERG20基因编码) 的作用下生成倍半萜化合物前体法尼基焦磷酸(FPP), 它可在不同的倍半萜合成酶催化下生成瓦伦烯、榄香烯等倍半萜类化合物(图 1)。

前期实验室已构建出过表达MVA途径相关基因的底盘菌YTT-T5, 该菌株能够显著增加酿酒酵母合成萜类化合物的通量。本研究采用蛋白质融合、高效基因元件筛选、调整限速酶基因的拷贝数、半乳糖诱导系统改造和发酵工艺优化等策略来提高酵母细胞工厂合成瓦伦烯的能力, 最终产量达到2.8 g·L^-1 (提高近2 500倍), 为瓦伦烯等倍半萜化合的生物合成奠定了基础。

材料与方法

收起

工具酶与试剂 PrimeSTAR GXL DNA聚合酶购自宝生物生物工程公司(大连); 质粒小量快速提取试剂盒购自美国Axygen公司; DNA回收试剂盒购自美国Biomiga公司; 酵母选择培养基购自北京泛基诺科技有限公司; 酵母DNA提取试剂盒购自北京康为世纪生物科技有限公司; 瓦伦烯标准品和5-氟乳清酸(5-FOA) 均购自美国Sigma-Aldrich公司。

质粒、菌株及引物 本研究所用的质粒和菌株信息见表 1。引物信息见表 2。

培养基 LB培养基: 1%蛋白胨、0.5%酵母粉、1%氯化钠、100 mg·L^-1氨苄青霉素; YPD培养基: 1%酵母粉、2%蛋白胨、2%葡萄糖; SD-Trp筛选培养基: 0.8%氨基酸选择培养组分(四缺, Ura-Trp-His-Leu)、2%葡萄糖、0.002%尿嘧啶、0.005%组氨酸和0.01%亮氨酸; SD-Trp-Ura筛选培养基: 0.8%氨基酸选择培养组分(四缺, Ura-Trp-His-Leu)、2%葡萄糖、0.005%组氨酸、0.01%亮氨酸; SD-Ura-Trp-His-Leu筛选培养基: 0.8%氨基酸选择培养组分(四缺, Ura-Trp-His-Leu)、2%葡萄糖; SD-Trp-5FOA筛选培养基: 0.8%氨基酸选择培养组分(四缺, Ura-Trp-His-Leu)、2%葡萄糖、0.002%尿嘧啶、0.005%组氨酸、0.01%亮氨酸、0.1%5-FOA。上述液体培养基加入2% Agar可配成相应的固体培养基。

高密度发酵培养基和补料培养基参照人参皂苷高密度发酵培养基配方配置^[12]。

瓦伦烯工程菌的构建 相关工程菌株构建均采用基于CRISPR/Cas9技术介导的同源重组, 原理如图 2所示。

VAL-01菌株的构建模块和引物的搭配方案见表 3, 用PrimeSTAR GXL高保真聚合酶扩增和切胶回收得到用于同源重组的各个片段, 用SD-Trp液体培养基活化YTT-T5后将各个片段同NDT80-gRNA共同转入醋酸锂方法制备的感受态中, 并用SD-Trp-Ura固体培养基筛选转化子。转化子分别用GAL10p-F(251bp)/AscI-CnVS-R、GAL1p-F(349)/ASC1-tHMG1-R、NDT80- OUT-F/NDT80-OUT-R等引物进行PCR验证后, 阳性克隆菌株命名为VAL-01。

工程菌VAL-02、VAL-03和VAL-04的构建模块和引物的搭配方案见表 4, 使用PrimeSTAR GXL高保真聚合酶扩增和切胶回收得到用于同源重组的各个片段, 用SD-Trp固体培养基活化YTT-T5后将各个片段转入醋酸锂方法制备的感受态中, 并用SD-Trp-Ura固体培养基筛选转化子。转化子分别用特异引物GAL10p-F(251bp)/AscI-CnVS-R、GAL10p-F(251bp)/CsVal-YZ-R、GAL10p-F(251bp)/EgVal-YZ-R(486), 以及共用引物GAL1p-F(349)/ASC1-tHMG1-R、NDT80-OUT-F/NDT80-OUT-R进行PCR验证后, 阳性克隆菌株分别命名为VAL-02、VAL-03和VAL-04。

工程菌VAL-05、VAL-06和VAL-07的构建模块和引物的搭配方案见表 5, 用PrimeSTAR GXL高保真聚合酶扩增和切胶回收得到用于同源重组的各个片段, 用SD-Trp液体培养基活化去除gRNA质粒的VAL-02后将各个片段同LEU2-gRNA共同转入醋酸锂方法制备的感受态中, 并用SD-Trp-Ura固体培养基筛选转化子。转化子分别用通过LEU-OUT-F/ASC1-CWP2t-F、ASC1-PRM9t-F/LEU-OUT-R、LEU-OUT-F/LEU-OUT-R引物进行PCR验证后阳性克隆命名为VAL-05。

用SD-Trp液体培养基活化去除gRNA质粒的VAL-05后将各个片段同HIS3-gRNA共同转入醋酸锂方法制备的感受态中, 并用SD-Trp-Ura固体培养基筛选转化子。用HIS3-OUT-F/ASC1-CWP2t-F、ASC1-PRM9t-F/HIS3-OUT-R、HIS3-OUT-F/HIS3-OUT-R引物进行PCR验证后阳性克隆命名为VAL-06。

用SD-Trp液体培养基活化去除gRNA质粒的VAL-06后将各个片段同HO-gRNA共同转入醋酸锂方法制备的感受态中, 并用SD-Trp-Ura固体培养基筛选转化子。用HO-OUT-F/ASC1-CWP2t-F、ASC1-PRM9t-F/HO-OUT-R、HO-OUT-F/HO-OUT-R引物进行PCR验证后阳性克隆命名为VAL-07。

工程菌VAL-08和VAL-09的构建模块和引物的搭配方案见表 6, 用PrimeSTAR GXL高保真聚合酶扩增和切胶回收得到用于同源重组的各个片段, 用SD-Trp液体培养基活化去除gRNA质粒的VAL-05后将ROX1-KO片段及ROX1-gRNA共同转入醋酸锂方法制备的感受态中, 并用SD-Trp-Ura固体培养基筛选转化子。转化子分别用ROX-YZ-F/ROX-YZ-R引物进行PCR验证后, 阳性克隆菌株命名VAL-08。

用SD-Trp液体培养基活化去除gRNA质粒的VAL-08后将HXK2-KO片段同HXK2-gRNA共同转入醋酸锂方法制备的感受态中, 使用HXK2-OUT-F/HXK2-OUT-R引物对转化子进行PCR验证后挑选其中一个阳性克隆, 去除gRNA后制备感受态, 加入GAL80-gRNA片段及P_GAL4-GAL4-T_GAL4, 并用SD-Trp-Ura固体培养基筛选转化子。转化子分别用GAL80-OUT-F/pGAL4-SexAI、GAL4t-F/GAL80-OUT-R、GAL80-OUT-F/GAL80-OUT-R引物进行PCR验证后, 阳性克隆菌株命名VAL-09。

工程菌VAL-10的构建使用同源重组法构建菌株VAL-10。用PrimeSTAR GXL高保真聚合酶扩增和切胶回收得到用于同源重组的各个片段, 将VAL-09去掉gRNA和Cas9质粒后用YPD液体培养基活化后用醋酸锂方法制备的感受态, 加入表 7所得的各个模块片段, 电击转化, 并用SD-Trp-Ura-Leu-Ura固体培养基筛选转化子。转化子再分别用PDR3-YZ-F(207)/TEF1-PAC1-R、TEF1p-YZ-F(350bp)/ASC-tHMG1-R、GAL1p-F(349)/INO2-R等引物进行PCR验证后, 得到工程菌VAL-10。

菌株发酵及产物提取 摇瓶发酵: 在平板上挑取单克隆于装有3 mL相应的液体培养基的试管中, 30 ℃、250 r·min^-1震荡培养过夜, 制备种子液。将种子液按照1% (v/v) 的接种量转移至含有15 mL相应液体选择培养基的100 mL三角瓶中, 加入1.5 mL正十二烷, 30 ℃、250 r·min^-1震荡培养6天。最后将三角瓶中的发酵液转移至50 mL离心管, 5 000 r·min ^-1离心5 min, 收集有机相, 用正己烷将其稀释10倍后, 过有机尼龙膜(0.22 μm) 后备用。高密度发酵: 经火焰接种环, 将200 mL种子液加入含2 L发酵培养基的5 L生物反应器中。发酵过程中参数设定值分别为: 温度30 ℃, pH 5.0, 溶氧30%, 空气流量3~9 L·min^-1, 搅拌转速300~1 000 r·min^-1, 溶氧与搅拌转速、通气相偶联。发酵24 h后向生物反应器中加入200 mL肉豆蔻酸异丙酯。

瓦伦烯的检测 分别使用GC-MS和GC进行样品的定性和定量分析^[12]。

GC-MS条件进样口温度250 ℃, 进样口体积1 μL, 不分流, 溶剂延时3 min; 色谱柱: HP-5ms (30 m × 0.25 mm × 0.25 μm); 色谱条件: 45 ℃, 1 min, 10 ℃·min^-1至300 ℃, 保温5 min; MS条件: Full Scan: 50~750 amu。

GC条件进样口温度250 ℃, 进样体积1 μL, 不分流, 色谱柱: HP-5 (30 m × 0.32 mm × 0.25 μm); 色谱条件: 45 ℃, 1 min, 10 ℃·min^-1至300 ℃, 保温5 min; FID检测器。

结果与分析

收起

1 瓦伦烯酿酒细胞工厂创建及产物鉴定

酿酒酵母能天然合成倍半萜类通用前体FPP, 自然界中FPP能被瓦伦烯合酶催化生成瓦伦烯。本实验室前期已构建了一株强化了MVA途径并弱化了ERG20基因表达的萜类底盘菌株YTT-T5。研究将北美金柏(Callitropsis nootkatensis) 来源的CnVS基因(经密码子优化) 置于半乳糖诱导型启动子P_GAL10下, 并插入到底盘菌YTT-T5的 NDT80基因位点, 经过缺陷培养基的筛选、PCR和测序验证正确后得到菌株VAL-01。VAL-01经过6天的摇瓶发酵后, 利用GC-MS对发酵产物进行定性和定量分析, 发现工程菌株VAL-01能在12.855 min出现与瓦伦烯标品相同质谱图的新峰, 其瓦伦烯产量为1.1 ± 0.6 mg·L^-1 (图 3)。

2 融合SmFPS和CnVS蛋白提高瓦伦烯产量

使用蛋白质融合技术拉近途径中相邻酶在空间上的距离, 能提高中间代谢产物的利用速率, 从而进一步提高整个途径的催化能力, 是代谢工程改造过程中的重要手段^{[13, 14]}。FPP是倍半萜类产物的中间体, 同时将被鲨烯合酶连接成为三萜通用前体鲨烯^[8]。将FPS和倍半萜合酶进行融合会提高倍半萜化合物的产量, 前期课题组张丽丽等^[13]使用连接肽GGGS将FPS与橙花叔醇合酶融合后使得工程菌株橙花叔醇产量提高了59.8倍。本研究中将来源丹参的法尼基焦磷酸合酶(SmFPS) 与CnVS进行融合后置于GAL1启动子下, 并整合到底盘菌YTT-T5中, 经过缺陷培养基的筛选、PCR和测序验证正确后得到菌株VAL-02。与VAL-01相比, VAL-02瓦伦烯的产量提高了343.2倍, 达到344.2 ± 17.6 mg·L^-1 (图 4)。

3 高效的瓦伦烯合酶的筛选

除北美金柏来源的CnVS外, 柑橘(Citrus sinensis)来源的Cstps1^[15], 葡萄(Vitis Vinifera) 来源的VvVal^[16]及冰川刺芹(Eryngium glaciale) 来源的EgVS均具有瓦伦烯合酶活性^[17]。本研究中将SmFPS分别与柑橘CsVal、冰川刺芹EgVS进行融合后, 转入YTT-T5菌株中评价融合酶的活性, 分别构建出的工程菌株VAL-03和VAL-04。摇瓶发酵6天后, 其中菌株VAL-02 (融合北美金柏来源的CnVS) 瓦伦烯的产量达344.2 ± 17.6 mg·L^-1, 是菌株VAL-03 (25.1 ± 6.2 mg·L^-1) 的13.7倍、VAL-04 (79.3 ± 22.3 mg·L^-1) 的4.3倍(图 4A), 表明SmFPS-CnVS融合酶的活性较其余二者更强。

4 调整基因拷贝数增强瓦伦烯生产

通过调节基因拷贝数来增加基因表达是提高目标代谢途径通量的有效方法^[18]。为了进一步提高瓦伦烯产量, 本研究调整了关键基因tHMG1以及SmFPS-CnVS的拷贝数, 分别获得了瓦伦烯合酶拷贝数为2、3、4的工程菌株VAL-05、VAL-06和VAL-07。其中过表达2拷贝的菌株VAL-05产量最高, 瓦伦烯产量可达436.4 ± 35.6 mg·L^-1 (图 4A)。在增加拷贝数时, 生物量显著降低, VAL-07较VAL-02生物量降低了33.3%, 推测可能过多增加基因表达将对细胞带来负荷^[19], 同时瓦伦烯对酿酒酵母细胞具有一定的毒性^[20]。

5 敲除调控因子ROX1提高细胞产量

在内源的麦角甾醇途径中, 编码血红素依赖的转录抑制因子Rox1p能与多个麦角甾醇途径基因(ERG) 启动子结合发挥直接抑制作用^{[21, 22]}。对ROX1进行敲除, 可以提高ERG基因的表达, 并显著提高类胡萝卜素、倍半萜类等萜类化合物产量^[23]。在本研究中, 进一步敲除了VAL-05菌株的ROX1基因, 获得工程菌株VAL-08, 其产量较VAL-05提高17.4%。

6 调控半乳糖诱导系统降低生产成本

虽然菌株VAL-08不能代谢半乳糖(其GAL1、GAL7和GAL10等半乳糖代谢基因被敲除), 但在发酵时还需要额外添加半乳糖作为诱导剂来生产瓦伦烯。半乳糖价格昂贵, 取消半乳糖的使用会极大地降低生产成本。酿酒酵母在高葡萄糖浓度时, 己糖激酶Hxk2p与Snf1p复合体相互作用, 阻断Mig1p磷酸化, 而Mig1p的磷酸化对于解除葡萄糖抑制基因(GAL基因) 是必不可少的^[24-27]。已有文献^[24]表明下调Hxk2p会大幅提高橙花叔醇的生产。Gal4p是半乳糖诱导系统的开关, 在添加半乳糖并缺乏抑制碳源(如葡萄糖) 的情况下, Gal4p可以大幅度诱导半乳糖代谢基因的表达(如GAL1/2/7/10表达量可以提高1 000倍以上)^[28]。Gal80p是半乳糖调控系统的负调控子, 通过与半乳糖诱导系统的开关Gal4p的C端转录激活域结合进而抑制GAL基因的表达^[29]。

因此进一步敲除了VAL-08菌株的HXK2和GAL80基因, 同时强化了GAL4的表达获得的工程菌VAL-09, 在仅以葡萄糖为碳源发酵VAL-09时, 其瓦伦烯的产量能成功达到259.3 ± 1.9 mg·L^-1。

7 工程菌修复及发酵工艺优化

为了进一步提高VAL-09工程菌的发酵性能, 增加瓦伦烯的产量及工程菌的耐受性, 在修复菌株VAL-09营养缺失marker基因LEU2、HIS3、URA3和TPR1的同时, 过表达PDR3^Q763L (PDR3^Q763L突变可能对提高萜类化合物的耐受性有益^[30])、tHMG1和INO2 (调控内质网大小的转录因子, 过表达INO2可以提高萜类化合物产量^[31]) 等功能模块, 得到菌株VAL-10。与VAL-09菌株相比, 其瓦伦烯产量进一步提高了13.8%, 达到295.3 ± 21.9 mg·L^-1。对VAL-10菌株进行发酵工艺优化, 在5 L生物反应器中发酵95 h, 瓦伦烯的产量达到2 798.6 ± 66.3 mg·L^-1, A_600nm达到329.9 (图 4B)。

讨论

收起

利用合成生物学技术生产具有高附加值天然化合物是合成生物学领域的热点及难点问题^[4]。瓦伦烯具有柑橘气味的特殊香气, 同时为诺卡酮的底物, 具有商业化前景。前期已有国际团队在多个方面进行异源生物合成的探索, 在宿主微生物选择方面, 酿酒酵母^[32]、谷氨酸棒状杆菌(Corynebacterium glutamicum)^[33]和集胞藻(Synechocystis sp. PCC 6803)^[34]等被用于瓦伦烯的生产; 在亚细胞区室化利用方面, 由于细胞中的线粒体、高尔基体、内质网等细胞器能为生物反应提供特定的催化环境, 利用导肽将法尼基焦磷酸合酶和瓦伦烯合酶定位到酵母细胞器线粒体中, 可以增加工程菌瓦伦烯的合成^[35]; 在碳源利用方面, 通过途径改造使工程菌能利用甘露醇来生产瓦伦烯, 甘露醇为一种更为环保、价格低廉的非发酵碳源^[36]。本研究通过蛋白质融合技术, 基因元件筛选, 关键基因拷贝数优化, 提高了瓦伦烯的产量; 进一步工作中, 为解决原料成本问题, 调控了半乳糖诱导系统相关基因表达, 最后结合调控系列转录因子的表达, 获得了高密度发酵瓦伦烯产量达到2 798.6 ± 66.3 mg·L^-1的酿酒酵母细胞工厂。研究最终获得的瓦伦烯工程菌摇瓶发酵产量为295.3 ± 21.9 mg·L^-1, 单位菌密度产量能达到26.9 mg·L^-1·A_600nm^-1, 但是高密度发酵时只有8.5 mg·L^-1·A_600nm^-1, 表明高密度发酵条件有待优化。此外, 前期研究发现在同一倍半萜酵母底盘菌株中进行酶催化能力评估时, 倍半萜化合物橙花叔醇和瓦伦烯的产量分别为900和5 mg·L^{-1 [37]}, 推断瓦伦烯合成酶的酶活可能是瓶颈, 因此可以进一步进行新酶筛选, 或利用AlphaFold2等计算机辅助设计^[38], 或通过酶定向进化^[39]等策略提高酶的活性。

作者贡献: 戴住波、张学礼和马文建设计实验并提供资金; 杨婷婷、王冬、李文豪、石玉松和李荣生参与实验、分析数据和撰写论文。

利益冲突: 所有作者均声明不存在利益冲突。

基金

收起

国家重点研发计划项目(2020YFA0908000)

参考文献

收起

文献

收起

参考文献引证文献

排序方式：

[1]

Hunter GLK, Brogden WB. Analysis of the terpene and sesquiterpene hydrocarbons in some citrus oils[J]. J Food Sci, 1965, 30: 383-387.

https://doi.org/10.1111/j.1365-2621.1965.tb01772.x

[2]

Ignea C, Trikka FA, Kourtzelis I, et al. Positive genetic interactors of HMG2 identify a new set of genetic perturbations for improving sesquiterpene production in Saccharomyces cerevisiae[J]. Microb Cell Fact, 2012, 11: 162.

https://doi.org/10.1186/1475-2859-11-162

[3]

Fraatz MA, Berger RG, Zorn H. Nootkatone-a biotechnological challenge[J]. Appl Microbiol Biotechnol, 2009, 83: 35-41.

https://doi.org/10.1007/s00253-009-1968-x

[4]

Dai Z, Wang Y, Zhou Z, et al. Synthetic biology for production of plant-derived natural products[J]. Bull Chin Acad Sci (中国科学院院刊), 2018, 33: 1228-1238.

[5]

Huang L, Gao W, Zhou Y. Application of synthetic biology to sustainable utilization of Chinese materia medica resources[J]. Acta Pharm Sin (药学学报), 2014, 49: 37-43. http://www.yxxb.com.cn/aps/cn/article/id/6b080d60-f6bb-4b84-abc2-75d989c2f531

[6]

Westfall PJ, Pitera DJ, Lenihan JR, et al. Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin[J]. Proc Natl Acad Sci USA, 2012, 109: E111-E118.

[7]

Luo XZ, Reiter MA, d'Espaux L, et al. Complete biosynthesis of cannabinoids and their unnatural analogues in yeast[J]. Nature, 2019, 567: 123-128.

https://doi.org/10.1038/s41586-019-0978-9

[8]

Wang D, Liu Y, Xu J, et al. Construction of efficient yeast cell factories for production of ginsenosides precursor dammarenediol-Ⅱ[J]. Acta Pharm Sin (药学学报), 2018, 53: 1233-1241. http://www.yxxb.com.cn/aps/cn/article/doi/10.16438/j.0513-4870.2018-0503

[9]

Zhang T, Liang H, Gong T, et al. Studies on the enzymatic synthesis of a new PPT-type ginsenoside via UDP-glycosyltransferase PgUGT74AE2 from Panax ginseng[J]. Acta Pharm Sin (药学学报), 2018, 53: 1565-1570. http://www.yxxb.com.cn/aps/cn/article/doi/10.16438/j.0513-4870.2018-0300

[10]

Luo H, Song J, Li X, et al. Cloning and expression analysis of a key device of HMGR gene involved in ginsenoside biosynthesis of Panax ginseng via synthetic biology approach[J]. Acta Pharm Sin (药学学报), 2013, 48: 219-227. http://www.yxxb.com.cn/aps/cn/article/id/0eb1cdd5-a23c-4792-b692-3d0cd2be265d

[11]

Liu X, Luo L, Ma Y, et al. Biopathway construction of plant natural products[J]. Acta Pharm Sin (药学学报), 2021, 56: 3285-3299. http://www.yxxb.com.cn/aps/cn/article/doi/10.16438/j.0513-4870.2021-1637

[12]

Dai Z, Liu Y, Zhang X, et al. Metabolic engineering of Saccharomyces cerevisiae for production of ginsenosides[J]. Metab Eng, 2013, 20: 146-156.

https://doi.org/10.1016/j.ymben.2013.10.004

[13]

Zhang L, Ma X, Wang D, et al. Construction of cell factories for high production of nerolidol in Saccharomyces cerevisiae[J]. China J Chin Mater Med (中国中药杂志), 2017, 42: 2962-2968.

[14]

Dai Z, Liu Y, Huang L, et al. Production of miltiradiene by metabolically engineered Saccharomyces cerevisiae[J]. Biotechnol Bioeng, 2012, 109: 2845-2853.

https://doi.org/10.1002/bit.24547

[15]

Sharon-Asa L, Shalit M, Frydman A, et al. Citrus fruit flavor and aroma biosynthesis: isolation, functional characterization, and developmental regulation of Cstps1, a key gene in the production of the sesquiterpene aroma compound valencene[J]. Plant J, 2003, 36: 664-674.

https://doi.org/10.1046/j.1365-313X.2003.01910.x

[16]

Lucker J, Bowen P, Bohlmann J. Vitis vinifera terpenoid cyclases: functional identification of two sesquiterpene synthase cDNAs encoding (+)-valencene synthase and (-)-germacrene D synthase and expression of mono- and sesquiterpene synthases in grapevine flowers and berries[J]. Phytochemistry, 2004, 65: 2649-2659.

https://doi.org/10.1016/j.phytochem.2004.08.017

[17]

Ye Z, Huang Y, Shi B, et al. Coupling cell growth and biochemical pathway induction in Saccharomyces cerevisiae for production of (+)-valencene and its chemical conversion to (+)-nootkatone[J]. Metab Eng, 2022, 72: 107-115.

https://doi.org/10.1016/j.ymben.2022.03.005

[18]

Meadows AL, Hawkins KM, Tsegaye Y, et al. Rewriting yeast central carbon metabolism for industrial isoprenoid production[J]. Nature, 2016, 537: 694-697.

https://doi.org/10.1038/nature19769

[19]

Ajikumar PK, Xiao WH, Tyo KE, et al. Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli[J]. Science, 2010, 330: 70-74.

https://doi.org/10.1126/science.1191652

[20]

Serra E, Hidalgo-Bastida LA, Verran J, et al. Antifungal activity of commercial essential oils and biocides against Candida albicans[J]. Pathogens, 2018, 7: 15.

https://doi.org/10.3390/pathogens7010015

[21]

Lowry CV, Zitomer RS. ROX1 encodes a heme-induced repression factor regulating ANB1 and CYC7 of Saccharomyces cerevisiae[J]. Mol Cell Biol, 1988, 8: 4651-4658.

[22]

Balasubramanian B, Lowry CV, Zitomer RS. The Rox1 repressor of the Saccharomyces cerevisiae hypoxic genes is a specific DNA-binding protein with a high-mobility-group motif[J]. Mol Cell Biol, 1993, 13: 6071-6078.

[23]

Ozaydin B, Burd H, Lee TS, et al. Carotenoid-based phenotypic screen of the yeast deletion collection reveals new genes with roles in isoprenoid production[J]. Metab Eng, 2013, 15: 174-183.

https://doi.org/10.1016/j.ymben.2012.07.010

[24]

Lu ZY, Peng BY, Ebert BE, et al. Auxin-mediated protein depletion for metabolic engineering in terpene-producing yeast[J]. Nat Commun, 2021, 12: 1051.

https://doi.org/10.1038/s41467-021-21313-1

[25]

Kummel A, Ewald JC, Fendt SM, et al. Differential glucose repression in common yeast strains in response to HXK2 deletion[J]. Fems Yeast Res, 2010, 10: 322-332.

https://doi.org/10.1111/j.1567-1364.2010.00609.x

[26]

Ahuatzi D, Riera A, Pelaez R, et al. Hxk2 regulates the phosphory-lation state of Mig1 and therefore its nucleocytoplasmic distribution[J]. J Biol Chem, 2007, 282: 4485-4493.

https://doi.org/10.1074/jbc.M606854200

[27]

Sanz P, Alms GR, Haystead TAJ, et al. Regulatory interactions between the Reg1-Glc7 protein phosphatase and the Snf1 protein kinase[J]. Mol Cell Biol, 2000, 20: 1321-1328.

https://doi.org/10.1128/MCB.20.4.1321-1328.2000

[28]

Mirisola MG, Gallo A, De Leo G. Ras-pathway has a dual role in yeast galactose metabolism[J]. FEBS Lett, 2007, 581: 2009-2016.

https://doi.org/10.1016/j.febslet.2007.04.018

[29]

Lohr D, Venkov P, Zlatanova J. Transcriptional regulation in the yeast gal gene family - a complex genetic network[J]. FASEB J, 1995, 9: 777-787.

https://doi.org/10.1096/fasebj.9.9.7601342

[30]

Brennan TCR, Williams TC, Schulz BL, et al. Evolutionary engineering improves tolerance for replacement jet fuels in Saccharomyces cerevisiae[J]. Appl Environ Microbiol, 2015, 81: 3316-3325.

https://doi.org/10.1128/AEM.04144-14

[31]

Kim JE, Jang IS, Son SH, et al. Tailoring the Saccharomyces cerevisiae endoplasmic reticulum for functional assembly of terpene synthesis pathway[J]. Metab Eng, 2019, 56: 50-59.

https://doi.org/10.1016/j.ymben.2019.08.013

[32]

Cao C, Cao X, Yu W, et al. Global metabolic rewiring of yeast enables overproduction of sesquiterpene (+)-valencene[J]. J Agric Food Chem, 2022, 70: 7180-7187.

https://doi.org/10.1021/acs.jafc.2c01394

[33]

Frohwitter J, Heider SA, Peters-Wendisch P, et al. Production of the sesquiterpene (+)-valencene by metabolically engineered Corynebacterium glutamicum[J]. J Biotechnol, 2014, 191: 205-213.

https://doi.org/10.1016/j.jbiotec.2014.05.032

[34]

Dietsch M, Behle A, Westhoff P, et al. Metabolic engineering of Synechocystis sp. PCC 6803 for the photoproduction of the sesquiterpene valencene[J]. Metab Eng Commun, 2021, 13: e00178.

https://doi.org/10.1016/j.mec.2021.e00178

[35]

Farhi M, Marhevka E, Masci T, et al. Harnessing yeast subcellular compartments for the production of plant terpenoids[J]. Metab Eng, 2011, 13: 474-481.

https://doi.org/10.1016/j.ymben.2011.05.001

[36]

Zhu CY, You X, Wu T, et al. Efficient utilization of carbon to produce aromatic valencene in Saccharomyces cerevisiae using mannitol as the substrate[J]. Green Chem, 2022, 24: 4614-4627.

https://doi.org/10.1039/D2GC00867J

[37]

Li RS, Wang K, Wang D, et al. Production of plant volatile terpenoids (rose oil) by yeast cell factories[J]. Green Chem, 2021, 23: 5088-5096.

https://doi.org/10.1039/D1GC00917F

[38]

Jumper J, Evans R, Pritzel A, et al. Highly accurate protein structure prediction with AlphaFold[J]. Nature, 2021, 596: 583-589.

https://doi.org/10.1038/s41586-021-03819-2

[39]

Xie W, Lv X, Ye L, et al. Construction of lycopene-overproducing Saccharomyces cerevisiae by combining directed evolution and metabolic engineering[J]. Metab Eng, 2015, 30: 69-78.

https://doi.org/10.1016/j.ymben.2015.04.009

2023年第58卷第6期

PDF下载

410

189

引用本文

BibTeX

文章信息

doi: 10.16438/j.0513-4870.2022-1382

接收时间：2022-12-21
首发时间：2025-11-21
出版时间：2023-06-12

补充材料

相关文章

文章信息

作者

出版历史

收稿日期：2022-12-21
修回日期：2023-01-18

基金

国家重点研发计划项目(2020YFA0908000)

作者信息

1.天津科技大学生物工程学院, 天津 300457

2.中国科学院天津工业生物技术研究所, 天津 300308

3.中国科学院系统微生物工程重点实验室, 天津 300308

通讯作者:

*戴住波, E-mail: dai_zb@tib.cas.cn

参考文献

分享链接

https://castjournals.cast.org.cn/joweb/yxxb/CN/10.16438/j.0513-4870.2022-1382

分享至

全文二维码

扫描看全文

引用本文

BibTeX

本文的引用情况

2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

关闭全屏

BibTeX
EndNote
RefWorks
TxT

Name	Description	Source
Plasmids
pEASY-Blunt simple	Cloning vector for blunt ligation, Amp, Km	TransGen Biotech
p-P_GAL7	Cloning P_GAL7 and T_SPG5 into pEASY-Blunt simple	Lab collection
p-P_GAL10	Cloning P_GAL10 and T_CWP2 into pEASY-Blunt simple	Lab collection
p-P_GAL1	Cloning P_GAL1 and T_PRM9 into pEASY-Blunt simple	Lab collection
pM3-tHMG1	Cloning P_TEF1-tHMG1-T_CYC1cassette into pEASY-Blunt simple	Lab collection
P_GAL7-cHMG2-T_SPG5	Cloning truncated HMG2 into p-P_GAL7	Lab collection
P_GAL10-HMGR-N-T_CWP2	Cloning HMGR-N into p-P_GAL10	Lab collection
P_GAL1-tHMG1-T_PRM9	Cloning tHMG1 into p-P_GAL1	Lab collection
P_GAL10-CnVS-T_CWP2	Cloning CnVS into p-P_GAL10	This study
P_GAL10-SmFPS-CsVal-T_CWP2	Cloning SmFPS-CsVal into p-P_GAL10	This study
P_GAL10-SmFPS-EgVal-T_CWP2	Cloning SmFPS-EgVal into p-P_GAL10	This study
P_GAL10-SmFPS-CnVS-T_CWP2	Cloning SmFPS-CnVS into p-P_GAL10	This study
pRS425-LEU2-TRP1-URA3-TEF1p-ObGES	Cloning LEU2, TRP1, URA3 makersand P_TEF1-ObGES-T_CYC1 cassette into pRS425	Lab collection
NDT80-HIS3	Cloning NDT80 and HIS3 marker into pEASY-Blunt simple	Lab collection
P_ACT3-PDR3m-T_PDR3	Cloning P_ACT3-PDR3^Q763L-T_PDR3 cassette into pEASY-Blunt simple	Lab collection
P_GAL1-INO2-T_PRM9	Cloning INO2 into p-P_GAL1	Lab collection
NDT80-gRNA	NDT80 disruption gRNA cassette, 2 micron, URA3 marker	Lab collection
LEU2-gRNA	LEU2 disruption gRNA cassette, 2 micron, URA3 marker	Lab collection
HIS3-gRNA	HIS3 disruption gRNA cassette, 2 micron, URA3 marker	Lab collection
HO-gRNA	HO disruption gRNA cassette, 2 micron, URA3 marker	Lab collection
GAL80-gRNA	GAL80 disruption gRNA cassette, 2 micron, URA3 marker	Lab collection
ROX1-gRNA	ROX1 disruption gRNA cassette, 2 micron, URA3 marker	Lab collection
HXK2-gRNA	HXK2 disruption gRNA cassette, 2 micron, URA3 marker	Lab collection
Strains
YTT-T5	A platform yeast for mono- and sesquiterpenoid production	Lab collection
VAL-01	P_GAL10-CnVS-T_CWP2 and P_GAL1-tHMG1-T_PRM9 cassettes were integrated into NDT80 site of YTT-T5	This study
VAL-02	P_GAL10-SmFPS-CnVS-T_CWP2 and P_GAL1-tHMG1-T_PRM9 cassettes were integrated into NDT80 site of YTT-T5	This study
VAL-03	P_GAL10-SmFPS-CsVal-T_CWP2 and P_GAL1-tHMG1-T_PRM9 cassettes were integrated into NDT80 site of YTT-T5	This study
VAL-04	P_GAL10-SmFPS-EgVal-T_CWP2 and P_GAL1-tHMG1-T_PRM9 cassettes were integrated into NDT80 site of YTT-T5	This study
VAL-05	P_GAL10-SmFPS-CnVS-T_CWP2 and P_GAL1-tHMG1-T_PRM9 cassettes were integrated into LEU2 site of VAL-02	This study
VAL-06	P_GAL10-SmFPS-CnVS-T_CWP2 and P_GAL1-tHMG1-T_PRM9 cassettes were integrated into HIS3 site of VAL-05	This study
VAL-07	P_GAL10-SmFPS-CnVS-T_CWP2 and P_GAL1-tHMG1-T_PRM9 cassettes were integrated into HO site of VAL-06	This study
VAL-08	ROX1 was deleted inVAL-05	This study
VAL-09	HXK2 was deleted in VAL-08 and subsequently P_GAL4-GAL4-T_GAL4 cassette was integrated into GAL80 site	This study
VAL-10	LEU2, TRP1, URA3, HIS3 makers, P_ACT3-PDR3m-T_PDR3, P_TEF1-tHMG1-T_CYC1 and P_GAL1-INO2-T_PRM9 were integrated into YPRCdelta15 site of VAL-09	This study

Name

Description

Source

Plasmids

pEASY-Blunt simple

Cloning vector for blunt ligation, Amp, Km

TransGen Biotech

p-P_GAL7

Cloning P_GAL7 and T_SPG5 into pEASY-Blunt simple

Lab collection

p-P_GAL10

Cloning P_GAL10 and T_CWP2 into pEASY-Blunt simple

Lab collection

p-P_GAL1

Cloning P_GAL1 and T_PRM9 into pEASY-Blunt simple

Lab collection

pM3-tHMG1

Cloning P_TEF1-tHMG1-T_CYC1cassette into pEASY-Blunt simple

Lab collection

P_GAL7-cHMG2-T_SPG5

Cloning truncated HMG2 into p-P_GAL7

Lab collection

P_GAL10-HMGR-N-T_CWP2

Cloning HMGR-N into p-P_GAL10

Lab collection

P_GAL1-tHMG1-T_PRM9

Cloning tHMG1 into p-P_GAL1

Lab collection

P_GAL10-CnVS-T_CWP2

Cloning CnVS into p-P_GAL10

This study

P_GAL10-SmFPS-CsVal-T_CWP2

Cloning SmFPS-CsVal into p-P_GAL10

This study

P_GAL10-SmFPS-EgVal-T_CWP2

Cloning SmFPS-EgVal into p-P_GAL10

This study

P_GAL10-SmFPS-CnVS-T_CWP2

Cloning SmFPS-CnVS into p-P_GAL10

This study

pRS425-LEU2-TRP1-URA3-TEF1p-ObGES

Cloning LEU2, TRP1, URA3 makersand P_TEF1-ObGES-T_CYC1 cassette into pRS425

Lab collection

NDT80-HIS3

Cloning NDT80 and HIS3 marker into pEASY-Blunt simple

Lab collection

P_ACT3-PDR3m-T_PDR3

Cloning P_ACT3-PDR3^Q763L-T_PDR3 cassette into pEASY-Blunt simple

Lab collection

P_GAL1-INO2-T_PRM9

Cloning INO2 into p-P_GAL1

Lab collection

NDT80-gRNA

NDT80 disruption gRNA cassette, 2 micron, URA3 marker

Lab collection

LEU2-gRNA

LEU2 disruption gRNA cassette, 2 micron, URA3 marker

Lab collection

HIS3-gRNA

HIS3 disruption gRNA cassette, 2 micron, URA3 marker

Lab collection

HO-gRNA

HO disruption gRNA cassette, 2 micron, URA3 marker

Lab collection

GAL80-gRNA

GAL80 disruption gRNA cassette, 2 micron, URA3 marker

Lab collection

ROX1-gRNA

ROX1 disruption gRNA cassette, 2 micron, URA3 marker

Lab collection

HXK2-gRNA

HXK2 disruption gRNA cassette, 2 micron, URA3 marker

Lab collection

Strains

YTT-T5

A platform yeast for mono- and sesquiterpenoid production

Lab collection

VAL-01

P_GAL10-CnVS-T_CWP2 and P_GAL1-tHMG1-T_PRM9 cassettes were integrated into NDT80 site of YTT-T5

This study

VAL-02

P_GAL10-SmFPS-CnVS-T_CWP2 and P_GAL1-tHMG1-T_PRM9 cassettes were integrated into NDT80 site of YTT-T5

This study

VAL-03

P_GAL10-SmFPS-CsVal-T_CWP2 and P_GAL1-tHMG1-T_PRM9 cassettes were integrated into NDT80 site of YTT-T5

This study

VAL-04

P_GAL10-SmFPS-EgVal-T_CWP2 and P_GAL1-tHMG1-T_PRM9 cassettes were integrated into NDT80 site of YTT-T5

This study

VAL-05

P_GAL10-SmFPS-CnVS-T_CWP2 and P_GAL1-tHMG1-T_PRM9 cassettes were integrated into LEU2 site of VAL-02

This study

VAL-06

P_GAL10-SmFPS-CnVS-T_CWP2 and P_GAL1-tHMG1-T_PRM9 cassettes were integrated into HIS3 site of VAL-05

This study

VAL-07

P_GAL10-SmFPS-CnVS-T_CWP2 and P_GAL1-tHMG1-T_PRM9 cassettes were integrated into HO site of VAL-06

This study

VAL-08

ROX1 was deleted inVAL-05

This study

VAL-09

HXK2 was deleted in VAL-08 and subsequently P_GAL4-GAL4-T_GAL4 cassette was integrated into GAL80 site

This study

VAL-10

LEU2, TRP1, URA3, HIS3 makers, P_ACT3-PDR3m-T_PDR3, P_TEF1-tHMG1-T_CYC1 and P_GAL1-INO2-T_PRM9 were integrated into YPRCdelta15 site of VAL-09

This study

Primer name	Sequence (5′ to 3′)
3G-1-M-GAL7p-CWP2t-R	CCCTTAGCTGCGATTAAAGAGTCAAGGCGTGAAAAACATAGGCGAAAGTGGTGTCACAGCGAATTTCCTCAC
3G-2-M-GAL7p-CWP2t-F	GAACTTGTAAACAATTCGGTCCCTACATGTGAGGAAATTCGCTGTGACACCACTTTCGCCTATGTTTTTCACGC
GAL1p-R	TATAGTTTTTTCTCCTTGACGTTAAAGTATAGAGGTAT
GAL1p-F	TTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGGATGG
NDT80-50-CWP2t-F	TACTAACCTTTCATTAAAGAGAAATAACAATATTATAAAAAGCGCTTAAACACTTTCGCCTATGTTTTTCACGC
NDT80-50-PRM9t-down	GATCCACAGAATTCGCATATTTTTTTAACGATTTAAAATCATTAGTTTATTCAACATCGTATTTTCCGAAGCG
LEU2-50-CWP2t-F	TTTACATTTCAGCAATATATATATATATATTTCAAGGATATACCATTCTACACTTTCGCCTATGTTTTTCACGC
LEU2-50-PRM9t-down	CGGTGGTACTGTTGGAACCACCTAAATCACCAGTTCTGATACCTGCATCCTCAACATCGTATTTTCCGAAGCG
HIS3-50-CWP2t-F	CTTCGAAGAATATACTAAAAAATGAGCAGGCAAGATAAACGAAGGCAAAGCACTTTCGCCTATGTTTTTCACGC
HIS3-50-PRM9t-down	TATACACATGTATATATATCGTATGCTGCAGCTTTAAATAATCGGTGTCATCAACATCGTATTTTCCGAAGCG
HO-50-CWP2t-F	TCTAAATCCATATCCTCATAAGCAGCAATCAATTCTATCTATACTTTAAACACTTTCGCCTATGTTTTTCACGC
HO-50-PRM9t-down	ATTAAATTTTACTTTTATTACATACAACTTTTTAAACTAATATACACATTTCAACATCGTATTTTCCGAAGCG
GAL80-GAL4p-F	CCAGCGTATACAATCTCGATAGTTGGTTTCCCGTTCTTTCCACTCCCGTCGACAGCATTCGCCCAGTATTTTTTTTATTC
GAL4t-GAL80-R	GTTTTTATAACGTTCGCTGCACTGGGGGCCAAGCACAGGGCAAGATGCTTTTAACGTTCTACTTTACACTGGTGGTAGG
delta15-LEU2t-F	GTAGTTTTAAAATTTCAAATCCGAACAACAGAGCATAGGGTTTCGCAAACCGTTGAGCCATTAGTATCAATTTGCTTAC
URA3t-HIS3p-R	CGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAACTCTTCCTTTTTCAATGGGTAATAACTG
URA3t-HIS3p-F	AGAGCTTCAATTTAATTATATCAGTTATTACCCATTGAAAAAGGAAGAGTTCGCGCGTTTCGGTGATGA
HIS3t-ACT3p-R	ATGTTAGAACCACCTGGGCCAACAATCATATTGAATCTTCTGGTATCAGCGTGTCACTACATAAGAACACCTTTGGTG
HIS3t-ACT3p-F	ATGGTACCAACGATGTTCCCTCCACCAAAGGTGTTCTTATGTAGTGACACGCTGATACCAGAAGATTCAATATGATTGTTG
PDR3t-TEF1p-R	GTAAAAAAGGAGTAGAAACATTTTGAAGCTATGGTGTGTGGGGGATCACTCTTTTTGAACAGCGCGCCCA
PDR3t-TEF1p-F	ACATAAGATCAACGATTTCTTCATTAATTTTGGGCGCGCTGTTCAAAAAGAGTGATCCCCCACACACCAT
CYC1t-GAL1p-R	CTTTGCGTCCATCCAAAAAAAAAGTAAGAATTTTTGAAAATTCAATATAAGCGCGTTGGCCGATTCATTA
CYC1t-GAL1p-F	GGCTTTAATTTGCAAGCTGCGGCCCTGCATTAATGAATCGGCCAACGCGCTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGGA
Delta15-50-PRM9t-R	TCTGTATTGTTCTTCTTAGTGCTTGTATATGCTCATCCCGACCTTCCATTTCAACATCGTATTTTCCGAAGCGT
ROX1-KO-up-F	CCGACAAATCCCCTGGATATCATTG
ROX1-KO-up-R	ACTTTTATTAAACAGGAGCCTGTTGATTGTCTAACTGCGTTCTTTTGT
ROX1-KO-down-F	ACGCAGTTAGACAATCAACAGGCTCCTGTTTAATAAAAGTTTAAATCGCC
ROX1-KO-down-R	CGATTGTGTCTATATCTACAATATGAACCTG
HXK2-KO-UP-F	ATGTTACTACGAGTTTTCTGAACCTCCTC
HXK2-KO-UP-R	GTAGAAAAAGGGCACCTTCTTGTTGTTCAAACTTAATTTACAAATTAAGTTTTATTTAATTAGCGTACTTATTATGTGTGGAGAATT
HXK2-KO-DOWN-F	GTTGTAGGAATATAATTCTCCACACATAATAAGTACGCTAATTAAATAAAACTTAATTTGTAAATTAAGTTTGAACAACAAGAAGGT
HXK2-KO-DOWN-R	CGCGTAAAAATCGCGGGTAT
GAL10p-F(251bp)	CGGAGGGCTGTCGCC
AscI-CnVS-R	GCGGCGCGCCTTAAGGGATGATTGGTTCAACGAAAAACT
CsVal-YZ-R	CTATATCGATTCTAACAGAGCAGATTTGC
EgVal-YZ-R(486)	CAAACCTCTCGCGTCGTTAAC
GAL1p-F(349)	GCCGCACTGCTCCGA
ASC1-tHMG1-R	GCGGCGCGCCTTAGGATTTAATGCAGGTGACGGACC
NDT80-OUT-F	TGTTAATTCCTTTGTGCCAGATAGTATTG
NDT80-OUT-R	CACCCAATCTTCATTATTCACCAAATATTCCC
LEU2-OUT-F	GCTTGCATCACAATACTTGAAGTTGAC
ASC1-CWP2t-F	GGCGCGCCGAAATCTCTGATTTTTTATAATATCTATATGGCTTTTTTC
ASC1-PRM9t-F	GGCGCGCCACAGAAGACGGGAGACACTAGC
LEU2-OUT-R	GTGCCCTCCTCCTTGTCAATATTAAT
HIS3-OUT-F	GCCTCGGTAATGATTTTCATTTTTTTTTTTCC
HIS3-OUT-R	GCGCCTCGTTCAGAATGACAC
HO-OUT-F	CTGGGCGTTATTAGGTGTGAAACC
HO-OUT-R	CGCGTAAATATTTAGCTTGATAACTGTTACTG
ROX1-YZ-F	CAGGTGCAGGCGTACTTTAAAG
ROX1-YZ-R	GCCACTTGAGCTTTCGGTTTG
HXK2-OUT-F	AACAAAGAAAAGGCAAGAGTAAATAGCG
HXK2-OUT-R	CGGTGTTCATCCAGGTGGTAATATC
GAL80-OUT-F	GTTGGCCTCTACTTACTCCATCGAC
pGAL4-SexAI	GCGACCTGGTCTTTCAGGAGGCTTGCTTCTCT
GAL4t-F	AATGAATCGTAGATACTGAAAAACCCC
GAL80-OUT-R	GGCATTATATGGCCGATTTGTATTAGTATCC
PDR3-YZ-F(207)	GGAGAGGAATCGTACACTCAAGAC
TEF1-PAC1-R	GCGTTAATTAATTTGTAATTAAAACTTAGATTAGATTGCTATGCTTTCTTTC
TEF1p-YZ-F(350bp)	GCCGTACCACTTCAAAACACC
INO2-R	TCAGGAATCATCCAGTATGTGCTGT

Primer name

Sequence (5′ to 3′)

3G-1-M-GAL7p-CWP2t-R

CCCTTAGCTGCGATTAAAGAGTCAAGGCGTGAAAAACATAGGCGAAAGTGGTGTCACAGCGAATTTCCTCAC

3G-2-M-GAL7p-CWP2t-F

GAACTTGTAAACAATTCGGTCCCTACATGTGAGGAAATTCGCTGTGACACCACTTTCGCCTATGTTTTTCACGC

GAL1p-R

TATAGTTTTTTCTCCTTGACGTTAAAGTATAGAGGTAT

GAL1p-F

TTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGGATGG

NDT80-50-CWP2t-F

TACTAACCTTTCATTAAAGAGAAATAACAATATTATAAAAAGCGCTTAAACACTTTCGCCTATGTTTTTCACGC

NDT80-50-PRM9t-down

GATCCACAGAATTCGCATATTTTTTTAACGATTTAAAATCATTAGTTTATTCAACATCGTATTTTCCGAAGCG

LEU2-50-CWP2t-F

TTTACATTTCAGCAATATATATATATATATTTCAAGGATATACCATTCTACACTTTCGCCTATGTTTTTCACGC

LEU2-50-PRM9t-down

CGGTGGTACTGTTGGAACCACCTAAATCACCAGTTCTGATACCTGCATCCTCAACATCGTATTTTCCGAAGCG

HIS3-50-CWP2t-F

CTTCGAAGAATATACTAAAAAATGAGCAGGCAAGATAAACGAAGGCAAAGCACTTTCGCCTATGTTTTTCACGC

HIS3-50-PRM9t-down

TATACACATGTATATATATCGTATGCTGCAGCTTTAAATAATCGGTGTCATCAACATCGTATTTTCCGAAGCG

HO-50-CWP2t-F

TCTAAATCCATATCCTCATAAGCAGCAATCAATTCTATCTATACTTTAAACACTTTCGCCTATGTTTTTCACGC

HO-50-PRM9t-down

ATTAAATTTTACTTTTATTACATACAACTTTTTAAACTAATATACACATTTCAACATCGTATTTTCCGAAGCG

GAL80-GAL4p-F

CCAGCGTATACAATCTCGATAGTTGGTTTCCCGTTCTTTCCACTCCCGTCGACAGCATTCGCCCAGTATTTTTTTTATTC

GAL4t-GAL80-R

GTTTTTATAACGTTCGCTGCACTGGGGGCCAAGCACAGGGCAAGATGCTTTTAACGTTCTACTTTACACTGGTGGTAGG

delta15-LEU2t-F

GTAGTTTTAAAATTTCAAATCCGAACAACAGAGCATAGGGTTTCGCAAACCGTTGAGCCATTAGTATCAATTTGCTTAC

URA3t-HIS3p-R

CGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAACTCTTCCTTTTTCAATGGGTAATAACTG

URA3t-HIS3p-F

AGAGCTTCAATTTAATTATATCAGTTATTACCCATTGAAAAAGGAAGAGTTCGCGCGTTTCGGTGATGA

HIS3t-ACT3p-R

ATGTTAGAACCACCTGGGCCAACAATCATATTGAATCTTCTGGTATCAGCGTGTCACTACATAAGAACACCTTTGGTG

HIS3t-ACT3p-F

ATGGTACCAACGATGTTCCCTCCACCAAAGGTGTTCTTATGTAGTGACACGCTGATACCAGAAGATTCAATATGATTGTTG

PDR3t-TEF1p-R

GTAAAAAAGGAGTAGAAACATTTTGAAGCTATGGTGTGTGGGGGATCACTCTTTTTGAACAGCGCGCCCA

PDR3t-TEF1p-F

ACATAAGATCAACGATTTCTTCATTAATTTTGGGCGCGCTGTTCAAAAAGAGTGATCCCCCACACACCAT

CYC1t-GAL1p-R

CTTTGCGTCCATCCAAAAAAAAAGTAAGAATTTTTGAAAATTCAATATAAGCGCGTTGGCCGATTCATTA

CYC1t-GAL1p-F

GGCTTTAATTTGCAAGCTGCGGCCCTGCATTAATGAATCGGCCAACGCGCTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGGA

Delta15-50-PRM9t-R

TCTGTATTGTTCTTCTTAGTGCTTGTATATGCTCATCCCGACCTTCCATTTCAACATCGTATTTTCCGAAGCGT

ROX1-KO-up-F

CCGACAAATCCCCTGGATATCATTG

ROX1-KO-up-R

ACTTTTATTAAACAGGAGCCTGTTGATTGTCTAACTGCGTTCTTTTGT

ROX1-KO-down-F

ACGCAGTTAGACAATCAACAGGCTCCTGTTTAATAAAAGTTTAAATCGCC

ROX1-KO-down-R

CGATTGTGTCTATATCTACAATATGAACCTG

HXK2-KO-UP-F

ATGTTACTACGAGTTTTCTGAACCTCCTC

HXK2-KO-UP-R

GTAGAAAAAGGGCACCTTCTTGTTGTTCAAACTTAATTTACAAATTAAGTTTTATTTAATTAGCGTACTTATTATGTGTGGAGAATT

HXK2-KO-DOWN-F

GTTGTAGGAATATAATTCTCCACACATAATAAGTACGCTAATTAAATAAAACTTAATTTGTAAATTAAGTTTGAACAACAAGAAGGT

HXK2-KO-DOWN-R

CGCGTAAAAATCGCGGGTAT

GAL10p-F(251bp)

CGGAGGGCTGTCGCC

AscI-CnVS-R

GCGGCGCGCCTTAAGGGATGATTGGTTCAACGAAAAACT

CsVal-YZ-R

CTATATCGATTCTAACAGAGCAGATTTGC

EgVal-YZ-R(486)

CAAACCTCTCGCGTCGTTAAC

GAL1p-F(349)

GCCGCACTGCTCCGA

ASC1-tHMG1-R

GCGGCGCGCCTTAGGATTTAATGCAGGTGACGGACC

NDT80-OUT-F

TGTTAATTCCTTTGTGCCAGATAGTATTG

NDT80-OUT-R

CACCCAATCTTCATTATTCACCAAATATTCCC

LEU2-OUT-F

GCTTGCATCACAATACTTGAAGTTGAC

ASC1-CWP2t-F

GGCGCGCCGAAATCTCTGATTTTTTATAATATCTATATGGCTTTTTTC

ASC1-PRM9t-F

GGCGCGCCACAGAAGACGGGAGACACTAGC

LEU2-OUT-R

GTGCCCTCCTCCTTGTCAATATTAAT

HIS3-OUT-F

GCCTCGGTAATGATTTTCATTTTTTTTTTTCC

HIS3-OUT-R

GCGCCTCGTTCAGAATGACAC

HO-OUT-F

CTGGGCGTTATTAGGTGTGAAACC

HO-OUT-R

CGCGTAAATATTTAGCTTGATAACTGTTACTG

ROX1-YZ-F

CAGGTGCAGGCGTACTTTAAAG

ROX1-YZ-R

GCCACTTGAGCTTTCGGTTTG

HXK2-OUT-F

AACAAAGAAAAGGCAAGAGTAAATAGCG

HXK2-OUT-R

CGGTGTTCATCCAGGTGGTAATATC

GAL80-OUT-F

GTTGGCCTCTACTTACTCCATCGAC

pGAL4-SexAI

GCGACCTGGTCTTTCAGGAGGCTTGCTTCTCT

GAL4t-F

AATGAATCGTAGATACTGAAAAACCCC

GAL80-OUT-R

GGCATTATATGGCCGATTTGTATTAGTATCC

PDR3-YZ-F(207)

GGAGAGGAATCGTACACTCAAGAC

TEF1-PAC1-R

GCGTTAATTAATTTGTAATTAAAACTTAGATTAGATTGCTATGCTTTCTTTC

TEF1p-YZ-F(350bp)

GCCGTACCACTTCAAAACACC

INO2-R

TCAGGAATCATCCAGTATGTGCTGT

Template	Primer name	Module
P_GAL10-CnVS-T_CWP2	NDT80-50-CWP2t-F	P_GAL10-CnVS-T_CWP2
GAL1p-R
P_GAL1-tHMG1-T_PRM9	GAL1p-F NDT80-50-PRM9t-R	P_GAL1-tHMG1-T_CWP2

Template

Primer name

Module

P_GAL10-CnVS-T_CWP2

NDT80-50-CWP2t-F

P_GAL10-CnVS-T_CWP2

GAL1p-R

P_GAL1-tHMG1-T_PRM9

GAL1p-F
NDT80-50-PRM9t-R

P_GAL1-tHMG1-T_CWP2

Strain	Template	Primer name	Module
VAL-02	P_GAL10-SmFPS-CnVS-T_CWP2	NDT80-50-CWP2t-F GAL1p-R	P_GAL10-SmFPS-CnVS-T_CWP2
P_GAL1-tHMG1-T_PRM9	GAL1p-F NDT80-50-PRM9t-R	P_GAL1-tHMG1-T_PRM9
VAL-03	P_GAL10-SmFPS-CsVal-T_CWP2	NDT80-50-CWP2t-F GAL1p-R	P_GAL10-SmFPS-CsVal-T_CWP2
P_GAL1-tHMG1-T_PRM9	GAL1p-F NDT80-50-PRM9t-R	P_GAL1-tHMG1-T_PRM9
VAL-04	P_GAL10-SmFPS-EgVal-T_CWP2	NDT80-50-CWP2t-F GAL1p-R	P_GAL10-SmFPS-EgVal-T_CWP2
P_GAL1-tHMG1-T_PRM9	GAL1p-F NDT80-50-PRM9t-R	P_GAL1-tHMG1-T_PRM9

Strain

Template

Primer name

Module

VAL-02

P_GAL10-SmFPS-CnVS-T_CWP2

NDT80-50-CWP2t-F
GAL1p-R

P_GAL10-SmFPS-CnVS-T_CWP2

P_GAL1-tHMG1-T_PRM9

GAL1p-F
NDT80-50-PRM9t-R

P_GAL1-tHMG1-T_PRM9

VAL-03

P_GAL10-SmFPS-CsVal-T_CWP2

NDT80-50-CWP2t-F
GAL1p-R

P_GAL10-SmFPS-CsVal-T_CWP2

P_GAL1-tHMG1-T_PRM9

GAL1p-F
NDT80-50-PRM9t-R

P_GAL1-tHMG1-T_PRM9

VAL-04

P_GAL10-SmFPS-EgVal-T_CWP2

NDT80-50-CWP2t-F
GAL1p-R

P_GAL10-SmFPS-EgVal-T_CWP2

P_GAL1-tHMG1-T_PRM9

GAL1p-F
NDT80-50-PRM9t-R

P_GAL1-tHMG1-T_PRM9

Strain	Template	Primer name	Module
VAL-05	P_GAL10-SmFPS-CnVS-T_CWP2	LEU2-50-CWP2t-F GAL1p-R	P_GAL10-SmFPS-CnVS-T_CWP2
P_GAL1-tHMG1-T_PRM9	GAL1p-F LEU2-50-PRM9t-R	P_GAL1-tHMG1-T_PRM9
VAL-06	P_GAL10-SmFPS-CnVS-T_CWP2	HIS3-50-CWP2t-F GAL1p-R	P_GAL10-SmFPS-CnVS-T_CWP2
P_GAL1-tHMG1-T_PRM9	GAL1p-F HIS3-50-PRM9t-R	P_GAL1-tHMG1-T_PRM9
VAL-07	P_GAL10-SmFPS-CnVS-T_CWP2	HO-50-CWP2t-F GAL1p-R	P_GAL10-SmFPS-CnVS-T_CWP2
P_GAL1-tHMG1-T_PRM9	GAL1p-F HO-50-PRM9t-R	P_GAL1-tHMG1-T_PRM9

Strain

Template

Primer name

Module

VAL-05

P_GAL10-SmFPS-CnVS-T_CWP2

LEU2-50-CWP2t-F
GAL1p-R

P_GAL10-SmFPS-CnVS-T_CWP2

P_GAL1-tHMG1-T_PRM9

GAL1p-F
LEU2-50-PRM9t-R

P_GAL1-tHMG1-T_PRM9

VAL-06

P_GAL10-SmFPS-CnVS-T_CWP2

HIS3-50-CWP2t-F
GAL1p-R

P_GAL10-SmFPS-CnVS-T_CWP2

P_GAL1-tHMG1-T_PRM9

GAL1p-F
HIS3-50-PRM9t-R

P_GAL1-tHMG1-T_PRM9

VAL-07

P_GAL10-SmFPS-CnVS-T_CWP2

HO-50-CWP2t-F
GAL1p-R

P_GAL10-SmFPS-CnVS-T_CWP2

P_GAL1-tHMG1-T_PRM9

GAL1p-F
HO-50-PRM9t-R

P_GAL1-tHMG1-T_PRM9

Template	Primer name	Module
Yeast genome	ROX1-KO-up-F ROX1-KO-up-R	ROX1-up
Yeast genome	ROX1-KO-down-F ROX1-KO-down-R	ROX1-down
ROX1-upand ROX1-down	ROX1-KO-up-F ROX1-KO-down-R	ROX1-KO
Yeast genome	HXK2-KO-UP-F HXK2-KO-UP-R	HXK2-up
Yeast genome	HXK2-KO-DOWN-F HXK2-KO-DOWN-R	HXK2-down
HXK2-upand HXK2-down	HXK2-KO-UP-F HXK2-KO-DOWN-R	HXK2-KO
Yeast genome	GAL80-GAL4p-F GAL4t-GAL80-R	P_GAL4-GAL4-T_GAL4

Template

Primer name

Module

Yeast genome

ROX1-KO-up-F
ROX1-KO-up-R

ROX1-up

Yeast genome

ROX1-KO-down-F
ROX1-KO-down-R

ROX1-down

ROX1-upand
ROX1-down

ROX1-KO-up-F
ROX1-KO-down-R

ROX1-KO

Yeast genome

HXK2-KO-UP-F
HXK2-KO-UP-R

HXK2-up

Yeast genome

HXK2-KO-DOWN-F
HXK2-KO-DOWN-R

HXK2-down

HXK2-upand
HXK2-down

HXK2-KO-UP-F
HXK2-KO-DOWN-R

HXK2-KO

Yeast genome

GAL80-GAL4p-F
GAL4t-GAL80-R

P_GAL4-GAL4-T_GAL4

Template	Primer name	Module
pRS425-LEU2-TRP1-URA3-TEF1p-ObGES	Delta15-LEU2t-F URA3t-HIS3p-R	LEU2-TRP1-URA3 Maker
NDT80-HIS3	URA3t-HIS3p-F HIS3t-ACT3p-R	HIS3 Maker
P_ACT3-PDR3m-T_PDR3	HIS3t-ACT3p-F PDR3t-TEF1p-R	P_ACT3-PDR3m-T_PDR3
pM3-tHMG1	PDR3t-TEF1p-F CYC1t-GAL1p-R	P_TEF1-tHMG1-T_CYC1
P_GAL1-INO2-T_PRM9	CYC1t-GAL1p-F Delta15-50-PRM9t-R	P_GAL1-INO2-T_PRM9

Template

Primer name

Module

pRS425-LEU2-TRP1-URA3-TEF1p-ObGES

Delta15-LEU2t-F
URA3t-HIS3p-R

LEU2-TRP1-URA3 Maker

NDT80-HIS3

URA3t-HIS3p-F
HIS3t-ACT3p-R

HIS3 Maker

P_ACT3-PDR3m-T_PDR3

HIS3t-ACT3p-F
PDR3t-TEF1p-R

P_ACT3-PDR3m-T_PDR3

pM3-tHMG1

PDR3t-TEF1p-F
CYC1t-GAL1p-R

P_TEF1-tHMG1-T_CYC1

P_GAL1-INO2-T_PRM9

CYC1t-GAL1p-F
Delta15-50-PRM9t-R

P_GAL1-INO2-T_PRM9